From Mechanism-Based Retaining Glycosidase Inhibitors to Activity-Based Glycosidase ProfilingClick to copy article linkArticle link copied!
- Marta ArtolaMarta ArtolaLeiden Institute of Chemistry, Leiden University, 2300 RA, Leiden, The NetherlandsMore by Marta Artola
- Johannes M. F. G. AertsJohannes M. F. G. AertsLeiden Institute of Chemistry, Leiden University, 2300 RA, Leiden, The NetherlandsMore by Johannes M. F. G. Aerts
- Gijsbert A. van der MarelGijsbert A. van der MarelLeiden Institute of Chemistry, Leiden University, 2300 RA, Leiden, The NetherlandsMore by Gijsbert A. van der Marel
- Carme RoviraCarme RoviraDepartament de Química Inorgànica I Orgànica & IQTCUB, Universitat de Barcelona, Barcelona 08028, SpainInstitució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08020, SpainMore by Carme Rovira
- Jeroen D. C. CodéeJeroen D. C. CodéeLeiden Institute of Chemistry, Leiden University, 2300 RA, Leiden, The NetherlandsMore by Jeroen D. C. Codée
- Gideon J. DaviesGideon J. DaviesDepartment of Chemistry, The University York, Heslington, York YO10 5DD, United KingdomMore by Gideon J. Davies
- Herman S. Overkleeft*Herman S. Overkleeft*Email: [email protected]Leiden Institute of Chemistry, Leiden University, 2300 RA, Leiden, The NetherlandsMore by Herman S. Overkleeft
Abstract
Activity-based protein profiling (ABPP) is an effective technology for the identification and functional annotation of enzymes in complex biological samples. ABP designs are normally directed to an enzyme active site nucleophile, and within the field of Carbohydrate-Active Enzymes (CAZymes), ABPP has been most successful for those enzymes that feature such a residue: retaining glycosidases (GHs). Several mechanism-based covalent and irreversible retaining GH inhibitors have emerged over the past sixty years. ABP designs based on these inhibitor chemistries appeared since the turn of the millennium, and we contributed to the field by designing a suite of retaining GH ABPs modeled on the structure and mode of action of the natural product, cyclophellitol. These ABPs enable the study of both exo- and endo-acting retaining GHs in human health and disease, for instance in genetic metabolic disorders in which retaining GHs are deficient. They are also finding increasing use in the study of GHs in gut microbiota and environmental microorganisms, both in the context of drug (de)toxification in the gut and that of biomass polysaccharide processing for future sustainable energy and chemistries. This account comprises the authors’ view on the history of mechanism-based retaining GH inhibitor design and discovery, on how these inhibitors served as blueprints for retaining GH ABP design, and on some current and future developments on how cyclophellitol-based ABPs may drive the discovery of retaining GHs and their inhibitors.
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
*Disclaimer
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
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Introduction
Figure 1
Figure 1. Mechanism of action of inverting β-glucosidases (A) and retaining β-glucosidases (B).
Mechanism-Based Retaining Glycosidase Inhibitor Designs
Figure 2
Figure 2. Two archetypal retaining glycosidase inhibitor designs: deoxyfluoroglycosides (A) and cyclitol epoxides (B).
Figure 3
Figure 3. Glycomimetic epoxides, aziridines and episulfides as mechanism-based, covalent and irreversible retaining glycosidase inhibitors.
The Advent of Activity-Based Retaining Glycosidase Profiling
Figure 4
Figure 4. Latent quinone methides (A) and activated fluorinated glycosides (B) as activity-based (retaining) GH probes. For the original images of the SDS-PAGE gels that are shown here and in the remainder of this account, please see the papers referred to in the text.
Cyclophellitol Aziridine Isosters in Activity-Based Retaining Exoglycosidase Profiling
Figure 5
Figure 5. Retaining GH ABPs based on cyclophellitol and cyclophellitol aziridine. A) Adaptation of the Madsen cyclophellitol synthesis to give cyclophellitol and cyclophellitol aziridine ABPs 44 and 48. B) Comparative ABPP of the human retaining β-glucosidases, GBA1, GBA2 and GBA3. C) Head-to-head comparison of activated fluorinated glucosides 49-52 and cyclophellitol 53 as GBA1 ABPs.
Cyclophellitol ABPs Act as Transition State Analogues in Respect of Conformation
Figure 6
Figure 6. A, B) Reaction coordinates by which retaining β-glucosidases (A) and retaining α-glucosidases (B) process their substrates. C) Computed free energy landscapes (FELs) of selected compounds.
Activity-Based Profiling of Retaining GHs Involved in Biomass Polysaccharide Turnover
Figure 7
Figure 7. A), Activity-based secretomes profiling of glucuronoarabinoxylan-degrading retaining arabinofuranosidases and xylanases. B) Xyloglucan-degrading retaining exoglucosidases, cellulases and xyloglucanases. Shown in the gel are samples of intact cells (C), cell extracts (L) and supernatant/secretomes (S) with ABPs 63-65.
Conclusion and Outlook
Acknowledgments
The work described in this review was supported by The Netherlands Organization for Scientific Research (NWO TOP grant 2018-714.018.002, to H.S.O.), the European Research Council (ERC-2011-AdG 290836 Chembiosphing, to H.S.O and ERC-2020-SyG 951231 Carbocentre, to H.S.O, C.R and G.J.D.), the Spanish Ministry of Science, Innovation and Universities (grants PID2020-118893GB-100MICIU/AEI /10.13039/501100011033 to C.R.), the Spanish Structures of Excellence María de Maeztu (CEX2021-001202-M, to C.R.), the Agency for Management of University and Research Grants of Catalonia (AGAUR, 2021-SGR-00680, to C.R.), and the Royal Society (Ken Murray Research Professorship to G.J.D.). We thank all undergraduate students, PhD students and postdoctoral researchers who did the actual work over the past decades and whose names are found on the cited papers. We thank Spencer J. Williams for his insightful and critical review of the manuscript. H.S.O. is thankful to the University of Melbourne (Australia) and the Norman Thomas Mortimer Wilsmore Research Fund for a Wilsmore Fellowship which enabled him to contemplate on mechanistic aspects on glycoside hydrolases and their inhibition. The Graphical abstract was created with Biorender. com.
References
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- 5Compounds that react within the enzyme active site to form a covalent and irreversible bond, thereby irreversibly inactivating the enzyme, are sometimes referred to as ‘targeted covalent inhibitors’, ‘suicide inhibitors’ or ‘activity-based inhibitors’. In line with other practitioners in the field (see also the titles of some of the references below) we have chosen to use the term ‘mechanism-based inhibitors’, which is also the term recommended by IUPAC. See also https://iupac.qmul.ac.uk/gtpoc/M.html.Google ScholarThere is no corresponding record for this reference.
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- 7Watts, A. F.; Damager, I.; Amaya, M. L.; Buschiazzo, A.; Alzari, P.; Frasch, A. C.; Withers, S. G. Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate. J. Am. Chem. Soc. 2003, 125, 7532– 7533, DOI: 10.1021/ja0344967Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktFGqt7c%253D&md5=b9728e3301bf0c453d7a2a3126413bd9Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: Tyrosine is the catalytic nucleophileWatts, Andrew G.; Damager, Iben; Amaya, Maria L.; Buschiazzo, Alejandro; Alzari, Pedro; Frasch, Alberto C.; Withers, Stephen G.Journal of the American Chemical Society (2003), 125 (25), 7532-7533CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Modified sialic acid substrates have been used to label Trypanosoma cruzi trans-sialidase, demonstrating that the enzyme catalyzes the transfer of sialic acid through a covalent glycosyl-enzyme intermediate, a mechanism common to most retaining glycosidases. Peptic digestion of labeled protein, followed by LC-MS/MS anal. of the digest, identified Tyr 342 as the catalytic nucleophile. This is the first such example of a retaining glycosidase utilizing an aryl glycoside intermediate. It is suggested that this alternative choice of nucleophile is a consequence of the chem. nature of sialic acid. A Tyr/Glu couple is invoked to relay charge from a remote glutamic acid, thereby avoiding electrostatic repulsion with the sialic acid carboxylate group.
- 8McGregor, N. G. S.; Coines, J.; Borlandelli, V.; Amaki, S.; Artola, M.; Nin-Hill, A.; Linzel, D.; Yamada, C.; Arakawa, T; Ishiwata, A.; Ito, Y.; van der Marel, G. A.; Codée, J. D. C.; Fushinobu, S.; Overkleeft, H. S.; Rovira, C.; Davies, G. J. Cysteine nucleophiles in glycosidase catalysis: application of a covalent β-L-arabinofuranosidase inhibitor. Angew. Chem., Int. Ed. 2021, 60, 5754– 5758, DOI: 10.1002/anie.202013920Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjtVSrurk%253D&md5=19db6aa6f66d9d81f9d5fddc61238a63Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β-L-Arabinofuranosidase InhibitorMcGregor, Nicholas G. S.; Coines, Joan; Borlandelli, Valentina; Amaki, Satoko; Artola, Marta; Nin-Hill, Alba; Linzel, Daniel; Yamada, Chihaya; Arakawa, Takatoshi; Ishiwata, Akihiro; Ito, Yukishige; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Fushinobu, Shinya; Overkleeft, Herman S.; Rovira, Carme; Davies, Gideon J.Angewandte Chemie, International Edition (2021), 60 (11), 5754-5758CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived β-L-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This β-L-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures detd. for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chem. of cyclophellitol derivs., the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.
- 9Dennis, R. J.; Taylor, E. J.; Macauley, M. S.; Stubbs, K. A.; Turkenburg, J. P.; Hart, S. J.; Black, G. N.; Vocadlo, D. J.; Davies, G. J. Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity. Nat. Struct. Mol. Biol. 2006, 13, 365– 371, DOI: 10.1038/nsmb1079Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xjs1OrtL8%253D&md5=2fec47f64f348ca3f6a143c37589ddcdStructure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activityDennis, Rebecca J.; Taylor, Edward J.; Macauley, Matthew S.; Stubbs, Keith A.; Turkenburg, Johan P.; Hart, Samuel J.; Black, Gary N.; Vocadlo, David J.; Davies, Gideon J.Nature Structural & Molecular Biology (2006), 13 (4), 365-371CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.
- 10Sobala, L. F.; Speciale, G.; Zhu, S.; Raich, L.; Sannikova, N.; Thompson, A. J.; Hakki, Z.; Lu, D.; Shansi Kazem Abadi, S.; Lewis, A. R.; Rojas-Cervellera, V.; Bernardo-Seisdedos, G.; Zhang, Y.; Millet, O.; Jiménez-Barbero, J.; Bennet, A. J.; Sollogoub, M.; Rovira, C.; Davies, G. J.; Williams, S. J. An epoxide intermediate in glycosidase catalysis. ACS Cent. Sci. 2020, 6, 760– 770, DOI: 10.1021/acscentsci.0c00111Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntFWhtL4%253D&md5=5d6812d3576863c1f633f6ea3b1534c3An epoxide intermediate in glycosidase catalysisSobala, Lukasz F.; Speciale, Gaetano; Zhu, Sha; Raich, Lluis; Sannikova, Natalia; Thompson, Andrew J.; Hakki, Zalihe; Lu, Dan; Shamsi Kazem Abadi, Saeideh; Lewis, Andrew R.; Rojas-Cervellera, Victor; Bernardo-Seisdedos, Ganeko; Zhang, Yongmin; Millet, Oscar; Jimenez-Barbero, Jesus; Bennet, Andrew J.; Sollogoub, Matthieu; Rovira, Carme; Davies, Gideon J.; Williams, Spencer J.ACS Central Science (2020), 6 (5), 760-770CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Retaining glycoside hydrolases cleave their substrates through stereochem. retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallog. snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/mol. mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/Km) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymic mechanism. Mannosidases of glycoside hydrolase family 99 use a neighboring-group participation mechanism involving the substrate 2-hydroxyl.
- 11Withers, S. G.; Street, I. P.; Bird, P.; Dolphin, D. H. 2-Deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitors. J. Am. Chem. Soc. 1987, 109, 7530– 7531, DOI: 10.1021/ja00258a047Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlajsbY%253D&md5=8ee52f1c5833b57cd6887aca4a0d80fb2-Deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitorsWithers, Stephen G.; Street, Ian P.; Bird, Paul; Dolphin, David H.Journal of the American Chemical Society (1987), 109 (24), 7530-1CODEN: JACSAT; ISSN:0002-7863.2,4-Dinitrophenyl 2-deoxy-2-fluoro β-D-glucopyranoside was synthesized and demonstrated to be a specific mechanism-based inhibitor for β-glucosidase from Alcaligenes faecalis. The inactivation of the enzyme followed pseudo-1st-order kinetics with a rate const. of 25 min-1 and a dissocn. const. of 0.05 mM. Protection against inactivation was shown by the competitive inhibitor isopropylthio β-D-glucopyranoside. Reactivation of the enzyme was obsd. as expected after removal of free inhibitor and incubation in the presence of substrate. The use of such inhibitors to investigate the intermediacy of a glucosyl enzyme intermediate and to identify the active site residue involved is projected.
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- 18Legler, G.; Bause, E. Epoxy-alkyl oligo-(1–4)-β-D-glucosides as active-site-directed inhibitors of cellulases. Carbohydr. Res. 1973, 28, 45– 52, DOI: 10.1016/S0008-6215(00)82855-4Google ScholarThere is no corresponding record for this reference.
- 19Rodriguez, E. B.; Scally, G. D.; Stick, R. V. The synthesis of optically pure epoxy-alkyl β-D-glucosides and β-cellobiosides as active-site directed inhibitors of some β-glucan hydrolases. Aust. J. Chem. 1990, 43, 1391– 1405, DOI: 10.1071/CH9901391Google ScholarThere is no corresponding record for this reference.
- 20Sulzenbacher, G.; Schülein, M.; Davies, G. J. Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Å resolution. Biochemistry 1997, 36, 5902– 5911, DOI: 10.1021/bi962963+Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXislSltrc%253D&md5=92928c9b648ae140ed1dbd23e0423183Structure of the endoglucanase I from Fusarium oxysporum: Native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Å resolutionSulzenbacher, Gerlind; Schuelein, Martin; Davies, Gideon J.Biochemistry (1997), 36 (19), 5902-5911CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The crystal structure of F. oxysporum cellulase endoglucanase I (I) of glycosyl hydrolase family 7 was solved at 2.3 Å resoln. In addn. to the native enzyme, structures were also detd. of I complexed with both the affinity label, 3,4-epoxybutyl β-D-cellobioside, and the reaction product, cellobiose. The affinity label was covalently bound, as expected, to the catalytic nucleophile, Glu-197, with clear evidence for binding of both the R and S stereoisomers. Cellobiose was found bound to the -2 and -1 subsites of I. In marked contrast to the previously reported crystal structure of I complexed with a nonhydrolyzable thiosaccharide analog, which spanned the -2, -1, and +1 subsites and which had a skew-boat conformation for the -1 subsite sugar, the I-cellobiose complex showed no pyranoside ring distortion in the -1 subsite, implying that strain is induced primarily by the addnl. +1 subsite interactions and that the product is found, as expected, in its unstrained conformation.
- 21Thomas, E. W.; McKelvy, J. F.; Sharon, N. Specific and irreversible inhibition of lysozyme by 2′,3′-epoxypropyl β-glycosides of N-acetyl-D-glucosamine oligomers. Nature 1969, 222, 485– 486, DOI: 10.1038/222485a0Google ScholarThere is no corresponding record for this reference.
- 22Havukainen, R.; Törrönen, A.; Laitinen, T.; Rouvinen, J. Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei. Biochemistry 1996, 35, 9617– 9624, DOI: 10.1021/bi953052nGoogle Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjvVynsb0%253D&md5=0d5a0a723343c9f0b9d54e297fcee8f3Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reeseiHavukainen, Riikka; Torronen, Anneli; Laitinen, Tuomo; Rouvinen, JuhaBiochemistry (1996), 35 (29), 9617-9624CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The three-dimensional structures of endo-1,4-xylanase II from (XYNII) Trichoderma reesei complexed with 4,5-epoxypentyl β-D-xyloside (X-O-C5), 3,4-epoxybutyl β-D-xyloside (X-O-C4), and 2,3-epoxypropyl β-D-xyloside (X-O-C3) were detd. by x-ray crystallog. High-resoln. measurement revealed clear electron densities for each ligand. Both X-O-C5 and X-O-C3 were found to form a covalent bond with the putative nucleophile Glu86. Unexpectedly, X-O-C4 was found to bind to the putative acid/base catalyst Glu177. In all three complexes, clear conformational changes were found in XYNII compared to the native structure. These changes were largest in the X-O-C3 complex structure.
- 23Liu, L.; Patricelli, M. P.; Cravatt, B. F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 1999, 96, 14694– 14699, DOI: 10.1073/pnas.96.26.14694Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhtFaitw%253D%253D&md5=d5d1a09328fc7715e9d30ca392733426Activity-based protein profiling: the serine hydrolasesLiu, Yongsheng; Patricelli, Matthew P.; Cravatt, Benjamin F.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (26), 14694-14699CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)With the postgenome era rapidly approaching, new strategies for the functional anal. of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chem. synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue exts., we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Addnl., we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
- 24Yariv, J.; Wilson, K. J.; Hildesheim, J.; Blumberg, S. Labelling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamine. FEBS Lett. 1971, 15, 24– 26, DOI: 10.1016/0014-5793(71)80070-4Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3MXkvVCksrk%253D&md5=d0807f7b2b5b9269861a9c58be849a22Labeling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamineYariv, Joseph; Wilson, Kenneth J.; Hildesheim, Jean; Blumberg, ShmaryahuFEBS Letters (1971), 15 (1), 24-6CODEN: FEBLAL; ISSN:0014-5793.The hydrolysis of o-nitrophenyl β-galactopyranoside by β-galactosidase was inhibited by incubating with labeled N-bromoacetyl β-D-galactopyranosylamine. The reaction followed 1st order kinetics leading to complete inactivation and 1 mole of reagent was bound to the enzyme per mole of site inactivated. The corresponding L-fucose deriv. did not inactivate the enzyme.
- 25Caron, G.; Withers, S. G. Conduritol aziridine: a new mechanism-based glucosidase inactivator. Biochem. Biophys. Res. Commun. 1989, 163, 495– 499, DOI: 10.1016/0006-291X(89)92164-5Google ScholarThere is no corresponding record for this reference.
- 26Tong, M. K.; Ganem, B. A potent new class of active-site-directed glycosidase inactivators. J. Am. Chem. Soc. 1988, 110, 312– 313, DOI: 10.1021/ja00209a062Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXjvVamtw%253D%253D&md5=86e63225e2f3b6e08ede5e42804dc0ecA potent new class of active-site-directed glycosidase inactivatorsTong, Michael K.; Ganem, BruceJournal of the American Chemical Society (1988), 110 (1), 312-13CODEN: JACSAT; ISSN:0002-7863.Aziridine I was prepd. from piperidine II in 6 steps. I showed potent inhibition of green coffee bean α-galactosidase, but had little or no effect on yeast α-glucosidase, jackbean α-mannosidase, or bovine β-galactosidase.
- 27Atsumi, S.; Umezawa, K.; Iinuma, H.; Naganawa, H.; Nakamura, H.; Iitaka, Y.; Takeuchi, T. Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus sp. J. Antibiot. 1990, 43, 49– 53, DOI: 10.7164/antibiotics.43.49Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhvFChu78%253D&md5=3d007c5da7425376dda4cb11e639c208Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus spAtsumi, Sonoko; Umezawa, Kazuo; Iinuma, Hironobu; Naganawa, Hiroshi; Nakamura, Hikaru; Iitaka, Yoichi; Takeuchi, TomioJournal of Antibiotics (1990), 43 (1), 49-53CODEN: JANTAJ; ISSN:0021-8820.A culture filtrate of a mushroom, Phellinus species, strongly inhibited β-glucosidase. The active substance was isolated through charcoal sepn., column chromatog. and crystn. Spectroscopic and crystallog. anal. revealed that it had a novel cyclitol structure, (1S,2R,3S,4R,5R,6R)-5-hydroxymethyl-7-oxabicyclo[4.1.0]heptane-2,3,4-triol; the compd. was named cyclophellitol (I). It inhibited almond-derived β-glucosidase with an IC50 of 0.8 μg/mL.
- 28Gloster, T. M.; Madsen, R.; Davies, G. J. Structural basis for cyclophellitol inhibition of a β-glucosidase. Org. Biomol. Chem. 2007, 5, 444– 446, DOI: 10.1039/B616590GGoogle Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXoslSnsw%253D%253D&md5=f1d62eeec2e2483e3fa386679b229622Structural basis for cyclophellitol inhibition of a β-glucosidaseGloster, Tracey M.; Madsen, Robert; Davies, Gideon J.Organic & Biomolecular Chemistry (2007), 5 (3), 444-446CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)The structural basis for β-glucosidase inhibition by cyclophellitol was demonstrated using x-ray crystallog., enzyme kinetics and mass spectrometry.
- 29Nakata, M.; Chong, C.; Niwata, Y.; Toshima, K.; Tatsuta, K. A family of cyclophellitol analogues: synthesis and evaluation. J. Antibiot. 1993, 46, 1919– 1922, DOI: 10.7164/antibiotics.46.1919Google ScholarThere is no corresponding record for this reference.
- 30Shing, T. K. M.; Tai, V. W.-F. (-)-Quinic acid in organic synthesis. Part 4. Synthesis of cyclophellitol and its (1R,6S)-, (2S)-, (1R,2S,6S)-diastereomers. J. Chem. Soc. Perkin Trans 1. 1994, 2017– 2025, DOI: 10.1039/P19940002017Google ScholarThere is no corresponding record for this reference.
- 31Greenbaum, F.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 2000, 7, 569– 581, DOI: 10.1016/S1074-5521(00)00014-4Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnt1WnsbY%253D&md5=4bfc0e299524198ec324c96c8981baf7Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery toolsGreenbaum, Doron; Medzihradszky, Katalin F.; Burlingame, Alma; Bogyo, MatthewChemistry & Biology (2000), 7 (8), 569-581CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Science Ltd.)Background: Anal. of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly detg. activities of an entire class of enzymes on a genome-wide scale would be of great utility. Results: We have engineered chem. probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes contg. a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivs. was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular exts. Finally, the affinity tag of DCG-04 allowed purifn. of modified proteases and identification by mass spectrometry. Conclusions: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixts. These probes were used to det. relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purifn./identification of targets.
- 32Janda, K. D.; Lo, L.-C.; Lo, C.-H. L.; Sim, M.-M.; Wang, R.; Wong, C.-H.; Lerner, R. A. Chemical selection for catalysis in combinatorial libraries. Science 1997, 275, 945– 948, DOI: 10.1126/science.275.5302.945Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXht1Cls7s%253D&md5=b03752f229221f9e2e2dd2e78cd9e5ddChemical selection for catalysis in combinatorial antibody librariesJanda, Kim D.; Lo, Lee-Chiang; Lo, Chih-Hung L.; Sim, Mui-Mui; Wang, Ruo; Wong, Chi-Huey; Lerner, Richard A.Science (Washington, D. C.) (1997), 275 (5302), 945-948CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)For the past decade the immune system has been exploited as a rich source of de novo catalysts. Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chem. reactions. In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes. Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function. A method that allows for the direct chem. selection for catalysis from antibody libraries was so devised, whereby the pos. aspects of hybridoma technol. were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis. This methodol. is based on a purely chem. selection process, making it more general than biol. based selection systems because it is not limited to reaction products that perturb cellular machinery.
- 33Tsai, C.-S.; Li, Y.-K.; Lo, L.-C. Design and synthesis of activity probes for glycosidases. Org. Lett. 2002, 4, 3607– 3610, DOI: 10.1021/ol0265315Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XnslansrY%253D&md5=a1afa3a47c57335d6ef7e75b405a7666Design and Synthesis of Activity Probes for GlycosidasesTsai, Charng-Sheng; Li, Yaw-Kuen; Lo, Lee-ChiangOrganic Letters (2002), 4 (21), 3607-3610CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)A new synthetic route was developed for the prepn. of glycoside I activity probe for β-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe I was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.
- 34Lu, C.-P.; Ren, C.-T.; Lai, Y.-N.; Wu, S.-H.; Wang, W.-M.; Chen, J.-Y.; Lo, L.-C. Design of a mechanism-based probe for neuraminidase to capture influenza viruses. Angew. Chem., Int. Ed. 2005, 44, 6888– 6892, DOI: 10.1002/anie.200501738Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1alsr7O&md5=b857fd16e902f49bdef55623818f8c0cDesign of a mechanism-based probe for neuraminidase to capture influenza virusesLu, Chun-Ping; Ren, Chien-Tai; Lai, Yi-Ning; Wu, Shih-Hsiung; Wang, Wei-Man; Chen, Jean-Yin; Lo, Lee-ChiangAngewandte Chemie, International Edition (2005), 44 (42), 6888-6892CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A mechanism-based probe for neuraminidase was developed. The probe consisted of a recognition head (N-acetylneuraminic acid), a trapping device (o-difluoromethylphenyl group), a linker, and a reporter group (biotin). It is capable of forming a biotinylated adduct with neuraminidase (NA) from Arthrobacter ureafaciens. It also displays an inhibitory effect on a no. of NA activities. ELISA expts. successfully demonstrated that influenza viruses can be selectively captured with this probe.
- 35Kwan, D. H.; Chen, H.-M.; Ratananikom, K.; Hancock, S. M.; Watanabe, Y.; Kongsaeree, P. T.; Samuels, A. L.; Withers, S. G. Self-immobilizing fluorogenic imaging agents of enzyme activity. Angew. Chem., Int. Ed. 2011, 50, 300– 303, DOI: 10.1002/anie.201005705Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1alu7rM&md5=ccaedba2806b8764cdcfaaf00f39ffccSelf-Immobilizing Fluorogenic Imaging Agents of Enzyme ActivityKwan, David H.; Chen, Hong-Ming; Ratananikom, Khakhanang; Hancock, Susan M.; Watanabe, Yoichiro; Kongsaeree, Prachumporn T.; Samuels, A. Lacey; Withers, Stephen G.Angewandte Chemie, International Edition (2011), 50 (1), 300-303, S300/1-S300/35CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)We have demonstrated a novel application for enzyme substrates that generate quinone methides. Such substrates have previously been used as enzyme inactivators and abused as proteomics probes; however, these compds. may find greater value as histol. or cell-labeling agents for the investigation of a wide variety of organisms, as illustrated by our results with plants, yeast, and bacteria.
- 36Vocadlo, D. J.; Bertozzi, C. R. A strategy for functional proteomic analysis of glycosidase activity from cell lysates. Angew. Chem., Int. Ed. 2004, 43, 5338– 5342, DOI: 10.1002/anie.200454235Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXptVanuro%253D&md5=784a8ee5408d8d25a1d46a771b8de249A strategy for functional proteomic analysis of glycosidase activity from cell lysatesVocadlo, David J.; Bertozzi, Carolyn R.Angewandte Chemie, International Edition (2004), 43 (40), 5338-5342CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)We have developed a strategy for activity-based labeling of retaining glycosidases by using the azide group as a sterically unobtrusive chem. tag. We prepd. 6-azido-2,6-dideoxy-2-fluoro-β-D-galactosyl fluoride (6Az2FgalF, 10, Scheme 2) as an activity-based probe for β-galactosidase. The high selectivity of both the inactivation with fluorosugars and the Staudinger ligation with phosphine probes allows detection of glycosidases in complex mixts. and the strategy can be used for profiling these enzyme activities in cell lysates. We have demonstrated that the approach can be used to tag several glycosidases from different glycoside hydrolase families. We anticipate that the strategy will find broad utility in proteomic anal. of these enzymes in prokaryotic and eukaryotic proteomes. The azide group might also be useful as a chem. tag within inactivators of other enzymes from entirely different families with sterically confining active sites.
- 37Gebler, J. C.; Aebersold, R.; Withers, S. G. Glu-537, not Glu-461, is the nucleophile in the active site of (LacZ) b-galactosidase from Escherichia coli. J. Biol. Chem. 1992, 267, 11126– 11130, DOI: 10.1016/S0021-9258(19)49884-0Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xls1KmtL0%253D&md5=42f3ec2cd10a75e454afb7eb65483be6Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) β-galactosidase from Escherichia coliGebler, John C.; Aebersold, Ruedi; Withers, Stephen G.Journal of Biological Chemistry (1992), 267 (16), 11126-30CODEN: JBCHA3; ISSN:0021-9258.The covalent intermediate formed during catalysis by the lac Z β-galactosidase from E. coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-β-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apoenzymes have been detd. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous β-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M.; Legler, G., 1984) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.
- 38Stubbs, K. A.; Scaffidi, A.; Debowski, A. W.; Mark, B. L.; Stick, R. V.; Vocadlo, D. J. Synthesis and use of mechanism-based protein profiling probes for retaining b-D-glucosaminidases facilitate identification of Pseudomonas aeruginosa NagZ. J. Am. Chem. Soc. 2008, 130, 327– 335, DOI: 10.1021/ja0763605Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhsVehurzE&md5=cf11627fe98c1f871d6a6711461194c4Synthesis and Use of Mechanism-Based Protein-Profiling Probes for Retaining β-D-Glucosaminidases Facilitate Identification of Pseudomonas aeruginosa NagZStubbs, Keith A.; Scaffidi, Adrian; Debowski, Aleksandra W.; Mark, Brian L.; Stick, Robert V.; Vocadlo, David J.Journal of the American Chemical Society (2008), 130 (1), 327-335CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The NagZ class of retaining exo-glucosaminidases play a crit. role in peptidoglycan recycling in Gram-neg. bacteria and the induction of resistance to β-lactams. Here we describe the concise synthesis of 2-azidoacetyl-2-deoxy-5-fluoro-β-D-glucopyranosyl fluoride as an activity-based proteomics probe for profiling these exo-glycosidases. This active-site directed reagent covalently inactivates this class of retaining N-acetylglucosaminidases with exquisite selectivity by stabilizing the glycosyl-enzyme intermediate. Inactivated Vibrio cholerae NagZ can be elaborated with biotin or a FLAG-peptide epitope using the Staudinger ligation or the Sharpless-Meldal click reaction and detected at nanogram levels. This ABPP enabled the profiling of the Pseudomonas aeruginosa proteome and identification at endogenous levels of a tagged protein with properties consistent with those of PA3005. Cloning of the gene encoding this hypothetical protein and biochem. characterization enabled unambiguous assignment of this hypothetical protein as a NagZ. The identification and cloning of this NagZ may facilitate the development of strategies to circumvent resistance to β-lactams in this human pathogen. As well, this general strategy, involving such 5-fluoro inactivators, may prove to be of general use for profiling proteomes and identifying glycoside hydrolases of medical importance or having desirable properties for biotechnol.
- 39Tsai, C.-S.; Yen, H.-Y.; Lin, M.-I.; Tsai, T.-I.; Wang, S.-Y.; Huang, W.-I.; Hsu, T.-L.; Cheng, Y. S. E.; Fang, J.-M.; Wong, C.-H. Cell-permeable probe for identification and imaging of sialidases. Proc. Natl. Acad. Sci. USA 2013, 110, 2466– 2471, DOI: 10.1073/pnas.1222183110Google ScholarThere is no corresponding record for this reference.
- 40Hekmat, O.; Kim, Y.-W.; Williams, S. J.; He, S.; Withers, S. G. Active-site “fingerprinting” of glycosidases in complex mixture by mass spectrometry. Discovery of a novel retaining b-1–4-glycanase in Cellulomonas fimi. J. Biol. Chem. 2005, 280, 35126– 35135, DOI: 10.1074/jbc.M508434200Google ScholarThere is no corresponding record for this reference.
- 41Chauvigné-Hines, L. M.; Anderson, L. N.; Weaver, H. M.; Brown, J. N.; Koech, P. K.; Nicora, C. D.; Hofstad, B. A.; Smith, R. D.; Wilkins, M. J.; Callister, S. J.; Wright, A. T. Suite of activity-based probes for cellulose-degrading enzymes. J. Am. Chem. Soc. 2012, 134, 20521– 20532, DOI: 10.1021/ja309790wGoogle Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhslalu77O&md5=f36917c45e720a70f786575722ae99fdSuite of Activity-Based Probes for Cellulose-Degrading EnzymesChauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.Journal of the American Chemical Society (2012), 134 (50), 20521-20532CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Microbial glycoside hydrolases play a dominant role in the biochem. conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits contg. cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degrdn. of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome-producing bacterium that couples rapid reprodn. rates to highly efficient degrdn. of cryst. cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycon, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chem. and to increase enzyme active site inclusion for liq. chromatog.-mass spectrometry (LC-MS) anal. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addn. to a large collection of integral cellulosome-contg. proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater understanding of the organismal role assocd. with biofuel development.
- 42Street, I. P.; Kempton, J. B.; Withers, S. G. Inactivation of a b-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-a-D-glucopyranosyl-enzyme intermediate: a detailed investigation. Biochemistry 1992, 31, 9970– 9978, DOI: 10.1021/bi00156a016Google ScholarThere is no corresponding record for this reference.
- 43Hansen, F. G.; Bundgaard, E.; Madsen, R. A short synthesis of (+)-cyclophellitol. J. Org. Chem. 2005, 70, 10139– 10142, DOI: 10.1021/jo051645qGoogle Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFahs7zF&md5=24abc432bcb3877a1c17ca1d3effc1c7A Short Synthesis of (+)-CyclophellitolHansen, Flemming Gundorph; Bundgaard, Eva; Madsen, RobertJournal of Organic Chemistry (2005), 70 (24), 10139-10142CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)A new synthesis of (+)-cyclophellitol, a potent β-glucosidase inhibitor, has been completed in nine steps from D-xylose. The key transformations involve a zinc-mediated fragmentation of benzyl-protected Me 5-deoxy-5-iodo-xylofuranoside followed by a highly diastereoselective indium-mediated coupling with Et 4-bromocrotonate. Subsequent ring-closing olefin metathesis, ester redn., olefin epoxidn., and deprotection then afford the natural product. This constitutes the shortest synthesis of (+)-cyclophellitol reported to date.
- 44Witte, M. D.; Kallemeijn, W. W.; Aten, J.; Li, K.-Y.; Strijland, A.; Donker-Koopman, W. E.; Blijlevens, B.; Kramer, G.; van den Nieuwendijk, A. M. C. H.; Florea, B. I.; Hooibrink, B.; Hollak, C. E. M.; Ottenhoff, R.; Boot, R. G.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. F. G. Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat. Chem. Biol. 2010, 6, 907– 913, DOI: 10.1038/nchembio.466Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVansb7N&md5=e9ead74400f958f1d4d2d5f5402d5c2fUltrasensitive in situ visualization of active glucocerebrosidase moleculesWitte, Martin D.; Kallemeijn, Wouter W.; Aten, Jan; Li, Kah-Yee; Strijland, Anneke; Donker-Koopman, Wilma E.; van den Nieuwendijk, Adrianus M. C. H.; Bleijlevens, Boris; Kramer, Gertjan; Florea, Bogdan I.; Hooibrink, Berend; Hollak, Carla E. M.; Ottenhoff, Roelof; Boot, Rolf G.; van der Marel, Gijsbert A.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.Nature Chemical Biology (2010), 6 (12), 907-913CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA mols. in situ. The authors here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA mols. in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, the authors obtained exclusive labeling of GBA mols. The authors present evidence from fluorescence-activated cell sorting anal., fluorescence microscopy and pulse-chase expts. of highly efficient labeling of GBA mols. in intact cells as well as tissues of mice. In addn., the authors illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.
- 45Li, K.-Y.; Jiang, J.; Witte, M. D.; Kallemeijn, W. W.; van den Elst, H.; Wong, C.-S.; Chander, S. D.; Hoogendoorn, S.; Beenakker, T. J. M.; Codée, J. D. C.; Aerts, J. M. F. G.; van der Marel, G. A.; Overkleeft, H. S. Synthesis of cyclophellitol, cyclophellitol aziridine, and their tagged derivatives. Eur. J. Org. Chem. 2014, 2014, 6030– 6043, DOI: 10.1002/ejoc.201402588Google ScholarThere is no corresponding record for this reference.
- 46Goddard-Borger, E. D.; Wennekes, T.; Withers, S. G. Getting lucky in the lysosome. Nat. Chem. Biol. 2010, 6, 881– 883, DOI: 10.1038/nchembio.470Google ScholarThere is no corresponding record for this reference.
- 47Wennekes, T.; van den Berg, R. J. B. H. N.; Donker, W.; van der Marel, G. A.; Strijland, A.; Aerts, J. M. F. G.; Overkleeft, H. S. Development of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivatives as selective inhibitors of glucosylceramide metabolism in man. J. Org. Chem. 2007, 72, 1088– 1097, DOI: 10.1021/jo061280pGoogle Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXot1Wgug%253D%253D&md5=8fb323274659673260e9aa98b90d7c05Development of Adamantan-1-yl-methoxy-Functionalized 1-Deoxynojirimycin Derivatives as Selective Inhibitors of Glucosylceramide Metabolism in ManWennekes, Tom; Van den Berg, Richard J. B. H. N.; Donker, Wilma; van der Marel, Gijsbert A.; Strijland, Anneke; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Journal of Organic Chemistry (2007), 72 (4), 1088-1097CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)The synthesis of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivs., e.g. I, via reductive ring opening, Swern oxidn. and reductive amination, is reported. The compds. reported are lipophilic iminosugar based on lead compd. II, a potent inhibitor of the three enzymes involved in the metab. of the glycosphingolipid glucosylceramide. The results demonstrate that relocating the lipophilic moiety from the nitrogen atom to other positions on the 1-deoxynojirimycin ring system does not lead to a more potent or selective inhibitor of glucosylceramide synthase. The β-aza-C-glycoside analog I retained the best inhibitory potency for glucosylceramide synthase and is a more potent inhibitor than the therapeutic agent N-butyl-1-deoxynojirimycin, marketed as treatment for Gaucher disease under the com. name Zavesca.
- 48Kallemeijn, W. W.; Li, K.-Y.; Witte, M. D.; Marques, A. R. A.; Aten, J.; Scheij, S.; Jiang, J.-B; Willems, L. I.; Voorn-Brouwer, T. M.; van Roomen, C. P. A. A.; Ottenhoff, R.; Boot, R. G.; van den Elst, H.; Walvoort, M. T. C.; Florea, B. I.; Codée, J. D. C.; van der Marel, G. A.; Aerts, J. M. F. G.; Overkleeft, H. S. Novel activity-based probes for broad-spectrum profiling of retaining beta-exoglucosidases in situ and in vivo. Angew. Chem., Int. Ed. 2012, 51, 12529– 12533, DOI: 10.1002/anie.201207771Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1GgsL3N&md5=6ce5afb5305786283c0abf83f603594eNovel Activity-Based Probes for Broad-Spectrum Profiling of Retaining β-Exoglucosidases In Situ and In VivoKallemeijn, Wouter W.; Li, Kah-Yee; Witte, Martin D.; Marques, Andre R. A.; Aten, Jan; Scheij, Saskia; Jiang, Jianbing; Willems, Lianne I.; Voorn-Brouwer, Tineke M.; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Boot, Rolf G.; van den Elst, Hans; Walvoort, Marthe T. C.; Florea, Bogdan I.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Angewandte Chemie, International Edition (2012), 51 (50), 12529-12533CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases. These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.
- 49Walvoort, M. T. C.; Kallemeijn, W. W.; Willems, L. I.; Witte, M. D.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. Tuning the leaving group in 2-deoxy-2-fluoroglucoside results in improved activity-based retaining beta-glucosidase probes. Chem. Commun. 2012, 48, 10386– 10388, DOI: 10.1039/c2cc35653hGoogle ScholarThere is no corresponding record for this reference.
- 50Yu, B.; Tao, H. Glycosyl trifluoroacetimidates. Part 1: preparation and application as new glycosyl donors. Tetrahedron Lett. 2001, 42, 2405– 2407, DOI: 10.1016/S0040-4039(01)00157-5Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhslOgs7w%253D&md5=187fb1007ca9f8cf9dcbc2aa4ffe0fbeGlycosyl trifluoroacetimidates. Part 1: Preparation and application as new glycosyl donorsYu, B.; Tao, H.Tetrahedron Letters (2001), 42 (12), 2405-2407CODEN: TELEAY; ISSN:0040-4039. (Elsevier Science Ltd.)Glycosyl (N-phenyl)trifluoroacetimidates, readily prepd. from 1-hydroxyl sugars by treatment with (N-phenyl)trifluoroacetimidoyl chloride in the presence of K2CO3, were demonstrated to be effective glycosyl donors. Glycosidation of these glycosyl fluoroacetimidates with alcs. is also reported.
- 51Witte, M. D.; Walvoort, M. T. C.; Li, K.-Y.; Kallemeijn, W. W.; Donker-Koopman, W. E.; Boot, R. G.; Aerts, J. M. F. G.; Codée, J. D. C.; van der Marel, G. A.; Overkleeft, H. S. Activity-based profiling of retaining beta-glucosidases: a comparative study. ChemBioChem 2011, 12, 1263– 1269, DOI: 10.1002/cbic.201000773Google ScholarThere is no corresponding record for this reference.
- 52Harrak, Y.; Barra, C. M.; Delgado, A.; Castaño, A. R.; Llebaria, A. Galacto-configured aminocyclitol phytoceramides are potent in vivo invariant natural killer T cell stimulators. J. Am. Chem. Soc. 2011, 133, 12079– 12084, DOI: 10.1021/ja202610xGoogle Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptFKls70%253D&md5=76a2544ab5fe19c786686ae69c99db1fGalacto-Configured Aminocyclitol Phytoceramides Are Potent in Vivo Invariant Natural Killer T Cell StimulatorsHarrak, Youssef; Barra, Carolina M.; Delgado, Antonio; Castano, A. Raul; Llebaria, AmadeuJournal of the American Chemical Society (2011), 133 (31), 12079-12084CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A new class of α-galactosylceramide (αGC) non-glycosidic analogs bearing galacto-configured aminocyclitols as sugar surrogates, e.g. I (R = H, OH), have been obtained. The aminocyclohexane having a hydroxyl substitution pattern similar to an α-galactoside is efficiently obtained by a sequence involving Evans aldol reaction and ring-closing metathesis with a Grubbs catalyst to give a key intermediate cyclohexene, which has been converted in galacto-aminocyclohexanes that are linked through a secondary amine to a phyto-ceramide lipid having a cerotyl N-acyl group. Natural Killer T (NKT) cellular assays have resulted in the identification of an active compd., HS161, which has been found to promote NKT cell expansion in vitro in a similar fashion but more weakly than αGC. This compd. stimulates the release of Interferon-γ (IFNγ) and Interleukin-4 (IL-4) in iNKT cell culture but with lower potency than αGC. The activation of Invariant Natural Killer T (iNKT) cells by this compd. has been confirmed in flow cytometry expts. Remarkably, when tested in mice, HS161 selectively induces a very strong prodn. of IFN-γ indicative of a potent Th1 cytokine profile. Overall, these data confirm the agonist activity of αGC lipid analogs having charged amino-substituted polar heads and their capacity to modulate the response arising from iNKT cell activation in vivo.
- 53Alcaide, A.; Trapero, A.; Pérez, Y.; Llebaria, A. Galacto configured N-aminoaziridines: a new type of irreversible inhibitor of b-galactosidases. Org. Biomol. Chem. 2015, 13, 5690– 5697, DOI: 10.1039/C5OB00532AGoogle ScholarThere is no corresponding record for this reference.
- 54Ofman, T. P.; Küllmer, F.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. An orthogonally protected cyclitol for the construction of nigerose- and dextran-mimetic cyclophellitols. Org. Lett. 2021, 23, 9516, DOI: 10.1021/acs.orglett.1c03723Google ScholarThere is no corresponding record for this reference.
- 55Jiang, J.; Kuo, C.-L.; Wu, L.; Franke, C.; Kallemeijn, W. W.; Florea, B. I.; van Meel, E.; van der Marel, G. A.; Codée, J. D. C.; Boot, R. G.; Davies, G. J.; Overkleeft, H. S.; Aerts, J. M. F. G. Detection of active mammalian GH31 alpha-glucosidases in health and disease using in-class, broad-spectrum activity-based probes. ACS Cent. Sci. 2016, 2, 351– 358, DOI: 10.1021/acscentsci.6b00057Google ScholarThere is no corresponding record for this reference.
- 56Armstrong, Z.; Kuo, C.-L.; Lahav, D.; Liu, B.; Johnson, R.; Beenakker, T. J. M.; de Boer, C.; Wong, C.-S.; van Rijssel, E. R.; Debets, M. F.; Florea, B. I.; Hissink, C.; Boot, R. G.; Geurink, P. P.; Ovaa, H.; van der Stelt, M.; van der Marel, G. A.; Codée, J. D. C.; Aerts, J. M. F. G.; Wu, L.; Overkleeft, H. S.; Davies, G. J. Manno-epi-cyclophellitols enable activity-based protein profiling of human α-mannosidases and discovery of new Golgi mannosidase II inhibitors. J. Am. Chem. Soc. 2020, 142, 13021– 13029, DOI: 10.1021/jacs.0c03880Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlSis7vK&md5=e5c95886b03aae55867ebe11fa828a1bManno-epi-cyclophellitols EnableActivity-Based Protein Profiling of Human α-Mannosidasesand Discovery of New Golgi Mannosidase II InhibitorsArmstrong, Zachary; Kuo, Chi-Lin; Lahav, Daniel; Liu, Bing; Johnson, Rachel; Beenakker, Thomas J. M.; de Boer, Casper; Wong, Chung-Sing; van Rijssel, Erwin R.; Debets, Marjoke F.; Florea, Bogdan I.; Hissink, Colin; Boot, Rolf G.; Geurink, Paul P.; Ovaa, Huib; van der Stelt, Mario; van der Marel, Gijsbert M.; Codee, Jeroen D. C.; Aerts, Johannes M. F. G.; Wu, Liang; Overkleeft, Herman S.; Davies, Gideon J.Journal of the American Chemical Society (2020), 142 (30), 13021-13029CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Golgi mannosidase II (GMII) catalyzes the sequential hydrolysisof two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans assocd. with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms.Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitolaziridine derivs. enabled activity-based protein profiling ofα-mannosidases from both human cell lysate and mouse tissue exts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallog. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.
- 57McGregor, N. G. S.; Kuo, C.-L.; Beenakker, T. J. M.; Wong, C.-S.; Armstrong, Z.; Florea, B. I.; Codée, J. D. C.; Overkleeft, H. S.; Aerts, J. M. F. G.; Davies, G. J. Synthesis of broad-specificity activity-based probes for exo-β-mannosidases. Org. Biomol. Chem. 2022, 20, 877– 886, DOI: 10.1039/D1OB02287CGoogle Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XpvVyktQ%253D%253D&md5=bf3b06d018229f253f6ec9d826861807Synthesis of broad-specificity activity-based probes for exo-β-mannosidasesMcGregor, Nicholas G. S.; Kuo, Chi-Lin; Beenakker, Thomas J. M.; Wong, Chun-Sing; Offen, Wendy A.; Armstrong, Zachary; Florea, Bogdan I.; Codee, Jeroen D. C.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.; Davies, Gideon J.Organic & Biomolecular Chemistry (2022), 20 (4), 877-886CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Exo-β-mannosidases are a broad class of stereochem. retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-β-mannosidases in complex biol. samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogs selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biol. samples. Towards the development of ABPs for exo-β-mannosidases, we present a concise synthesis of β-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-β-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric center to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine deriv. by specifically labeling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogs hold promise for detecting exo-β-mannosidases in biol. and biomedical research.
- 58Jiang, J.; Kallemeijn, W. W.; Wright, D. W.; van den Nieuwendijk, A. M. C. H.; Coco Rohde, V.; Folch, E. C.; van den Elst, H.; Florea, B. I.; Scheij, S.; Donker-Koopman, W. E.; Verhoek, M.; Li, N.; Schürmann, M.; Mink, D.; Boot, R. G.; Codée, J. D. C.; van der Marel, G. A.; Davies, G. J.; Aerts, J. M. F. G.; Overkleeft, H. S. In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 alpha-L-fucosidases. Chem. Sci. 2015, 6, 2782– 2789, DOI: 10.1039/C4SC03739AGoogle Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitlaktrs%253D&md5=ba96b62c03ff47fb7375b48e854fb055In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 α-L-fucosidasesJiang, Jianbing; Kallemeijn, Wouter W.; Wright, Daniel W.; van den Nieuwendijk, Adrianus M. C. H.; Rohde, Veronica Coco; Folch, Elisa Colomina; van den Elst, Hans; Florea, Bogdan I.; Scheij, Saskia; Donker-Koopman, Wilma E.; Verhoek, Marri; Li, Nan; Schuermann, Martin; Mink, Daniel; Boot, Rolf G.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Davies, Gideon J.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Chemical Science (2015), 6 (5), 2782-2789CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)GH29 α-L-fucosidases catalyze the hydrolysis of α-L-fucosidic linkages. Deficiency in human lysosomal α-L-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein, we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 α-L-fucosidases from bacteria, mice and man. Crystallog. anal. on bacterial α-L-fucosidase confirms that the ABPs act by covalent modification of the active site nucleophile. Competitive activity-based protein profiling identified L-fuconojirimycin as the single GH29 α-L-fucosidase inhibitor from eight configurational isomers.
- 59Willems, L. I.; Beenakker, T. J. M.; Murray, B.; Scheij, S.; Kallemeijn, W. W.; Boot, R. G.; Verhoek, M.; Donker-Koopman, W. E.; Ferraz, M. J.; van Rijssel, E. R.; Florea, B. I.; Codée, J. D. C.; van der Marel, G. A.; Aerts, J. M. F. G.; Overkleeft, H. S. Potent and selective activity-based probes for GH27 human retaining alpha-galactosidases. J. Am. Chem. Soc. 2014, 136, 11622– 11625, DOI: 10.1021/ja507040nGoogle Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlaisrfF&md5=647ba661e02788a4b0494048be633236Potent and Selective Activity-Based Probes for GH27 Human Retaining α-GalactosidasesWillems, Lianne I.; Beenakker, Thomas J. M.; Murray, Benjamin; Scheij, Saskia; Kallemeijn, Wouter W.; Boot, Rolf G.; Verhoek, Marri; Donker-Koopman, Wilma E.; Ferraz, Maria J.; van Rijssel, Erwin R.; Florea, Bogdan I.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Journal of the American Chemical Society (2014), 136 (33), 11622-11625CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Lysosomal degrdn. of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases. The fluorescently labeled aziridine-based probes inhibit the two human retaining α-galactosidases αGal A and αGal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both α-galactosidases in cell exts., thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells vs. those derived from Fabry patients.
- 60Kuo, C.-L.; Su, Q.; van den Nieuwendijk, A. M. C. H.; Beenakker, T. J. M.; Offen, W. A.; Willems, L. I.; Boot, R. G.; Sarris, A. J.; Marques, A. R. A.; Codée, J. D. C.; van der Marel, G. A.; Florea, B. I.; Davies, G. J.; Overkleeft, H. S.; Aerts, J. M. F. G. The development of a broad-spectrum retaining beta-exogalactosidase activity-based probe. Org. Biomol. Chem. 2023, 21, 7813– 7820, DOI: 10.1039/D3OB01261AGoogle ScholarThere is no corresponding record for this reference.
- 61Artola, M.; Kuo, C.-L.; McMahon, S. A.; Hansen, T.; van der Lienden, M.; He, X.; van den Elst, H.; Florea, B. I.; Kermode, A. R.; van der Marel, G. A.; Gloster, T.; Codée, J. D. C.; Overkleeft, H. S.; Aerts, J. M. F. G. New irreversible α-L-iduronidase inhibitors and activity-based probes. Chem. Eur. J. 2018, 24, 19081– 19088, DOI: 10.1002/chem.201804662Google ScholarThere is no corresponding record for this reference.
- 62Wu, L.; Jiang, J.; Jin, Y.; Kallemeijn, W. W.; Kuo, C.-L.; Artola, M.; Dai, W.; van Elk, C.; van Eijk, M.; van der Marel, G. A.; Codée, J. D. C.; Florea, B. I.; Aerts, J. M. F. G.; Overkleeft, H. S.; Davies, G. J. Activity-based probes for functional interrogation of retaining beta-glucuronidases. Nat. Chem. Biol. 2017, 13, 867– 873, DOI: 10.1038/nchembio.2395Google ScholarThere is no corresponding record for this reference.
- 63Biarnés, X.; Ardèvol, A.; Planas, A.; Rovira, C.; Laio, A.; Parrinello, M. The conformational free energy landscape of b-D-glucopyranose. Implications for substrate preactivation in b-glucoside hydrolysis. J. Am. Chem. Soc. 2007, 129, 10686– 10693, DOI: 10.1021/ja068411oGoogle Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXovF2mtLs%253D&md5=3470037854668863bd12f3f0093b84c0The Conformational Free Energy Landscape of β-D-Glucopyranose. Implications for Substrate Preactivation in β-Glucoside HydrolasesBiarnes, Xevi; Ardevol, Albert; Planas, Antoni; Rovira, Carme; Laio, Alessandro; Parrinello, MicheleJournal of the American Chemical Society (2007), 129 (35), 10686-10693CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Using ab initio metadynamics the conformational free energy landscape of β-D-glucopyranose as a function of the puckering coordinates has been computed. It is shown that the correspondence between the free energy and the Stoddard's pseudorotational itinerary for the system is rather poor. The no. of free energy min. (9) is smaller than the no. of ideal structures (13). Moreover, only six min. correspond to a canonical conformation. The structural features, the electronic properties, and the relative stability of the predicted conformers permit the rationalization of the occurrence of distorted sugar conformations in all the available X-ray structures of β-glucoside hydrolase Michaelis complexes. It is shown that these enzymes recognize the most stable distorted conformers of the isolated substrate and at the same time the ones better prepd. for catalysis in terms of bond elongation/shrinking and charge distribution. This suggests that the factors governing the distortions present in these complexes are largely dictated by the intrinsic properties of a single glucose unit.
- 64Davies, G. J.; Planas, R.; Rovira, C. Conformational analysis of the reaction coordinate of glycosidases. Acc. Chem. Res. 2012, 45, 308– 316, DOI: 10.1021/ar2001765Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFyksrvP&md5=2c4a164a3dfed4c096cce1e3d046bf1dConformational Analyses of the Reaction Coordinate of GlycosidasesDavies, Gideon J.; Planas, Antoni; Rovira, CarmeAccounts of Chemical Research (2012), 45 (2), 308-316CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The enzymic hydrolysis of the glycosidic bond is catalyzed by diverse enzymes generically termed glycoside hydrolases (hereafter GHs) or glycosidases. The many sequence-based families of glycosidases have served as a rich hunting ground for enzymologists for years. Not only are these enzymes of fundamental interest, providing paradigms for enzymic catalysis that extend beyond the bounds of carbohydrate chem., but the enzymes themselves play myriad essential roles in diverse biol. processes. The wide utility of glycosidases, from their industrial harnessing in the hydrolysis of plant biomass to their roles in human physiol. and disease, has engendered a large scientific constituency with an interest in glycosidase chem. A fascinating thread of this research, and one with major impact on the design of enzyme inhibitors, is the conformational anal. of reaction pathways within the diverse families. These GH families provide a large pallet of enzymes with which chemists have attempted to depict the conformational landscape of glycosidase action. In this Account, we review three-dimensional insight into the conformational changes directed by glycosidases, primarily from structural observations of the stable enzyme-ligand species adjacent to the transition state (or states) and of enzyme-inhibitor complexes. We further show how recent computational advances dovetail with structural insight to provide a quantum mech. basis for glycosidase action. The glycosidase-mediated hydrolysis of the acetal or ketal bond in a glycoside may occur with either inversion or retention of the configuration of the anomeric carbon. Inversion involves a single step and transition state, whereas retention, often referred to as the double displacement, is a two-step process with two transition states. The single transition state for the inverting enzymes and the two transition states (those flanking the covalent intermediate) in the double displacement have been shown to have substantial oxocarbenium ion character. The dissociative nature of these transition states results in significant relative pos. charge accumulation on the pyranose ring. The delocalization of lone-pair electrons from the ring oxygen that stabilizes the cationic transition state implies that at, or close to, the transition states the pyranose will be distorted away from its lowest energy conformation to one that favors orbital overlap. Over the preceding decade, research has highlighted the harnessing of noncovalent interactions to aid this distortion of the sugar substrates from their lowest energy chair conformation to a variety of different boat, skew boat, and half-chair forms, each of which favors catalysis with a given enzyme and substrate. Crystallog. observation of stable species that flank the transition state (or states), of both retaining and inverting glycosidases, has allowed a description of their conformational itineraries, illustrating how enzymes facilitate the "electrophilic migration" of the anomeric center along the reaction coordinate. The blossom of computational approaches, such as ab initio metadynamics, has underscored the quantum mech. basis for glycoside hydrolysis. Conformational analyses highlight not only the itineraries used by enzymes, enabling their inhibition, but are also reflected in the nonenzymic synthesis of glycosides, wherein chemists mimic strategies found in nature.
- 65Vasella, A.; Davies, G. J.; Böhm, M. Glycosidase mechanisms. Curr. Opin. Chem. Biol. 2002, 6, 619– 629, DOI: 10.1016/S1367-5931(02)00380-0Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xot1agsbs%253D&md5=17e2373cd971486dec70981975dbdb61Glycosidase mechanismsVasella, Andrea; Davies, Gideon J.; Boehm, MatthiasCurrent Opinion in Chemical Biology (2002), 6 (5), 619-629CODEN: COCBF4; ISSN:1367-5931. (Elsevier Science Ltd.)A review with 99 refs. The 3-dimensional structure of glycosidases and of their complexes and the study of transition-state mimics reveal structural details that correlate with their catalytic mechanism. Of particular interest are the transition-state conformations harnessed by individual enzymes and the substrate distortion obsd. in enzyme-ligand complexes. The 3-dimensional structure in synergy with transition-state mimicry opens the way for mechanistic interpretation of enzyme inhibition and for the development of therapeutic agents.
- 66Beenakker, T. J. M.; Wander, D. P. A.; Offen, W. A.; Artola, M.; Raich, L.; Ferraz, M. J.; Li, K.-Y.; Houben, J. H. P. M.; van Rijssel, E. R.; Hansen, T.; van der Marel, G. A.; Codée, J. D. C.; Aerts, J. M. F. G.; Rovira, C.; Davies, G. J.; Overkleeft, H. S. Carba-cyclophellitols are neutral retaining-glucosidase inhibitors. J. Am. Chem. Soc. 2017, 139, 6534– 6537, DOI: 10.1021/jacs.7b01773Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXntVGgtb8%253D&md5=4ca99d2e4c4a4a9755db80b8d3ec8b61Carba-cyclophellitols are neutral retaining-glucosidase inhibitorsBeenakker, Thomas J. M.; Wander, Dennis P. A.; Offen, Wendy A.; Artola, Marta; Raich, Lluis; Ferraz, Maria J.; Li, Kah-Yee; Houben, Judith H. P. M.; van Rijssel, Erwin R.; Hansen, Thomas; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Aerts, Johannes M. F. G.; Rovira, Carme; Davies, Gideon J.; Overkleeft, Herman S.Journal of the American Chemical Society (2017), 139 (19), 6534-6537CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The conformational anal. of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine, both covalent retaining-β-glucosidase inhibitors, we postulated that the corresponding carba "cyclopropyl" analog would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the 4H3 transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors showed a strong bias for the 4H3 conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (Ki = 8.2 nM) of Thermotoga maritima (TmGH1) β-glucosidase. Three-dimensional structural anal. and comparison with unreacted epoxides showed that this compd. indeed bound in the 4H3 conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
- 67de Boer, C.; Armstrong, Z.; Lit, V. A. J.; Barash, U.; Ruijgrok, G.; Boyango, I.; Weitzenberg, M. M.; Schröder, S. P.; Sarris, A. J. C.; Meeuwenoord, N. J.; Bule, P.; Kayal, Y.; Ilan, N.; Codée, J. D. C.; Vlodavsky, I.; Overkleeft, H. S.; Davies, G. J.; Wu, L. Mechanism based heparanase inhibitors reduce cancer metastasis in vivo. Proc. Natl. Acad. Sci. USA 2022, 119, e2203167119 DOI: 10.1073/pnas.2203167119Google ScholarThere is no corresponding record for this reference.
- 68Artola, M.; Wu, L.; Ferraz, M. J.; Kuo, C.-L.; Raich, L.; Breen, I. Z.; Offen, W. A.; Codée, J. D. C.; van der Marel, G. A.; Rovira, C.; Aerts, J. M. F. G.; Davies, G. J.; Overkleeft, H. S. 1,6-Cyclophellitol cyclosulfates: a new class of irreversible glycosidase inhibitor. ACS Cent. Sci. 2017, 3, 784– 793, DOI: 10.1021/acscentsci.7b00214Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFGltrfK&md5=b8aaf5f955c0d78618aded623375f92e1,6-Cyclophellitol cyclosulfates: A new class of irreversible glycosidase inhibitorArtola, Marta; Wu, Liang; Ferraz, Maria J.; Kuo, Chi-Lin; Raich, Lluis; Breen, Imogen Z.; Offen, Wendy A.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Rovira, Carme; Aerts, Johannes M. F. G.; Davies, Gideon J.; Overkleeft, Herman S.ACS Central Science (2017), 3 (7), 784-793CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)The essential biol. roles played by glycosidases, coupled with the diverse therapeutic benefits of pharmacol. targeting of these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivs. of epoxides in org. synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here, we present the synthesis, conformational anal., and application of novel 1,6-cyclophellitol cyclosulfates. We showed that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matched that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacted more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
- 69Li, Z.; Pickles, I. B.; Sharma, M.; Melling, B.; Pallasdies, L.; Codée, J. D. C.; Williams, S. J.; Overkleeft, H. S.; Davies, G. J. Detection of sulfoquinovosidase activity in cell lysates using activity-based probes. Angew. Chem., Int. Ed. 2024, 63, e202401358 DOI: 10.1002/anie.202401358Google ScholarThere is no corresponding record for this reference.
- 70McGregor, N.; Artola, M.; Nin-Hill, A.; Linzel, D.; Haon, M.; Reijngoud, J.; Ram, A. F. J.; Rosso, M.-N.; van der Marel, G. A.; Codée, J. D. C.; van Wezel, G. P.; Berrin, J.-G.; Rovira, C.; Overkleeft, H. S.; Davies, G. J. Rational design of mechanism-based inhibitors and activity-based probes for the identification of retaining α-L-arabinofuranosidases. J. Am. Chem. Soc. 2020, 142, 4648– 4662, DOI: 10.1021/jacs.9b11351Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivFyis7s%253D&md5=5e8b4fa22bcc8f05667f308ed9516636Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-L-ArabinofuranosidasesMcGregor, Nicholas G. S.; Artola, Marta; Nin-Hill, Alba; Linzel, Daniel; Haon, Mireille; Reijngoud, Jos; Ram, Arthur; Rosso, Marie-Noelle; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; van Wezel, Gilles P.; Berrin, Jean-Guy; Rovira, Carme; Overkleeft, Herman S.; Davies, Gideon J.Journal of the American Chemical Society (2020), 142 (10), 4648-4662CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Identifying and characterizing the enzymes responsible for an obsd. activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the prodn. of α-L-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-L-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-L-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural expts., the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-L-arabinofuranosidases.
- 71Schröder, S. P.; de Boer, C.; McGregor, N. G. S.; Rowland, R. J.; Moroz, O.; Blagova, E.; Reijngoud, J.; Arentshorst, M.; Osborn, D.; Morant, M. D.; Abbate, E.; Stringer, M. A.; Krogh, K. B. R. M.; Raich, L.; Rovira, C.; Berrin, J.-G.; van Wezel, G. P.; Ram, A. F. J.; Florea, B. I.; van der Marel, G. A.; Codée, J. D. C.; Wilson, K. S.; Wu, L.; Davies, G. J.; Overkleeft, H. S. Dynamic and functional profiling of xylan-degrading enzymes in Aspergillus secretomes using activity-based probes. ACS Cent. Sci. 2019, 5, 1067– 1078, DOI: 10.1021/acscentsci.9b00221Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MzjtFykug%253D%253D&md5=bf680edfbc4663d4b7a12cbcacd8db15Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Aspergillus Secretomes Using Activity-Based ProbesSchroder Sybrin P; de Boer Casper; Florea Bogdan I; van der Marel Gijsbert A; Codee Jeroen D C; Overkleeft Herman S; McGregor Nicholas G S; Rowland Rhianna J; Moroz Olga; Blagova Elena; Wilson Keith S; Wu Liang; Davies Gideon J; Reijngoud Jos; Arentshorst Mark; van Wezel Gilles P; Ram Arthur F J; Osborn David; Abbate Eric; Morant Marc D; Stringer Mary A; Krogh Kristian B R M; Raich Lluis; Rovira Carme; Rovira Carme; Berrin Jean-GuyACS central science (2019), 5 (6), 1067-1078 ISSN:2374-7943.Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
- 72McGregor, N. G. S.; de Boer, C.; Foucart, Q. P. O.; Beenakker, T.; Offen, W. A.; Codée, J. D. C.; Willems, L. I.; Overkleeft, H. S.; Davies, G. J. A multiplexing activity-based protein profiling platform for dissection of a native bacterial xyloglucan-degrading system. ACS Cent. Sci. 2023, 9, 2306– 2314, DOI: 10.1021/acscentsci.3c00831Google ScholarThere is no corresponding record for this reference.
- 73van Meel, E.; Bos, E.; van den Lienden, M. J. C.; Overkleeft, H. S.; van Kasteren, S. I.; Koster, A. J.; Aerts, J. M. F. G. Localization of active endogenous and exogenous beta-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts. Traffic 2019, 20, 346– 356, DOI: 10.1111/tra.12641Google ScholarThere is no corresponding record for this reference.
- 74Schröder, S. P.; Petracca, R.; Minnee, H.; Artola, M.; Aerts, J. M. F. G.; Codée, J. D. C.; van der Marel, G. A.; Overkleeft, H. S. A divergent synthesis of L-arabino and D-xylo-configured cyclophellitol epoxides and aziridines. Eur. J. Org. Chem. 2016, 2016, 4787– 4794, DOI: 10.1002/ejoc.201600983Google ScholarThere is no corresponding record for this reference.
- 75Jariwala, P. B.; Pellock, S. J.; Goldfarb, D.; Cloer, E. C.; Artola, M.; Simpson, J. B.; Bhatt, A. P.; Walton, W. G.; Roberts, L. R.; Major, M. B.; Davies, G. J.; Overkleeft, H. S.; Redinbo, M. R. Discovering the microbial enzymes driving drug toxicity with activity-based protein profiling. ACS Chem. Biol. 2020, 15, 217– 225, DOI: 10.1021/acschembio.9b00788Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1CrsrjM&md5=6fad6eccb2851f48c279eaac95afe6a2Discovering the Microbial Enzymes Driving Drug Toxicity with Activity-Based Protein ProfilingJariwala, Parth B.; Pellock, Samuel J.; Goldfarb, Dennis; Cloer, Erica W.; Artola, Marta; Simpson, Joshua B.; Bhatt, Aadra P.; Walton, William G.; Roberts, Lee R.; Major, Michael B.; Davies, Gideon J.; Overkleeft, Herman S.; Redinbo, Matthew R.ACS Chemical Biology (2020), 15 (1), 217-225CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)It is increasingly clear that interindividual variability in human gut microbial compn. contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not obsd. in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial β-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug-glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with ex vivo kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the mol. basis for differential gut bacterial GUS inhibition obsd. between human fecal samples. Taken together, this work provides an unprecedented tech. and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.
- 76Lipsh-Sokolik, R.; Khersonsky, O.; Schröder, S. P.; de Boer, C.; Hoch, S.-Y.; Davies, G. J.; Overkleeft, H. S.; Fleishman, S. J. Combinatorial assembly and design of enzymes. Science 2023, 379, 195– 201, DOI: 10.1126/science.ade9434Google ScholarThere is no corresponding record for this reference.
- 77Lahav, D.; Liu, B.; van den Berg, R. J. B. H. N.; van den Nieuwendijk, A. M. C. H.; Wennekes, T.; Ghisaidoobe, A. T.; Breen, I.; Ferraz, M. J.; Kuo, C.-L.; Wu, L.; Geurink, P. P.; Ovaa, H.; van der Marel, G. A.; van der Stelt, M.; Boot, R. G.; Davies, G. J.; Aerts, J. M. F. G.; Overkleeft, H. S. A fluorescence polarization activity-based protein profiling assay in the discovery of potent, selective inhibitors for human non-lysosomal glucosylceramidase. J. Am. Chem. Soc. 2017, 139, 14192– 14197, DOI: 10.1021/jacs.7b07352Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFCis7vO&md5=0eda44ab424f5685fa536d64be93f91eA fluorescence polarization activity-based protein profiling assay in the discovery of potent, selective inhibitors for human nonlysosomal glucosylceramidaseLahav, Daniel; Liu, Bing; van den Berg, Richard J. B. H. N.; van den Nieuwendijk, Adrianus M. C. H.; Wennekes, Tom; Ghisaidoobe, Amar T.; Breen, Imogen; Ferraz, Maria J.; Kuo, Chi-Lin; Wu, Liang; Geurink, Paul P.; Ovaa, Huib; van der Marel, Gijsbert A.; van der Stelt, Mario; Boot, Rolf G.; Davies, Gideon J.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Journal of the American Chemical Society (2017), 139 (40), 14192-14197CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chem. tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell exts. contg. recombinant, overexpressed glycosidase as the easily accessible enzyme source.
- 78Ndeh, D.; Rogowski, A.; Cartmell, A.; Luis, A. S.; Baslé, A.; Gray, J.; Venditto, I.; Briggs, J.; Zhang, X.; Labourel, A.; Terrapon, N.; Buffetto, F.; Nepogodiev, S.; Xiao, Y.; Field, R. A.; Zhu, Y.; O’Neil, M.; Urbanowicz, B. R.; York, W. S.; Davies, G. J.; Abbott, D. W.; Ralet, M.-C.; Martens, E. C.; Henrissat, B.; Gilbert, H. J. Complex pectin metabolism by gut bacteria reveals novel catalytic functions. Nature 2017, 544, 65– 70, DOI: 10.1038/nature21725Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltVWnurY%253D&md5=8a779b1beb1d7d394bcae22b635488cfComplex pectin metabolism by gut bacteria reveals novel catalytic functionsNdeh, Didier; Rogowski, Artur; Cartmell, Alan; Luis, Ana S.; Basle, Arnaud; Gray, Joseph; Venditto, Immacolata; Briggs, Jonathon; Zhang, Xiaoyang; Labourel, Aurore; Terrapon, Nicolas; Buffetto, Fanny; Nepogodiev, Sergey; Xiao, Yao; Field, Robert A.; Zhu, Yanping; O'Neill, Malcolm A.; Urbanowicz, Breeanna R.; York, William S.; Davies, Gideon J.; Abbott, D. Wade; Ralet, Marie-Christine; Martens, Eric C.; Henrissat, Bernard; Gilbert, Harry J.Nature (London, United Kingdom) (2017), 544 (7648), 65-70CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The metab. of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degrdn. involves previously undiscovered enzyme families and catalytic activities. The degrdn. system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.
- 79Ren, W.; Pengelly, R.; Farren-Dai, M.; Shamsi Kazem Abadi, S.; Oehiler, V.; Akintola, O.; Draper, J.; Meanwell, M.; Chakladar, S.; Swiderek, K.; Moliner, V.; Britton, R.; Gloster, T. M.; Bennet, A. J. Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level. Nat. Commun. 2018, 9, 3243, DOI: 10.1038/s41467-018-05702-7Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c7nslyrtw%253D%253D&md5=dd3282d4c9a0eb8fe4c7805dc4d29118Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic levelRen Weiwu; Farren-Dai Marco; Akintola Oluwafemi; Draper Jason; Meanwell Michael; Chakladar Saswati; Britton Robert; Bennet Andrew J; Pengelly Robert; Oehler Verena; Gloster Tracey M; Shamsi Kazem Abadi Saeideh; Swiderek Katarzyna; Moliner VicentNature communications (2018), 9 (1), 3243 ISSN:.Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate's structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair ((2)H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair ((2)H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.
- 80Jain, N.; Tamura, K.; Déjean, G.; van Petegem, F.; Brumer, H. Orthogonal active-site labels for mixed-linkage endo-b-glucanases. ACS Chem. Biol. 2021, 16, 1968– 1984, DOI: 10.1021/acschembio.1c00063Google ScholarThere is no corresponding record for this reference.
- 81Schröder, S. P.; Kallemeijn, W. W.; Debets, M. F.; Hansen, T.; Sobala, L. F.; Hakki, Z.; Williams, S. J.; Beenakker, T. J. M.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Davies, G. J.; Overkleeft, H. S. Spiro-epoxyglycosides as activity-based probes for glycoside hydrolase family 99 endomannosidase/endomannanase. Chem. Eur. J. 2018, 24, 9983– 9992, DOI: 10.1002/chem.201801902Google ScholarThere is no corresponding record for this reference.
- 82Thaler, M.; Ofman, T. P.; Kok, K.; Heming, J. J. A.; Moran, E.; Pickles, I.; Leijs, A. A.; van den Nieuwendijk, A. M. C. H.; van den Berg, R. J. B. H. N.; Ruijgrok, G.; Armstrong, Z.; Salgado-Benvindo, C.; Ninaber, D. K.; Snijder, E. J.; van Boeckel, C. A. A.; Artola, A.; Davies, G. J.; Overkleeft, H. S.; van Hemert, M. J. Epi-cyclophellitol cyclosulfate, a mechanism-based ER α-glucosidase II inhibitor, blocks replication of SARS-CoV-2 and other coronaviruses. ACS Cent. Sci. 2024, 10, 1594, DOI: 10.1021/acscentsci.4c00506Google ScholarThere is no corresponding record for this reference.
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Abstract
Figure 1
Figure 1. Mechanism of action of inverting β-glucosidases (A) and retaining β-glucosidases (B).
Figure 2
Figure 2. Two archetypal retaining glycosidase inhibitor designs: deoxyfluoroglycosides (A) and cyclitol epoxides (B).
Figure 3
Figure 3. Glycomimetic epoxides, aziridines and episulfides as mechanism-based, covalent and irreversible retaining glycosidase inhibitors.
Figure 4
Figure 4. Latent quinone methides (A) and activated fluorinated glycosides (B) as activity-based (retaining) GH probes. For the original images of the SDS-PAGE gels that are shown here and in the remainder of this account, please see the papers referred to in the text.
Figure 5
Figure 5. Retaining GH ABPs based on cyclophellitol and cyclophellitol aziridine. A) Adaptation of the Madsen cyclophellitol synthesis to give cyclophellitol and cyclophellitol aziridine ABPs 44 and 48. B) Comparative ABPP of the human retaining β-glucosidases, GBA1, GBA2 and GBA3. C) Head-to-head comparison of activated fluorinated glucosides 49-52 and cyclophellitol 53 as GBA1 ABPs.
Figure 6
Figure 6. A, B) Reaction coordinates by which retaining β-glucosidases (A) and retaining α-glucosidases (B) process their substrates. C) Computed free energy landscapes (FELs) of selected compounds.
Figure 7
Figure 7. A), Activity-based secretomes profiling of glucuronoarabinoxylan-degrading retaining arabinofuranosidases and xylanases. B) Xyloglucan-degrading retaining exoglucosidases, cellulases and xyloglucanases. Shown in the gel are samples of intact cells (C), cell extracts (L) and supernatant/secretomes (S) with ABPs 63-65.
References
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- 2Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P. M.; Henrissat, B. The carbohydrate active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014, 42, D490– D495, DOI: 10.1093/nar/gkt11782https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXoslWn&md5=fa419a15f2d86a1359d4657f079401e1The carbohydrate-active enzymes database (CAZy) in 2013Lombard, Vincent; Golaconda Ramulu, Hemalatha; Drula, Elodie; Coutinho, Pedro M.; Henrissat, BernardNucleic Acids Research (2014), 42 (D1), D490-D495CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The Carbohydrate-Active Enzymes database (CAZy; http://www.cazy.org) provides online and continuously updated access to a sequence-based family classification linking the sequence to the specificity and 3D structure of the enzymes that assemble, modify and breakdown oligo- and polysaccharides. Functional and 3D structural information is added and curated on a regular basis based on the available literature. In addn. to the use of the database by enzymologists seeking curated information on CAZymes, the dissemination of a stable nomenclature for these enzymes is probably a major contribution of CAZy. The past few years have seen the expansion of the CAZy classification scheme to new families, the development of subfamilies in several families and the power of CAZy for the anal. of genomes and metagenomes. This article outlines the changes that have occurred in CAZy during the past 5 years and presents our novel effort to display the resoln. and the carbohydrate ligands in crystallog. complexes of CAZymes.
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- 4Koshland, D. E. Stereochemistry and the mechanism of enzymatic reactions. Biol. Rev. 1953, 28, 416– 436, DOI: 10.1111/j.1469-185X.1953.tb01386.x4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG2cXhtFCrsQ%253D%253D&md5=9cf02ad1ef0d343b609b063e819d9e8bStereochemistry and the mechanism of enzymic reactionsKoshland, D. E., Jr.Biological Reviews of the Cambridge Philosophical Society (1953), 28 (), 416-36CODEN: BRCPAH; ISSN:0006-3231.In those enzymic reactions involving substitutions of a group X at an asymmetric C atom by a group Y, the product has either the same configuration as the initial substrate or has an inverted configuration. Examples of each are listed. The mechanisms accounting for the retention or inversion of the stereochem. configuration are discussed. 90 references.
- 5Compounds that react within the enzyme active site to form a covalent and irreversible bond, thereby irreversibly inactivating the enzyme, are sometimes referred to as ‘targeted covalent inhibitors’, ‘suicide inhibitors’ or ‘activity-based inhibitors’. In line with other practitioners in the field (see also the titles of some of the references below) we have chosen to use the term ‘mechanism-based inhibitors’, which is also the term recommended by IUPAC. See also https://iupac.qmul.ac.uk/gtpoc/M.html.There is no corresponding record for this reference.
- 6Vickers, C.; Liu, F.; Abe, K.; Salama-Alber, O.; Jenkins, M.; Springate, C. M. K.; Burke, J. E.; Withers, S. H.; Boraston, A. B. Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-L-fucosidases from GH29. J. Biol. Chem. 2018, 293, 18296– 18308, DOI: 10.1074/jbc.RA118.0051346https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit12ksrzM&md5=682741ddc53cdf0bd1f751680697fc85Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-L-fucosidases from GH29Vickers, Chelsea; Liu, Feng; Abe, Kento; Salama-Alber, Orly; Jenkins, Meredith; Springate, Christopher M. K.; Burke, John E.; Withers, Stephen G.; Boraston, Alisdair B.Journal of Biological Chemistry (2018), 293 (47), 18296-18308CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Fucoidans are chem. complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochem. and biol. activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymg. enzymes have been identified, the mol. basis of their activity on these chem. complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymd. by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallog. and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-L-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the mol. details underpinning the biol. processing of fucoidans. The nucleotide sequence(s) reported in this paper have been submitted to the GenBankTM/EBI Data Bank with accession nos. MH707445 and MH707446.
- 7Watts, A. F.; Damager, I.; Amaya, M. L.; Buschiazzo, A.; Alzari, P.; Frasch, A. C.; Withers, S. G. Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate. J. Am. Chem. Soc. 2003, 125, 7532– 7533, DOI: 10.1021/ja03449677https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktFGqt7c%253D&md5=b9728e3301bf0c453d7a2a3126413bd9Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: Tyrosine is the catalytic nucleophileWatts, Andrew G.; Damager, Iben; Amaya, Maria L.; Buschiazzo, Alejandro; Alzari, Pedro; Frasch, Alberto C.; Withers, Stephen G.Journal of the American Chemical Society (2003), 125 (25), 7532-7533CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Modified sialic acid substrates have been used to label Trypanosoma cruzi trans-sialidase, demonstrating that the enzyme catalyzes the transfer of sialic acid through a covalent glycosyl-enzyme intermediate, a mechanism common to most retaining glycosidases. Peptic digestion of labeled protein, followed by LC-MS/MS anal. of the digest, identified Tyr 342 as the catalytic nucleophile. This is the first such example of a retaining glycosidase utilizing an aryl glycoside intermediate. It is suggested that this alternative choice of nucleophile is a consequence of the chem. nature of sialic acid. A Tyr/Glu couple is invoked to relay charge from a remote glutamic acid, thereby avoiding electrostatic repulsion with the sialic acid carboxylate group.
- 8McGregor, N. G. S.; Coines, J.; Borlandelli, V.; Amaki, S.; Artola, M.; Nin-Hill, A.; Linzel, D.; Yamada, C.; Arakawa, T; Ishiwata, A.; Ito, Y.; van der Marel, G. A.; Codée, J. D. C.; Fushinobu, S.; Overkleeft, H. S.; Rovira, C.; Davies, G. J. Cysteine nucleophiles in glycosidase catalysis: application of a covalent β-L-arabinofuranosidase inhibitor. Angew. Chem., Int. Ed. 2021, 60, 5754– 5758, DOI: 10.1002/anie.2020139208https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjtVSrurk%253D&md5=19db6aa6f66d9d81f9d5fddc61238a63Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β-L-Arabinofuranosidase InhibitorMcGregor, Nicholas G. S.; Coines, Joan; Borlandelli, Valentina; Amaki, Satoko; Artola, Marta; Nin-Hill, Alba; Linzel, Daniel; Yamada, Chihaya; Arakawa, Takatoshi; Ishiwata, Akihiro; Ito, Yukishige; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Fushinobu, Shinya; Overkleeft, Herman S.; Rovira, Carme; Davies, Gideon J.Angewandte Chemie, International Edition (2021), 60 (11), 5754-5758CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived β-L-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This β-L-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures detd. for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chem. of cyclophellitol derivs., the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.
- 9Dennis, R. J.; Taylor, E. J.; Macauley, M. S.; Stubbs, K. A.; Turkenburg, J. P.; Hart, S. J.; Black, G. N.; Vocadlo, D. J.; Davies, G. J. Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity. Nat. Struct. Mol. Biol. 2006, 13, 365– 371, DOI: 10.1038/nsmb10799https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xjs1OrtL8%253D&md5=2fec47f64f348ca3f6a143c37589ddcdStructure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activityDennis, Rebecca J.; Taylor, Edward J.; Macauley, Matthew S.; Stubbs, Keith A.; Turkenburg, Johan P.; Hart, Samuel J.; Black, Gary N.; Vocadlo, David J.; Davies, Gideon J.Nature Structural & Molecular Biology (2006), 13 (4), 365-371CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.
- 10Sobala, L. F.; Speciale, G.; Zhu, S.; Raich, L.; Sannikova, N.; Thompson, A. J.; Hakki, Z.; Lu, D.; Shansi Kazem Abadi, S.; Lewis, A. R.; Rojas-Cervellera, V.; Bernardo-Seisdedos, G.; Zhang, Y.; Millet, O.; Jiménez-Barbero, J.; Bennet, A. J.; Sollogoub, M.; Rovira, C.; Davies, G. J.; Williams, S. J. An epoxide intermediate in glycosidase catalysis. ACS Cent. Sci. 2020, 6, 760– 770, DOI: 10.1021/acscentsci.0c0011110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntFWhtL4%253D&md5=5d6812d3576863c1f633f6ea3b1534c3An epoxide intermediate in glycosidase catalysisSobala, Lukasz F.; Speciale, Gaetano; Zhu, Sha; Raich, Lluis; Sannikova, Natalia; Thompson, Andrew J.; Hakki, Zalihe; Lu, Dan; Shamsi Kazem Abadi, Saeideh; Lewis, Andrew R.; Rojas-Cervellera, Victor; Bernardo-Seisdedos, Ganeko; Zhang, Yongmin; Millet, Oscar; Jimenez-Barbero, Jesus; Bennet, Andrew J.; Sollogoub, Matthieu; Rovira, Carme; Davies, Gideon J.; Williams, Spencer J.ACS Central Science (2020), 6 (5), 760-770CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Retaining glycoside hydrolases cleave their substrates through stereochem. retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallog. snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/mol. mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/Km) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymic mechanism. Mannosidases of glycoside hydrolase family 99 use a neighboring-group participation mechanism involving the substrate 2-hydroxyl.
- 11Withers, S. G.; Street, I. P.; Bird, P.; Dolphin, D. H. 2-Deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitors. J. Am. Chem. Soc. 1987, 109, 7530– 7531, DOI: 10.1021/ja00258a04711https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlajsbY%253D&md5=8ee52f1c5833b57cd6887aca4a0d80fb2-Deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitorsWithers, Stephen G.; Street, Ian P.; Bird, Paul; Dolphin, David H.Journal of the American Chemical Society (1987), 109 (24), 7530-1CODEN: JACSAT; ISSN:0002-7863.2,4-Dinitrophenyl 2-deoxy-2-fluoro β-D-glucopyranoside was synthesized and demonstrated to be a specific mechanism-based inhibitor for β-glucosidase from Alcaligenes faecalis. The inactivation of the enzyme followed pseudo-1st-order kinetics with a rate const. of 25 min-1 and a dissocn. const. of 0.05 mM. Protection against inactivation was shown by the competitive inhibitor isopropylthio β-D-glucopyranoside. Reactivation of the enzyme was obsd. as expected after removal of free inhibitor and incubation in the presence of substrate. The use of such inhibitors to investigate the intermediacy of a glucosyl enzyme intermediate and to identify the active site residue involved is projected.
- 12Rempel, B. P.; Withers, S. G. Covalent inhibitors of glycosidases and their applications in biochemistry and biology. Glycobiol. 2008, 18, 570– 586, DOI: 10.1093/glycob/cwn041There is no corresponding record for this reference.
- 13Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 2001, 412, 835– 838, DOI: 10.1038/3509060213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXms1aqs74%253D&md5=7b43460d032eac35eda551b6dbea7ca6Catalysis by hen egg-white lysozyme proceeds via a covalent intermediateVocadlo, David J.; Davies, Gideon J.; Laine, Roger; Withers, Stephen G.Nature (London, United Kingdom) (2001), 412 (6849), 835-839CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure detd. by x-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of β-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric center. Studies with other retaining β-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here the authors show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. The authors also show the three-dimensional structure of this intermediate as detd. by x-ray diffraction. The authors formulate a general catalytic mechanism for all retaining β-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.
- 14Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, I. Darstellung und Eigenschaften spezifischer Inaktivatoren. Hoppe-Seyler’s Z. Physiol. Chem. 1966, 345, 197– 214, DOI: 10.1515/bchm2.1966.345.1.197There is no corresponding record for this reference.
- 15Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, II. Isolierung und enzymatische Eigenschaften from zwei β-Glucosidasen aus Aspergillus wentii. Hoppe-Seyler’s Z. Physiol. Chem. 1967, 348, 1359– 1366, DOI: 10.1515/bchm2.1967.348.1.1359There is no corresponding record for this reference.
- 16Legler, G. Untersuchungen zum Wirkunsmechanismus glycosidspaltender Enzyme, III. Markierung des aktiven Zentrums einer β-Glucosidase aus Aspergillus wentii mit [14C]Condurit-B-epoxid. Hoppe-Seyler’s Z. Physiol. Chem. 1968, 349, 767– 774, DOI: 10.1515/bchm2.1968.349.1.767There is no corresponding record for this reference.
- 17Braun, H.; Legler, G.; Deshusses, J.; Semenza, G. Stereoselective ring opening of conduritol-B-epoxide by an active site aspartate residue of sucrose-isomaltase. Biochim. Biophys. Acta 1977, 483, 135– 140, DOI: 10.1016/0005-2744(77)90015-8There is no corresponding record for this reference.
- 18Legler, G.; Bause, E. Epoxy-alkyl oligo-(1–4)-β-D-glucosides as active-site-directed inhibitors of cellulases. Carbohydr. Res. 1973, 28, 45– 52, DOI: 10.1016/S0008-6215(00)82855-4There is no corresponding record for this reference.
- 19Rodriguez, E. B.; Scally, G. D.; Stick, R. V. The synthesis of optically pure epoxy-alkyl β-D-glucosides and β-cellobiosides as active-site directed inhibitors of some β-glucan hydrolases. Aust. J. Chem. 1990, 43, 1391– 1405, DOI: 10.1071/CH9901391There is no corresponding record for this reference.
- 20Sulzenbacher, G.; Schülein, M.; Davies, G. J. Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Å resolution. Biochemistry 1997, 36, 5902– 5911, DOI: 10.1021/bi962963+20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXislSltrc%253D&md5=92928c9b648ae140ed1dbd23e0423183Structure of the endoglucanase I from Fusarium oxysporum: Native, cellobiose, and 3,4-epoxybutyl β-D-cellobioside-inhibited forms, at 2.3 Å resolutionSulzenbacher, Gerlind; Schuelein, Martin; Davies, Gideon J.Biochemistry (1997), 36 (19), 5902-5911CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The crystal structure of F. oxysporum cellulase endoglucanase I (I) of glycosyl hydrolase family 7 was solved at 2.3 Å resoln. In addn. to the native enzyme, structures were also detd. of I complexed with both the affinity label, 3,4-epoxybutyl β-D-cellobioside, and the reaction product, cellobiose. The affinity label was covalently bound, as expected, to the catalytic nucleophile, Glu-197, with clear evidence for binding of both the R and S stereoisomers. Cellobiose was found bound to the -2 and -1 subsites of I. In marked contrast to the previously reported crystal structure of I complexed with a nonhydrolyzable thiosaccharide analog, which spanned the -2, -1, and +1 subsites and which had a skew-boat conformation for the -1 subsite sugar, the I-cellobiose complex showed no pyranoside ring distortion in the -1 subsite, implying that strain is induced primarily by the addnl. +1 subsite interactions and that the product is found, as expected, in its unstrained conformation.
- 21Thomas, E. W.; McKelvy, J. F.; Sharon, N. Specific and irreversible inhibition of lysozyme by 2′,3′-epoxypropyl β-glycosides of N-acetyl-D-glucosamine oligomers. Nature 1969, 222, 485– 486, DOI: 10.1038/222485a0There is no corresponding record for this reference.
- 22Havukainen, R.; Törrönen, A.; Laitinen, T.; Rouvinen, J. Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reesei. Biochemistry 1996, 35, 9617– 9624, DOI: 10.1021/bi953052n22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjvVynsb0%253D&md5=0d5a0a723343c9f0b9d54e297fcee8f3Covalent binding of three epoxyalkyl xylosides to the active site of endo-1,4-xylanase II from Trichoderma reeseiHavukainen, Riikka; Torronen, Anneli; Laitinen, Tuomo; Rouvinen, JuhaBiochemistry (1996), 35 (29), 9617-9624CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The three-dimensional structures of endo-1,4-xylanase II from (XYNII) Trichoderma reesei complexed with 4,5-epoxypentyl β-D-xyloside (X-O-C5), 3,4-epoxybutyl β-D-xyloside (X-O-C4), and 2,3-epoxypropyl β-D-xyloside (X-O-C3) were detd. by x-ray crystallog. High-resoln. measurement revealed clear electron densities for each ligand. Both X-O-C5 and X-O-C3 were found to form a covalent bond with the putative nucleophile Glu86. Unexpectedly, X-O-C4 was found to bind to the putative acid/base catalyst Glu177. In all three complexes, clear conformational changes were found in XYNII compared to the native structure. These changes were largest in the X-O-C3 complex structure.
- 23Liu, L.; Patricelli, M. P.; Cravatt, B. F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 1999, 96, 14694– 14699, DOI: 10.1073/pnas.96.26.1469423https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhtFaitw%253D%253D&md5=d5d1a09328fc7715e9d30ca392733426Activity-based protein profiling: the serine hydrolasesLiu, Yongsheng; Patricelli, Matthew P.; Cravatt, Benjamin F.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (26), 14694-14699CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)With the postgenome era rapidly approaching, new strategies for the functional anal. of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chem. synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue exts., we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Addnl., we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
- 24Yariv, J.; Wilson, K. J.; Hildesheim, J.; Blumberg, S. Labelling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamine. FEBS Lett. 1971, 15, 24– 26, DOI: 10.1016/0014-5793(71)80070-424https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3MXkvVCksrk%253D&md5=d0807f7b2b5b9269861a9c58be849a22Labeling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamineYariv, Joseph; Wilson, Kenneth J.; Hildesheim, Jean; Blumberg, ShmaryahuFEBS Letters (1971), 15 (1), 24-6CODEN: FEBLAL; ISSN:0014-5793.The hydrolysis of o-nitrophenyl β-galactopyranoside by β-galactosidase was inhibited by incubating with labeled N-bromoacetyl β-D-galactopyranosylamine. The reaction followed 1st order kinetics leading to complete inactivation and 1 mole of reagent was bound to the enzyme per mole of site inactivated. The corresponding L-fucose deriv. did not inactivate the enzyme.
- 25Caron, G.; Withers, S. G. Conduritol aziridine: a new mechanism-based glucosidase inactivator. Biochem. Biophys. Res. Commun. 1989, 163, 495– 499, DOI: 10.1016/0006-291X(89)92164-5There is no corresponding record for this reference.
- 26Tong, M. K.; Ganem, B. A potent new class of active-site-directed glycosidase inactivators. J. Am. Chem. Soc. 1988, 110, 312– 313, DOI: 10.1021/ja00209a06226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXjvVamtw%253D%253D&md5=86e63225e2f3b6e08ede5e42804dc0ecA potent new class of active-site-directed glycosidase inactivatorsTong, Michael K.; Ganem, BruceJournal of the American Chemical Society (1988), 110 (1), 312-13CODEN: JACSAT; ISSN:0002-7863.Aziridine I was prepd. from piperidine II in 6 steps. I showed potent inhibition of green coffee bean α-galactosidase, but had little or no effect on yeast α-glucosidase, jackbean α-mannosidase, or bovine β-galactosidase.
- 27Atsumi, S.; Umezawa, K.; Iinuma, H.; Naganawa, H.; Nakamura, H.; Iitaka, Y.; Takeuchi, T. Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus sp. J. Antibiot. 1990, 43, 49– 53, DOI: 10.7164/antibiotics.43.4927https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhvFChu78%253D&md5=3d007c5da7425376dda4cb11e639c208Production, isolation and structure determination of a novel β-glucosidase inhibitor, cyclophellitol, from Phellinus spAtsumi, Sonoko; Umezawa, Kazuo; Iinuma, Hironobu; Naganawa, Hiroshi; Nakamura, Hikaru; Iitaka, Yoichi; Takeuchi, TomioJournal of Antibiotics (1990), 43 (1), 49-53CODEN: JANTAJ; ISSN:0021-8820.A culture filtrate of a mushroom, Phellinus species, strongly inhibited β-glucosidase. The active substance was isolated through charcoal sepn., column chromatog. and crystn. Spectroscopic and crystallog. anal. revealed that it had a novel cyclitol structure, (1S,2R,3S,4R,5R,6R)-5-hydroxymethyl-7-oxabicyclo[4.1.0]heptane-2,3,4-triol; the compd. was named cyclophellitol (I). It inhibited almond-derived β-glucosidase with an IC50 of 0.8 μg/mL.
- 28Gloster, T. M.; Madsen, R.; Davies, G. J. Structural basis for cyclophellitol inhibition of a β-glucosidase. Org. Biomol. Chem. 2007, 5, 444– 446, DOI: 10.1039/B616590G28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXoslSnsw%253D%253D&md5=f1d62eeec2e2483e3fa386679b229622Structural basis for cyclophellitol inhibition of a β-glucosidaseGloster, Tracey M.; Madsen, Robert; Davies, Gideon J.Organic & Biomolecular Chemistry (2007), 5 (3), 444-446CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)The structural basis for β-glucosidase inhibition by cyclophellitol was demonstrated using x-ray crystallog., enzyme kinetics and mass spectrometry.
- 29Nakata, M.; Chong, C.; Niwata, Y.; Toshima, K.; Tatsuta, K. A family of cyclophellitol analogues: synthesis and evaluation. J. Antibiot. 1993, 46, 1919– 1922, DOI: 10.7164/antibiotics.46.1919There is no corresponding record for this reference.
- 30Shing, T. K. M.; Tai, V. W.-F. (-)-Quinic acid in organic synthesis. Part 4. Synthesis of cyclophellitol and its (1R,6S)-, (2S)-, (1R,2S,6S)-diastereomers. J. Chem. Soc. Perkin Trans 1. 1994, 2017– 2025, DOI: 10.1039/P19940002017There is no corresponding record for this reference.
- 31Greenbaum, F.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 2000, 7, 569– 581, DOI: 10.1016/S1074-5521(00)00014-431https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnt1WnsbY%253D&md5=4bfc0e299524198ec324c96c8981baf7Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery toolsGreenbaum, Doron; Medzihradszky, Katalin F.; Burlingame, Alma; Bogyo, MatthewChemistry & Biology (2000), 7 (8), 569-581CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Science Ltd.)Background: Anal. of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly detg. activities of an entire class of enzymes on a genome-wide scale would be of great utility. Results: We have engineered chem. probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes contg. a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivs. was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular exts. Finally, the affinity tag of DCG-04 allowed purifn. of modified proteases and identification by mass spectrometry. Conclusions: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixts. These probes were used to det. relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purifn./identification of targets.
- 32Janda, K. D.; Lo, L.-C.; Lo, C.-H. L.; Sim, M.-M.; Wang, R.; Wong, C.-H.; Lerner, R. A. Chemical selection for catalysis in combinatorial libraries. Science 1997, 275, 945– 948, DOI: 10.1126/science.275.5302.94532https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXht1Cls7s%253D&md5=b03752f229221f9e2e2dd2e78cd9e5ddChemical selection for catalysis in combinatorial antibody librariesJanda, Kim D.; Lo, Lee-Chiang; Lo, Chih-Hung L.; Sim, Mui-Mui; Wang, Ruo; Wong, Chi-Huey; Lerner, Richard A.Science (Washington, D. C.) (1997), 275 (5302), 945-948CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)For the past decade the immune system has been exploited as a rich source of de novo catalysts. Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chem. reactions. In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes. Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function. A method that allows for the direct chem. selection for catalysis from antibody libraries was so devised, whereby the pos. aspects of hybridoma technol. were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis. This methodol. is based on a purely chem. selection process, making it more general than biol. based selection systems because it is not limited to reaction products that perturb cellular machinery.
- 33Tsai, C.-S.; Li, Y.-K.; Lo, L.-C. Design and synthesis of activity probes for glycosidases. Org. Lett. 2002, 4, 3607– 3610, DOI: 10.1021/ol026531533https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XnslansrY%253D&md5=a1afa3a47c57335d6ef7e75b405a7666Design and Synthesis of Activity Probes for GlycosidasesTsai, Charng-Sheng; Li, Yaw-Kuen; Lo, Lee-ChiangOrganic Letters (2002), 4 (21), 3607-3610CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)A new synthetic route was developed for the prepn. of glycoside I activity probe for β-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe I was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.
- 34Lu, C.-P.; Ren, C.-T.; Lai, Y.-N.; Wu, S.-H.; Wang, W.-M.; Chen, J.-Y.; Lo, L.-C. Design of a mechanism-based probe for neuraminidase to capture influenza viruses. Angew. Chem., Int. Ed. 2005, 44, 6888– 6892, DOI: 10.1002/anie.20050173834https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1alsr7O&md5=b857fd16e902f49bdef55623818f8c0cDesign of a mechanism-based probe for neuraminidase to capture influenza virusesLu, Chun-Ping; Ren, Chien-Tai; Lai, Yi-Ning; Wu, Shih-Hsiung; Wang, Wei-Man; Chen, Jean-Yin; Lo, Lee-ChiangAngewandte Chemie, International Edition (2005), 44 (42), 6888-6892CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A mechanism-based probe for neuraminidase was developed. The probe consisted of a recognition head (N-acetylneuraminic acid), a trapping device (o-difluoromethylphenyl group), a linker, and a reporter group (biotin). It is capable of forming a biotinylated adduct with neuraminidase (NA) from Arthrobacter ureafaciens. It also displays an inhibitory effect on a no. of NA activities. ELISA expts. successfully demonstrated that influenza viruses can be selectively captured with this probe.
- 35Kwan, D. H.; Chen, H.-M.; Ratananikom, K.; Hancock, S. M.; Watanabe, Y.; Kongsaeree, P. T.; Samuels, A. L.; Withers, S. G. Self-immobilizing fluorogenic imaging agents of enzyme activity. Angew. Chem., Int. Ed. 2011, 50, 300– 303, DOI: 10.1002/anie.20100570535https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1alu7rM&md5=ccaedba2806b8764cdcfaaf00f39ffccSelf-Immobilizing Fluorogenic Imaging Agents of Enzyme ActivityKwan, David H.; Chen, Hong-Ming; Ratananikom, Khakhanang; Hancock, Susan M.; Watanabe, Yoichiro; Kongsaeree, Prachumporn T.; Samuels, A. Lacey; Withers, Stephen G.Angewandte Chemie, International Edition (2011), 50 (1), 300-303, S300/1-S300/35CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)We have demonstrated a novel application for enzyme substrates that generate quinone methides. Such substrates have previously been used as enzyme inactivators and abused as proteomics probes; however, these compds. may find greater value as histol. or cell-labeling agents for the investigation of a wide variety of organisms, as illustrated by our results with plants, yeast, and bacteria.
- 36Vocadlo, D. J.; Bertozzi, C. R. A strategy for functional proteomic analysis of glycosidase activity from cell lysates. Angew. Chem., Int. Ed. 2004, 43, 5338– 5342, DOI: 10.1002/anie.20045423536https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXptVanuro%253D&md5=784a8ee5408d8d25a1d46a771b8de249A strategy for functional proteomic analysis of glycosidase activity from cell lysatesVocadlo, David J.; Bertozzi, Carolyn R.Angewandte Chemie, International Edition (2004), 43 (40), 5338-5342CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)We have developed a strategy for activity-based labeling of retaining glycosidases by using the azide group as a sterically unobtrusive chem. tag. We prepd. 6-azido-2,6-dideoxy-2-fluoro-β-D-galactosyl fluoride (6Az2FgalF, 10, Scheme 2) as an activity-based probe for β-galactosidase. The high selectivity of both the inactivation with fluorosugars and the Staudinger ligation with phosphine probes allows detection of glycosidases in complex mixts. and the strategy can be used for profiling these enzyme activities in cell lysates. We have demonstrated that the approach can be used to tag several glycosidases from different glycoside hydrolase families. We anticipate that the strategy will find broad utility in proteomic anal. of these enzymes in prokaryotic and eukaryotic proteomes. The azide group might also be useful as a chem. tag within inactivators of other enzymes from entirely different families with sterically confining active sites.
- 37Gebler, J. C.; Aebersold, R.; Withers, S. G. Glu-537, not Glu-461, is the nucleophile in the active site of (LacZ) b-galactosidase from Escherichia coli. J. Biol. Chem. 1992, 267, 11126– 11130, DOI: 10.1016/S0021-9258(19)49884-037https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xls1KmtL0%253D&md5=42f3ec2cd10a75e454afb7eb65483be6Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) β-galactosidase from Escherichia coliGebler, John C.; Aebersold, Ruedi; Withers, Stephen G.Journal of Biological Chemistry (1992), 267 (16), 11126-30CODEN: JBCHA3; ISSN:0021-9258.The covalent intermediate formed during catalysis by the lac Z β-galactosidase from E. coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-β-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apoenzymes have been detd. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous β-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M.; Legler, G., 1984) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.
- 38Stubbs, K. A.; Scaffidi, A.; Debowski, A. W.; Mark, B. L.; Stick, R. V.; Vocadlo, D. J. Synthesis and use of mechanism-based protein profiling probes for retaining b-D-glucosaminidases facilitate identification of Pseudomonas aeruginosa NagZ. J. Am. Chem. Soc. 2008, 130, 327– 335, DOI: 10.1021/ja076360538https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhsVehurzE&md5=cf11627fe98c1f871d6a6711461194c4Synthesis and Use of Mechanism-Based Protein-Profiling Probes for Retaining β-D-Glucosaminidases Facilitate Identification of Pseudomonas aeruginosa NagZStubbs, Keith A.; Scaffidi, Adrian; Debowski, Aleksandra W.; Mark, Brian L.; Stick, Robert V.; Vocadlo, David J.Journal of the American Chemical Society (2008), 130 (1), 327-335CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The NagZ class of retaining exo-glucosaminidases play a crit. role in peptidoglycan recycling in Gram-neg. bacteria and the induction of resistance to β-lactams. Here we describe the concise synthesis of 2-azidoacetyl-2-deoxy-5-fluoro-β-D-glucopyranosyl fluoride as an activity-based proteomics probe for profiling these exo-glycosidases. This active-site directed reagent covalently inactivates this class of retaining N-acetylglucosaminidases with exquisite selectivity by stabilizing the glycosyl-enzyme intermediate. Inactivated Vibrio cholerae NagZ can be elaborated with biotin or a FLAG-peptide epitope using the Staudinger ligation or the Sharpless-Meldal click reaction and detected at nanogram levels. This ABPP enabled the profiling of the Pseudomonas aeruginosa proteome and identification at endogenous levels of a tagged protein with properties consistent with those of PA3005. Cloning of the gene encoding this hypothetical protein and biochem. characterization enabled unambiguous assignment of this hypothetical protein as a NagZ. The identification and cloning of this NagZ may facilitate the development of strategies to circumvent resistance to β-lactams in this human pathogen. As well, this general strategy, involving such 5-fluoro inactivators, may prove to be of general use for profiling proteomes and identifying glycoside hydrolases of medical importance or having desirable properties for biotechnol.
- 39Tsai, C.-S.; Yen, H.-Y.; Lin, M.-I.; Tsai, T.-I.; Wang, S.-Y.; Huang, W.-I.; Hsu, T.-L.; Cheng, Y. S. E.; Fang, J.-M.; Wong, C.-H. Cell-permeable probe for identification and imaging of sialidases. Proc. Natl. Acad. Sci. USA 2013, 110, 2466– 2471, DOI: 10.1073/pnas.1222183110There is no corresponding record for this reference.
- 40Hekmat, O.; Kim, Y.-W.; Williams, S. J.; He, S.; Withers, S. G. Active-site “fingerprinting” of glycosidases in complex mixture by mass spectrometry. Discovery of a novel retaining b-1–4-glycanase in Cellulomonas fimi. J. Biol. Chem. 2005, 280, 35126– 35135, DOI: 10.1074/jbc.M508434200There is no corresponding record for this reference.
- 41Chauvigné-Hines, L. M.; Anderson, L. N.; Weaver, H. M.; Brown, J. N.; Koech, P. K.; Nicora, C. D.; Hofstad, B. A.; Smith, R. D.; Wilkins, M. J.; Callister, S. J.; Wright, A. T. Suite of activity-based probes for cellulose-degrading enzymes. J. Am. Chem. Soc. 2012, 134, 20521– 20532, DOI: 10.1021/ja309790w41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhslalu77O&md5=f36917c45e720a70f786575722ae99fdSuite of Activity-Based Probes for Cellulose-Degrading EnzymesChauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.Journal of the American Chemical Society (2012), 134 (50), 20521-20532CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Microbial glycoside hydrolases play a dominant role in the biochem. conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits contg. cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degrdn. of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome-producing bacterium that couples rapid reprodn. rates to highly efficient degrdn. of cryst. cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycon, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chem. and to increase enzyme active site inclusion for liq. chromatog.-mass spectrometry (LC-MS) anal. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addn. to a large collection of integral cellulosome-contg. proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater understanding of the organismal role assocd. with biofuel development.
- 42Street, I. P.; Kempton, J. B.; Withers, S. G. Inactivation of a b-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-a-D-glucopyranosyl-enzyme intermediate: a detailed investigation. Biochemistry 1992, 31, 9970– 9978, DOI: 10.1021/bi00156a016There is no corresponding record for this reference.
- 43Hansen, F. G.; Bundgaard, E.; Madsen, R. A short synthesis of (+)-cyclophellitol. J. Org. Chem. 2005, 70, 10139– 10142, DOI: 10.1021/jo051645q43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFahs7zF&md5=24abc432bcb3877a1c17ca1d3effc1c7A Short Synthesis of (+)-CyclophellitolHansen, Flemming Gundorph; Bundgaard, Eva; Madsen, RobertJournal of Organic Chemistry (2005), 70 (24), 10139-10142CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)A new synthesis of (+)-cyclophellitol, a potent β-glucosidase inhibitor, has been completed in nine steps from D-xylose. The key transformations involve a zinc-mediated fragmentation of benzyl-protected Me 5-deoxy-5-iodo-xylofuranoside followed by a highly diastereoselective indium-mediated coupling with Et 4-bromocrotonate. Subsequent ring-closing olefin metathesis, ester redn., olefin epoxidn., and deprotection then afford the natural product. This constitutes the shortest synthesis of (+)-cyclophellitol reported to date.
- 44Witte, M. D.; Kallemeijn, W. W.; Aten, J.; Li, K.-Y.; Strijland, A.; Donker-Koopman, W. E.; Blijlevens, B.; Kramer, G.; van den Nieuwendijk, A. M. C. H.; Florea, B. I.; Hooibrink, B.; Hollak, C. E. M.; Ottenhoff, R.; Boot, R. G.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. F. G. Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat. Chem. Biol. 2010, 6, 907– 913, DOI: 10.1038/nchembio.46644https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVansb7N&md5=e9ead74400f958f1d4d2d5f5402d5c2fUltrasensitive in situ visualization of active glucocerebrosidase moleculesWitte, Martin D.; Kallemeijn, Wouter W.; Aten, Jan; Li, Kah-Yee; Strijland, Anneke; Donker-Koopman, Wilma E.; van den Nieuwendijk, Adrianus M. C. H.; Bleijlevens, Boris; Kramer, Gertjan; Florea, Bogdan I.; Hooibrink, Berend; Hollak, Carla E. M.; Ottenhoff, Roelof; Boot, Rolf G.; van der Marel, Gijsbert A.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.Nature Chemical Biology (2010), 6 (12), 907-913CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA mols. in situ. The authors here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA mols. in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, the authors obtained exclusive labeling of GBA mols. The authors present evidence from fluorescence-activated cell sorting anal., fluorescence microscopy and pulse-chase expts. of highly efficient labeling of GBA mols. in intact cells as well as tissues of mice. In addn., the authors illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.
- 45Li, K.-Y.; Jiang, J.; Witte, M. D.; Kallemeijn, W. W.; van den Elst, H.; Wong, C.-S.; Chander, S. D.; Hoogendoorn, S.; Beenakker, T. J. M.; Codée, J. D. C.; Aerts, J. M. F. G.; van der Marel, G. A.; Overkleeft, H. S. Synthesis of cyclophellitol, cyclophellitol aziridine, and their tagged derivatives. Eur. J. Org. Chem. 2014, 2014, 6030– 6043, DOI: 10.1002/ejoc.201402588There is no corresponding record for this reference.
- 46Goddard-Borger, E. D.; Wennekes, T.; Withers, S. G. Getting lucky in the lysosome. Nat. Chem. Biol. 2010, 6, 881– 883, DOI: 10.1038/nchembio.470There is no corresponding record for this reference.
- 47Wennekes, T.; van den Berg, R. J. B. H. N.; Donker, W.; van der Marel, G. A.; Strijland, A.; Aerts, J. M. F. G.; Overkleeft, H. S. Development of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivatives as selective inhibitors of glucosylceramide metabolism in man. J. Org. Chem. 2007, 72, 1088– 1097, DOI: 10.1021/jo061280p47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXot1Wgug%253D%253D&md5=8fb323274659673260e9aa98b90d7c05Development of Adamantan-1-yl-methoxy-Functionalized 1-Deoxynojirimycin Derivatives as Selective Inhibitors of Glucosylceramide Metabolism in ManWennekes, Tom; Van den Berg, Richard J. B. H. N.; Donker, Wilma; van der Marel, Gijsbert A.; Strijland, Anneke; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Journal of Organic Chemistry (2007), 72 (4), 1088-1097CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)The synthesis of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivs., e.g. I, via reductive ring opening, Swern oxidn. and reductive amination, is reported. The compds. reported are lipophilic iminosugar based on lead compd. II, a potent inhibitor of the three enzymes involved in the metab. of the glycosphingolipid glucosylceramide. The results demonstrate that relocating the lipophilic moiety from the nitrogen atom to other positions on the 1-deoxynojirimycin ring system does not lead to a more potent or selective inhibitor of glucosylceramide synthase. The β-aza-C-glycoside analog I retained the best inhibitory potency for glucosylceramide synthase and is a more potent inhibitor than the therapeutic agent N-butyl-1-deoxynojirimycin, marketed as treatment for Gaucher disease under the com. name Zavesca.
- 48Kallemeijn, W. W.; Li, K.-Y.; Witte, M. D.; Marques, A. R. A.; Aten, J.; Scheij, S.; Jiang, J.-B; Willems, L. I.; Voorn-Brouwer, T. M.; van Roomen, C. P. A. A.; Ottenhoff, R.; Boot, R. G.; van den Elst, H.; Walvoort, M. T. C.; Florea, B. I.; Codée, J. D. C.; van der Marel, G. A.; Aerts, J. M. F. G.; Overkleeft, H. S. Novel activity-based probes for broad-spectrum profiling of retaining beta-exoglucosidases in situ and in vivo. Angew. Chem., Int. Ed. 2012, 51, 12529– 12533, DOI: 10.1002/anie.20120777148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1GgsL3N&md5=6ce5afb5305786283c0abf83f603594eNovel Activity-Based Probes for Broad-Spectrum Profiling of Retaining β-Exoglucosidases In Situ and In VivoKallemeijn, Wouter W.; Li, Kah-Yee; Witte, Martin D.; Marques, Andre R. A.; Aten, Jan; Scheij, Saskia; Jiang, Jianbing; Willems, Lianne I.; Voorn-Brouwer, Tineke M.; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Boot, Rolf G.; van den Elst, Hans; Walvoort, Marthe T. C.; Florea, Bogdan I.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Angewandte Chemie, International Edition (2012), 51 (50), 12529-12533CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases. These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.
- 49Walvoort, M. T. C.; Kallemeijn, W. W.; Willems, L. I.; Witte, M. D.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. Tuning the leaving group in 2-deoxy-2-fluoroglucoside results in improved activity-based retaining beta-glucosidase probes. Chem. Commun. 2012, 48, 10386– 10388, DOI: 10.1039/c2cc35653hThere is no corresponding record for this reference.
- 50Yu, B.; Tao, H. Glycosyl trifluoroacetimidates. Part 1: preparation and application as new glycosyl donors. Tetrahedron Lett. 2001, 42, 2405– 2407, DOI: 10.1016/S0040-4039(01)00157-550https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhslOgs7w%253D&md5=187fb1007ca9f8cf9dcbc2aa4ffe0fbeGlycosyl trifluoroacetimidates. Part 1: Preparation and application as new glycosyl donorsYu, B.; Tao, H.Tetrahedron Letters (2001), 42 (12), 2405-2407CODEN: TELEAY; ISSN:0040-4039. (Elsevier Science Ltd.)Glycosyl (N-phenyl)trifluoroacetimidates, readily prepd. from 1-hydroxyl sugars by treatment with (N-phenyl)trifluoroacetimidoyl chloride in the presence of K2CO3, were demonstrated to be effective glycosyl donors. Glycosidation of these glycosyl fluoroacetimidates with alcs. is also reported.
- 51Witte, M. D.; Walvoort, M. T. C.; Li, K.-Y.; Kallemeijn, W. W.; Donker-Koopman, W. E.; Boot, R. G.; Aerts, J. M. F. G.; Codée, J. D. C.; van der Marel, G. A.; Overkleeft, H. S. Activity-based profiling of retaining beta-glucosidases: a comparative study. ChemBioChem 2011, 12, 1263– 1269, DOI: 10.1002/cbic.201000773There is no corresponding record for this reference.
- 52Harrak, Y.; Barra, C. M.; Delgado, A.; Castaño, A. R.; Llebaria, A. Galacto-configured aminocyclitol phytoceramides are potent in vivo invariant natural killer T cell stimulators. J. Am. Chem. Soc. 2011, 133, 12079– 12084, DOI: 10.1021/ja202610x52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptFKls70%253D&md5=76a2544ab5fe19c786686ae69c99db1fGalacto-Configured Aminocyclitol Phytoceramides Are Potent in Vivo Invariant Natural Killer T Cell StimulatorsHarrak, Youssef; Barra, Carolina M.; Delgado, Antonio; Castano, A. Raul; Llebaria, AmadeuJournal of the American Chemical Society (2011), 133 (31), 12079-12084CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A new class of α-galactosylceramide (αGC) non-glycosidic analogs bearing galacto-configured aminocyclitols as sugar surrogates, e.g. I (R = H, OH), have been obtained. The aminocyclohexane having a hydroxyl substitution pattern similar to an α-galactoside is efficiently obtained by a sequence involving Evans aldol reaction and ring-closing metathesis with a Grubbs catalyst to give a key intermediate cyclohexene, which has been converted in galacto-aminocyclohexanes that are linked through a secondary amine to a phyto-ceramide lipid having a cerotyl N-acyl group. Natural Killer T (NKT) cellular assays have resulted in the identification of an active compd., HS161, which has been found to promote NKT cell expansion in vitro in a similar fashion but more weakly than αGC. This compd. stimulates the release of Interferon-γ (IFNγ) and Interleukin-4 (IL-4) in iNKT cell culture but with lower potency than αGC. The activation of Invariant Natural Killer T (iNKT) cells by this compd. has been confirmed in flow cytometry expts. Remarkably, when tested in mice, HS161 selectively induces a very strong prodn. of IFN-γ indicative of a potent Th1 cytokine profile. Overall, these data confirm the agonist activity of αGC lipid analogs having charged amino-substituted polar heads and their capacity to modulate the response arising from iNKT cell activation in vivo.
- 53Alcaide, A.; Trapero, A.; Pérez, Y.; Llebaria, A. Galacto configured N-aminoaziridines: a new type of irreversible inhibitor of b-galactosidases. Org. Biomol. Chem. 2015, 13, 5690– 5697, DOI: 10.1039/C5OB00532AThere is no corresponding record for this reference.
- 54Ofman, T. P.; Küllmer, F.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. An orthogonally protected cyclitol for the construction of nigerose- and dextran-mimetic cyclophellitols. Org. Lett. 2021, 23, 9516, DOI: 10.1021/acs.orglett.1c03723There is no corresponding record for this reference.
- 55Jiang, J.; Kuo, C.-L.; Wu, L.; Franke, C.; Kallemeijn, W. W.; Florea, B. I.; van Meel, E.; van der Marel, G. A.; Codée, J. D. C.; Boot, R. G.; Davies, G. J.; Overkleeft, H. S.; Aerts, J. M. F. G. Detection of active mammalian GH31 alpha-glucosidases in health and disease using in-class, broad-spectrum activity-based probes. ACS Cent. Sci. 2016, 2, 351– 358, DOI: 10.1021/acscentsci.6b00057There is no corresponding record for this reference.
- 56Armstrong, Z.; Kuo, C.-L.; Lahav, D.; Liu, B.; Johnson, R.; Beenakker, T. J. M.; de Boer, C.; Wong, C.-S.; van Rijssel, E. R.; Debets, M. F.; Florea, B. I.; Hissink, C.; Boot, R. G.; Geurink, P. P.; Ovaa, H.; van der Stelt, M.; van der Marel, G. A.; Codée, J. D. C.; Aerts, J. M. F. G.; Wu, L.; Overkleeft, H. S.; Davies, G. J. Manno-epi-cyclophellitols enable activity-based protein profiling of human α-mannosidases and discovery of new Golgi mannosidase II inhibitors. J. Am. Chem. Soc. 2020, 142, 13021– 13029, DOI: 10.1021/jacs.0c0388056https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlSis7vK&md5=e5c95886b03aae55867ebe11fa828a1bManno-epi-cyclophellitols EnableActivity-Based Protein Profiling of Human α-Mannosidasesand Discovery of New Golgi Mannosidase II InhibitorsArmstrong, Zachary; Kuo, Chi-Lin; Lahav, Daniel; Liu, Bing; Johnson, Rachel; Beenakker, Thomas J. M.; de Boer, Casper; Wong, Chung-Sing; van Rijssel, Erwin R.; Debets, Marjoke F.; Florea, Bogdan I.; Hissink, Colin; Boot, Rolf G.; Geurink, Paul P.; Ovaa, Huib; van der Stelt, Mario; van der Marel, Gijsbert M.; Codee, Jeroen D. C.; Aerts, Johannes M. F. G.; Wu, Liang; Overkleeft, Herman S.; Davies, Gideon J.Journal of the American Chemical Society (2020), 142 (30), 13021-13029CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Golgi mannosidase II (GMII) catalyzes the sequential hydrolysisof two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans assocd. with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms.Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitolaziridine derivs. enabled activity-based protein profiling ofα-mannosidases from both human cell lysate and mouse tissue exts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallog. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.
- 57McGregor, N. G. S.; Kuo, C.-L.; Beenakker, T. J. M.; Wong, C.-S.; Armstrong, Z.; Florea, B. I.; Codée, J. D. C.; Overkleeft, H. S.; Aerts, J. M. F. G.; Davies, G. J. Synthesis of broad-specificity activity-based probes for exo-β-mannosidases. Org. Biomol. Chem. 2022, 20, 877– 886, DOI: 10.1039/D1OB02287C57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XpvVyktQ%253D%253D&md5=bf3b06d018229f253f6ec9d826861807Synthesis of broad-specificity activity-based probes for exo-β-mannosidasesMcGregor, Nicholas G. S.; Kuo, Chi-Lin; Beenakker, Thomas J. M.; Wong, Chun-Sing; Offen, Wendy A.; Armstrong, Zachary; Florea, Bogdan I.; Codee, Jeroen D. C.; Overkleeft, Herman S.; Aerts, Johannes M. F. G.; Davies, Gideon J.Organic & Biomolecular Chemistry (2022), 20 (4), 877-886CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Exo-β-mannosidases are a broad class of stereochem. retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-β-mannosidases in complex biol. samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogs selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biol. samples. Towards the development of ABPs for exo-β-mannosidases, we present a concise synthesis of β-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-β-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric center to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine deriv. by specifically labeling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogs hold promise for detecting exo-β-mannosidases in biol. and biomedical research.
- 58Jiang, J.; Kallemeijn, W. W.; Wright, D. W.; van den Nieuwendijk, A. M. C. H.; Coco Rohde, V.; Folch, E. C.; van den Elst, H.; Florea, B. I.; Scheij, S.; Donker-Koopman, W. E.; Verhoek, M.; Li, N.; Schürmann, M.; Mink, D.; Boot, R. G.; Codée, J. D. C.; van der Marel, G. A.; Davies, G. J.; Aerts, J. M. F. G.; Overkleeft, H. S. In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 alpha-L-fucosidases. Chem. Sci. 2015, 6, 2782– 2789, DOI: 10.1039/C4SC03739A58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitlaktrs%253D&md5=ba96b62c03ff47fb7375b48e854fb055In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 α-L-fucosidasesJiang, Jianbing; Kallemeijn, Wouter W.; Wright, Daniel W.; van den Nieuwendijk, Adrianus M. C. H.; Rohde, Veronica Coco; Folch, Elisa Colomina; van den Elst, Hans; Florea, Bogdan I.; Scheij, Saskia; Donker-Koopman, Wilma E.; Verhoek, Marri; Li, Nan; Schuermann, Martin; Mink, Daniel; Boot, Rolf G.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Davies, Gideon J.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Chemical Science (2015), 6 (5), 2782-2789CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)GH29 α-L-fucosidases catalyze the hydrolysis of α-L-fucosidic linkages. Deficiency in human lysosomal α-L-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein, we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 α-L-fucosidases from bacteria, mice and man. Crystallog. anal. on bacterial α-L-fucosidase confirms that the ABPs act by covalent modification of the active site nucleophile. Competitive activity-based protein profiling identified L-fuconojirimycin as the single GH29 α-L-fucosidase inhibitor from eight configurational isomers.
- 59Willems, L. I.; Beenakker, T. J. M.; Murray, B.; Scheij, S.; Kallemeijn, W. W.; Boot, R. G.; Verhoek, M.; Donker-Koopman, W. E.; Ferraz, M. J.; van Rijssel, E. R.; Florea, B. I.; Codée, J. D. C.; van der Marel, G. A.; Aerts, J. M. F. G.; Overkleeft, H. S. Potent and selective activity-based probes for GH27 human retaining alpha-galactosidases. J. Am. Chem. Soc. 2014, 136, 11622– 11625, DOI: 10.1021/ja507040n59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlaisrfF&md5=647ba661e02788a4b0494048be633236Potent and Selective Activity-Based Probes for GH27 Human Retaining α-GalactosidasesWillems, Lianne I.; Beenakker, Thomas J. M.; Murray, Benjamin; Scheij, Saskia; Kallemeijn, Wouter W.; Boot, Rolf G.; Verhoek, Marri; Donker-Koopman, Wilma E.; Ferraz, Maria J.; van Rijssel, Erwin R.; Florea, Bogdan I.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Journal of the American Chemical Society (2014), 136 (33), 11622-11625CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Lysosomal degrdn. of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases. The fluorescently labeled aziridine-based probes inhibit the two human retaining α-galactosidases αGal A and αGal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both α-galactosidases in cell exts., thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells vs. those derived from Fabry patients.
- 60Kuo, C.-L.; Su, Q.; van den Nieuwendijk, A. M. C. H.; Beenakker, T. J. M.; Offen, W. A.; Willems, L. I.; Boot, R. G.; Sarris, A. J.; Marques, A. R. A.; Codée, J. D. C.; van der Marel, G. A.; Florea, B. I.; Davies, G. J.; Overkleeft, H. S.; Aerts, J. M. F. G. The development of a broad-spectrum retaining beta-exogalactosidase activity-based probe. Org. Biomol. Chem. 2023, 21, 7813– 7820, DOI: 10.1039/D3OB01261AThere is no corresponding record for this reference.
- 61Artola, M.; Kuo, C.-L.; McMahon, S. A.; Hansen, T.; van der Lienden, M.; He, X.; van den Elst, H.; Florea, B. I.; Kermode, A. R.; van der Marel, G. A.; Gloster, T.; Codée, J. D. C.; Overkleeft, H. S.; Aerts, J. M. F. G. New irreversible α-L-iduronidase inhibitors and activity-based probes. Chem. Eur. J. 2018, 24, 19081– 19088, DOI: 10.1002/chem.201804662There is no corresponding record for this reference.
- 62Wu, L.; Jiang, J.; Jin, Y.; Kallemeijn, W. W.; Kuo, C.-L.; Artola, M.; Dai, W.; van Elk, C.; van Eijk, M.; van der Marel, G. A.; Codée, J. D. C.; Florea, B. I.; Aerts, J. M. F. G.; Overkleeft, H. S.; Davies, G. J. Activity-based probes for functional interrogation of retaining beta-glucuronidases. Nat. Chem. Biol. 2017, 13, 867– 873, DOI: 10.1038/nchembio.2395There is no corresponding record for this reference.
- 63Biarnés, X.; Ardèvol, A.; Planas, A.; Rovira, C.; Laio, A.; Parrinello, M. The conformational free energy landscape of b-D-glucopyranose. Implications for substrate preactivation in b-glucoside hydrolysis. J. Am. Chem. Soc. 2007, 129, 10686– 10693, DOI: 10.1021/ja068411o63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXovF2mtLs%253D&md5=3470037854668863bd12f3f0093b84c0The Conformational Free Energy Landscape of β-D-Glucopyranose. Implications for Substrate Preactivation in β-Glucoside HydrolasesBiarnes, Xevi; Ardevol, Albert; Planas, Antoni; Rovira, Carme; Laio, Alessandro; Parrinello, MicheleJournal of the American Chemical Society (2007), 129 (35), 10686-10693CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Using ab initio metadynamics the conformational free energy landscape of β-D-glucopyranose as a function of the puckering coordinates has been computed. It is shown that the correspondence between the free energy and the Stoddard's pseudorotational itinerary for the system is rather poor. The no. of free energy min. (9) is smaller than the no. of ideal structures (13). Moreover, only six min. correspond to a canonical conformation. The structural features, the electronic properties, and the relative stability of the predicted conformers permit the rationalization of the occurrence of distorted sugar conformations in all the available X-ray structures of β-glucoside hydrolase Michaelis complexes. It is shown that these enzymes recognize the most stable distorted conformers of the isolated substrate and at the same time the ones better prepd. for catalysis in terms of bond elongation/shrinking and charge distribution. This suggests that the factors governing the distortions present in these complexes are largely dictated by the intrinsic properties of a single glucose unit.
- 64Davies, G. J.; Planas, R.; Rovira, C. Conformational analysis of the reaction coordinate of glycosidases. Acc. Chem. Res. 2012, 45, 308– 316, DOI: 10.1021/ar200176564https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFyksrvP&md5=2c4a164a3dfed4c096cce1e3d046bf1dConformational Analyses of the Reaction Coordinate of GlycosidasesDavies, Gideon J.; Planas, Antoni; Rovira, CarmeAccounts of Chemical Research (2012), 45 (2), 308-316CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The enzymic hydrolysis of the glycosidic bond is catalyzed by diverse enzymes generically termed glycoside hydrolases (hereafter GHs) or glycosidases. The many sequence-based families of glycosidases have served as a rich hunting ground for enzymologists for years. Not only are these enzymes of fundamental interest, providing paradigms for enzymic catalysis that extend beyond the bounds of carbohydrate chem., but the enzymes themselves play myriad essential roles in diverse biol. processes. The wide utility of glycosidases, from their industrial harnessing in the hydrolysis of plant biomass to their roles in human physiol. and disease, has engendered a large scientific constituency with an interest in glycosidase chem. A fascinating thread of this research, and one with major impact on the design of enzyme inhibitors, is the conformational anal. of reaction pathways within the diverse families. These GH families provide a large pallet of enzymes with which chemists have attempted to depict the conformational landscape of glycosidase action. In this Account, we review three-dimensional insight into the conformational changes directed by glycosidases, primarily from structural observations of the stable enzyme-ligand species adjacent to the transition state (or states) and of enzyme-inhibitor complexes. We further show how recent computational advances dovetail with structural insight to provide a quantum mech. basis for glycosidase action. The glycosidase-mediated hydrolysis of the acetal or ketal bond in a glycoside may occur with either inversion or retention of the configuration of the anomeric carbon. Inversion involves a single step and transition state, whereas retention, often referred to as the double displacement, is a two-step process with two transition states. The single transition state for the inverting enzymes and the two transition states (those flanking the covalent intermediate) in the double displacement have been shown to have substantial oxocarbenium ion character. The dissociative nature of these transition states results in significant relative pos. charge accumulation on the pyranose ring. The delocalization of lone-pair electrons from the ring oxygen that stabilizes the cationic transition state implies that at, or close to, the transition states the pyranose will be distorted away from its lowest energy conformation to one that favors orbital overlap. Over the preceding decade, research has highlighted the harnessing of noncovalent interactions to aid this distortion of the sugar substrates from their lowest energy chair conformation to a variety of different boat, skew boat, and half-chair forms, each of which favors catalysis with a given enzyme and substrate. Crystallog. observation of stable species that flank the transition state (or states), of both retaining and inverting glycosidases, has allowed a description of their conformational itineraries, illustrating how enzymes facilitate the "electrophilic migration" of the anomeric center along the reaction coordinate. The blossom of computational approaches, such as ab initio metadynamics, has underscored the quantum mech. basis for glycoside hydrolysis. Conformational analyses highlight not only the itineraries used by enzymes, enabling their inhibition, but are also reflected in the nonenzymic synthesis of glycosides, wherein chemists mimic strategies found in nature.
- 65Vasella, A.; Davies, G. J.; Böhm, M. Glycosidase mechanisms. Curr. Opin. Chem. Biol. 2002, 6, 619– 629, DOI: 10.1016/S1367-5931(02)00380-065https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xot1agsbs%253D&md5=17e2373cd971486dec70981975dbdb61Glycosidase mechanismsVasella, Andrea; Davies, Gideon J.; Boehm, MatthiasCurrent Opinion in Chemical Biology (2002), 6 (5), 619-629CODEN: COCBF4; ISSN:1367-5931. (Elsevier Science Ltd.)A review with 99 refs. The 3-dimensional structure of glycosidases and of their complexes and the study of transition-state mimics reveal structural details that correlate with their catalytic mechanism. Of particular interest are the transition-state conformations harnessed by individual enzymes and the substrate distortion obsd. in enzyme-ligand complexes. The 3-dimensional structure in synergy with transition-state mimicry opens the way for mechanistic interpretation of enzyme inhibition and for the development of therapeutic agents.
- 66Beenakker, T. J. M.; Wander, D. P. A.; Offen, W. A.; Artola, M.; Raich, L.; Ferraz, M. J.; Li, K.-Y.; Houben, J. H. P. M.; van Rijssel, E. R.; Hansen, T.; van der Marel, G. A.; Codée, J. D. C.; Aerts, J. M. F. G.; Rovira, C.; Davies, G. J.; Overkleeft, H. S. Carba-cyclophellitols are neutral retaining-glucosidase inhibitors. J. Am. Chem. Soc. 2017, 139, 6534– 6537, DOI: 10.1021/jacs.7b0177366https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXntVGgtb8%253D&md5=4ca99d2e4c4a4a9755db80b8d3ec8b61Carba-cyclophellitols are neutral retaining-glucosidase inhibitorsBeenakker, Thomas J. M.; Wander, Dennis P. A.; Offen, Wendy A.; Artola, Marta; Raich, Lluis; Ferraz, Maria J.; Li, Kah-Yee; Houben, Judith H. P. M.; van Rijssel, Erwin R.; Hansen, Thomas; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; Aerts, Johannes M. F. G.; Rovira, Carme; Davies, Gideon J.; Overkleeft, Herman S.Journal of the American Chemical Society (2017), 139 (19), 6534-6537CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The conformational anal. of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine, both covalent retaining-β-glucosidase inhibitors, we postulated that the corresponding carba "cyclopropyl" analog would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the 4H3 transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors showed a strong bias for the 4H3 conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (Ki = 8.2 nM) of Thermotoga maritima (TmGH1) β-glucosidase. Three-dimensional structural anal. and comparison with unreacted epoxides showed that this compd. indeed bound in the 4H3 conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
- 67de Boer, C.; Armstrong, Z.; Lit, V. A. J.; Barash, U.; Ruijgrok, G.; Boyango, I.; Weitzenberg, M. M.; Schröder, S. P.; Sarris, A. J. C.; Meeuwenoord, N. J.; Bule, P.; Kayal, Y.; Ilan, N.; Codée, J. D. C.; Vlodavsky, I.; Overkleeft, H. S.; Davies, G. J.; Wu, L. Mechanism based heparanase inhibitors reduce cancer metastasis in vivo. Proc. Natl. Acad. Sci. USA 2022, 119, e2203167119 DOI: 10.1073/pnas.2203167119There is no corresponding record for this reference.
- 68Artola, M.; Wu, L.; Ferraz, M. J.; Kuo, C.-L.; Raich, L.; Breen, I. Z.; Offen, W. A.; Codée, J. D. C.; van der Marel, G. A.; Rovira, C.; Aerts, J. M. F. G.; Davies, G. J.; Overkleeft, H. S. 1,6-Cyclophellitol cyclosulfates: a new class of irreversible glycosidase inhibitor. ACS Cent. Sci. 2017, 3, 784– 793, DOI: 10.1021/acscentsci.7b0021468https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFGltrfK&md5=b8aaf5f955c0d78618aded623375f92e1,6-Cyclophellitol cyclosulfates: A new class of irreversible glycosidase inhibitorArtola, Marta; Wu, Liang; Ferraz, Maria J.; Kuo, Chi-Lin; Raich, Lluis; Breen, Imogen Z.; Offen, Wendy A.; Codee, Jeroen D. C.; van der Marel, Gijsbert A.; Rovira, Carme; Aerts, Johannes M. F. G.; Davies, Gideon J.; Overkleeft, Herman S.ACS Central Science (2017), 3 (7), 784-793CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)The essential biol. roles played by glycosidases, coupled with the diverse therapeutic benefits of pharmacol. targeting of these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivs. of epoxides in org. synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here, we present the synthesis, conformational anal., and application of novel 1,6-cyclophellitol cyclosulfates. We showed that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matched that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacted more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
- 69Li, Z.; Pickles, I. B.; Sharma, M.; Melling, B.; Pallasdies, L.; Codée, J. D. C.; Williams, S. J.; Overkleeft, H. S.; Davies, G. J. Detection of sulfoquinovosidase activity in cell lysates using activity-based probes. Angew. Chem., Int. Ed. 2024, 63, e202401358 DOI: 10.1002/anie.202401358There is no corresponding record for this reference.
- 70McGregor, N.; Artola, M.; Nin-Hill, A.; Linzel, D.; Haon, M.; Reijngoud, J.; Ram, A. F. J.; Rosso, M.-N.; van der Marel, G. A.; Codée, J. D. C.; van Wezel, G. P.; Berrin, J.-G.; Rovira, C.; Overkleeft, H. S.; Davies, G. J. Rational design of mechanism-based inhibitors and activity-based probes for the identification of retaining α-L-arabinofuranosidases. J. Am. Chem. Soc. 2020, 142, 4648– 4662, DOI: 10.1021/jacs.9b1135170https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivFyis7s%253D&md5=5e8b4fa22bcc8f05667f308ed9516636Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-L-ArabinofuranosidasesMcGregor, Nicholas G. S.; Artola, Marta; Nin-Hill, Alba; Linzel, Daniel; Haon, Mireille; Reijngoud, Jos; Ram, Arthur; Rosso, Marie-Noelle; van der Marel, Gijsbert A.; Codee, Jeroen D. C.; van Wezel, Gilles P.; Berrin, Jean-Guy; Rovira, Carme; Overkleeft, Herman S.; Davies, Gideon J.Journal of the American Chemical Society (2020), 142 (10), 4648-4662CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Identifying and characterizing the enzymes responsible for an obsd. activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the prodn. of α-L-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-L-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-L-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural expts., the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-L-arabinofuranosidases.
- 71Schröder, S. P.; de Boer, C.; McGregor, N. G. S.; Rowland, R. J.; Moroz, O.; Blagova, E.; Reijngoud, J.; Arentshorst, M.; Osborn, D.; Morant, M. D.; Abbate, E.; Stringer, M. A.; Krogh, K. B. R. M.; Raich, L.; Rovira, C.; Berrin, J.-G.; van Wezel, G. P.; Ram, A. F. J.; Florea, B. I.; van der Marel, G. A.; Codée, J. D. C.; Wilson, K. S.; Wu, L.; Davies, G. J.; Overkleeft, H. S. Dynamic and functional profiling of xylan-degrading enzymes in Aspergillus secretomes using activity-based probes. ACS Cent. Sci. 2019, 5, 1067– 1078, DOI: 10.1021/acscentsci.9b0022171https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MzjtFykug%253D%253D&md5=bf680edfbc4663d4b7a12cbcacd8db15Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Aspergillus Secretomes Using Activity-Based ProbesSchroder Sybrin P; de Boer Casper; Florea Bogdan I; van der Marel Gijsbert A; Codee Jeroen D C; Overkleeft Herman S; McGregor Nicholas G S; Rowland Rhianna J; Moroz Olga; Blagova Elena; Wilson Keith S; Wu Liang; Davies Gideon J; Reijngoud Jos; Arentshorst Mark; van Wezel Gilles P; Ram Arthur F J; Osborn David; Abbate Eric; Morant Marc D; Stringer Mary A; Krogh Kristian B R M; Raich Lluis; Rovira Carme; Rovira Carme; Berrin Jean-GuyACS central science (2019), 5 (6), 1067-1078 ISSN:2374-7943.Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
- 72McGregor, N. G. S.; de Boer, C.; Foucart, Q. P. O.; Beenakker, T.; Offen, W. A.; Codée, J. D. C.; Willems, L. I.; Overkleeft, H. S.; Davies, G. J. A multiplexing activity-based protein profiling platform for dissection of a native bacterial xyloglucan-degrading system. ACS Cent. Sci. 2023, 9, 2306– 2314, DOI: 10.1021/acscentsci.3c00831There is no corresponding record for this reference.
- 73van Meel, E.; Bos, E.; van den Lienden, M. J. C.; Overkleeft, H. S.; van Kasteren, S. I.; Koster, A. J.; Aerts, J. M. F. G. Localization of active endogenous and exogenous beta-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts. Traffic 2019, 20, 346– 356, DOI: 10.1111/tra.12641There is no corresponding record for this reference.
- 74Schröder, S. P.; Petracca, R.; Minnee, H.; Artola, M.; Aerts, J. M. F. G.; Codée, J. D. C.; van der Marel, G. A.; Overkleeft, H. S. A divergent synthesis of L-arabino and D-xylo-configured cyclophellitol epoxides and aziridines. Eur. J. Org. Chem. 2016, 2016, 4787– 4794, DOI: 10.1002/ejoc.201600983There is no corresponding record for this reference.
- 75Jariwala, P. B.; Pellock, S. J.; Goldfarb, D.; Cloer, E. C.; Artola, M.; Simpson, J. B.; Bhatt, A. P.; Walton, W. G.; Roberts, L. R.; Major, M. B.; Davies, G. J.; Overkleeft, H. S.; Redinbo, M. R. Discovering the microbial enzymes driving drug toxicity with activity-based protein profiling. ACS Chem. Biol. 2020, 15, 217– 225, DOI: 10.1021/acschembio.9b0078875https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1CrsrjM&md5=6fad6eccb2851f48c279eaac95afe6a2Discovering the Microbial Enzymes Driving Drug Toxicity with Activity-Based Protein ProfilingJariwala, Parth B.; Pellock, Samuel J.; Goldfarb, Dennis; Cloer, Erica W.; Artola, Marta; Simpson, Joshua B.; Bhatt, Aadra P.; Walton, William G.; Roberts, Lee R.; Major, Michael B.; Davies, Gideon J.; Overkleeft, Herman S.; Redinbo, Matthew R.ACS Chemical Biology (2020), 15 (1), 217-225CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)It is increasingly clear that interindividual variability in human gut microbial compn. contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not obsd. in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial β-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug-glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with ex vivo kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the mol. basis for differential gut bacterial GUS inhibition obsd. between human fecal samples. Taken together, this work provides an unprecedented tech. and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.
- 76Lipsh-Sokolik, R.; Khersonsky, O.; Schröder, S. P.; de Boer, C.; Hoch, S.-Y.; Davies, G. J.; Overkleeft, H. S.; Fleishman, S. J. Combinatorial assembly and design of enzymes. Science 2023, 379, 195– 201, DOI: 10.1126/science.ade9434There is no corresponding record for this reference.
- 77Lahav, D.; Liu, B.; van den Berg, R. J. B. H. N.; van den Nieuwendijk, A. M. C. H.; Wennekes, T.; Ghisaidoobe, A. T.; Breen, I.; Ferraz, M. J.; Kuo, C.-L.; Wu, L.; Geurink, P. P.; Ovaa, H.; van der Marel, G. A.; van der Stelt, M.; Boot, R. G.; Davies, G. J.; Aerts, J. M. F. G.; Overkleeft, H. S. A fluorescence polarization activity-based protein profiling assay in the discovery of potent, selective inhibitors for human non-lysosomal glucosylceramidase. J. Am. Chem. Soc. 2017, 139, 14192– 14197, DOI: 10.1021/jacs.7b0735277https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFCis7vO&md5=0eda44ab424f5685fa536d64be93f91eA fluorescence polarization activity-based protein profiling assay in the discovery of potent, selective inhibitors for human nonlysosomal glucosylceramidaseLahav, Daniel; Liu, Bing; van den Berg, Richard J. B. H. N.; van den Nieuwendijk, Adrianus M. C. H.; Wennekes, Tom; Ghisaidoobe, Amar T.; Breen, Imogen; Ferraz, Maria J.; Kuo, Chi-Lin; Wu, Liang; Geurink, Paul P.; Ovaa, Huib; van der Marel, Gijsbert A.; van der Stelt, Mario; Boot, Rolf G.; Davies, Gideon J.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.Journal of the American Chemical Society (2017), 139 (40), 14192-14197CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chem. tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell exts. contg. recombinant, overexpressed glycosidase as the easily accessible enzyme source.
- 78Ndeh, D.; Rogowski, A.; Cartmell, A.; Luis, A. S.; Baslé, A.; Gray, J.; Venditto, I.; Briggs, J.; Zhang, X.; Labourel, A.; Terrapon, N.; Buffetto, F.; Nepogodiev, S.; Xiao, Y.; Field, R. A.; Zhu, Y.; O’Neil, M.; Urbanowicz, B. R.; York, W. S.; Davies, G. J.; Abbott, D. W.; Ralet, M.-C.; Martens, E. C.; Henrissat, B.; Gilbert, H. J. Complex pectin metabolism by gut bacteria reveals novel catalytic functions. Nature 2017, 544, 65– 70, DOI: 10.1038/nature2172578https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltVWnurY%253D&md5=8a779b1beb1d7d394bcae22b635488cfComplex pectin metabolism by gut bacteria reveals novel catalytic functionsNdeh, Didier; Rogowski, Artur; Cartmell, Alan; Luis, Ana S.; Basle, Arnaud; Gray, Joseph; Venditto, Immacolata; Briggs, Jonathon; Zhang, Xiaoyang; Labourel, Aurore; Terrapon, Nicolas; Buffetto, Fanny; Nepogodiev, Sergey; Xiao, Yao; Field, Robert A.; Zhu, Yanping; O'Neill, Malcolm A.; Urbanowicz, Breeanna R.; York, William S.; Davies, Gideon J.; Abbott, D. Wade; Ralet, Marie-Christine; Martens, Eric C.; Henrissat, Bernard; Gilbert, Harry J.Nature (London, United Kingdom) (2017), 544 (7648), 65-70CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The metab. of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degrdn. involves previously undiscovered enzyme families and catalytic activities. The degrdn. system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.
- 79Ren, W.; Pengelly, R.; Farren-Dai, M.; Shamsi Kazem Abadi, S.; Oehiler, V.; Akintola, O.; Draper, J.; Meanwell, M.; Chakladar, S.; Swiderek, K.; Moliner, V.; Britton, R.; Gloster, T. M.; Bennet, A. J. Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level. Nat. Commun. 2018, 9, 3243, DOI: 10.1038/s41467-018-05702-779https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c7nslyrtw%253D%253D&md5=dd3282d4c9a0eb8fe4c7805dc4d29118Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic levelRen Weiwu; Farren-Dai Marco; Akintola Oluwafemi; Draper Jason; Meanwell Michael; Chakladar Saswati; Britton Robert; Bennet Andrew J; Pengelly Robert; Oehler Verena; Gloster Tracey M; Shamsi Kazem Abadi Saeideh; Swiderek Katarzyna; Moliner VicentNature communications (2018), 9 (1), 3243 ISSN:.Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate's structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair ((2)H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair ((2)H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.
- 80Jain, N.; Tamura, K.; Déjean, G.; van Petegem, F.; Brumer, H. Orthogonal active-site labels for mixed-linkage endo-b-glucanases. ACS Chem. Biol. 2021, 16, 1968– 1984, DOI: 10.1021/acschembio.1c00063There is no corresponding record for this reference.
- 81Schröder, S. P.; Kallemeijn, W. W.; Debets, M. F.; Hansen, T.; Sobala, L. F.; Hakki, Z.; Williams, S. J.; Beenakker, T. J. M.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Davies, G. J.; Overkleeft, H. S. Spiro-epoxyglycosides as activity-based probes for glycoside hydrolase family 99 endomannosidase/endomannanase. Chem. Eur. J. 2018, 24, 9983– 9992, DOI: 10.1002/chem.201801902There is no corresponding record for this reference.
- 82Thaler, M.; Ofman, T. P.; Kok, K.; Heming, J. J. A.; Moran, E.; Pickles, I.; Leijs, A. A.; van den Nieuwendijk, A. M. C. H.; van den Berg, R. J. B. H. N.; Ruijgrok, G.; Armstrong, Z.; Salgado-Benvindo, C.; Ninaber, D. K.; Snijder, E. J.; van Boeckel, C. A. A.; Artola, A.; Davies, G. J.; Overkleeft, H. S.; van Hemert, M. J. Epi-cyclophellitol cyclosulfate, a mechanism-based ER α-glucosidase II inhibitor, blocks replication of SARS-CoV-2 and other coronaviruses. ACS Cent. Sci. 2024, 10, 1594, DOI: 10.1021/acscentsci.4c00506There is no corresponding record for this reference.