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Comparison of Cellular Target Engagement Methods for the Tubulin Deacetylases Sirt2 and HDAC6: NanoBRET, CETSA, Tubulin Acetylation, and PROTACs
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Comparison of Cellular Target Engagement Methods for the Tubulin Deacetylases Sirt2 and HDAC6: NanoBRET, CETSA, Tubulin Acetylation, and PROTACs
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ACS Pharmacology & Translational Science

Cite this: ACS Pharmacol. Transl. Sci. 2022, 5, 2, 138–140
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https://doi.org/10.1021/acsptsci.2c00004
Published January 27, 2022

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is available under these Terms of Use.

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Abstract

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The tubulin deacetylases Sirt2 and HDAC6 have been associated with the development of various diseases. Herein, we discuss recent approaches that enable cellular target engagement studies for these deacetylases and thus play a critical role in the evaluation of small molecule inhibitors of Sirt2 or HDAC6 as potential therapeutic agents.

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Copyright © 2022 The Authors. Published by American Chemical Society

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Special Issue

Published as part of the ACS Pharmacology & Translational Science special issue “Epigenetics 2022”.

Both histone deacetylase 6 (HDAC6) and Sirtuin 2 (Sirt2) are protein deacylases that cleave off acetyl as well as other acyl groups from the ε-amino group of lysines in their substrate proteins. While HDAC6 is a Zn2+-dependent lysine deacylase and belongs to class IIb of HDACs, Sirt2 features an NAD+-dependent catalytic mechanism and has been classified as a class III HDAC, the so-called Sirtuin family. Despite the fact that both deacylases have been assigned as histone deacetylases (HDACs), they share acetylated α-tubulin (α-tubulin K40ac) as a major substrate and are hence frequently referred to as tubulin deacetylases. (1,2) Dysregulation of both Sirt2 and HDAC6 activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. This has prompted intense efforts in the development of small molecule inhibitors of Sirt2 and HDAC6, which are reviewed elsewhere. (3,4) A critical step in preclinical drug discovery is the assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment. (5) This step, also referred to as cellular target engagement, is highly important for successfully delivering compounds with the desired biological and ultimately clinical effects, as the on-target activity of small molecules can be changed significantly when transitioning from a biochemical to a cellular environment. A loss of activity in a cellular environment can be attributed to various factors, including low cell permeability, compound efflux, off-target protein binding, or a change in target protein’s structure/accessibility. Herein, we review different approaches that have recently been applied to study cellular target engagement for Sirt2 and HDAC6, thereby playing a critical role in the evaluation of small molecule inhibitors for these two tubulin deacetylases.

As already mentioned, Sirt2 and HDAC6 are tubulin deacetylases. Therefore, α-tubulin acetylation has widely been applied to confirm cellular inhibition of Sirt2- or HDAC6-mediated deacetylation. The increase in tubulin acetylation is most commonly detected with antibody-based techniques (e.g., Western blot, ELISA, immunofluorescence microscopy). An important benefit of using tubulin acetylation as a readout for cellular Sirt2 or HDAC6 target engagement is that this method does not require any context-specific modification of the targeted proteins. However, α-tubulin acetylation is influenced not only by Sirt2 and HDAC6 activity but also by other factors such as α-tubulin N-acetyltransferase (ATAT1) activity, oxidative stress or high glucose levels. Thus, effects of certain compounds on tubulin acetylation must not necessarily be a consequence of cellular Sirt2 or HDAC6 inhibition. Furthermore, the overall effect of selective Sirt2 inhibition on tubulin acetylation is not highly pronounced and often challenging to demonstrate via Western blot, due to its low dynamic range of detection. This might be one reason, why several recently published studies preferred immunofluorescence microscopy over Western blotting to prove cellular inhibition of Sirt2-mediated α-tubulin deacetylation. (6,7) Additionally, antibody-based methods for the detection of α-tubulin acetylation are time and labor intensive because of their heterogeneous assay protocols, thereby limiting the throughput of these methods. Due to the aforementioned drawbacks of using α-tubulin acetylation as a readout for cellular inhibition of Sirt2- or HDAC6-mediated deacetylation, several alternative methods have been recently established to demonstrate cellular target engagement for Sirt2 and HDAC6.

Cellular thermal shift assays (CETSA) enable the assessment of cellular target engagement by quantifying the changes in the thermal stability of a targeted protein upon ligand binding in intact cells. Similar to the aforementioned methods based on α-tubulin acetylation, CETSA is a label-free technique and is usually combined with an immunosorbent assay (e.g., Western blot) for detecting the amount of stabile protein remaining in solution at a given temperature. In contrast to α-tubulin acetylation, which can be influenced by various factors (see above), ligand-induced shifts of thermal protein stability are a direct and exclusive consequence of ligand-target-binding interactions. Due to its broad applicability, the CETSA technique has revolutionized cell-based target engagement studies and has recently been successfully used for Sirt2 (7) as well as HDAC6. (8) However, it should be noted that not all ligand-protein interactions result in a significant change of thermal protein stability, depending on the nature of binding interaction. Thus, negative results from CETSA-based target engagement studies should be verified by an orthogonal method, to rule out false negatives. More recent approaches to increase the throughput of CETSA, so-called high-throughput CETSA (HT-CETSA), use methods other than Western blot (e.g., β-galactosidase and NanoLuciferase reporters, AlphaLISA) for protein detection, but as of now they have not been applied for Sirt2- or HDAC6-based target engagement studies.

The approach of targeted protein degradation, induced by means of so-called proteolysis-targeting chimeras (PROTACs), has recently gained much traction due to a number of key advantages compared to standard inhibition of protein function by small molecules. Two major advantages of PROTACs are their catalytic mode of action and a durable inhibition of protein function as a consequence of irreversible target protein degradation. Besides these pharmacological benefits, which have implications for basic research and clinical applications, PROTACs can also be used as molecular tools to study cellular target engagement. By displacing the PROTAC from its target protein binding site, an unlabeled small molecule competitor can prevent PROTAC-induced protein degradation, which can be assessed via Western blot analysis. (9,10) This experiment is commonly performed in the course of PROTAC validation, but it can just as well be used as a method for cellular target engagement. Similar to tubulin acetylation- and CETSA-based approaches for cellular target engagement, the PROTAC-based protocol does not require specific protein modifications. Of course, the availability of PROTACs that are able to induce a significant reduction of target protein levels is required for such an approach. In the case of Sirt2 (9) and HDAC6, (10,11) potent and selective degraders have already been reported, thereby laying the key basis for PROTAC-mediated target engagement studies for these two deacylases. Of note, ligands showing noncompetitive binding toward the employed PROTAC cannot be detected with the approach described above. For ligands that show a reduction of PROTAC-mediated protein degradation, an orthogonal target engagement assay should be performed, as an inhibition of the employed E3 ligase or the proteasome might lead to false positive results.

The NanoBRET technique is proximity-based and relies on bioluminescence resonance energy transfer (BRET) from a donor (e.g., Nanoluciferase (Nluc)-labeled fusion protein) to an acceptor (e.g., fluorescently labeled ligand). Thus, the NanoBRET technology requires both a modified ligand and a modified, non-native target protein (Table 1). If used in a displacement setup, the binding of an unlabeled small molecule ligand to the targeted binding site can be detected via the displacement of the fluorescent tracer, thereby resulting in a reduced BRET signal. As with all tracer-dependent assay techniques, there is a potential risk of false negative results for ligands that do not show competitive binding behavior toward the tracer molecule. In contrast to the other methods applied for studying cellular target engagement for Sirt2 or HDAC6 (see above), NanoBRET assays can be performed in a microtiter plate format following a straightforward homogeneous assay protocol, which does not require any antibodies, cell lysis/permeabilization or washing steps. Therefore, the NanoBRET technology allows a quantitative real-time detection of protein–ligand interactions in live cells. Moreover, the assay readout can be performed with a plate reader in a highly accurate and high-throughput manner. Whereas NanoBRET-based target engagement assays for Zn2+-dependent HDACs, including HDAC6, have been available for several years, (12) the first method for a member of the NAD+-dependent HDACs, Sirt2, has only very recently been reported. (6) Application of the NanoBRET-based Sirt2 target engagement assay enabled the development of small molecule inhibitors with low nanomolar Sirt2 affinities in cells. Moreover, this method was used to prove cellular target engagement for several literature-known Sirt2 inhibitors and provided additional evidence of the low on-target specificity of the broadly used Sirt2 probe Sirtinol. (6)

Table 1. Comparison of Available Cellular Target Engagement Methods for the Tubulin Deacetylases Sirt2 and HDAC6
 method
 α-tubulin acetylationCETSAPROTACNanoBRET
principleactivity-basedthermal stabilityproximity-basedproximity-based
secondary detection methodWestern blot, fluorescence microscopyWestern blotWestern blotnot required
modified ligand (tracer)not requirednot requiredrequiredrequired
modified proteinnot requirednot requirednot requiredrequired
assay protocolheterogeneousheterogeneousheterogeneoushomogeneous

Cellular target engagement studies are of fundamental importance in order to confirm relevant drug targets and to evaluate the cellular on-target activity of biologically active compounds. For the tubulin deacetylases Sirt2 and HDAC6, which are both relevant drug targets, substantial progress in establishing methods to study cellular target engagement has been made in recent years. Currently, researchers can choose between a few techniques to assess cellular target engagement for Sirt2 and HDAC6, including tubulin acetylation, CETSA, as well as PROTAC- and NanoBRET-based approaches. Systematic application of different orthogonal target engagement methods will further aid the development of high-quality probes and drug candidates with a well-validated mechanism of action.

Author Information

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  • Corresponding Author
  • Authors
    • Anja Vogelmann - Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstraße 25, 79104 Freiburg im Breisgau, Germany
    • Manfred Jung - Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstraße 25, 79104 Freiburg im Breisgau, Germany;  Orcidhttps://orcid.org/0000-0002-6361-7716
    • Finn K. Hansen - Department of Pharmaceutical & Medicinal Chemistry, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany;  Orcidhttps://orcid.org/0000-0001-9765-5975
  • Author Contributions

    The manuscript was written through contributions of all authors and all authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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M.S. (Li 204/04) is supported by the Verband der Chemischen Industrie (VCI). A.V. and M.J. were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through SFB992 (Project ID 192904750). We thank M.E. Huber for the preparation of the Table of Contents Graphic.

Abbreviations

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ATAT1

α-tubulin N-acetyltransferase

BRET

bioluminescence resonance energy transfer

CETSA

cellular thermal shift assays

ELISA

enzyme-linked immunosorbent assay

HDAC

histone deacetylase

NAD

Nicotinamide adenine dinucleotide

Nluc

nanoluciferase

PROTAC

proteolysis-targeting chimera

References

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This article references 12 other publications.

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    12
    The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines
    Kolbinger, Fiona R.; Koeneke, Emily; Ridinger, Johannes; Heimburg, Tino; Mueller, Michael; Bayer, Theresa; Sippl, Wolfgang; Jung, Manfred; Gunkel, Nikolas; Miller, Aubry K.; Westermann, Frank; Witt, Olaf; Oehme, Ina
    Archives of Toxicology (2018), 92 (8), 2649-2664CODEN: ARTODN; ISSN:0340-5761. (Springer)
    High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concns. up to 25μM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concn.-dependent manner in several human neuroblastoma cell lines. In addn. to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.

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      The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase
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      The silent information regulator 2 protein (Sir2p) of Saccharomyces cerevisiae is an NAD-dependent histone deacetylase that plays a crit. role in transcriptional silencing. Here, we report that a human ortholog of Sir2p, sirtuin type 2 (SIRT2), is a predominantly cytoplasmic protein that colocalizes with microtubules. SIRT2 deacetylates lysine-40 of α-tubulin both in vitro and in vivo. Knockdown of SIRT2 via siRNA results in tubulin hyperacetylation. SIRT2 colocalizes and interacts in vivo with HDAC6, another tubulin deacetylase. Enzymic anal. of recombinant SIRT2 in comparison to a yeast homolog of Sir2 protein (Hst2p) shows a striking preference of SIRT2 for acetylated tubulin peptide as a substrate relative to acetylated histone H3 peptide. These observations establish SIRT2 as a bona fide tubulin deacetylase.
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      Reversible acetylation of α-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated α-tubulin is tightly controlled and stereotypic. Acetylated α-tubulin is most abundant in stable microtubules but is absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts. However, the enzymes responsible for regulating tubulin acetylation and deacetylation are not known. Here we report that a member of the histone deacetylase family, HDAC6, functions as a tubulin deacetylase. HDAC6 is localized exclusively in the cytoplasm, where it assocs. with microtubules and localizes with the microtubule motor complex contg. p150glued. In vivo, the overexpression of HDAC6 leads to a global deacetylation of α-tubulin, whereas a decrease in HDAC6 increases α-tubulin acetylation. In vitro, purified HDAC6 potently deacetylates α-tubulin in assembled microtubules. Furthermore, overexpression of HDAC6 promotes chemotactic cell movement, supporting the idea that HDAC6-mediated deacetylation regulates microtubule-dependent cell motility. Our results show that HDAC6 is the tubulin deacetylase, and provide evidence that reversible acetylation regulates important biol. processes beyond histone metab. and gene transcription.
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      Yang, W.; Chen, W.; Su, H.; Li, R.; Song, C.; Wang, Z.; Yang, L. Recent advances in the development of histone deacylase SIRT2 inhibitors. RSC Adv. 2020, 10, 37382– 37390,  DOI: 10.1039/D0RA06316A
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      Recent advances in the development of histone deacylase SIRT2 inhibitors
      Yang, Wenyu; Chen, Wei; Su, Huilin; Li, Rong; Song, Chen; Wang, Zhouyu; Yang, Lingling
      RSC Advances (2020), 10 (61), 37382-37390CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      A review. Sirtuin 2 (SIRT2) is an important and special member of the atypical histone deacetylase Sirtuin (SIRT) family. Due to its extensive catalytic effects, SIRT2 can regulate autophagy, myelination, immunity, inflammation and other physiol. processes. Recent evidence revealed that dysregulation of human SIRT2 activity is assocd. with the pathogenesis and prognosis of cancers, Parkinson's disease and other disorders thus SIRT2 is a promising target for potential therapeutic intervention. This review presents a systematic summary of nine chemotypes of small-mol. SIRT2 inhibitors, particularly including the discovery and structural optimization strategies, which will be useful for future efforts to develop new inhibitors targeting SIRT2 and assocd. target proteins.
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      Zhang, X. H.; Qin, M.; Wu, H. P.; Khamis, M. Y.; Li, Y. H.; Ma, L. Y.; Liu, H. M. A Review of Progress in Histone Deacetylase 6 Inhibitors Research: Structural Specificity and Functional Diversity. J. Med. Chem. 2021, 64, 1362– 1391,  DOI: 10.1021/acs.jmedchem.0c01782
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      A Review of Progress in Histone Deacetylase 6 Inhibitors Research: Structural Specificity and Functional Diversity
      Zhang, Xin-Hui; Qin-Ma; Wu, Hui-Pan; Khamis, Mussa Yussuf; Li, Yi-Han; Ma, Li-Ying; Liu, Hong-Min
      Journal of Medicinal Chemistry (2021), 64 (3), 1362-1391CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. Histone deacetylases (HDACs) are essential for maintaining homeostasis by catalyzing histone deacetylation. Aberrant expression of HDACs is assocd. with various human diseases. Although HDAC inhibitors are used as effective chemotherapeutic agents in clin. practice, their applications remain limited due to assocd. side effects induced by weak isoform selectivity. HDAC6 displays unique structure and cellular localization as well as diverse substrates and exhibits a wider range of biol. functions than other isoforms. HDAC6 inhibitors have been effectively used to treat cancers, neurodegenerative diseases, and autoimmune disorders without exerting significant toxic effects. Progress has been made in defining the crystal structures of HDAC6 catalytic domains which has influenced the structure-based drug design of HDAC6 inhibitors. This review summarizes recent literature on HDAC6 inhibitors with particular ref. to structural specificity and functional diversity. It may provide up-to-date guidance for the development of HDAC6 inhibitors and perspectives for optimization of therapeutic applications.
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      Stefaniak, J.; Huber, K. V. M. Importance of Quantifying Drug-Target Engagement in Cells. ACS Med. Chem. Lett. 2020, 11, 403– 406,  DOI: 10.1021/acsmedchemlett.9b00570
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      Importance of Quantifying Drug-Target Engagement in Cells
      Stefaniak, Jakub; Huber, Kilian V. M.
      ACS Medicinal Chemistry Letters (2020), 11 (4), 403-406CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      A review. Measuring and quantifying the binding of a drug to a protein target inside living cells and thereby correlating biochem. or biophys. activity with target engagement in cells or tissue represents a key step in target validation and drug development. A prototypic target engagement assay should allow for unbiased detn. of small mol.-protein interactions in order to confirm cellular mechanism-of-action (MoA) while avoiding major artificial perturbations of cellular homeostasis and integrity. Recently, several new addns. to the chem. biol. toolbox have expanded our ability to study drug action in intact cells and enabled surveying of intracellular residence time and binding kinetics, which are particularly important for potent receptor ligands and therapeutic moieties with limited therapeutic index.
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      Vogelmann, A.; Schiedel, M.; Wössner, N.; Merz, A.; Herp, D.; Hammelmann, S.; Colcerasa, A.; Komaniecki, G.; Hong, J. Y.; Sum, M.; Metzger, E.; Neuwirt, E.; Zhang, L.; Einsle, O.; Groß, O.; Schüle, R.; Lin, H.; Sippl, W.; Jung, M. Development of a NanoBRET Assay to Validate Dual Inhibitors of Sirt2-Mediated Lysine Deacetylation and Defatty-Acylation That Block Prostate Cancer Cell Migration. ChemRxiv, December 16, 2021, ver. 1.  DOI: 10.26434/chemrxiv-2021-1r1dn .
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      Nielsen, A. L.; Rajabi, N.; Kudo, N.; Lundo, K.; Moreno-Yruela, C.; Baek, M.; Fontenas, M.; Lucidi, A.; Madsen, A. S.; Yoshida, M.; Olsen, C. A. Mechanism-based inhibitors of SIRT2: structure-activity relationship, X-ray structures, target engagement, regulation of alpha-tubulin acetylation and inhibition of breast cancer cell migration. RSC Chem. Biol. 2021, 2, 612– 626,  DOI: 10.1039/D0CB00036A
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      Mechanism-based inhibitors of SIRT2: structure-activity relationship, X-ray structures, target engagement, regulation of α-tubulin acetylation and inhibition of breast cancer cell migration
      Nielsen, Alexander L.; Rajabi, Nima; Kudo, Norio; Lundoe, Kathrine; Moreno-Yruela, Carlos; Baek, Michael; Fontenas, Martin; Lucidi, Alessia; Madsen, Andreas S.; Yoshida, Minoru; Olsen, Christian A.
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      Sirtuin 2 (SIRT2) is a protein deacylase enzyme that removes acetyl groups and longer chain acyl groups from post-translationally modified lysine residues. It affects diverse biol. functions in the cell and has been considered a drug target in relation to both neurodegenerative diseases and cancer. Therefore, access to well-characterized and robust tool compds. is essential for the continued investigation of the complex functions of this enzyme. Here, we report a collection of chem. probes that are potent, selective, stable in serum, water-sol., and inhibit SIRT2-mediated deacetylation and demyristoylation in cells. Compared to the current landscape of SIRT2 inhibitors, this is a unique ensemble of features built into a single compd. We expect the developed chemotypes to find broad application in the interrogation of SIRT2 functions in both healthy and diseased cells, and to provide a foundation for the development of future therapeutics.
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      Thakur, A.; Tawa, G. J.; Henderson, M. J.; Danchik, C.; Liu, S.; Shah, P.; Wang, A. Q.; Dunn, G.; Kabir, M.; Padilha, E. C.; Xu, X.; Simeonov, A.; Kharbanda, S.; Stone, R.; Grewal, G. Design, Synthesis, and Biological Evaluation of Quinazolin-4-one-Based Hydroxamic Acids as Dual PI3K/HDAC Inhibitors. J. Med. Chem. 2020, 63, 4256– 4292,  DOI: 10.1021/acs.jmedchem.0c00193
      8
      Design, Synthesis, and Biological Evaluation of Quinazolin-4-one-Based Hydroxamic Acids as Dual PI3K/HDAC Inhibitors
      Thakur, Ashish; Tawa, Gregory J.; Henderson, Mark J.; Danchik, Carina; Liu, Suiyang; Shah, Pranav; Wang, Amy Q.; Dunn, Garrett; Kabir, Md.; Padilha, Elias C.; Xu, Xin; Simeonov, Anton; Kharbanda, Surender; Stone, Richard; Grewal, Gurmit
      Journal of Medicinal Chemistry (2020), 63 (8), 4256-4292CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A series of quinazolin-4-one based hydroxamic acids was rationally designed and synthesized as novel dual PI3K/HDAC inhibitors by incorporating an HDAC pharmacophore into a PI3K inhibitor (Idelalisib) via an optimized linker. Several of these dual inhibitors were highly potent (IC50 < 10 nM) and selective against PI3Kγ, δ and HDAC6 enzymes and exhibited good antiproliferative activity against multiple cancer cell lines. The lead compd. 48c, induced necrosis in several mutant and FLT3-resistant AML cell lines and primary blasts from AML patients, while showing no cytotoxicity against normal PBMCs, NIH3T3, and HEK293 cells. Target engagement of PI3Kδ and HDAC6 by 48c was demonstrated in MV411 cells using the cellular thermal shift assay (CETSA). Compd. 48c showed good pharmacokinetics properties in mice via i.p. administration and provides a means to examine the biol. effects of inhibiting these two important enzymes with a single mol., either in vitro or in vivo.
    9. 9
      Schiedel, M.; Herp, D.; Hammelmann, S.; Swyter, S.; Lehotzky, A.; Robaa, D.; Olah, J.; Ovadi, J.; Sippl, W.; Jung, M. Chemically induced degradation of sirtuin 2 (Sirt2) by a proteolysis targeting chimera (PROTAC) based on sirtuin rearranging ligands (SirReals). J. Med. Chem. 2018, 61, 482– 491,  DOI: 10.1021/acs.jmedchem.6b01872
      9
      Chemically Induced Degradation of Sirtuin 2 (Sirt2) by a Proteolysis Targeting Chimera (PROTAC) Based on Sirtuin Rearranging Ligands (SirReals)
      Schiedel, Matthias; Herp, Daniel; Hammelmann, Soeren; Swyter, Soeren; Lehotzky, Attila; Robaa, Dina; Olah, Judit; Ovadi, Judit; Sippl, Wolfgang; Jung, Manfred
      Journal of Medicinal Chemistry (2018), 61 (2), 482-491CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Here the authors report the development of a proteolysis targeting chimera (PROTAC) I based on the combination of the unique features of the sirtuin rearranging ligands (SirReals) as highly potent and isotype-selective Sirt2 inhibitors with thalidomide, a bona fide cereblon ligand. For the first time, the authors report the formation of a PROTAC by Cu(I)-catalyzed cycloaddn. of a thalidomide-derived azide to an alkynylated inhibitor. This thalidomide-derived azide as well as the highly versatile linking strategy can be readily adapted to alkynylated ligands of other targets. In HeLa cells, the SirReal-based PROTAC induced isotype-selective Sirt2 degrdn. that results in the hyperacetylation of the microtubule network coupled with enhanced process elongation. Thus, the SirReal-based PROTAC is the first example of a probe that is able to chem. induce the degrdn. of an epigenetic eraser protein.
    10. 10
      Yang, K.; Wu, H.; Zhang, Z.; Leisten, E. D.; Nie, X.; Liu, B.; Wen, Z.; Zhang, J.; Cunningham, M. D.; Tang, W. Development of Selective Histone Deacetylase 6 (HDAC6) Degraders Recruiting Von Hippel-Lindau (VHL) E3 Ubiquitin Ligase. ACS Med. Chem. Lett. 2020, 11, 575– 581,  DOI: 10.1021/acsmedchemlett.0c00046
      10
      Development of selective histone deacetylase 6 (HDAC6) degraders recruiting Von Hippel-Lindau (VHL) E3 ubiquitin ligase
      Yang, Ka; Wu, Hao; Zhang, Zhongrui; Leisten, Eric D.; Nie, Xueqing; Liu, Binkai; Wen, Zhi; Zhang, Jing; Cunningham, Michael D.; Tang, Weiping
      ACS Medicinal Chemistry Letters (2020), 11 (4), 575-581CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      Histone deacetylase 6 (HDAC6) is involved in multiple cellular processes such as aggresome formation, protein stability, and cell motility. Numerous HDAC6-selective inhibitors have been developed as cellular chem. tools to elucidate the function of HDAC6. Since HDAC6 has multiple domains that cannot be studied by HDAC6-selective inhibitors, CRISPR-CAS9 and siRNA/shRNA have been employed to elucidate the nonenzymic functions of HDAC6. However, these genetic methods have many limitations. Proteolysis targeting chimera (PROTAC) is an emerging technol. for the development of small mols. that can quickly remove the entire protein in cells. We previously developed multifunctional HDAC6 degraders that can recruit cereblon (CRBN) E3 ubiquitin ligase. These HDAC6 degraders can degrade not only HDAC6 but also neo-substrates of CRBN. They are excellent candidates for the development of anticancer therapeutics, but the multifunctional nature of the CRBN-based HDAC6 degraders has limited their utility as specific chem. probes for the study of HDAC6-related cellular pathways. Herein we report the development of the first cell-permeable HDAC6-selective degraders employing Von Hippel-Lindau (VHL) E3 ubiquitin ligase, which does not have any known neo-substrates. The DC50's of the most potent compd. 3j are 7.1 nM and 4.3 nM in human MM1S and mouse 4935 cell lines, resp. The Dmax's of 3j in these two cell lines are 90% and 57%, resp.
    11. 11
      Sinatra, L.; Bandolik, J. J.; Roatsch, M.; Sonnichsen, M.; Schoeder, C. T.; Hamacher, A.; Scholer, A.; Borkhardt, A.; Meiler, J.; Bhatia, S.; Kassack, M. U.; Hansen, F. K. Hydroxamic Acids Immobilized on Resins (HAIRs): Synthesis of Dual-Targeting HDAC Inhibitors and HDAC Degraders (PROTACs). Angew. Chem., Int. Ed. Engl. 2020, 59, 22494– 22499,  DOI: 10.1002/anie.202006725
      11
      Hydroxamic Acids Immobilized on Resins (HAIRs): Synthesis of Dual-Targeting HDAC Inhibitors and HDAC Degraders (PROTACs)
      Sinatra Laura; Roatsch Martin; Scholer Andrea; Meiler Jens; Hansen Finn K; Bandolik Jan J; Hamacher Alexandra; Kassack Matthias U; Roatsch Martin; Sonnichsen Melf; Borkhardt Arndt; Bhatia Sanil; Schoeder Clara T; Meiler Jens; Hansen Finn K
      Angewandte Chemie (International ed. in English) (2020), 59 (50), 22494-22499 ISSN:.
      Inhibition of more than one cancer-related pathway by multi-target agents is an emerging approach in modern anticancer drug discovery. Here, based on the well-established synergy between histone deacetylase inhibitors (HDACi) and alkylating agents, we present the discovery of a series of alkylating HDACi using a pharmacophore-linking strategy. For the parallel synthesis of the target compounds, we developed an efficient solid-phase-supported protocol using hydroxamic acids immobilized on resins (HAIRs) as stable and versatile building blocks for the preparation of functionalized HDACi. The most promising compound, 3 n, was significantly more active in apoptosis induction, activation of caspase 3/7, and formation of DNA damage (γ-H2AX) than the sum of the activities of either active principle alone. Furthermore, to demonstrate the utility of our preloaded resins, the HAIR approach was successfully extended to the synthesis of a proof-of-concept proteolysis-targeting chimera (PROTAC), which efficiently degrades histone deacetylases.
    12. 12
      Kolbinger, F. R.; Koeneke, E.; Ridinger, J.; Heimburg, T.; Muller, M.; Bayer, T.; Sippl, W.; Jung, M.; Gunkel, N.; Miller, A. K.; Westermann, F.; Witt, O.; Oehme, I. The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines. Arch. Toxicol. 2018, 92, 2649– 2664,  DOI: 10.1007/s00204-018-2234-8
      12
      The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines
      Kolbinger, Fiona R.; Koeneke, Emily; Ridinger, Johannes; Heimburg, Tino; Mueller, Michael; Bayer, Theresa; Sippl, Wolfgang; Jung, Manfred; Gunkel, Nikolas; Miller, Aubry K.; Westermann, Frank; Witt, Olaf; Oehme, Ina
      Archives of Toxicology (2018), 92 (8), 2649-2664CODEN: ARTODN; ISSN:0340-5761. (Springer)
      High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concns. up to 25μM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concn.-dependent manner in several human neuroblastoma cell lines. In addn. to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.