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OFF-State-Specific Inhibition of the Proprotein Convertase Furin
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OFF-State-Specific Inhibition of the Proprotein Convertase Furin
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Cite this: ACS Chem. Biol. 2021, 16, 9, 1692–1700
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https://doi.org/10.1021/acschembio.1c00411
Published August 20, 2021

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Abstract

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The pro-protein convertase furin is a highly specific serine protease involved in the proteolytic maturation of many proteins in the secretory pathway. It also activates surface proteins of many viruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furin inhibitors effectively suppress viral replication and thus are promising antiviral therapeutics with broad application potential. Polybasic substrate-like ligands typically trigger conformational changes shifting furin’s active site cleft from the OFF-state to the ON-state. Here, we solved the X-ray structures of furin in complex with four different arginine mimetic compounds with reduced basicity. These guanylhydrazone-based inhibitor complexes showed for the first time an active site-directed binding mode to furin’s OFF-state conformation. The compounds undergo unique interactions within the S1 pocket, largely different compared to substrate-like ligands. A second binding site was identified at the S4/S5 pocket of furin. Crystallography-based titration experiments confirmed the S1 site as the primary binding pocket. We also tested the proprotein convertases PC5/6 and PC7 for inhibition by guanylhydrazones and found an up to 7-fold lower potency for PC7. Interestingly, the observed differences in the Ki values correlated with the sequence conservation of the PCs at the allosteric sodium binding site. Therefore, OFF-state-specific targeting of furin can serve as a valuable strategy for structure-based development of PC-selective small-molecule inhibitors.

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Introduction

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In addition to physiological protein maturation, furin activates the surface proteins of many human-pathogenic viruses, (1,2) including the severe acute respiratory syndrome coronavirus 2 (SARS-Cov2). (3−5) Acquisition of a furin cleavage site is regarded as a general pathogenicity factor for known viruses, and newly emerging viruses could also rely on this mechanism. Thus, furin inhibitors are regarded as promising antiviral therapeutics with a broad application potential. (6,7)
Furin belongs to the proprotein convertases (PCs), a family of serine proteases that share structural homology with the bacterial protease subtilisin. Largely different from subtilisin, the PCs are characterized by stringent substrate specificity. A subset of the PCs (PC1, PC2, Furin, PC4, PC5/6, PACE4, PC7) recognizes multibasic sequence motifs of the type (R/K)-Xn-(R/K)↓ (n = 0, 2, 4; ↓ indicates the cleavage site; arginine is preferred at the P1 position). (8) The typical consensus motif R–X–(R/K)–R↓ (9,10) has been utilized for the development of numerous substrate-like peptidic furin inhibitors (e.g., refs (11−19)). Structures of such inhibitors in complex with furin revealed typical side-chain interaction patterns at the P1, P2, and P4 positions driven by electrostatic interactions and extensive hydrogen-bond networks. (20) Optimized substrate-mimicking inhibitors yielded very high potencies with Ki values in the low picomolar range. (21) These compounds also revealed potent antiviral effects in cell cultures infected with several furin-dependent viruses and were well tolerated in mice and rats. (21)
Binding of substrate-like inhibitors to furin induces the substrate binding cleft to switch its conformation from the OFF-state to the ON-state. (22) In the OFF-state, the alignment template region (also called the edge strand) between Ser253 and Pro256 adopts a specific conformation in which its typical beta-sheet-like interaction pattern with the substrate’s peptide backbone is prohibited. The S1 pocket is also partially blocked by Ser253 in the OFF-state (Figure 1A). Structural changes at furin’s S1 pocket are connected to an allosteric sodium-binding site. In the OFF-state, the sodium ion adopts an octahedral coordination sphere. Ligand binding induces a reconfiguration of hydrogen bonds, resulting in a change of the coordination sphere to tetragonal pyramidal in the ON-state (Figure 1A). The central players in this relay are the side chains of Thr309 and the Ser316, whereby the latter acts as sodium ligand in the OFF-state only. We concluded that the more stable octahedral coordination of the sodium binding site stabilizes the OFF-state. (22) The gatekeeper function of the alignment template and especially Ser253 prevents the binding of monobasic peptides and is crucial for the stringent substrate specificity of furin. Capitalizing on this selection mechanism, we have proposed that specific targeting of the OFF-state might be utilized for the development of furin inhibitors. (22)

Figure 1

Figure 1. (A) Ligand binding to furin induces a conformational shift of furin from the ON- to the OFF-state. Superposition of furin in the ON- (gray, PDB ID: 5JXH (22)) and OFF-states (yellow, PDB ID: 5JXG (22)). Dashed lines highlight important interactions. Big and small spheres represent the sodium ion and water molecules, respectively. Instead of the P1 residue of the inhibitor, in the OFF-state a water molecule occupies two alternative sites (H2O A and B) at the bottom of the S1 pocket. (B) Chemical structure of guanabenz. (C) Chemical structures of the inhibitors used in this study.

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Several classes of nonpeptidic furin inhibitors have been reported in the literature. (23−27) Small molecule inhibitors facilitate binding modes beyond the canonical substrate specificity pockets, as reported for 2,5-dideoxystreptamine derived compounds. (28) Structural investigations revealed an unusual interaction of such inhibitors with the active site residues. Guanylhydrazone-derived compounds are less basic than guanidino or amidino compounds. For instance, the pKa of the orally available antihypertensive guanylhydrazone-based drug guanabenz (Figure 1B) is 8.1 compared to 13 for guanidine. (29) Another example is the anti-inflammatory guanylhydrazone Semapimod (also called CNI-1493, ref (30)), which reached phase 2 clinical trials. Recently, we showed that the reduction of the basicity of furin inhibitors resulted in increased bioavailability and reduced toxicity. (3,21) Thus, guanylhydrazones are promising lead compounds for the development of next-generation furin inhibitors. However, the binding mode of these inhibitors is completely unknown so far.
In this study, we elucidated the structural basis of the interaction of guanylhydrazone inhibitors with furin. Our structural studies revealed characteristic binding patterns that require and stabilize the OFF-state conformation of the protease.

Results and Discussion

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OFF-State-Specific Inhibition of Furin

Crystals of ligand-free furin usually grow under high salt conditions containing ∼3 M sodium chloride. (22) Until now, attempts to investigate inhibitors with Ki values in the micromolar range or less, i.e., weaker inhibitors, by soaking constantly failed. We assumed that the high ionic strength shields furin’s highly charged active site cleft and competes with the electrostatic binding of ligands. Thus, we established new soaking conditions with reduced ionic strength. Supplementing sodium chloride with PEG8000 allowed us to reduce the sodium chloride concentration to 1 M at 10% PEG8000 or 0.25 M at 20% PEG8000. Crystals are stable over long time periods under the 1 M sodium chloride conditions, whereas the incubation time had to be reduced to ≤30 min at 0.25 M sodium chloride to prevent a decline of the diffraction power.
Using these newly developed soaking protocols, we investigated the structural basis of the interaction of four guanylhydrazones (Figure 1C) with furin. The structures of furin in complex with compounds 1 and 2 (Table 1, Figure 2A,B, stereo representation in Figure S1A,B) revealed a unique interaction mode of the inhibitors at the S1 pocket (Figure 3A,B). One of the guanylhydrazone moieties of the inhibitors specifically interacts with the alignment template of the protease (Figure 3A,B). This involves the carbonyl oxygens of Ser253 and Pro256 forming hydrogen bonds to the guanylhydrazone group. One of the terminal nitrogen atoms of the inhibitor’s guanylhydrazone moiety also mediates an electrostatic contact with Asp306. The other terminal nitrogen atom mediates a hydrogen bond to a specific water molecule at the S1 pocket of furin. This water molecule is bound to the enzyme by hydrogen bonds to the side-chain carboxylate group of Asp306 and to the carbonyl oxygen of Ala292 (Figure 3A,B). Taken together, this is a largely different interaction network than observed for substrate-like inhibitors requiring the ON-state of the protease (Figure 4A, Figure S2A). The P1 residue of a substrate-like inhibitor inserts more deeply into the S1 pocket, and thereby a nitrogen atom of the amidine expels and replaces the water molecule. In the ON-state, the peptide bond between Ser253 and Trp254 is also rotated by 90°, uncovering the S1 pocket and allowing β sheet-like interactions with the ligand backbone at the same time. (22) Other residues that are involved in the OFF-state to ON-state transition also adopt a typical OFF-state conformation with 1 and 2 bound (Figure S3A,B). This includes the sodium-binding site showing the OFF-state-specific coordination sphere (Figure S4A,B). The side chain of Ser316 contributes to the octahedral coordination of sodium. By contrast, in the substrate-bound ON-state, the hydroxyl group of Ser316 flipped 120° away from the sodium ion adopting a tetragonal pyramidal coordination (Figure 4A). The reorientation of Ser316 also prevents its interaction with Thr309. Thus, in the ON-state, Thr309 mediates a hydrogen bond with the carbonyl oxygen of Asp306 instead. The side-chain flip of Thr309 in the OFF-state also provides more space on the bottom of the S1 pocket and thus facilitates binding of the water molecule to Asp306 and Ala292 (Figure 4A). In the ON-state orientation of Thr309, however, its methyl group would clash with this water molecule. The carbonyl oxygen of Ala292 also moves by 0.7 Å toward the S1 pocket to avoid a clash with the methyl group of Thr309 in the ON-state.

Figure 2

Figure 2. Structures of furin bound to guanylhydrazone-based inhibitors. The protease is shown as a cartoon representation (golden), catalytic residues as sticks with carbon atoms in cyan and the inhibitors as ball-and-stick models, respectively. The specificity pockets are labeled (S1–S4). The Fo–Fc annealed omit electron density map of the inhibitors is shown as green mesh and is contoured at 3.0 σ. Furin in complex with inhibitors 1 (A), 2 (B), 3 (C), and 4 (D). Inhibitor 1 was found in two conformations shown with carbons in dark and light gray. (E,F) Two binding sites of 2 (E) and 3 (F) were found at the active site cleft of furin. The binding sites 1 and 2 are labeled I-1 and I-2, respectively.

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Figure 3

Figure 3. Binding mode of the OFF-state-specific furin inhibitors. Specific furin residues and the inhibitors are shown as stick and ball-and-stick models, respectively. Important interactions between the inhibitor and furin are marked with dashed lines. The furin backbone is given as a cartoon representation (yellow). The inhibitors 1 (A, two conformations are indicated by light and dark colors), 2 (B), 3 (C), and 4 (D) are shown with carbons in green, orange, brown, and magenta, respectively. The active site residues are shown with carbons in cyan. The specificity pockets are labeled (S1–S4). Ligand-free furin (PDB-ID: 5jxg (22)) is superimposed on the furin-inhibitor complex structures and is shown as a cartoon in black.

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Figure 4

Figure 4. Allosteric connection between the S1 pocket and the sodium binding site. (A) Superposition of furin (carbons in yellow) bound to 1 (colored according to Figure 3A, OFF-state) and furin in the ON-state bound the substrate-like inhibitor 3-guanidinomethyl-phenylacetyl-RVR-4-aminomethyl-benzamidine (gray, PDB ID: 5jxh (22)). Competing space requirements of the water molecule at the S1 pocket and the side chain of Thr306 are highlighted by red dashes. Dashed lines highlight important interactions. For clarity, in the structure of furin bound to the substrate-like ligand, dashed lines, the Na+ ion and the H2O ligand are shown in gray. (B) Sequence alignment of the sodium-binding loop of furin-like PCs. Positions with sequence identities of 100% and >50% are highlighted in black and gray, respectively. Sodium binding residues are framed and numbered.

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Table 1. Data Collection and Refinement Statistics
inhibitor12234
data collection statistics
PDB ID7O1U7O1W7O1Y7O207O22
soaking concentration (mM)20204010040
wavelength (Å)0.9184
space groupP6522
unit cell parameters: a = b (Å), c (Å)131.7, 155.7131.6, 156.0131.1, 156.5130.3, 156.0131.0, 155.5
resolution rangea (Å)47.2–1.7 (1.80–1.70)47.3–1.8 (1.91–1.80)47.4–1.7 (1.80–1.70)47.2–1.8 (1.91–1.80)47.1–1.8 (1.91–1.80)
Rmeasa (%)14.8 (187.9)14.8 (187.9)11.2 (193.1)16.5 (276.3)19.1 (345.0)
I/sigIa18.7 (2.0)16.6 (1.7)15.1 (1.3)16.3 (1.2)15.2 (1.1)
CC1/2 (%), (44),a99.9 (79.1)99.9 (75.6)99.9 (61.2)99.9 (60.4)99.9 (55.0)
completenessa98.7 (97.6)99.1 (98.0)99.1 (98.5)98.5 (97.0)99.6 (99.0)
no. of observations (total/unique)1 734 173/86 7321 457 796/73 502668 083/86 4051 434 222/71 8881 458 226/72 880
refinement statistics
no. of non-hydrogen atoms43754335432141904130
protein/inhibitor/water/other3776/42/519/803816/54/424/953732/54/475/1143718/52/376/963646/21/427/57
Rwork/Rfree15.6/16.716.1/17.916.3/17.716.3/17.617.0/18.3
B-factors (Å2)
overall/Wilson plot28.8/26.231.2/29.431.4/29.535.1/33.335.1/33.5
protein/inhibitor/water/other24.9/26.6/35.0/32.328.4/32.6/36.5/36.428.1/32.4/38.5/37.632.5/32.2/39.2/38.633.5/31.5/39.9/40.2
RMSD bond length (Å)0.0050.0090.0090.0090.006
RMSD bonded B-factors (Å2)3.33.43.72.62.3
a

Values of the highest resolution shell are given in parentheses

The guanylhydrazone moieties of the inhibitors 1 and 2 exhibit meta- and para configurations, respectively. The second meta-substituted guanylhydrazone moiety of 1 is found in two different, nearly equally occupied conformations (Figure 3A, Table S1). In conformation A (Figure 3A, light green carbons), it contacts the carbonyl oxygens of Gly229 and Leu227. In conformation B (Figure 3A, dark green carbons), the elongated guanylhydrazone moiety of 1 more efficiently covers the hydrophobic surface of the side-chain of Leu227. The second guanylhydrazone moiety increases the positive net charge, which is in general favorable at the highly negatively charged binding site. The aniline-like amino group of 1 interacts only indirectly through water-mediated hydrogen bonds with the side chains of Ser253 and Asp153. Inhibitor 1 also contains an additional methyl group at the guanylhydrazone moiety, and the rather flat molecule efficiently covers the hydrophobic surface at the rim of furin’s S1 pocket.
The more elongated para configuration of 2 facilitates charge-assisted hydrogen bonds to the side chains of Asp153 and Asp154. It should be noted that Asp153 is part of the catalytic triad and Asp154 usually contributes to S2–P2 interactions with substrate-like inhibitors. To mediate these additional interactions, the central phenyl ring and the second guanylhydrazone moiety of 2 are rotated by ∼60° compared to 1 (Figure 5A). In this orientation, inhibitor 2 mediates less hydrophobic contacts with furin compared to inhibitor 1. The more extensive hydrogen-bond pattern of 2 in comparison to 1 might thus be compensated by weaker hydrophobic interactions. This observation could explain the similar Ki values for furin by inhibitors 1 and 2 of 3.3 μM and 3.1 μM, respectively (Table 2).

Figure 5

Figure 5. Comparison of the binding poses of guanylhydrazones. Stereo representations of structural superpositions of furin-inhibitor complex structures. Specific furin residues and the inhibitors are shown as stick and ball-and-stick models, respectively. Important interactions between the inhibitor and furin are marked with dashed lines. The inhibitors are colored according to Figure 3. Furin residues and the furin backbone are colored according to the inhibitors in green (1), orange (2), and magenta (4). (A) Superposition of furin in complex with 1 and 2. (B) Superposition of furin in complex with 1 and 4.

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Table 2. Ki of Inhibitors 1 and 2 for Selected PCs
inhibitorfurin [μM]PC5/6 [μM]PC7 [μM]
13.3 ± 0.13.6 ± 0.222.4 ± 1.9
23.1 ± 0.21.7 ± 0.210.4 ± 0.7
According to our structural data, a minimal OFF-state-specific binder should comprise a phenyl group with a single guanylhydrazone substituent. To prove this assumption, we solved the structure of furin in complex with compound 3 (Table 1, Figure 2C, stereo representation in Figure S1C). Indeed, the structure revealed a largely similar S1-binding pattern as observed for inhibitors 1 and 2 (Figure 3C). For compound 3, a Ki value of 273 μM has been reported. (26) The largely reduced potency is well explained by the missing interactions of the second guanylhydrazone group, which is lacking in 3.

Involvement of the S1′ Pocket in Alternative OFF-State-Specific Binding by Inhibitor 4

Inhibitor 4 (Figure 1C), initially published by Komiyama and co-workers, contains a single guanylhydrazone substituent. (24) Different from derivative 3, however, the reported Ki values of 11.8 μM (24) or 25.3 μM (26) revealed a 1 order of magnitude stronger furin inhibition. In 4, an additional para-chlorobenzyloxy substituent is attached in the ortho position to the guanylhydrazone arm. According to the binding modes observed for 1–3, we were expecting that the ortho-attachment of 4 would result in steric clashes at the S1 pocket. Therefore, we assumed that 4 must adopt a different binding pose compared to the other inhibitors. To verify this hypothesis, we solved the structure of furin in complex with analogue 4 (Table 1, Figure 2D, stereo representation in Figure S1D). Here, the guanylhydrazone moiety is tilted by ∼40° compared to 1 toward the catalytic serine (Figure 3D, Figure 5B). The direct interactions with Asp306 and Pro256 are lost and instead substituted by a salt bridge to the carboxyl group of Asp258. The hydrogen-bond to the carbonyl oxygen of Ser253, however, is maintained as well as the water-mediated interaction to the carbonyl oxygen of Ala292 and the carboxylate group of Asp306 (Figure 3D, Figure S3D). This is facilitated by a displacement of the alignment template, resulting in a relocation of Ser253 by 1.3 Å (based on Cα) and a rotation of its side chain by 120°. The side-chain flip of Ser253 enables a hydrogen bond to the carbonyl oxygen of the catalytic Ser368 (Figure 3D). We also observed a hydrogen-bond between the amide nitrogen of Asn295 from furin’s oxy-anion hole and the oxygen of the p-chlorobenzyl ether moiety of inhibitor 4. The chlorophenyl ring inserts into a small hydrophobic pocket surrounded by Gly366, Tyr329, Trp328, and Asn295, the putative S1′ site of furin (Figure 3D). The relocations of the alignment template and catalytic residues induced by compound 4 impressively demonstrate the structural plasticity of furin’s active site cleft.

Second Binding Site of Inhibitor 2 and 3 at the Substrate Binding Cleft of Furin

Interestingly, we found a second binding site of 2 and 3 at furin’s substrate binding cleft involving residues of the S4 pocket (Figure 6A,B, Figure S5A, C). This second binding site revealed similar occupancies of the inhibitors but increased B-factors compared to the binding site at the S1 pocket (Table S1). One guanylhydrazone group mediates salt bridges to Glu236 and Asp264 as well as a hydrogen bond to Tyr308. The phenyl ring covers the hydrophobic stretch build up by Val231, Leu227, Trp254, and Gly255. The inhibitor molecule bound at the S1 pocket further extends this hydrophobic surface. The second guanylhydrazone group of 2, which is bound to the S4 pocket, is also aligned to the other molecule at S1 (Figure 6A). In addition, it mediates a hydrogen bond to the carbonyl oxygen of Leu227. Superposition of furin in complex with inhibitor 2 and a substrate-like inhibitor revealed different salt bridge configurations with Asp264 (Figure S6A). P4–arginine interacts in an end-on bidentate configuration with Asp264. In complex with inhibitor 2, the Cα of Asp264 is displaced by 0.6 Å away from the S4 pocket. Thus, only one carboxyl oxygen atom remains in interaction distance with the guanylhydrazone moiety. This orientation of Asp264 was also observed in the structure of furin in complex with inhibitor 1 (Figure S6B,C) and in ligand free furin. (22) We conclude that this effect might be a specific OFF-state feature rather than a ligand dependent effect at the S4 pocket.

Figure 6

Figure 6. Second binding site of 2 and 3. (A–E) The furin backbone is given as a cartoon representation (yellow). Specific furin residues and the inhibitors are shown as stick and ball-and-stick models, respectively. The active site residues are shown with carbons in cyan. The specificity pockets are labeled (S1, S4). (A, B) The second molecule of 2 (A) or 3 (B) is shown with gray carbons (I-2). Inhibitor molecules bound to the S1 pocket are colored according to Figure 1 and Figure 2. Important interactions between the inhibitor and furin are marked with dashed lines. Ligand-free furin (PDB-ID: 5jxg (22)) is superimposed and is shown as a cartoon in black. (C–E) Titration of furin at low ionic strength (250 mM NaCl) with 0.4 mM (C), 4 mM (D), and 40 mM (E) of inhibitor 2. The Fo–Fc annealed omit electron density map of the inhibitors is shown as a green mesh and is contoured at 3.0 σ.

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Because of the lower number of specific interactions, we assumed that the binding affinity of 2 for the S4 pocket is lower as compared to the S1 pocket. To prove this hypothesis, we titrated crystals with increasing inhibitor concentrations. The strength of the electron density directly correlates to the occupation of the binding sites with the inhibitor. Indeed, we found the S1 pocket already occupied by 2 at 0.4 mM as indicated by the occupancy of 75% from crystallographic refinement (Table S2). In contrast, very weak electron density of this compound was observed at S4 (Figure 6C). A partial occupation of 63% by 2 at S4 was observed at a 4 mM concentration (Figure 6D) and an occupation of 82% was reached at 40 mM (Figure 6E). The refined occupancy of inhibitor 2 at the S1 pocket at 40 mM concentration was 0.94. It should be noted that water molecules occupy the substrate binding pockets in absence of any ligand. (22) The binding sites of these water molecules overlap with the binding sites of the inhibitors and affect crystallographic occupancy refinement. Thus, the refined occupation should always be considered in context with the electron density (Figure 6C–E). Nonetheless, these results show that 2 binds much tighter at S1 compared to S4. We also observed a third binding site of 2 and 3 outside of the substrate binding cleft. Here, two inhibitor molecules are stacked between two arginine residues of a crystal contact. This binding mode is specific for the crystal lattice and thus is not expected to occur in solution (Figure S5B, D).
On the basis of the inhibitor titration experiments, we estimate the apparent dissociation constant of 2 at the S4 binding site in the low millimolar range. Because of the very similar binding mode, a similar affinity can be expected for 3. Thus, it is tempting to speculate that the chemical merging of 2 and 3 to target the S1, S2, and S4 pockets simultaneously would result in a more potent inhibitor. This strategy might increase the affinity of follow-up compounds up to several orders of magnitude.

OFF-State-Specific Inhibitors As Structural Probes for PCs

Due to a lack of structural information, it is not yet clear whether ligand binding also induces an ON-to-OFF-state transition in other PCs. On the basis of sequence alignments, the residues that participate in the conformational transition at the S1 pocket are conserved in all PCs. (31) This includes the residues involved in the direct interaction with the inhibitors 1 and 2. In previous studies, we could show that the structural differences between the OFF- and ON-states also involve allosteric sites in furin. (22) The S1 pocket and the Na+-binding site seem to be connected to allow an efficient cross-talk between both sites (Figure 4A). The central players of this communication line are Thr309 and Ser316 (see above). For all herein investigated inhibitors, furin adopted the octahedral OFF-state coordination of the sodium-binding site (Figure S3A–D and Figure S4A–D). Therefore, we concluded that the more stable octahedral Na+ coordination sphere supposedly stabilizes the OFF-state. (22) Interestingly, the crucial amino acids Thr309 and Ser316 are replaced by Ala and Thr in PC7 (Figure 4B). If the stability of the OFF-state is affected by this relay, one would expect an influence on binding of the guanylhydrazone inhibitors and specifically a reduction of their affinity to PC7. To test this hypothesis, we determined the Ki values of inhibitors 1 and 2 for PC7 and used furin as well as PC5/6 as control proteins. The sodium binding residues, including Thr309 and Ser316, are identical in furin and PC5/6 (Figure 4B). Thus, we would expect similar affinities of both inhibitors for these PCs. Indeed, inhibitor 1 was less potent for PC7 as indicated by a ∼7-fold potency loss of the Ki (Table 2). While inhibitor 1 interacts almost exclusively with the S1 pocket, inhibitor 2 also interacts with Asp153 and Asp154 in the S2 pocket. The conformation of these residues is not OFF-state-specific. In conclusion, we would expect a less pronounced effect on the Ki of compound 2. In excellent agreement with this hypothesis, we observed only a ∼3-fold increase of the Ki of inhibitor 2 for PC7 compared with furin (Table 2). In contrast, 1 and 2 showed similar Ki values for PC5/6 and furin. Inhibitor 2 was even more potent for PC5/6 compared to furin, which might indicate that the interaction of 2 with Asp153 and Asp154 contributes more strongly for this protease. Taken together, our data suggest that PC7 is capable of adopting the OFF-state conformation, although it seems to be less stable compared to furin and PC5/6. These data also emphasize the importance of the sodium-binding site and its influence on ligand binding by the different PCs. Although the amino acids of the S1 pocket are 100% conserved among the PC family members, their conformation could be influenced by an allosteric effect of distant sites, as shown for the sodium-binding site. Because these amino acids are less conserved, conformation-specific inhibitors could be more selective for certain PC family members. Thus, OFF-state-specific interactions could be utilized to increase the specificity of next generation furin inhibitors.

Methods

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X-ray Crystallography

Equal volumes of homogeneously glycosylated human furin (ref (20), ∼10 mg mL–1 in 10 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM CaCl2) and crystallization solution (100 mM MES, 200 mM K/NaH2PO4, pH 5.5–6.0, and 2 M NaCl) were mixed and equilibrated against the reservoir (3.0–3.2 M NaCl) in vapor diffusion experiments at 18–20 °C as described previously. (32,22)
The optimized soaking solutions contained either 1.0 M NaCl, 200 mM Mes/NaOH, at pH 5.5, 10% (w/v) PEG8000, and 20% (v/v) DMSO or 0.25 M NaCl, 200 mM Mes/NaOH, at pH 5.5, 20% (w/v) PEG8000, and 20% (v/v) DMSO and were supplemented with the specific inhibitors. Inhibitors 1 and 2 were soaked at 20 mM concentration in 1 M NaCl containing soaking solution for 0.5 h. Inhibitor 3 and 4 were soaked in 0.25 M NaCl containing soaking solution for 0.5 h at an inhibitor concentration of 100 mM and 20 mM, respectively. For the inhibitor titration experiments with 2, 40 mM, 4 mM, or 0.4 mM of the inhibitor was soaked for 0.5 h in 0.25 M NaCl-containing soaking solution. Soaked crystals were flash cooled in liquid N2. Diffraction data collection was performed at the synchrotron beamline BL14.2 (33) (BESSY-II) of Helmholtz-Zentrum Berlin (HZB). The data were processed using XDS (34) with XDS-APP (35) (v2.0) and the CCP4 program suite (36) (v.7.1.001). COOT (37) (v.0.8.9.2) was used for model building. Refinement was performed in PHENIX (38) (v1.18.2) using the PDB ID 5JXG (22) as an initial model. One specific Rfree-set (initially generated up to 1.0 Å, ref (22)) was transferred to the data sets prior refinement start. Geometry restraints of the inhibitors were obtained from the PRODRG server (39) and from the GRADE Web server (http://grade.globalphasing.org). All guanylhydrazones were modeled in the E configuration, which is in agreement with previous studies with model compounds. (40) Electron density omit maps were calculated in PHENIX (38) (v1.18.2). PYMOL was used for molecular graphics (http://www.pymol.org) and structural alignments. In the case of inhibitor titration experiments with 2, the structure was first built and refined for a crystal soaked at 40 mM. This structure was used to calculate refinements with (for occupancy calculation) and without (for omit map calculation) an inhibitor in the model for three additional crystals independently soaked at 40 mM, 4 mM, and 0.4 mM (Table S2) obtaining R/Rfree-values of 17.3/19.0, 17.5/19.1, and 17.7/19.3, respectively. The occupancy refinement yielded consistent B-factors between the three differently soaked structures (Table S2). Structure factors and coordinates of the complexes of furin with inhibitors 1, 2, at 1 M NaCl; 2 at 0.25 M NaCl; and 3 and 4 have been deposited as PDB entries 7O1U, 7O1W, 7O1Y, 7O20, and 7O22, respectively.

Protein Expression and Purification

HEK293S cells were stably transfected with the coding sequences of human furin, (20) PC5/6 (Arg33-Gly634, UNP (41) Q92824) and PC7 (Pro44-Thr667, UNP (41) Q16549) cloned into the pCDNA3.1(+) vector (ThermoFisher). Cells were grown in DMEM (4.5 g L–1 glucose, 2 mM l-glutamine, 110 mg l–1 sodium pyruvate, 3.7 g L–1 NaHCO3, nonessential amino acids) supplemented with 10% (v/v) FBS. Polyclonal selection of stable cell lines was conducted in the presence of 500 μg mL–1 G418 (Geneticin). The cells were grown in multilayer flasks (HyperFlask M, Corning) without G418 for large-scale protein expression. The medium of confluent expression cultures was exchanged with DMEM supplemented with 2 mM sodium butyrate as well as with FBS (2% (v/v), furin) or without FBS (PC5/6 and PC7). The medium exchange was repeated five (furin) or two (PC5/6 and PC7) times, and the centrifuged medium (20 min, 5500 g, 4 °C) was stored at −80 °C until further use.
The purification of human furin from a conditioned medium has been shown previously. (20) PC5/6 and PC7 were purified by immobilized metal affinity chromatography and gel permeation chromatography (GPC), as shown for furin but excluding the inhibitor affinity purification step. (20) PC7 (50 μg mL–1) was fully activated in 50 mM Tris/HCl, at pH 7.5, 10 mM CaCl2, and 0.15 M NaCl, at pH 7.5, in the presence of 2.0 μg mL–1 thermolysin for 16 h at 37 °C. To remove thermolysin, the reaction mixture was concentrated in an ultrafiltration device (30 kDa cutoff, amicon, Millipore) and subjected to GPC (both steps at 4 °C). Purified PC5/6 and PC7 were stored at −20 °C.

Enzyme Kinetics

Enzyme kinetic measurements were performed in 100 mM HEPES/NaOH, at pH 7.0, 2 mM CaCl2, and 0.2% (v/v) Triton X 100 with the fluorogenic substrate pyr-ERTKR-7-amino-4-methylcoumarin (Bachem) at 37 °C. The 100 μL reactions were prepared in 96-well black half-area plates (Corning), preincubated for 15 min at 37 °C and measured in a 96-well plate reader (Tecan infinite 200) at excitation and emission wavelengths of 380 and 460 nm. Because of the autofluorescence of inhibitor 1, the Ki values were determined at constant inhibitor concentrations (1: 20 μM for furin, 15 μM for PC5/6 and 50 μM for PC7; 2: 10 μM for furin, 5 μM for PC5/6 and 15 μM for PC7) and 1:2 dilution series of the substrate (Figure S7). The KM values were determined with 1:2 dilution series of the substrate in the absence of an inhibitor (4.7 ± 0.4 μM for furin, 2.5 ± 0.2 μM for PC5/6, and 19.3 ± 0.8 μM for PC7; Figure S7). Enzyme kinetic data were evaluated with GRAPHPAD PRISM (GraphPad Software) using the prebuild “Michaelis–Menten” model to calculate KM and a competitive inhibition model (v = Vmax × S/[S + KM(1 + I/Ki)]; v = reaction velocity, Vmax = maximum velocity, S = substrate concentration, KM = Michaelis–Menten constant, Ki = inhibition constant) for Ki determination. All measurements were performed in triplicate.

Sequence Alignments

Sequence alignments of the Uniprot (41) entries for furin (P09958), PC1 (P29120), PC2 (P16519), PC4 (Q6UW60), PC5/6 (Q92824), PACE4 (P29122), and PC7 (Q16549) were calculated with EMBL-EBI Clustal Omega (42) and modified with the ALINE software. (43)

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.1c00411.

  • Supporting Figures S1–S7 as well as Supporting Tables S1 and S2 (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Tanja Haider - Department of Biosciences, University of Salzburg, Hellbrunnerstraße 34, A-5020 Salzburg, Austria
    • Gerhard Klebe - Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
    • Torsten Steinmetzer - Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany;  Orcidhttps://orcid.org/0000-0001-6523-4754
    • Hans Brandstetter - Department of Biosciences, University of Salzburg, Hellbrunnerstraße 34, A-5020 Salzburg, Austria;  Orcidhttps://orcid.org/0000-0002-6089-3045
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We acknowledge Helmholtz Zentrum Berlin (BESSY II) for provision of synchrotron radiation at the beamline BL 14.2 and thank the scientific staff for assistance. We thank E. Dall for critical reading of the manuscript. The authors thank F. Sielaff for help with the synthesis of the inhibitors. The research leading to these results has been supported by the project CALIPSOplus under Grant Agreement 730872 from the EU Framework Programme for Research and Innovation HORIZON 2020. Funding was provided by the Austrian Science Fund (FWF) to S.O.D. (M 2730), by Bundesministerium für Bildung und Forschung (BMBF, Frag4Lead, FKZ: 05K16RM1) to G.K., and by the LOEWE Center DRUID (Novel Drug Targets against Poverty-Related and Neglected Tropical Infectious Diseases) to T.S.

References

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    Lewandowska-Goch, Monika A.; Kwiatkowska, Anna; Lepek, Teresa; Ly, Kevin; Navals, Pauline; Gagnon, Hugo; Dory, Yves L.; Prahl, Adam; Day, Robert
    ACS Medicinal Chemistry Letters (2021), 12 (3), 365-372CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
    Furin plays an important role in various pathol. states, esp. in bacterial and viral infections. A detailed understanding of the structural requirements for inhibitors targeting this enzyme is crucial to develop new therapeutic strategies in infectious diseases, including an urgent unmet need for SARS-CoV-2 infection. Previously, we have identified a potent furin inhibitor, peptide Ac-RARRRKKRT-NH2 (CF1), based on the highly pathogenic avian influenza hemagglutinin. The goal of this study was to det. how its N-terminal part (the P8-P5 positions) affects its activity profile. To do so, the positional-scanning libraries of individual peptides modified at the selected positions with natural amino acids were generated. Subsequently, the best substitutions were combined together and/or replaced by unnatural residues to expand our investigations. The results reveal that the affinity of CF1 can be improved (2-2.5-fold) by substituting its P5 position with the small hydrophobic residues (Ile or Val) or a basic Lys.
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  14. 14
    Ramos-Molina, B.; Lick, A. N.; Nasrolahi Shirazi, A.; Oh, D.; Tiwari, R.; El-Sayed, N. S.; Parang, K.; Lindberg, I. Cationic Cell-Penetrating Peptides Are Potent Furin Inhibitors. PLoS One 2015, 10, e0130417,  DOI: 10.1371/journal.pone.0130417
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    14
    Cationic cell-penetrating peptides are potent furin inhibitors
    Ramos-Molina, Bruno; Lick, Adam N.; Shirazi, Amir Nasrolahi; Oh, Donghoon; Tiwari, Rakesh; El-Sayed, Naglaa Salem; Parang, Keykavous; Lindberg, Iris
    PLoS One (2015), 10 (6), e0130417/1-e0130417/11CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Cationic cell-penetrating peptides have been widely used to enhance the intracellular delivery of various types of cargoes, such as drugs and proteins. These reagents are chem. similar to the multi-basic peptides that are known to be potent proprotein convertase inhibi- tors. Here, we report that both HIV-1 TAT47-57 peptide and the Chariot reagent are micromo- lar inhibitors of furin activity in vitro. In agreement, HIV-1 TAT47-57 reduced HT1080 cell migration, thought to be mediated by proprotein convertases, by 25%. In addn., cyclic polyarginine peptides contg. hydrophobic moieties which have been previously used as transfection reagents also exhibited potent furin inhibition in vitro and also inhibited intracellular convertases. Our finding that cationic cell-penetrating peptides exert potent effects on cellular convertase activity should be taken into account when biol. effects are assessed.
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  15. 15
    Fittler, H.; Depp, A.; Avrutina, O.; Dahms, S. O.; Than, M. E.; Empting, M.; Kolmar, H. Engineering a Constrained Peptidic Scaffold towards Potent and Selective Furin Inhibitors. ChemBioChem 2015, 16, 2441– 2444,  DOI: 10.1002/cbic.201500447
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    15
    Engineering a Constrained Peptidic Scaffold towards Potent and Selective Furin Inhibitors
    Fittler, Heiko; Depp, Alexander; Avrutina, Olga; Dahms, Sven O.; Than, Manuel E.; Empting, Martin; Kolmar, Harald
    ChemBioChem (2015), 16 (17), 2441-2444CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We report the engineering of the monocyclic sunflower trypsin inhibitor (SFTI-1[1,14]) into a potent furin inhibitor. In a rational approach, we converted the native scaffold of this trypsin-like serine protease inhibitor into a subtilisin-like one by substitutions in the canonical and, particularly, in the substrate-binding loop. Although the substrate sequence for furin is Arg-X-Arg/Lys-Arg↓, the most potent inhibitor had a lysine at position P1. C-terminally truncated versions demonstrated the strongest activity, thus suggesting a lack of interaction between this motif and the surface of furin. This observation was further supported by mol. modeling. With an inhibition const. of 0.49 nΜ, the engineered peptide H-KRCKKSIPPICF-NH2 is a promising compd. for further development of furin inhibitors aimed at controlling the activity of this protease in vitro and in vivo.
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  16. 16
    Becker, G. L.; Lu, Y.; Hardes, K.; Strehlow, B.; Levesque, C.; Lindberg, I.; Sandvig, K.; Bakowsky, U.; Day, R.; Garten, W.; Steinmetzer, T. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. J. Biol. Chem. 2012, 287, 21992– 22003,  DOI: 10.1074/jbc.M111.332643
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    16
    Highly Potent Inhibitors of Proprotein Convertase Furin as Potential Drugs for Treatment of Infectious Diseases
    Becker, Gero L.; Lu, Yinghui; Hardes, Kornelia; Strehlow, Boris; Levesque, Christine; Lindberg, Iris; Sandvig, Kirsten; Bakowsky, Udo; Day, Robert; Garten, Wolfgang; Steinmetzer, Torsten
    Journal of Biological Chemistry (2012), 287 (26), 21992-22003CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compds. contg. an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with Ki values of 16 and 8 pm, resp. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compds. with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivs. were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.
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  17. 17
    Becker, G. L.; Sielaff, F.; Than, M. E.; Lindberg, I.; Routhier, S.; Day, R.; Lu, Y.; Garten, W.; Steinmetzer, T. Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics. J. Med. Chem. 2010, 53, 1067– 1075,  DOI: 10.1021/jm9012455
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    17
    Potent Inhibitors of Furin and Furin-like Proprotein Convertases Containing Decarboxylated P1 Arginine Mimetics
    Becker, Gero L.; Sielaff, Frank; Than, Manuel E.; Lindberg, Iris; Routhier, Sophie; Day, Robert; Lu, Ying-Hui; Garten, Wolfgang; Steinmetzer, Torsten
    Journal of Medicinal Chemistry (2010), 53 (3), 1067-1075CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiol. and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analog peptidic inhibitors, we could identify highly potent furin inhibitors. The most potent compd., phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a Ki value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Mol. modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.
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  18. 18
    Hardes, K.; Becker, G. L.; Lu, Y.; Dahms, S. O.; Köhler, S.; Beyer, W.; Sandvig, K.; Yamamoto, H.; Lindberg, I.; Walz, L.; von Messling, V.; Than, M. E.; Garten, W.; Steinmetzer, T. Novel Furin Inhibitors with Potent Anti-infectious Activity. ChemMedChem 2015, 10, 1218– 1231,  DOI: 10.1002/cmdc.201500103
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    18
    Novel Furin Inhibitors with Potent Anti-infectious Activity
    Hardes, Kornelia; Becker, Gero L.; Lu, Yinghui; Dahms, Sven O.; Koehler, Susanne; Beyer, Wolfgang; Sandvig, Kirsten; Yamamoto, Hiroyuki; Lindberg, Iris; Walz, Lisa; von Messling, Veronika; Than, Manuel E.; Garten, Wolfgang; Steinmetzer, Torsten
    ChemMedChem (2015), 10 (7), 1218-1231CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
    New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compd. 4-guanidinomethyl-phenylacteyl-Arg-Tle-Arg 4-amidinobenzylamide (MI-1148) inhibits furin with a Ki value of 5.5 pM. The derivs. also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was obsd. in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI-1148 was crystd. in complex with human furin. Its N-terminal guanidinomethyl group in the para position of the P5 Ph ring occupies the same position as that found previously for a structurally related inhibitor contg. this substitution in the meta position, thereby maintaining all of the important P5 interactions. The authors' results confirm that the inhibition of furin is a promising strategy for a short-term treatment of acute infectious diseases.
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  19. 19
    Ivanova, T.; Hardes, K.; Kallis, S.; Dahms, S. O.; Than, M. E.; Kunzel, S.; Böttcher-Friebertshäuser, E.; Lindberg, I.; Jiao, G. S.; Bartenschlager, R.; Steinmetzer, T. Optimization of Substrate-Analogue Furin Inhibitors. ChemMedChem 2017, 12, 1953– 1968,  DOI: 10.1002/cmdc.201700596
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    19
    Optimization of Substrate-Analogue Furin Inhibitors
    Ivanova, Teodora; Hardes, Kornelia; Kallis, Stephanie; Dahms, Sven O.; Than, Manuel E.; Kuenzel, Sebastian; Boettcher-Friebertshaeuser, Eva; Lindberg, Iris; Jiao, Guan-Sheng; Bartenschlager, Ralf; Steinmetzer, Torsten
    ChemMedChem (2017), 12 (23), 1953-1968CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The proprotein convertase furin is a potential target for drug design, esp. for the inhibition of furin-dependent virus replication. All effective synthetic furin inhibitors identified thus far are multibasic compds.; the highest potency was found for our previously developed inhibitor 4-(guanidinomethyl)phenylacetyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148). An initial study in mice revealed a narrow therapeutic range for this tetrabasic compd., while significantly reduced toxicity was obsd. for some tribasic analogs. This suggests that the toxicity depends at least to some extent on the overall multibasic character of this inhibitor. Therefore, in a first approach, the C-terminal benzamidine of MI-1148 was replaced by less basic P1 residues. Despite decreased potency, a few compds. still inhibit furin in the low nanomolar range, but display negligible efficacy in cells. In a second approach, the P2 arginine was replaced by lysine; compared to MI-1148, this furin inhibitor has slightly decreased potency, but exhibits similar antiviral activity against West Nile and Dengue virus in cell culture and decreased toxicity in mice. These results provide a promising starting point for the development of efficacious and well-tolerated furin inhibitors.
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  20. 20
    Dahms, S. O.; Hardes, K.; Becker, G. L.; Steinmetzer, T.; Brandstetter, H.; Than, M. E. X-ray Structures of Human Furin in Complex with Competitive Inhibitors. ACS Chem. Biol. 2014, 9, 1113– 1118,  DOI: 10.1021/cb500087x
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    20
    X-ray Structures of Human Furin in Complex with Competitive Inhibitors
    Dahms, Sven O.; Hardes, Kornelia; Becker, Gero L.; Steinmetzer, Torsten; Brandstetter, Hans; Than, Manuel E.
    ACS Chemical Biology (2014), 9 (5), 1113-1118CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Furin inhibitors are promising therapeutics for the treatment of cancer and numerous infections caused by bacteria and viruses, including the highly lethal Bacillus anthracis or the pandemic influenza virus. Development and improvement of inhibitors for pharmacol. use require a detailed knowledge of the protease's substrate and inhibitor binding properties. Here we present a novel prepn. of human furin and the first crystal structures of this enzyme in complex with noncovalent inhibitors. We show the inhibitor exchange by soaking, allowing the investigation of addnl. inhibitors and substrate analogs. Thus, our work provides a basis for the rational design of furin inhibitors.
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  21. 21
    Lam van, T. V.; Heindl, M. R.; Schlutt, C.; Böttcher-Friebertshäuser, E.; Bartenschlager, R.; Klebe, G.; Brandstetter, H.; Dahms, S. O.; Steinmetzer, T. The Basicity Makes the Difference: Improved Canavanine-Derived Inhibitors of the Proprotein Convertase Furin. ACS Med. Chem. Lett. 2021, 12, 426– 432,  DOI: 10.1021/acsmedchemlett.0c00651
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    21
    The Basicity Makes the Difference: Improved Canavanine-Derived Inhibitors of the Proprotein Convertase Furin
    Lam van, Thuy Van; Heindl, Miriam Ruth; Schlutt, Christine; Boettcher-Friebertshaeuser, Eva; Bartenschlager, Ralf; Klebe, Gerhard; Brandstetter, Hans; Dahms, Sven O.; Steinmetzer, Torsten
    ACS Medicinal Chemistry Letters (2021), 12 (3), 426-432CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
    A review. Furin activates numerous viral glycoproteins, and its inhibition prevents virus replication and spread. Through the replacement of arginine by the less basic canavanine, new inhibitors targeting furin in the trans-Golgi network were developed. These inhibitors exert potent antiviral activity in cell culture with much lower toxicity than arginine-derived analogs, most likely due to their reduced protonation in the blood circulation. Thus, despite its important physiol. functions, furin might be a suitable antiviral drug target.
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  22. 22
    Dahms, S. O.; Arciniega, M.; Steinmetzer, T.; Huber, R.; Than, M. E. Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 11196– 11201,  DOI: 10.1073/pnas.1613630113
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    22
    Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism
    Dahms, Sven O.; Arciniega, Marcelino; Steinmetzer, Torsten; Huber, Robert; Than, Manuel E.
    Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (40), 11196-11201CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolemia, and infectious diseases. Novel protein crystallog. structures of the prototypical PC family member furin in different functional states were detd. to 1.8-2.0 Å. These, together with biochem. data and modeling by mol. dynamics calcns., suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the "off state," incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active "on state" seen in inhibitor-bound furin; and the resp. (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The mol. dynamics simulations of the four structural states reflect the exptl. observations in general and provide approxns. of the resp. stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and dets. the rotamer state of the oxyanion hole-forming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacol. intervention targeting furin.
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  23. 23
    Jiao, G. S.; Cregar, L.; Wang, J.; Millis, S. Z.; Tang, C.; O’Malley, S.; Johnson, A. T.; Sareth, S.; Larson, J.; Thomas, G. Synthetic small molecule furin inhibitors derived from 2,5-dideoxystreptamine. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 19707– 19712,  DOI: 10.1073/pnas.0606555104
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    23
    Synthetic small molecule furin inhibitors derived from 2,5-dideoxystreptamine
    Jiao, Guan-Sheng; Cregar, Lynne; Wang, Jinzhi; Millis, Sherri Z.; Tang, Cho; O'Malley, Sean; Johnson, Alan T.; Sareth, Sina; Larson, Jason; Thomas, Gary
    Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (52), 19707-19712CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small mol. furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addn. of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochem. cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Mol. modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH2Cl. The compds. presented here are bona fide synthetic small mol. furin inhibitors that exhibit potency in the nano-molar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furin-dependent diseases.
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  24. 24
    Komiyama, T.; Coppola, J. M.; Larsen, M. J.; van Dort, M. E.; Ross, B. D.; Day, R.; Rehemtulla, A.; Fuller, R. S. Inhibition of furin/proprotein convertase-catalyzed surface and intracellular processing by small molecules. J. Biol. Chem. 2009, 284, 15729– 15738,  DOI: 10.1074/jbc.M901540200
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    24
    Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules
    Komiyama, Tomoko; Coppola, Julia M.; Larsen, Martha J.; van Dort, Marcian E.; Ross, Brian D.; Day, Robert; Rehemtulla, Alnawaz; Fuller, Robert S.
    Journal of Biological Chemistry (2009), 284 (23), 15729-15738CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Furin is a ubiquitously expressed proprotein convertase (PC) that plays a vital role in numerous disease processes including cancer metastasis, bacterial toxin activation (e.g. anthrax and Pseudomonas), and viral propagation (e.g. avian influenza and human immunodeficiency virus). To identify small mol. inhibitors of furin and related processing enzymes, we performed high-throughput screens of chem. diversity libraries utilizing both enzyme-based and cell-based assays. The screens identified partially overlapping sets of compds. that were further characterized for affinity, mechanism, and efficacy in addnl. cellular processing assays. Dicoumarols were identified as a class of compds. that inhibited furin non-competitively and reversibly with Ki values in the micromolar range. These compds. inhibited furin/furin-like activity both at the cell surface (protecting against anthrax toxin) and in the secretory pathway (blocking processing of the metastasis factor membrane-type 1 matrix metalloproteinase/MT1-MMP) at concns. close to Ki values. Compds. tested exhibited distinct patterns of inhibition of other furin-family PCs (rat PACE4, human PC5/6 and human PC7), showing that dicoumarol derivs. might be developed as either generic or selective inhibitors of the PCs. The extensive clin. use, high bioavailability and relatively low toxicity of dicoumarols suggests that the dicoumarol structure will be a good starting point for development of drug-like inhibitors of furin and other PCs that can act both intracellularly and at the cell surface.
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  25. 25
    Ramos-Molina, B.; Lick, A. N.; Blanco, E. H.; Posada-Salgado, J. A.; Martinez-Mayorga, K.; Johnson, A. T.; Jiao, G. S.; Lindberg, I. Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays. Biochem. Pharmacol. 2015, 96, 107– 118,  DOI: 10.1016/j.bcp.2015.05.008
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    25
    Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays
    Ramos-Molina, Bruno; Lick, Adam N.; Blanco, Elias H.; Posada-Salgado, J. Alejandro; Martinez-Mayorga, Karina; Johnson, Alan T.; Jiao, Guan-Sheng; Lindberg, Iris
    Biochemical Pharmacology (Amsterdam, Netherlands) (2015), 96 (2), 107-118CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
    The proprotein convertase furin is implicated in a variety of pathogenic processes such as bacterial toxin activation, viral propagation, and cancer. Several groups have identified non-peptide compds. with high inhibitory potency against furin in vitro, although their efficacy in various cell-based assays is largely unknown. In this study certain guanidinylated 2,5-dideoxystreptamine derivs. exhibit interesting ex vivo properties. Compd. 1b (1,1'-(4-((2,4-diguanidino-5-(4-guanidinophenoxy)cyclohexyl)oxy)-1,3-phenylene)diguanidine) is a potent and cell-permeable inhibitor of cellular furin, since it was able to retard tumor cell migration, block release of a Golgi reporter, and protect cells against Bacillus anthracis (anthrax) and Pseudomonas aeruginosa intoxication, with no evident cell toxicity. Other compds. based on the 2,5-dideoxystreptamine scaffold, such as compd. 1g (1,1'-(4,6-bis(4-guanidinophenoxy)cyclohexane-1,3-diyl)diguanidine) also efficiently protected cells against anthrax, but displayed only moderate protection against Pseudomonas exotoxin A and did not inhibit cell migration, suggesting poor cell permeability. Certain bis-guanidinophenyl ether derivs. such as 2f (1,3-bis(2,4-diguanidinophenoxy) benzene) exhibited micromolar potency against furin in vitro, low cell toxicity, and highly efficient protection against anthrax toxin; this compd. only slightly inhibited intracellular furin. Thus, compds. 1g and 2f both represent potent furin inhibitors at the cell surface with low intracellular inhibitory action, and these particular compds. might therefore be of preferred therapeutic interest in the treatment of certain bacterial and viral infections.
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    Sielaff, F.; Than, M. E.; Bevec, D.; Lindberg, I.; Steinmetzer, T. New furin inhibitors based on weakly basic amidinohydrazones. Bioorg. Med. Chem. Lett. 2011, 21, 836– 840,  DOI: 10.1016/j.bmcl.2010.11.092
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    26
    New furin inhibitors based on weakly basic amidinohydrazones
    Sielaff, Frank; Than, Manuel E.; Bevec, Dorian; Lindberg, Iris; Steinmetzer, Torsten
    Bioorganic & Medicinal Chemistry Letters (2011), 21 (2), 836-840CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
    A novel series of amidinohydrazone-derived furin inhibitors was prepd.; the most potent compds. I and II inhibit furin with Ki values of 0.46 and 0.59 μM, resp. In contrast to inhibitor I, which still contains a guanidino residue, compd. II possesses only weakly basic amidinohydrazone groups.
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  27. 27
    Essalmani, R.; Jain, J.; Susan-Resiga, D.; Andréo, U.; Evagelidis, A.; Derbali, R. M.; Huynh, D. N.; Dallaire, F.; Laporte, M.; Delpal, A.; Sutto-Ortiz, P.; Coutard, B.; Mapa, C.; Wilcoxen, K.; Decroly, E.; Pham, T. N. Q.; Cohen, E. A.; Seidah, N. G. Implications of Spike-glycoprotein processing at S1/S2 by Furin, at S2’ by Furin and/or TMPRSS2 and shedding of ACE2: cell-to-cell fusion, cell entry and infectivity of SARS-CoV-2. bioRxiv 2020.
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    Dahms, S. O.; Jiao, G. S.; Than, M. E. Structural Studies Revealed Active Site Distortions of Human Furin by a Small Molecule Inhibitor. ACS Chem. Biol. 2017, 12, 1211– 1216,  DOI: 10.1021/acschembio.6b01110
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    Structural Studies Revealed Active Site Distortions of Human Furin by a Small Molecule Inhibitor
    Dahms, Sven O.; Jiao, Guan-Sheng; Than, Manuel E.
    ACS Chemical Biology (2017), 12 (5), 1211-1216CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of several pathologies including cancer, atherosclerosis, hypercholesterolemia, and infectious diseases. Here we present the first exptl. complex of furin with a non substrate-like small mol. inhibitor, and the X-ray structure of the enzyme complexed to the small mol. inhibitor I at 1.9 Å resoln. Two mols. of inhibitor 1 were found to interact with furin. One is anchored at the S4 pocket of the enzyme and interferes directly with the conformation and function of the catalytic triade; the other mol. shows weaker binding and interacts with a distant, less conserved region of furin. The obsd. binding modes represent a new inhibition strategy of furin and imply the possibility to attain specificity among the PCs providing an innovative starting point of structure guided inhibitor development for furin.
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  29. 29
    Soll, R. M.; Lu, T.; Tomczuk, B.; Illig, C. R.; Fedde, C.; Eisennagel, S.; Bone, R.; Murphy, L.; Spurlino, J.; Salemme, F. R. Amidinohydrazones as guanidine bioisosteres: application to a new class of potent, selective and orally bioavailable, non-amide-based small-molecule thrombin inhibitors. Bioorg. Med. Chem. Lett. 2000, 10, 1– 4,  DOI: 10.1016/S0960-894X(99)00632-0
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    Amidinohydrazones as guanidine bioisosteres: application to a new class of potent, selective and orally bioavailable, non-amide-based small-molecule thrombin inhibitors
    Soll, Richard M.; Lu, Tianobao; Tomczuk, Bruce; Illig, Carl R.; Fedde, Cynthia; Eisennagel, Stephen; Bone, Roger; Murphy, Larry; Spurlino, John; Salemme, F. Raymond
    Bioorganic & Medicinal Chemistry Letters (2000), 10 (1), 1-4CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
    We describe a new class of potent, non-amide-based small mol. thrombin inhibitors in which an amidinohydrazone is used as a guanidine bioisostere on a non-peptide scaffold. Compd. 4 exhibits nM inhibition of thrombin, is selective for thrombin, and shows 60 and 23% bioavailability in rabbits and dogs, resp. Crystallog. anal. of 4 bound to thrombin confirmed the amindinohydrazone binding mode.
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    Specht, S.; Sarite, S. R.; Hauber, I.; Hauber, J.; Gorbig, U. F.; Meier, C.; Bevec, D.; Hoerauf, A.; Kaiser, A. The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase. Parasitol. Res. 2008, 102, 1177– 1184,  DOI: 10.1007/s00436-008-0891-x
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    The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase
    Specht Sabine; Sarite Salem Ramadan; Hauber Ilona; Hauber Joachim; Gorbig Ulf F; Meier Chris; Bevec Dorian; Hoerauf Achim; Kaiser Annette
    Parasitology research (2008), 102 (6), 1177-84 ISSN:0932-0113.
    Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.
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    Henrich, S.; Lindberg, I.; Bode, W.; Than, M. E. Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity. J. Mol. Biol. 2005, 345, 211– 227,  DOI: 10.1016/j.jmb.2004.10.050
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    Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity
    Henrich Stefan; Lindberg Iris; Bode Wolfram; Than Manuel E
    Journal of molecular biology (2005), 345 (2), 211-27 ISSN:0022-2836.
    In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs). These PCs cleave their protein substrates very specifically following multiple basic residues. The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain. The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern.
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    Dahms, S. O.; Hardes, K.; Steinmetzer, T.; Than, M. E. X-ray structures of the proprotein convertase furin bound with substrate analog inhibitors reveal substrate specificity determinants beyond the S4 pocket. Biochemistry 2018, 57, 925,  DOI: 10.1021/acs.biochem.7b01124
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    X-ray Structures of the Proprotein Convertase Furin Bound with Substrate Analogue Inhibitors Reveal Substrate Specificity Determinants beyond the S4 Pocket
    Dahms, Sven O.; Hardes, Kornelia; Steinmetzer, Torsten; Than, Manuel E.
    Biochemistry (2018), 57 (6), 925-934CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The proprotein convertase furin is a highly specific serine protease modifying and thereby activating proteins in the secretory pathway by proteolytic cleavage. Its substrates are involved in many diseases, including cancer and infections caused by bacteria and viruses. Understanding furin's substrate specificity is crucially important for the development of pharmacol. applicable inhibitors. Using protein X-ray crystallog., we investigated the extended substrate binding site of furin in complex with three peptide-derived inhibitors at up to 1.9 Å resoln. The structure of the protease bound with a hexapeptide inhibitor revealed mol. details of its S6 pocket, which remained completely unknown so far. The arginine residue at P6 induced an unexpected turnlike conformation of the inhibitor backbone, which is stabilized by intra- and intermol. H-bonds. In addn., we confirmed the binding of arginine to the previously proposed S5 pocket (S51). An alternative S5 site (S52) could be utilized by shorter side chains as demonstrated for a 4-aminomethyl-phenylacetyl residue, which shows steric properties similar to those of a lysine side chain. Interestingly, we also obsd. binding of a peptide with citrulline at P4 substituting for the highly conserved arginine. The structural data might indicate an unusual protonation state of Asp264 maintaining the interaction with uncharged citrulline. The herein identified mol. interaction sites at P5 and P6 can be utilized to improve next-generation furin inhibitors. Our data will also help to predict furin substrates more precisely on the basis of the addnl. specificity determinants obsd. for P5 and P6.
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    Mueller, U.; Darowski, N.; Fuchs, M. R.; Forster, R.; Hellmig, M.; Paithankar, K. S.; Puhringer, S.; Steffien, M.; Zocher, G.; Weiss, M. S. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. J. Synchrotron Radiat. 2012, 19, 442– 449,  DOI: 10.1107/S0909049512006395
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    Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin
    Mueller, Uwe; Darowski, Nora; Fuchs, Martin R.; Foerster, Ronald; Hellmig, Michael; Paithankar, Karthik S.; Puehringer, Sandra; Steffien, Michael; Zocher, Georg; Weiss, Manfred S.
    Journal of Synchrotron Radiation (2012), 19 (3), 442-449CODEN: JSYRES; ISSN:0909-0495. (International Union of Crystallography)
    Three macromol. crystallog. (MX) beamlines at the Helmholtz-Zentrum Berlin (HZB) are available for the regional, national and international structural biol. user community. The state-of-the-art synchrotron beamlines for MX BL14.1, BL14.2 and BL14.3 are located within the low-β section of the BESSY II electron storage ring. All beamlines are fed from a superconducting 7 T wavelength-shifter insertion device. BL14.1 and BL14.2 are energy tunable in the range 5-16 keV, while BL14.3 is a fixed-energy side station operated at 13.8 keV. All three beamlines are equipped with CCD detectors. BL14.1 and BL14.2 are in regular user operation providing about 200 beam days per yr and about 600 user shifts to approx. 50 research groups across Europe. BL14.3 has initially been used as a test facility and was brought into regular user mode operation during the year 2010. BL14.1 has recently been upgraded with a microdiffractometer including a mini-κ goniometer and an automated sample changer. Addnl. user facilities include office space adjacent to the beamlines, a sample prepn. lab., a biol. lab. (safety level 1) and high-end computing resources. In this article the instrumentation of the beamlines is described, and a summary of the exptl. possibilities of the beamlines and the provided ancillary equipment for the user community is given.
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    Kabsch, W. Xds. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132,  DOI: 10.1107/S0907444909047337
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    Software XDS for image rotation, recognition and crystal symmetry assignment
    Kabsch, Wolfgang
    Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
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    Krug, M.; Weiss, M. S.; Heinemann, U.; Mueller, U. XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS. J. Appl. Crystallogr. 2012, 45, 568– 572,  DOI: 10.1107/S0021889812011715
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    XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS
    Krug, Michael; Weiss, Manfred S.; Heinemann, Udo; Mueller, Uwe
    Journal of Applied Crystallography (2012), 45 (3), 568-572CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
    XDSAPP is a Tcl/Tk-based graphical user interface for the easy and convenient processing of diffraction data sets using XDS. It provides easy access to all XDS functionalities, automates the data processing and generates graphical plots of various data set statistics provided by XDS. By incorporating addnl. software, further information on certain features of the data set, such as radiation decay during data collection or the presence of pseudo-translational symmetry and/or twinning, can be obtained. Intensity files suitable for CCP4, CNS and SHELX are generated.
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    Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S. Overview of the CCP4 suite and current developments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 235– 242,  DOI: 10.1107/S0907444910045749
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    Overview of the CCP4 suite and current developments
    Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.; Dodson, Eleanor J.; Emsley, Paul; Evans, Phil R.; Keegan, Ronan M.; Krissinel, Eugene B.; Leslie, Andrew G. W.; McCoy, Airlie; McNicholas, Stuart J.; Murshudov, Garib N.; Pannu, Navraj S.; Potterton, Elizabeth A.; Powell, Harold R.; Read, Randy J.; Vagin, Alexei; Wilson, Keith S.
    Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 235-242CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    A review. The CCP4 (Collaborative Computational Project, No. 4) software suite is a collection of programs and assocd. data and software libraries which can be used for macromol. structure detn. by X-ray crystallog. The suite is designed to be flexible, allowing users a no. of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by std. file formats, data objects and graphical interfaces. Structure soln. by macromol. crystallog. is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.
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    Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486– 501,  DOI: 10.1107/S0907444910007493
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    Features and development of Coot
    Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.
    Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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    Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213– 221,  DOI: 10.1107/S0907444909052925
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    PHENIX: a comprehensive Python-based system for macromolecular structure solution
    Adams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.
    Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
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    Schuttelkopf, A. W.; van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 1355– 1363,  DOI: 10.1107/S0907444904011679
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    PRODRG: a tool for high-throughput crystallography of protein-ligand complexes
    Schuttelkopf Alexander W; van Aalten Daan M F
    Acta crystallographica. Section D, Biological crystallography (2004), 60 (Pt 8), 1355-63 ISSN:0907-4449.
    The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray refinement of protein-ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force field used, structures with good geometries are generated. X-ray refinement in X-PLOR/CNS, REFMAC and SHELX using PRODRG-generated topologies produces results comparable to refinement with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.
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    Györgydeák, Z.; Holzer, W.; Mereiter, K. Guanylhydrazones of (Hetero)Aryl Methyl Ketones: Structure and Reaction with Acetic Anhydride. Monatsh. Chem. 1999, 130, 899– 913,  DOI: 10.1007/PL00010271
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    Guanylhydrazones of (hetero)aryl methyl ketones. Structure and reaction with acetic anhydride
    Gyorgydeak, Zoltan; Holzer, Wolfgang; Mereiter, Kurt
    Monatshefte fuer Chemie (1999), 130 (7), 899-913CODEN: MOCMB7; ISSN:0026-9247. (Springer-Verlag Wien)
    The synthesis of guanylhydrazones of (hetero)aryl Me ketones is described. Successive reaction with hot Ac2O leads to the corresponding N,N'-diacetyl derivs. Structural assignments of all novel compds. and those of some already known congeners were achieved by NMR (1H, 13C) and x-ray structure anal.
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    Li, W.; Cowley, A.; Uludag, M.; Gur, T.; McWilliam, H.; Squizzato, S.; Park, Y. M.; Buso, N.; Lopez, R. The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Res. 2015, 43, W580– 584,  DOI: 10.1093/nar/gkv279
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    The EMBL-EBI bioinformatics web and programmatic tools framework
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    Nucleic Acids Research (2015), 43 (W1), W580-W584CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    A review. Since 2009 the EMBL-EBI Job Dispatcher framework has provided free access to a range of mainstream sequence anal. applications. These include sequence similarity search services such as BLAST, FASTA and PSI-Search, multiple sequence alignment tools such as Clustal Omega, MAFFT and T-Coffee, and other sequence anal. tools such as InterProScan. Through these services users can search mainstream sequence databases such as ENA, UniProt and Ensembl Genomes, utilizing a uniform web interface or systematically through Web Services interfaces using common programming languages, and obtain enriched results with novel visualizations. Integration with EBI Search and the dbfetch retrieval service further expands the usefulness of the framework. New tools and updates such as NCBI BLAST+, InterProScan 5 and PfamScan, new categories such as RNA anal. tools, new databases such as ENA non-coding, WormBase ParaSite, Pfam and Rfam, and new workflow methods, together with the retirement of depreciated services, ensure that the framework remains relevant to today's biol. community.
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    Bond, C. S.; Schuttelkopf, A. W. ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 510– 512,  DOI: 10.1107/S0907444909007835
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    ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments
    Bond, Charles Simon; Schuettelkopf, Alexander Wolfgang
    Acta Crystallographica, Section D: Biological Crystallography (2009), 65 (5), 510-512CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    Marked-up sequence alignments typically provide the central figure in articles describing proteins, whether in the fields of biochem., bioinformatics or structural biol. The generation of these figures is often unwieldy: interactive programs are often aesthetically limited and the use of batch programs requires the repetitive iterative editing of scripts. ALINE is a portable interactive graphical sequence-alignment editor implemented in Perl/Tk which produces publication-quality sequence-alignment figures where 'what you see is what you get'. ALINE is freely available for download from http://crystal.bcs.uwa.edu.au/px/charlie/software/aline/.
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    Linking Crystallographic Model and Data Quality
    Karplus, P. Andrew; Diederichs, Kay
    Science (Washington, DC, United States) (2012), 336 (6084), 1030-1033CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
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Cited By

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This article is cited by 7 publications.

  1. Surasak Wichaiyo, Pimpisid Koonyosying, Noppawan Phumala Morales. Functional Roles of Furin in Cardio-Cerebrovascular Diseases. ACS Pharmacology & Translational Science 2024, 7 (3) , 570-585. https://doi.org/10.1021/acsptsci.3c00325
  2. Sven O. Dahms, Gisela Schnapp, Martin Winter, Frank H. Büttner, Marco Schlepütz, Christian Gnamm, Alexander Pautsch, Hans Brandstetter. Dichlorophenylpyridine-Based Molecules Inhibit Furin through an Induced-Fit Mechanism. ACS Chemical Biology 2022, 17 (4) , 816-821. https://doi.org/10.1021/acschembio.2c00103
  3. Zhenni Lu, Dongya Liu, Peng Wei, Tao Yi. Activated aggregation strategies to construct size‐increasing nanoparticles for cancer therapy. WIREs Nanomedicine and Nanobiotechnology 2023, 15 (2) https://doi.org/10.1002/wnan.1848
  4. Dongyan Feng, Le Ren, Jiaqi Wu, Lingling Guo, Zhitao Han, Jingjing Yang, Wei Xie, Yanbing Wang, Fanxing Xu, Xin Su, Dahong Li, Hao Cao. Permethrin as a Potential Furin Inhibitor through a Novel Non-Competitive Allosteric Inhibition. Molecules 2023, 28 (4) , 1883. https://doi.org/10.3390/molecules28041883
  5. Sang-Nam Lee, Joo-Heon Yoon. The Role of Proprotein Convertases in Upper Airway Remodeling. Molecules and Cells 2022, 45 (6) , 353-361. https://doi.org/10.14348/molcells.2022.0019
  6. Michèle Reboud-Ravaux, Chahrazade El Amri. COVID-19 Therapies: Protease Inhibitions and Novel Degrader Strategies. Frontiers in Drug Discovery 2022, 2 https://doi.org/10.3389/fddsv.2022.892057
  7. Kasi Pandima Devi, Mahmoud Reza Pourkarim, Marijn Thijssen, Antoni Sureda, Maryam Khayatkashani, Cosmin Andrei Cismaru, Ioana Berindan Neagoe, Solomon Habtemariam, Soha Razmjouei, Hamid Reza Khayat Kashani. A perspective on the applications of furin inhibitors for the treatment of SARS-CoV-2. Pharmacological Reports 2022, 74 (2) , 425-430. https://doi.org/10.1007/s43440-021-00344-x
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  • Abstract

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    Figure 1

    Figure 1. (A) Ligand binding to furin induces a conformational shift of furin from the ON- to the OFF-state. Superposition of furin in the ON- (gray, PDB ID: 5JXH (22)) and OFF-states (yellow, PDB ID: 5JXG (22)). Dashed lines highlight important interactions. Big and small spheres represent the sodium ion and water molecules, respectively. Instead of the P1 residue of the inhibitor, in the OFF-state a water molecule occupies two alternative sites (H2O A and B) at the bottom of the S1 pocket. (B) Chemical structure of guanabenz. (C) Chemical structures of the inhibitors used in this study.

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    Figure 2

    Figure 2. Structures of furin bound to guanylhydrazone-based inhibitors. The protease is shown as a cartoon representation (golden), catalytic residues as sticks with carbon atoms in cyan and the inhibitors as ball-and-stick models, respectively. The specificity pockets are labeled (S1–S4). The Fo–Fc annealed omit electron density map of the inhibitors is shown as green mesh and is contoured at 3.0 σ. Furin in complex with inhibitors 1 (A), 2 (B), 3 (C), and 4 (D). Inhibitor 1 was found in two conformations shown with carbons in dark and light gray. (E,F) Two binding sites of 2 (E) and 3 (F) were found at the active site cleft of furin. The binding sites 1 and 2 are labeled I-1 and I-2, respectively.

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    Figure 3

    Figure 3. Binding mode of the OFF-state-specific furin inhibitors. Specific furin residues and the inhibitors are shown as stick and ball-and-stick models, respectively. Important interactions between the inhibitor and furin are marked with dashed lines. The furin backbone is given as a cartoon representation (yellow). The inhibitors 1 (A, two conformations are indicated by light and dark colors), 2 (B), 3 (C), and 4 (D) are shown with carbons in green, orange, brown, and magenta, respectively. The active site residues are shown with carbons in cyan. The specificity pockets are labeled (S1–S4). Ligand-free furin (PDB-ID: 5jxg (22)) is superimposed on the furin-inhibitor complex structures and is shown as a cartoon in black.

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    Figure 4

    Figure 4. Allosteric connection between the S1 pocket and the sodium binding site. (A) Superposition of furin (carbons in yellow) bound to 1 (colored according to Figure 3A, OFF-state) and furin in the ON-state bound the substrate-like inhibitor 3-guanidinomethyl-phenylacetyl-RVR-4-aminomethyl-benzamidine (gray, PDB ID: 5jxh (22)). Competing space requirements of the water molecule at the S1 pocket and the side chain of Thr306 are highlighted by red dashes. Dashed lines highlight important interactions. For clarity, in the structure of furin bound to the substrate-like ligand, dashed lines, the Na+ ion and the H2O ligand are shown in gray. (B) Sequence alignment of the sodium-binding loop of furin-like PCs. Positions with sequence identities of 100% and >50% are highlighted in black and gray, respectively. Sodium binding residues are framed and numbered.

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    Figure 5

    Figure 5. Comparison of the binding poses of guanylhydrazones. Stereo representations of structural superpositions of furin-inhibitor complex structures. Specific furin residues and the inhibitors are shown as stick and ball-and-stick models, respectively. Important interactions between the inhibitor and furin are marked with dashed lines. The inhibitors are colored according to Figure 3. Furin residues and the furin backbone are colored according to the inhibitors in green (1), orange (2), and magenta (4). (A) Superposition of furin in complex with 1 and 2. (B) Superposition of furin in complex with 1 and 4.

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    Figure 6

    Figure 6. Second binding site of 2 and 3. (A–E) The furin backbone is given as a cartoon representation (yellow). Specific furin residues and the inhibitors are shown as stick and ball-and-stick models, respectively. The active site residues are shown with carbons in cyan. The specificity pockets are labeled (S1, S4). (A, B) The second molecule of 2 (A) or 3 (B) is shown with gray carbons (I-2). Inhibitor molecules bound to the S1 pocket are colored according to Figure 1 and Figure 2. Important interactions between the inhibitor and furin are marked with dashed lines. Ligand-free furin (PDB-ID: 5jxg (22)) is superimposed and is shown as a cartoon in black. (C–E) Titration of furin at low ionic strength (250 mM NaCl) with 0.4 mM (C), 4 mM (D), and 40 mM (E) of inhibitor 2. The Fo–Fc annealed omit electron density map of the inhibitors is shown as a green mesh and is contoured at 3.0 σ.

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  • References

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      Recently it was shown that furin, a mammalian homolog of the yeast precursor-processing endoprotease Kex2, is involved in precursor cleavage at sites marked by the Arg-X-Lys/Arg-Arg motif within the constitutive secretory pathway. In this study, mol. and enzymic properties of furin expressed in Chinese hamster ovary cells were analyzed using gene transfer techniques. COOH-terminal truncation analyses indicate that the polypeptide region significantly conserved among the Kex2 family members is required for the endoprotease activity of furin, while the COOH-terminal unconserved region contg. the Cys-rich domain and the transmembrane domain is dispensable. A mutant of furin truncated up to the transmembrane domain from the COOH-terminus was secreted into the culture medium as an active form. The sequence requirements for precursor cleavage of this truncated furin detd. in vitro were similar to those of wild-type furin detd. by expression studies in cultured cells. It had a strong resemblance to the Kex2 protease in the inhibitor profile and pH dependency. These observations support the notion that furin is the endogenous endoprotease involved in precursor cleaved at Arg-X-Lys/Arg-Arg sites.
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      Polyarginines are potent furin inhibitors
      Cameron, Angus; Appel, Jon; Houghten, Richard A.; Lindberg, Iris
      Journal of Biological Chemistry (2000), 275 (47), 36741-36749CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The ubiquitous serine endoprotease furin has been implicated in the activation of bacterial toxins and viral glycoproteins as well as in the metastatic progression of certain tumors. Although high mol. mass bioengineered serpin inhibitors have been well characterized, no small nontoxic nanomolar inhibitors have been reported to date. Here the authors describe the identification of such inhibitors using positional scanning amidated and acetylated synthetic L- and D-hexapeptide combinatorial libraries. The results indicated that L-Arg or L-Lys in all positions generated the most potent inhibitors. However, further investigation revealed that the peptide terminating groups hindered inhibition. Consequently, a series of non-amidated and acetylated polyarginines was synthesized. The most potent inhibitor identified, nona-L-arginine, had a Ki for furin of 40 nM. The Ki values for the related convertases PACE4 and prohormone convertase-1 (PC1) were 110 nM and 2.5 μM, resp. Although nona-L-arginine was cleaved by furin, the major products after a 6-h incubation at 37° were hexa-and hepta-L-arginines, both of which retained the great majority of their potency and specificity against furin. Hexa-D-arginine was as potent and specific a furin inhibitor as hexa-L-arginine (Ki values of hexa-D-arginine: 106 nM, 580 nM, and 13.2 μM for furin, PACE4, and PC1, resp.). PC2 was not inhibited by any polyarginine tested; indeed, PC2 showed an increase in activity of ≤140% of the control in the presence of L-polyarginines. Data are also presented that show extended subsite recognition by furin and PC2. Whereas N-terminal acetylation was found to reduce the inhibitory potency of the L-hexapeptide LLRVKR against furin 8-fold, C-terminal amidation reduced the potency <2-fold. Conversely, N-terminal acetylation increased the potency against PC2 nearly 3-fold, whereas C-terminal amidation of the same peptide increased the potency by a factor of 1.6. Our data indicate that non-acetylated, poly-D-arginine-derived mols. may represent excellent lead compds. for the development of therapeutically useful furin inhibitors.
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      Levesque, C.; Fugere, M.; Kwiatkowska, A.; Couture, F.; Desjardins, R.; Routhier, S.; Moussette, P.; Prahl, A.; Lammek, B.; Appel, J. R.; Houghten, R. A.; D’Anjou, F.; Dory, Y. L.; Neugebauer, W.; Day, R. The Multi-Leu peptide inhibitor discriminates between PACE4 and furin and exhibits antiproliferative effects on prostate cancer cells. J. Med. Chem. 2012, 55, 10501– 10511,  DOI: 10.1021/jm3011178
      12
      The Multi-Leu Peptide Inhibitor Discriminates Between PACE4 and Furin And Exhibits Antiproliferative Effects On Prostate Cancer Cells
      Levesque, Christine; Fugere, Martin; Kwiatkowska, Anna; Couture, Frederic; Desjardins, Roxane; Routhier, Sophie; Moussette, Philippe; Prahl, Adam; Lammek, Bernard; Appel, Jon R.; Houghten, Richard A.; DAnjou, Francois; Dory, Yves L.; Neugebauer, Witold; Day, Robert
      Journal of Medicinal Chemistry (2012), 55 (23), 10501-10511CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The proprotein convertases (PCs) play an important role in protein precursor activation through processing at paired basic residues. However, significant substrate cleavage redundancy has been reported between PCs. The question remains whether specific PC inhibitors can be designed. This study describes the identification of the sequence LLLLRVKR, named Multi-Leu (ML)-peptide, that displayed a 20-fold selectivity on PACE4 over furin, two enzymes with similar structural characteristics. We have previously demonstrated that PACE4 plays an important role in prostate cancer and could be a druggable target. The present study demonstrates that the ML-peptide significantly reduced the proliferation of DU145 and LNCaP prostate cancer-derived cell lines and induced G0/G1 cell cycle arrest. However, the ML-peptide must enter the cell to inhibit proliferation. It is concluded that peptide-based inhibitors can yield specific PC inhibitors and that the ML-peptide is an important lead compd. that could potentially have applications in prostate cancer.
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    13. 13
      Lewandowska-Goch, M. A.; Kwiatkowska, A.; Lepek, T.; Ly, K.; Navals, P.; Gagnon, H.; Dory, Y. L.; Prahl, A.; Day, R. Design and Structure-Activity Relationship of a Potent Furin Inhibitor Derived from Influenza Hemagglutinin. ACS Med. Chem. Lett. 2021, 12, 365– 372,  DOI: 10.1021/acsmedchemlett.0c00386
      13
      Design and structure-activity relationship of a potent furin inhibitor derived from influenza hemagglutinin
      Lewandowska-Goch, Monika A.; Kwiatkowska, Anna; Lepek, Teresa; Ly, Kevin; Navals, Pauline; Gagnon, Hugo; Dory, Yves L.; Prahl, Adam; Day, Robert
      ACS Medicinal Chemistry Letters (2021), 12 (3), 365-372CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      Furin plays an important role in various pathol. states, esp. in bacterial and viral infections. A detailed understanding of the structural requirements for inhibitors targeting this enzyme is crucial to develop new therapeutic strategies in infectious diseases, including an urgent unmet need for SARS-CoV-2 infection. Previously, we have identified a potent furin inhibitor, peptide Ac-RARRRKKRT-NH2 (CF1), based on the highly pathogenic avian influenza hemagglutinin. The goal of this study was to det. how its N-terminal part (the P8-P5 positions) affects its activity profile. To do so, the positional-scanning libraries of individual peptides modified at the selected positions with natural amino acids were generated. Subsequently, the best substitutions were combined together and/or replaced by unnatural residues to expand our investigations. The results reveal that the affinity of CF1 can be improved (2-2.5-fold) by substituting its P5 position with the small hydrophobic residues (Ile or Val) or a basic Lys.
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    14. 14
      Ramos-Molina, B.; Lick, A. N.; Nasrolahi Shirazi, A.; Oh, D.; Tiwari, R.; El-Sayed, N. S.; Parang, K.; Lindberg, I. Cationic Cell-Penetrating Peptides Are Potent Furin Inhibitors. PLoS One 2015, 10, e0130417,  DOI: 10.1371/journal.pone.0130417
      14
      Cationic cell-penetrating peptides are potent furin inhibitors
      Ramos-Molina, Bruno; Lick, Adam N.; Shirazi, Amir Nasrolahi; Oh, Donghoon; Tiwari, Rakesh; El-Sayed, Naglaa Salem; Parang, Keykavous; Lindberg, Iris
      PLoS One (2015), 10 (6), e0130417/1-e0130417/11CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Cationic cell-penetrating peptides have been widely used to enhance the intracellular delivery of various types of cargoes, such as drugs and proteins. These reagents are chem. similar to the multi-basic peptides that are known to be potent proprotein convertase inhibi- tors. Here, we report that both HIV-1 TAT47-57 peptide and the Chariot reagent are micromo- lar inhibitors of furin activity in vitro. In agreement, HIV-1 TAT47-57 reduced HT1080 cell migration, thought to be mediated by proprotein convertases, by 25%. In addn., cyclic polyarginine peptides contg. hydrophobic moieties which have been previously used as transfection reagents also exhibited potent furin inhibition in vitro and also inhibited intracellular convertases. Our finding that cationic cell-penetrating peptides exert potent effects on cellular convertase activity should be taken into account when biol. effects are assessed.
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    15. 15
      Fittler, H.; Depp, A.; Avrutina, O.; Dahms, S. O.; Than, M. E.; Empting, M.; Kolmar, H. Engineering a Constrained Peptidic Scaffold towards Potent and Selective Furin Inhibitors. ChemBioChem 2015, 16, 2441– 2444,  DOI: 10.1002/cbic.201500447
      15
      Engineering a Constrained Peptidic Scaffold towards Potent and Selective Furin Inhibitors
      Fittler, Heiko; Depp, Alexander; Avrutina, Olga; Dahms, Sven O.; Than, Manuel E.; Empting, Martin; Kolmar, Harald
      ChemBioChem (2015), 16 (17), 2441-2444CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We report the engineering of the monocyclic sunflower trypsin inhibitor (SFTI-1[1,14]) into a potent furin inhibitor. In a rational approach, we converted the native scaffold of this trypsin-like serine protease inhibitor into a subtilisin-like one by substitutions in the canonical and, particularly, in the substrate-binding loop. Although the substrate sequence for furin is Arg-X-Arg/Lys-Arg↓, the most potent inhibitor had a lysine at position P1. C-terminally truncated versions demonstrated the strongest activity, thus suggesting a lack of interaction between this motif and the surface of furin. This observation was further supported by mol. modeling. With an inhibition const. of 0.49 nΜ, the engineered peptide H-KRCKKSIPPICF-NH2 is a promising compd. for further development of furin inhibitors aimed at controlling the activity of this protease in vitro and in vivo.
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    16. 16
      Becker, G. L.; Lu, Y.; Hardes, K.; Strehlow, B.; Levesque, C.; Lindberg, I.; Sandvig, K.; Bakowsky, U.; Day, R.; Garten, W.; Steinmetzer, T. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. J. Biol. Chem. 2012, 287, 21992– 22003,  DOI: 10.1074/jbc.M111.332643
      16
      Highly Potent Inhibitors of Proprotein Convertase Furin as Potential Drugs for Treatment of Infectious Diseases
      Becker, Gero L.; Lu, Yinghui; Hardes, Kornelia; Strehlow, Boris; Levesque, Christine; Lindberg, Iris; Sandvig, Kirsten; Bakowsky, Udo; Day, Robert; Garten, Wolfgang; Steinmetzer, Torsten
      Journal of Biological Chemistry (2012), 287 (26), 21992-22003CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compds. contg. an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with Ki values of 16 and 8 pm, resp. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compds. with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivs. were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.
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    17. 17
      Becker, G. L.; Sielaff, F.; Than, M. E.; Lindberg, I.; Routhier, S.; Day, R.; Lu, Y.; Garten, W.; Steinmetzer, T. Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics. J. Med. Chem. 2010, 53, 1067– 1075,  DOI: 10.1021/jm9012455
      17
      Potent Inhibitors of Furin and Furin-like Proprotein Convertases Containing Decarboxylated P1 Arginine Mimetics
      Becker, Gero L.; Sielaff, Frank; Than, Manuel E.; Lindberg, Iris; Routhier, Sophie; Day, Robert; Lu, Ying-Hui; Garten, Wolfgang; Steinmetzer, Torsten
      Journal of Medicinal Chemistry (2010), 53 (3), 1067-1075CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiol. and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analog peptidic inhibitors, we could identify highly potent furin inhibitors. The most potent compd., phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a Ki value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Mol. modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.
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    18. 18
      Hardes, K.; Becker, G. L.; Lu, Y.; Dahms, S. O.; Köhler, S.; Beyer, W.; Sandvig, K.; Yamamoto, H.; Lindberg, I.; Walz, L.; von Messling, V.; Than, M. E.; Garten, W.; Steinmetzer, T. Novel Furin Inhibitors with Potent Anti-infectious Activity. ChemMedChem 2015, 10, 1218– 1231,  DOI: 10.1002/cmdc.201500103
      18
      Novel Furin Inhibitors with Potent Anti-infectious Activity
      Hardes, Kornelia; Becker, Gero L.; Lu, Yinghui; Dahms, Sven O.; Koehler, Susanne; Beyer, Wolfgang; Sandvig, Kirsten; Yamamoto, Hiroyuki; Lindberg, Iris; Walz, Lisa; von Messling, Veronika; Than, Manuel E.; Garten, Wolfgang; Steinmetzer, Torsten
      ChemMedChem (2015), 10 (7), 1218-1231CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compd. 4-guanidinomethyl-phenylacteyl-Arg-Tle-Arg 4-amidinobenzylamide (MI-1148) inhibits furin with a Ki value of 5.5 pM. The derivs. also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was obsd. in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI-1148 was crystd. in complex with human furin. Its N-terminal guanidinomethyl group in the para position of the P5 Ph ring occupies the same position as that found previously for a structurally related inhibitor contg. this substitution in the meta position, thereby maintaining all of the important P5 interactions. The authors' results confirm that the inhibition of furin is a promising strategy for a short-term treatment of acute infectious diseases.
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    19. 19
      Ivanova, T.; Hardes, K.; Kallis, S.; Dahms, S. O.; Than, M. E.; Kunzel, S.; Böttcher-Friebertshäuser, E.; Lindberg, I.; Jiao, G. S.; Bartenschlager, R.; Steinmetzer, T. Optimization of Substrate-Analogue Furin Inhibitors. ChemMedChem 2017, 12, 1953– 1968,  DOI: 10.1002/cmdc.201700596
      19
      Optimization of Substrate-Analogue Furin Inhibitors
      Ivanova, Teodora; Hardes, Kornelia; Kallis, Stephanie; Dahms, Sven O.; Than, Manuel E.; Kuenzel, Sebastian; Boettcher-Friebertshaeuser, Eva; Lindberg, Iris; Jiao, Guan-Sheng; Bartenschlager, Ralf; Steinmetzer, Torsten
      ChemMedChem (2017), 12 (23), 1953-1968CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The proprotein convertase furin is a potential target for drug design, esp. for the inhibition of furin-dependent virus replication. All effective synthetic furin inhibitors identified thus far are multibasic compds.; the highest potency was found for our previously developed inhibitor 4-(guanidinomethyl)phenylacetyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148). An initial study in mice revealed a narrow therapeutic range for this tetrabasic compd., while significantly reduced toxicity was obsd. for some tribasic analogs. This suggests that the toxicity depends at least to some extent on the overall multibasic character of this inhibitor. Therefore, in a first approach, the C-terminal benzamidine of MI-1148 was replaced by less basic P1 residues. Despite decreased potency, a few compds. still inhibit furin in the low nanomolar range, but display negligible efficacy in cells. In a second approach, the P2 arginine was replaced by lysine; compared to MI-1148, this furin inhibitor has slightly decreased potency, but exhibits similar antiviral activity against West Nile and Dengue virus in cell culture and decreased toxicity in mice. These results provide a promising starting point for the development of efficacious and well-tolerated furin inhibitors.
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    20. 20
      Dahms, S. O.; Hardes, K.; Becker, G. L.; Steinmetzer, T.; Brandstetter, H.; Than, M. E. X-ray Structures of Human Furin in Complex with Competitive Inhibitors. ACS Chem. Biol. 2014, 9, 1113– 1118,  DOI: 10.1021/cb500087x
      20
      X-ray Structures of Human Furin in Complex with Competitive Inhibitors
      Dahms, Sven O.; Hardes, Kornelia; Becker, Gero L.; Steinmetzer, Torsten; Brandstetter, Hans; Than, Manuel E.
      ACS Chemical Biology (2014), 9 (5), 1113-1118CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Furin inhibitors are promising therapeutics for the treatment of cancer and numerous infections caused by bacteria and viruses, including the highly lethal Bacillus anthracis or the pandemic influenza virus. Development and improvement of inhibitors for pharmacol. use require a detailed knowledge of the protease's substrate and inhibitor binding properties. Here we present a novel prepn. of human furin and the first crystal structures of this enzyme in complex with noncovalent inhibitors. We show the inhibitor exchange by soaking, allowing the investigation of addnl. inhibitors and substrate analogs. Thus, our work provides a basis for the rational design of furin inhibitors.
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    21. 21
      Lam van, T. V.; Heindl, M. R.; Schlutt, C.; Böttcher-Friebertshäuser, E.; Bartenschlager, R.; Klebe, G.; Brandstetter, H.; Dahms, S. O.; Steinmetzer, T. The Basicity Makes the Difference: Improved Canavanine-Derived Inhibitors of the Proprotein Convertase Furin. ACS Med. Chem. Lett. 2021, 12, 426– 432,  DOI: 10.1021/acsmedchemlett.0c00651
      21
      The Basicity Makes the Difference: Improved Canavanine-Derived Inhibitors of the Proprotein Convertase Furin
      Lam van, Thuy Van; Heindl, Miriam Ruth; Schlutt, Christine; Boettcher-Friebertshaeuser, Eva; Bartenschlager, Ralf; Klebe, Gerhard; Brandstetter, Hans; Dahms, Sven O.; Steinmetzer, Torsten
      ACS Medicinal Chemistry Letters (2021), 12 (3), 426-432CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      A review. Furin activates numerous viral glycoproteins, and its inhibition prevents virus replication and spread. Through the replacement of arginine by the less basic canavanine, new inhibitors targeting furin in the trans-Golgi network were developed. These inhibitors exert potent antiviral activity in cell culture with much lower toxicity than arginine-derived analogs, most likely due to their reduced protonation in the blood circulation. Thus, despite its important physiol. functions, furin might be a suitable antiviral drug target.
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    22. 22
      Dahms, S. O.; Arciniega, M.; Steinmetzer, T.; Huber, R.; Than, M. E. Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 11196– 11201,  DOI: 10.1073/pnas.1613630113
      22
      Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism
      Dahms, Sven O.; Arciniega, Marcelino; Steinmetzer, Torsten; Huber, Robert; Than, Manuel E.
      Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (40), 11196-11201CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolemia, and infectious diseases. Novel protein crystallog. structures of the prototypical PC family member furin in different functional states were detd. to 1.8-2.0 Å. These, together with biochem. data and modeling by mol. dynamics calcns., suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the "off state," incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active "on state" seen in inhibitor-bound furin; and the resp. (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The mol. dynamics simulations of the four structural states reflect the exptl. observations in general and provide approxns. of the resp. stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and dets. the rotamer state of the oxyanion hole-forming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacol. intervention targeting furin.
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    23. 23
      Jiao, G. S.; Cregar, L.; Wang, J.; Millis, S. Z.; Tang, C.; O’Malley, S.; Johnson, A. T.; Sareth, S.; Larson, J.; Thomas, G. Synthetic small molecule furin inhibitors derived from 2,5-dideoxystreptamine. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 19707– 19712,  DOI: 10.1073/pnas.0606555104
      23
      Synthetic small molecule furin inhibitors derived from 2,5-dideoxystreptamine
      Jiao, Guan-Sheng; Cregar, Lynne; Wang, Jinzhi; Millis, Sherri Z.; Tang, Cho; O'Malley, Sean; Johnson, Alan T.; Sareth, Sina; Larson, Jason; Thomas, Gary
      Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (52), 19707-19712CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small mol. furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addn. of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochem. cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Mol. modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH2Cl. The compds. presented here are bona fide synthetic small mol. furin inhibitors that exhibit potency in the nano-molar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furin-dependent diseases.
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    24. 24
      Komiyama, T.; Coppola, J. M.; Larsen, M. J.; van Dort, M. E.; Ross, B. D.; Day, R.; Rehemtulla, A.; Fuller, R. S. Inhibition of furin/proprotein convertase-catalyzed surface and intracellular processing by small molecules. J. Biol. Chem. 2009, 284, 15729– 15738,  DOI: 10.1074/jbc.M901540200
      24
      Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules
      Komiyama, Tomoko; Coppola, Julia M.; Larsen, Martha J.; van Dort, Marcian E.; Ross, Brian D.; Day, Robert; Rehemtulla, Alnawaz; Fuller, Robert S.
      Journal of Biological Chemistry (2009), 284 (23), 15729-15738CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Furin is a ubiquitously expressed proprotein convertase (PC) that plays a vital role in numerous disease processes including cancer metastasis, bacterial toxin activation (e.g. anthrax and Pseudomonas), and viral propagation (e.g. avian influenza and human immunodeficiency virus). To identify small mol. inhibitors of furin and related processing enzymes, we performed high-throughput screens of chem. diversity libraries utilizing both enzyme-based and cell-based assays. The screens identified partially overlapping sets of compds. that were further characterized for affinity, mechanism, and efficacy in addnl. cellular processing assays. Dicoumarols were identified as a class of compds. that inhibited furin non-competitively and reversibly with Ki values in the micromolar range. These compds. inhibited furin/furin-like activity both at the cell surface (protecting against anthrax toxin) and in the secretory pathway (blocking processing of the metastasis factor membrane-type 1 matrix metalloproteinase/MT1-MMP) at concns. close to Ki values. Compds. tested exhibited distinct patterns of inhibition of other furin-family PCs (rat PACE4, human PC5/6 and human PC7), showing that dicoumarol derivs. might be developed as either generic or selective inhibitors of the PCs. The extensive clin. use, high bioavailability and relatively low toxicity of dicoumarols suggests that the dicoumarol structure will be a good starting point for development of drug-like inhibitors of furin and other PCs that can act both intracellularly and at the cell surface.
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    25. 25
      Ramos-Molina, B.; Lick, A. N.; Blanco, E. H.; Posada-Salgado, J. A.; Martinez-Mayorga, K.; Johnson, A. T.; Jiao, G. S.; Lindberg, I. Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays. Biochem. Pharmacol. 2015, 96, 107– 118,  DOI: 10.1016/j.bcp.2015.05.008
      25
      Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays
      Ramos-Molina, Bruno; Lick, Adam N.; Blanco, Elias H.; Posada-Salgado, J. Alejandro; Martinez-Mayorga, Karina; Johnson, Alan T.; Jiao, Guan-Sheng; Lindberg, Iris
      Biochemical Pharmacology (Amsterdam, Netherlands) (2015), 96 (2), 107-118CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
      The proprotein convertase furin is implicated in a variety of pathogenic processes such as bacterial toxin activation, viral propagation, and cancer. Several groups have identified non-peptide compds. with high inhibitory potency against furin in vitro, although their efficacy in various cell-based assays is largely unknown. In this study certain guanidinylated 2,5-dideoxystreptamine derivs. exhibit interesting ex vivo properties. Compd. 1b (1,1'-(4-((2,4-diguanidino-5-(4-guanidinophenoxy)cyclohexyl)oxy)-1,3-phenylene)diguanidine) is a potent and cell-permeable inhibitor of cellular furin, since it was able to retard tumor cell migration, block release of a Golgi reporter, and protect cells against Bacillus anthracis (anthrax) and Pseudomonas aeruginosa intoxication, with no evident cell toxicity. Other compds. based on the 2,5-dideoxystreptamine scaffold, such as compd. 1g (1,1'-(4,6-bis(4-guanidinophenoxy)cyclohexane-1,3-diyl)diguanidine) also efficiently protected cells against anthrax, but displayed only moderate protection against Pseudomonas exotoxin A and did not inhibit cell migration, suggesting poor cell permeability. Certain bis-guanidinophenyl ether derivs. such as 2f (1,3-bis(2,4-diguanidinophenoxy) benzene) exhibited micromolar potency against furin in vitro, low cell toxicity, and highly efficient protection against anthrax toxin; this compd. only slightly inhibited intracellular furin. Thus, compds. 1g and 2f both represent potent furin inhibitors at the cell surface with low intracellular inhibitory action, and these particular compds. might therefore be of preferred therapeutic interest in the treatment of certain bacterial and viral infections.
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    26. 26
      Sielaff, F.; Than, M. E.; Bevec, D.; Lindberg, I.; Steinmetzer, T. New furin inhibitors based on weakly basic amidinohydrazones. Bioorg. Med. Chem. Lett. 2011, 21, 836– 840,  DOI: 10.1016/j.bmcl.2010.11.092
      26
      New furin inhibitors based on weakly basic amidinohydrazones
      Sielaff, Frank; Than, Manuel E.; Bevec, Dorian; Lindberg, Iris; Steinmetzer, Torsten
      Bioorganic & Medicinal Chemistry Letters (2011), 21 (2), 836-840CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
      A novel series of amidinohydrazone-derived furin inhibitors was prepd.; the most potent compds. I and II inhibit furin with Ki values of 0.46 and 0.59 μM, resp. In contrast to inhibitor I, which still contains a guanidino residue, compd. II possesses only weakly basic amidinohydrazone groups.
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    27. 27
      Essalmani, R.; Jain, J.; Susan-Resiga, D.; Andréo, U.; Evagelidis, A.; Derbali, R. M.; Huynh, D. N.; Dallaire, F.; Laporte, M.; Delpal, A.; Sutto-Ortiz, P.; Coutard, B.; Mapa, C.; Wilcoxen, K.; Decroly, E.; Pham, T. N. Q.; Cohen, E. A.; Seidah, N. G. Implications of Spike-glycoprotein processing at S1/S2 by Furin, at S2’ by Furin and/or TMPRSS2 and shedding of ACE2: cell-to-cell fusion, cell entry and infectivity of SARS-CoV-2. bioRxiv 2020.
      There is no corresponding record for this reference.
    28. 28
      Dahms, S. O.; Jiao, G. S.; Than, M. E. Structural Studies Revealed Active Site Distortions of Human Furin by a Small Molecule Inhibitor. ACS Chem. Biol. 2017, 12, 1211– 1216,  DOI: 10.1021/acschembio.6b01110
      28
      Structural Studies Revealed Active Site Distortions of Human Furin by a Small Molecule Inhibitor
      Dahms, Sven O.; Jiao, Guan-Sheng; Than, Manuel E.
      ACS Chemical Biology (2017), 12 (5), 1211-1216CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of several pathologies including cancer, atherosclerosis, hypercholesterolemia, and infectious diseases. Here we present the first exptl. complex of furin with a non substrate-like small mol. inhibitor, and the X-ray structure of the enzyme complexed to the small mol. inhibitor I at 1.9 Å resoln. Two mols. of inhibitor 1 were found to interact with furin. One is anchored at the S4 pocket of the enzyme and interferes directly with the conformation and function of the catalytic triade; the other mol. shows weaker binding and interacts with a distant, less conserved region of furin. The obsd. binding modes represent a new inhibition strategy of furin and imply the possibility to attain specificity among the PCs providing an innovative starting point of structure guided inhibitor development for furin.
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    29. 29
      Soll, R. M.; Lu, T.; Tomczuk, B.; Illig, C. R.; Fedde, C.; Eisennagel, S.; Bone, R.; Murphy, L.; Spurlino, J.; Salemme, F. R. Amidinohydrazones as guanidine bioisosteres: application to a new class of potent, selective and orally bioavailable, non-amide-based small-molecule thrombin inhibitors. Bioorg. Med. Chem. Lett. 2000, 10, 1– 4,  DOI: 10.1016/S0960-894X(99)00632-0
      29
      Amidinohydrazones as guanidine bioisosteres: application to a new class of potent, selective and orally bioavailable, non-amide-based small-molecule thrombin inhibitors
      Soll, Richard M.; Lu, Tianobao; Tomczuk, Bruce; Illig, Carl R.; Fedde, Cynthia; Eisennagel, Stephen; Bone, Roger; Murphy, Larry; Spurlino, John; Salemme, F. Raymond
      Bioorganic & Medicinal Chemistry Letters (2000), 10 (1), 1-4CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
      We describe a new class of potent, non-amide-based small mol. thrombin inhibitors in which an amidinohydrazone is used as a guanidine bioisostere on a non-peptide scaffold. Compd. 4 exhibits nM inhibition of thrombin, is selective for thrombin, and shows 60 and 23% bioavailability in rabbits and dogs, resp. Crystallog. anal. of 4 bound to thrombin confirmed the amindinohydrazone binding mode.
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    30. 30
      Specht, S.; Sarite, S. R.; Hauber, I.; Hauber, J.; Gorbig, U. F.; Meier, C.; Bevec, D.; Hoerauf, A.; Kaiser, A. The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase. Parasitol. Res. 2008, 102, 1177– 1184,  DOI: 10.1007/s00436-008-0891-x
      30
      The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase
      Specht Sabine; Sarite Salem Ramadan; Hauber Ilona; Hauber Joachim; Gorbig Ulf F; Meier Chris; Bevec Dorian; Hoerauf Achim; Kaiser Annette
      Parasitology research (2008), 102 (6), 1177-84 ISSN:0932-0113.
      Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.
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    31. 31
      Henrich, S.; Lindberg, I.; Bode, W.; Than, M. E. Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity. J. Mol. Biol. 2005, 345, 211– 227,  DOI: 10.1016/j.jmb.2004.10.050
      31
      Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity
      Henrich Stefan; Lindberg Iris; Bode Wolfram; Than Manuel E
      Journal of molecular biology (2005), 345 (2), 211-27 ISSN:0022-2836.
      In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs). These PCs cleave their protein substrates very specifically following multiple basic residues. The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain. The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern.
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    32. 32
      Dahms, S. O.; Hardes, K.; Steinmetzer, T.; Than, M. E. X-ray structures of the proprotein convertase furin bound with substrate analog inhibitors reveal substrate specificity determinants beyond the S4 pocket. Biochemistry 2018, 57, 925,  DOI: 10.1021/acs.biochem.7b01124
      32
      X-ray Structures of the Proprotein Convertase Furin Bound with Substrate Analogue Inhibitors Reveal Substrate Specificity Determinants beyond the S4 Pocket
      Dahms, Sven O.; Hardes, Kornelia; Steinmetzer, Torsten; Than, Manuel E.
      Biochemistry (2018), 57 (6), 925-934CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The proprotein convertase furin is a highly specific serine protease modifying and thereby activating proteins in the secretory pathway by proteolytic cleavage. Its substrates are involved in many diseases, including cancer and infections caused by bacteria and viruses. Understanding furin's substrate specificity is crucially important for the development of pharmacol. applicable inhibitors. Using protein X-ray crystallog., we investigated the extended substrate binding site of furin in complex with three peptide-derived inhibitors at up to 1.9 Å resoln. The structure of the protease bound with a hexapeptide inhibitor revealed mol. details of its S6 pocket, which remained completely unknown so far. The arginine residue at P6 induced an unexpected turnlike conformation of the inhibitor backbone, which is stabilized by intra- and intermol. H-bonds. In addn., we confirmed the binding of arginine to the previously proposed S5 pocket (S51). An alternative S5 site (S52) could be utilized by shorter side chains as demonstrated for a 4-aminomethyl-phenylacetyl residue, which shows steric properties similar to those of a lysine side chain. Interestingly, we also obsd. binding of a peptide with citrulline at P4 substituting for the highly conserved arginine. The structural data might indicate an unusual protonation state of Asp264 maintaining the interaction with uncharged citrulline. The herein identified mol. interaction sites at P5 and P6 can be utilized to improve next-generation furin inhibitors. Our data will also help to predict furin substrates more precisely on the basis of the addnl. specificity determinants obsd. for P5 and P6.
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    33. 33
      Mueller, U.; Darowski, N.; Fuchs, M. R.; Forster, R.; Hellmig, M.; Paithankar, K. S.; Puhringer, S.; Steffien, M.; Zocher, G.; Weiss, M. S. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. J. Synchrotron Radiat. 2012, 19, 442– 449,  DOI: 10.1107/S0909049512006395
      33
      Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin
      Mueller, Uwe; Darowski, Nora; Fuchs, Martin R.; Foerster, Ronald; Hellmig, Michael; Paithankar, Karthik S.; Puehringer, Sandra; Steffien, Michael; Zocher, Georg; Weiss, Manfred S.
      Journal of Synchrotron Radiation (2012), 19 (3), 442-449CODEN: JSYRES; ISSN:0909-0495. (International Union of Crystallography)
      Three macromol. crystallog. (MX) beamlines at the Helmholtz-Zentrum Berlin (HZB) are available for the regional, national and international structural biol. user community. The state-of-the-art synchrotron beamlines for MX BL14.1, BL14.2 and BL14.3 are located within the low-β section of the BESSY II electron storage ring. All beamlines are fed from a superconducting 7 T wavelength-shifter insertion device. BL14.1 and BL14.2 are energy tunable in the range 5-16 keV, while BL14.3 is a fixed-energy side station operated at 13.8 keV. All three beamlines are equipped with CCD detectors. BL14.1 and BL14.2 are in regular user operation providing about 200 beam days per yr and about 600 user shifts to approx. 50 research groups across Europe. BL14.3 has initially been used as a test facility and was brought into regular user mode operation during the year 2010. BL14.1 has recently been upgraded with a microdiffractometer including a mini-κ goniometer and an automated sample changer. Addnl. user facilities include office space adjacent to the beamlines, a sample prepn. lab., a biol. lab. (safety level 1) and high-end computing resources. In this article the instrumentation of the beamlines is described, and a summary of the exptl. possibilities of the beamlines and the provided ancillary equipment for the user community is given.
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    34. 34
      Kabsch, W. Xds. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132,  DOI: 10.1107/S0907444909047337
      34
      Software XDS for image rotation, recognition and crystal symmetry assignment
      Kabsch, Wolfgang
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
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    35. 35
      Krug, M.; Weiss, M. S.; Heinemann, U.; Mueller, U. XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS. J. Appl. Crystallogr. 2012, 45, 568– 572,  DOI: 10.1107/S0021889812011715
      35
      XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS
      Krug, Michael; Weiss, Manfred S.; Heinemann, Udo; Mueller, Uwe
      Journal of Applied Crystallography (2012), 45 (3), 568-572CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
      XDSAPP is a Tcl/Tk-based graphical user interface for the easy and convenient processing of diffraction data sets using XDS. It provides easy access to all XDS functionalities, automates the data processing and generates graphical plots of various data set statistics provided by XDS. By incorporating addnl. software, further information on certain features of the data set, such as radiation decay during data collection or the presence of pseudo-translational symmetry and/or twinning, can be obtained. Intensity files suitable for CCP4, CNS and SHELX are generated.
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    36. 36
      Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S. Overview of the CCP4 suite and current developments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 235– 242,  DOI: 10.1107/S0907444910045749
      36
      Overview of the CCP4 suite and current developments
      Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.; Dodson, Eleanor J.; Emsley, Paul; Evans, Phil R.; Keegan, Ronan M.; Krissinel, Eugene B.; Leslie, Andrew G. W.; McCoy, Airlie; McNicholas, Stuart J.; Murshudov, Garib N.; Pannu, Navraj S.; Potterton, Elizabeth A.; Powell, Harold R.; Read, Randy J.; Vagin, Alexei; Wilson, Keith S.
      Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 235-242CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      A review. The CCP4 (Collaborative Computational Project, No. 4) software suite is a collection of programs and assocd. data and software libraries which can be used for macromol. structure detn. by X-ray crystallog. The suite is designed to be flexible, allowing users a no. of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by std. file formats, data objects and graphical interfaces. Structure soln. by macromol. crystallog. is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.
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    37. 37
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486– 501,  DOI: 10.1107/S0907444910007493
      37
      Features and development of Coot
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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    38. 38
      Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213– 221,  DOI: 10.1107/S0907444909052925
      38
      PHENIX: a comprehensive Python-based system for macromolecular structure solution
      Adams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
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    39. 39
      Schuttelkopf, A. W.; van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 1355– 1363,  DOI: 10.1107/S0907444904011679
      39
      PRODRG: a tool for high-throughput crystallography of protein-ligand complexes
      Schuttelkopf Alexander W; van Aalten Daan M F
      Acta crystallographica. Section D, Biological crystallography (2004), 60 (Pt 8), 1355-63 ISSN:0907-4449.
      The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray refinement of protein-ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force field used, structures with good geometries are generated. X-ray refinement in X-PLOR/CNS, REFMAC and SHELX using PRODRG-generated topologies produces results comparable to refinement with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.
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    40. 40
      Györgydeák, Z.; Holzer, W.; Mereiter, K. Guanylhydrazones of (Hetero)Aryl Methyl Ketones: Structure and Reaction with Acetic Anhydride. Monatsh. Chem. 1999, 130, 899– 913,  DOI: 10.1007/PL00010271
      40
      Guanylhydrazones of (hetero)aryl methyl ketones. Structure and reaction with acetic anhydride
      Gyorgydeak, Zoltan; Holzer, Wolfgang; Mereiter, Kurt
      Monatshefte fuer Chemie (1999), 130 (7), 899-913CODEN: MOCMB7; ISSN:0026-9247. (Springer-Verlag Wien)
      The synthesis of guanylhydrazones of (hetero)aryl Me ketones is described. Successive reaction with hot Ac2O leads to the corresponding N,N'-diacetyl derivs. Structural assignments of all novel compds. and those of some already known congeners were achieved by NMR (1H, 13C) and x-ray structure anal.
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    41. 41
      The UniProt Consortium UniProt: a hub for protein information. Nucleic Acids Res. 2015, 43, D204– D212,  DOI: 10.1093/nar/gku989
      There is no corresponding record for this reference.
    42. 42
      Li, W.; Cowley, A.; Uludag, M.; Gur, T.; McWilliam, H.; Squizzato, S.; Park, Y. M.; Buso, N.; Lopez, R. The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Res. 2015, 43, W580– 584,  DOI: 10.1093/nar/gkv279
      42
      The EMBL-EBI bioinformatics web and programmatic tools framework
      Li, Weizhong; Cowley, Andrew; Uludag, Mahmut; Gur, Tamer; McWilliam, Hamish; Squizzato, Silvano; Park, Young Mi; Buso, Nicola; Lopez, Rodrigo
      Nucleic Acids Research (2015), 43 (W1), W580-W584CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      A review. Since 2009 the EMBL-EBI Job Dispatcher framework has provided free access to a range of mainstream sequence anal. applications. These include sequence similarity search services such as BLAST, FASTA and PSI-Search, multiple sequence alignment tools such as Clustal Omega, MAFFT and T-Coffee, and other sequence anal. tools such as InterProScan. Through these services users can search mainstream sequence databases such as ENA, UniProt and Ensembl Genomes, utilizing a uniform web interface or systematically through Web Services interfaces using common programming languages, and obtain enriched results with novel visualizations. Integration with EBI Search and the dbfetch retrieval service further expands the usefulness of the framework. New tools and updates such as NCBI BLAST+, InterProScan 5 and PfamScan, new categories such as RNA anal. tools, new databases such as ENA non-coding, WormBase ParaSite, Pfam and Rfam, and new workflow methods, together with the retirement of depreciated services, ensure that the framework remains relevant to today's biol. community.
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    43. 43
      Bond, C. S.; Schuttelkopf, A. W. ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 510– 512,  DOI: 10.1107/S0907444909007835
      43
      ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments
      Bond, Charles Simon; Schuettelkopf, Alexander Wolfgang
      Acta Crystallographica, Section D: Biological Crystallography (2009), 65 (5), 510-512CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Marked-up sequence alignments typically provide the central figure in articles describing proteins, whether in the fields of biochem., bioinformatics or structural biol. The generation of these figures is often unwieldy: interactive programs are often aesthetically limited and the use of batch programs requires the repetitive iterative editing of scripts. ALINE is a portable interactive graphical sequence-alignment editor implemented in Perl/Tk which produces publication-quality sequence-alignment figures where 'what you see is what you get'. ALINE is freely available for download from http://crystal.bcs.uwa.edu.au/px/charlie/software/aline/.
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    44. 44
      Karplus, P. A.; Diederichs, K. Linking crystallographic model and data quality. Science 2012, 336, 1030– 1033,  DOI: 10.1126/science.1218231
      44
      Linking Crystallographic Model and Data Quality
      Karplus, P. Andrew; Diederichs, Kay
      Science (Washington, DC, United States) (2012), 336 (6084), 1030-1033CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      In macromol. x-ray crystallog., refinement R values measure the agreement between obsd. and calcd. data. Analogously, Rmerge values reporting on the agreement between multiple measurements of a given reflection are used to assess data quality. Here, we show that despite their widespread use, Rmerge values are poorly suited for detg. the high-resoln. limit and that current std. protocols discard much useful data. We introduce a statistic that ests. the correlation of an obsd. data set with the underlying (not measurable) true signal; this quantity, CC*, provides a single statistically valid guide for deciding which data are useful. CC* also can be used to assess model and data quality on the same scale, and this reveals when data quality is limiting model improvement.
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  • Supporting Information

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