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Structural determinants of affinity and selectivity in the binding of inhibitors to histone deacetylase 6
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Digest
Structural determinants of affinity and selectivity in the binding of inhibitors to histone deacetylase 6 Jeremy D. Osko, David W. Christianson
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Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, United States
ARTICLE INFO
ABSTRACT
Keywords: Enzyme Inhibitor X-ray crystallography
Histone deacetylase 6 (HDAC6) is associated with multiple neurological disorders as well as aggressive cancers, making its selective inhibition highly desirable for therapeutic purposes. The basic molecular design of an effective HDAC6 inhibitor consists of a zinc-binding group, a linker, and a capping group capable of making interactions at the mouth of the active site. To date, more than 50 high-resolution X-ray crystal structures of HDAC6-inhibitor complexes have been reported, many of which reveal intermolecular interactions that contribute to isozyme affinity and selectivity. Here, we review the key features of HDAC6 inhibitor design illuminated by these structural studies.
X-ray crystallography is a powerful tool that provides a direct, atomic-resolution view of proteins and their complexes with small molecules. Such structural studies can inform and accelerate the discovery of new chemical entities that regulate protein function, a critical goal of medicinal chemistry. In this Digest, we focus on X-ray crystallographic studies of the tubulin deacetylase domain of histone deacetylase 6 (HDAC6; Fig. 1a)1,2 and its complexes with small molecule inhibitors. Inhibitors of HDAC6 are generally tripartite in design (Fig. 1b), consisting of a zinc-binding group that coordinates to the catalytic zinc ion, a flexible or rigid molecular linker, and a capping group capable of making interactions in the outer active site cleft. Since the crystal structures of human and zebrafish HDAC6 catalytic domains were first reported,3,4 more than 50 X-ray crystal structures of HDAC6inhibitor complexes have been determined and deposited in the Protein Data Bank (www.rcsb.org).5–13 These structures provide a wealth of information regarding the molecular basis of effective inhibition. Four classes of zinc-dependent HDAC isozymes are found in humans: class I (HDACs 1, 2, 3 and 8), class IIa (HDACs 4, 5, 7, and 9), class IIb (HDACs 6 and 10), and class IV (HDAC 11).14 These HDACs adopt the characteristic α/β arginase-deacetylase fold with general conservation of active site residues required for the chemistry of deacetylation.15,16 Most HDAC isozymes catalyze the hydrolysis of acetyllysine-containing protein and peptide substrates to generate products lysine and acetate.17–20 However, there are exceptions: HDAC10 is a polyamine deacetylase,21 and HDAC11 is a lysine fatty acid deacylase,22,23 an activity that has also been observed with HDAC8.24 Focusing solely on the chemistry and biology of lysine
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deacetylation, it is notable that there are approximately 40,000 unique protein acetylation sites identified to date (the “acetylome”) according to the PhosphoSite Plus database.25 Precise regulation of protein acetylation levels is implicated in numerous biological processes such as epigenetics,26,27 metabolism,28,29 and cell signaling.30 Moreover, aberrant regulation of protein acetylation is associated with neurodegenerative disease and cancer.31,32 The HDAC inhibitors Vorinostat (suberanilohydroxamic acid, SAHA), Belinostat, Panobinostat, and Romidepsin have been developed and approved for clinical use in humans.33 However, these inhibitors do not exhibit appreciable selectivity for any particular HDAC isozyme, and as such their use can lead to unwanted off-target effects.34–38 Due to their generally broad activity against all zinc-dependent HDAC isozymes, these inhibitors are often referred to as “pan-HDAC” inhibitors. Structure, function, and inhibition of HDAC6 With 1215 amino acids, human HDAC6 (Uniprot Q9UBN7) is the largest member of the zinc-dependent HDAC family.1,2 HDAC6 is additionally unique in that it contains two catalytic domains, designated CD1 and CD2 (Fig. 1a).39,40 HDAC6 CD2 is the tubulin deacetylase, whereas the biological function of HDAC6 CD1 is less well understood. Enzymological measurements using purified protein constructs corresponding to CD1 and CD2 reveal that CD2 exhibits broad substrate specificity, whereas CD1 exhibits much narrower specificity for peptide substrates bearing C-terminal acetyllysine residues.3 A more recent study utilizing full-length human HDAC6 in acetylome peptide
Corresponding author. E-mail address: [email protected] (D.W. Christianson).
https://doi.org/10.1016/j.bmcl.2020.127023 Received 20 December 2019; Received in revised form 7 February 2020; Accepted 8 February 2020 0960-894X/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Jeremy D. Osko and David W. Christianson, Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2020.127023
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In recent years, X-ray crystallographic studies of HDAC6 CD2 complexed with pan-HDAC inhibitors and isozyme-selective inhibitors have illuminated structural aspects of affinity and isozyme selectivity. The general contributions of the principle components of the tripartite inhibitors – the zinc-binding group, the linker, and the capping group – are now outlined as observed in recent X-ray crystallographic studies of complexes with HDAC6 CD2. All structures discussed below are deposited in the Protein Data Bank (www.rcsb.org) and are publicly available. For reference, molecular structures of selected inhibitors discussed in this Digest are shown in Table 1. Zinc-binding groups X-ray crystallographic studies have yielded key structural insights on the catalytic mechanism of HDAC6 CD2 (Fig. 3), and these insights include the coordination chemistry of the catalytic zinc ion. X-ray crystal structures of Y745F and H574A HDAC6 CD2 complexed with an acetyllysine substrate reveal that the scissile amide carbonyl coordinates to the catalytic zinc ion and accepts a hydrogen bond from Y745.3 Together, these interactions polarize and activate the carbonyl group for nucleophilic attack by a zinc-bound water molecule to yield a tetrahedral intermediate that collapses to yield products lysine and acetate. Tandem histidine residues H573 and H574 function as general base-general acid catalysts in this chemical mechanism, as shown in Fig. 3.3 These structural-mechanistic studies show that the catalytic zinc ion has two possible coordination sites for nonprotein ligands, as represented by the coordination sites of the substrate carbonyl and the zinc-bound water molecule. Ketone-based substrate analogues such as HC Toxin or 7-[(3-aminopropyl)amino]-1,1,1-trifluoroheptan-2-one (AAT) undergo a chemical reaction upon binding to HDAC6 CD2 that mimics the first step of the catalytic mechanism.3 If the ketone carbonyl is isosteric with the scissile carbonyl of the acetyllysine substrate, the ketone undergoes nucleophilic attack by a water molecule to yield a tetrahedral gemdiolate (Fig. 4a) that mimics the actual tetrahedral intermediate in catalysis (Fig. 3c).3 Thus, the binding of these reactive substrate analogues reflects the preferential binding of the tetrahedral transition state-like structure rather than the planar ketone substrate-like structure. This is a common feature observed for the binding of ketone-based inhibitors to HDAC4,43 HDAC8,44 and HDAC10.45 This hydration reaction was first observed in the binding of ketone- and aldehyde-based substrate analogues to the zinc enzyme carboxypeptidase A – regardless of whether or not the carbonyl group is activated by an adjacent trifluoromethyl group, the inhibitor was always observed to bind as the tetrahedral gem-diol(ate).46–48 Since an unactivated ketone exists to less than 0.2% gem-diol in aqueous solution, the measured Kd or IC50 values for the actual inhibiting species can be ~500 fold lower than the measured value, taking into account the low concentration of the inhibiting species in aqueous solution. Hydroxamic acids are the zinc-binding groups most often utilized in HDAC6 CD2 inhibitors. Trichostatin A (TSA) is a natural product bearing a hydroxamic acid moiety that is a pan-HDAC inhibitor. The first HDAC structure determined in complex with TSA was that of HDAC8.49 Suberanilohydroxamic acid (SAHA) is another pan-HDAC inhibitor, currently in use as the cancer chemotherapy drug Vorinostat. The first human HDAC structure determined in complex with SAHA was that of HDAC8.49 The crystal structures of HDAC6 complexes with these pan-HDAC inhibitors were recently reported.3,4 A hydroxamic acid zinc-binding group ionizes to form a negativelycharged hydroxamate upon binding to the enzyme. Hydroxamate-zinc coordination is often bidentate, with zinc chelation by the hydroxamate C]O and NeO– groups (Fig. 4b). This coordination geometry also enables hydrogen bond formation with tandem histidine residues, such that the zinc-bound oxyanion of the hydroxamate NeO– group accepts a hydrogen bond from H573 and the hydroxamate NH group donates a hydrogen bond to H574. The hydroxamate carbonyl group additionally
Fig. 1. (a) Crystal structure of the di-domain construct of zebrafish HDAC6 (PDB 5G0J) in which catalytic domain 1 (CD1, tan) and catalytic domain 2 (CD2, blue) are connected by a 23-residue linker (red). Catalytic zinc ions appear as gray spheres. (b) The general structure of an HDAC inhibitor is tripartite, consisting of a zinc-binding group, a linker, and a capping group that typically occupies the L1 pocket at the mouth of the active site. Inhibitors with bifurcated capping groups can occupy both the L1 and L2 pockets at the mouth of the active site.
microarrays indicates that internal acetyllysine residues in certain peptide sequences can be hydrolyzed by full-length human HDAC6 CD1,41 and recent measurements with zebrafish HDAC6 CD1 reveal weak catalytic activity against peptide substrates containing internal acetyllysine residues flanked by smaller residues.42 The recently-reported X-ray crystal structures of human HDAC6 CD2 from Homo sapiens (human),3 and HDAC6 CD1 and CD2 from Danio rerio (zebrafish),3,4 reveal that the active site contour of HDAC6 CD2 exhibits certain structural differences in comparison with other HDAC isozymes of known structure, indicating significant potential for the structure-based design of HDAC6-selective inhibitors. Comparison of human and zebrafish HDAC6 CD2 structures reveals very similar active sites and similar binding modes for the natural product inhibitor Trichostatin A (Fig. 2).3 Because the zebrafish enzyme is much more amenable to the generation of crystalline enzyme-inhibitor complexes that diffract X-rays to high resolution, zebrafish HDAC6 CD2 is most often used for structural studies aimed at understanding inhibitor structure-affinity and isozyme selectivity relationships. Selectivity for HDAC6 inhibition is most often desired and defined relative to the class I isozymes, HDACs 1–3 and 8. In contrast with pan-HDAC inhibitors, HDAC6 CD2-selective inhibitors can exhibit improved pharmacokinetic properties and fewer off-target effects. The first crystal structures of HDAC6 CD1 complexes with panHDAC inhibitors have only recently been reported.3,42 Slight differences in the active sites of HDAC6 CD1 and CD2 were noted when their crystal structures were first reported,3 suggesting the possibility of selective inhibition of either catalytic domain. A key difference in the active site of HDAC6 CD1 is K330, which corresponds to a leucine residue in HDAC6 CD2. The positively charged side chain of K330 is thought to confer specificity for the negatively charged carboxylate of a C-terminal acetyllysine residue in a peptide substrate.3 Since K330 can interact with bound inhibitors,3,42 interactions with this residue could be exploited by a selective inhibitor. 2
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Fig. 2. (a) The crystal structure of human HDAC6 CD2 (yellow) was determined as a fusion with maltose binding protein (MBP, cyan) (PDB 5EDU). The helix-loop linker (red) at the interface of the two proteins was engineered to optimize their association. Maltose is bound to MBP, and Trichostatin A (TSA) is bound in the active site of human HDAC6 CD2 (Zn2+ = gray sphere, K+ = purple spheres). (b) Superposition of the human HDAC6 CD2-TSA complex (wheat; orange TSA; PDB 5EDU) and the zebrafish HDAC6 CD2-TSA complex (light blue; blue TSA; PDB 5EEK). The Zn2+ ion and K+ ions in the human enzyme are white and purple spheres, respectively; the corresponding metal ions in the zebrafish enzyme are blue spheres. Only two active site residues differ between the two enzymes: N530 and N645 at the mouth of the active site of zebrafish HDAC6 CD2 appear as D567 and M682 in human HDAC6 CD2. (c) Comparison of human HDAC6 CD2 (left) and zebrafish HDAC6 CD2 (right), color-coded by electrostatic surface potential. Bound TSA molecules are represented in ball-and-stick fashion. Reprinted with permission from ref. 3.
Table 1 Selected inhibitors of HDAC6 CD2. (See below-mentioned references for further information.).
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Fig. 3. Snapshots of catalysis by HDAC6 CD2 captured in X-ray crystallographic studies. The active site of the unliganded enzyme in (a) reveals a nucleophilic Zn2+-bound water molecule hydrogen bonded with H573 and H574; a vacant coordination site on the Zn2+ ion is occupied by the scissile carbonyl of acetyllysine in the enzyme-substrate complex in (b). The substrate cannot react in this complex due to the Y745F substitution. In (c), the substrate carbonyl is fully activated and undergoes nucleophilic attack by the Zn2+-bound water molecule to yield a tetrahedral intermediate, but this intermediate cannot collapse due to the loss of the required proton donor through the H574A substitution. The oxyanion of this intermediate is stabilized by Zn2+ and Y745. Collapse of the tetrahedral intermediate yields products lysine and acetate, and the acetate complex in (d) represents a product complex following lysine dissociation. Reprinted with permission from ref. 3.
hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB)3,50 and has since been observed for other inhibitors such as ACY-1083.5 The monodentate coordination mode is only 0.5 kcal/mol less stable than the bidentate coordination mode, based on the 30:70 mixture of binding conformations observed for Trichostatin A in the ultrahigh resolution crystal structure of its complex with HDAC6 CD2.5 Either of the two coordination modes for the hydroxamate zinc-binding group may be favored based on the overall steric demands of the inhibitor in the active site. A third zinc-binding mode is feasible for a hydroxamate moiety, in which metal coordination is achieved by an ionized hydroxamate NH
accepts a hydrogen bond from Y745. This constellation of interactions is well-suited for the hydroxamate moiety and likely contributes to the high affinity of hydroxamate-bearing inhibitors. An alternative hydroxamate-zinc binding mode is sometimes observed that involves monodentate zinc coordination through the hydroxamate NeO– group, which also accepts a hydrogen bond from Y745 (Fig. 4c). The hydroxamate C]O group accepts a hydrogen bond from the zinc-bound water molecule, which is not displaced by inhibitor binding and retains its hydrogen bond interactions with H573 and H574. This monodentate hydroxamate-zinc binding mode was first observed in the HDAC6 CD2 complex with N-hydroxy-4-(2-[(24
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Fig. 4. Overlays of HDAC6 CD2-inhibitor complexes: (a) zinc-bound gem-diolates (PDB accession codes 5EFH, 5EFJ, 5EFN); bidentate hydroxamates (PDB accession codes 5EDU, 5EEI, 5EEK, 5EEM, 5EEN, 5EF8, 5EFB, 5G0H, 5WGL, 6CSP, 6CSQ, 6CSR, 6CSS, 6DVO, 6R0K, 6PZR, 6Q0Z, 6PZO, 6PZU; (c) monodentate hydroxamates (PDB accession codes 5EF7, 5W5K, 5WGK, 5WGM, 6CGP, 6CW8, 6DVL, 6DVM, 6DVN, 6PZO and 6PZS); zinc-bound thiolate (PDB accession code 6MR5).
group as observed for the binding of methylhydroxamic acid and trifluoromethylhydroxamic acid to human carbonic anhydrase II.51 However, this binding mode is not observed for hydroxamate binding to HDAC6. Despite the predominance of hydroxamate zinc-binding groups in HDAC6 inhibitors, the hydroxamate moiety is not particularly ideal for incorporation into a drug molecule due to its susceptibility to degradation. Hydroxamates can undergo Lossen rearrangement to yield a reactive isocyanate that can covalently modify proteins or nucleic acids. This undesirable chemistry underlies the genotoxicity of hydroxamatebased HDAC inhibitors and motivates the search for inhibitors bearing alternative zinc-binding groups with improved safety profiles.52,53 Mercaptoacetamide zinc-binding groups are roughly isosteric with hydroxamates but do not exhibit genotoxicity, and accordingly have been incorporated into HDAC6-selective inhibitors.54–56 The crystal structure of a complex between HDAC6 CD2 and N-(5-(5,6-dichloro-1Hindol-1-yl)pentyl)-2-mercaptoacetamide56 reveals that the zinc-bound
thiolate moiety displaces the zinc-bound water molecule and accepts a hydrogen bond from H573.8 The mercaptoacetamide carbonyl group accepts a hydrogen bond from Y745, and the mercaptoacetamide NH group donates a hydrogen bond to H574 (Fig. 4d). Thus, the mercaptoacetamide group makes the same array of hydrogen bond interactions as observed for a hydroxamate moiety. While this is the only crystal structure of an HDAC6 CD2 complex with a thiol-based inhibitor, the zinc coordination polyhedron is identical to that of HDAC8 and HDAC10 complexes with inhibitors bearing thiolate zinc-binding groups.45,57,58 Notably, thiol zinc-binding groups are also found in pan-HDAC inhibitors, including the FDA-approved drug Romidepsin. Romidepsin is structurally similar to Largazole,59 except that the free thiol of Romidepsin is released by reduction of the internal disulfide group and the free thiol of Largazole is liberated by thioester hydrolysis (Fig. 5). Simple thiols are subject to oxidation and inactivation in aqueous solution, e.g., forming disulfides, as recently observed for thiol-based 5
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Fig. 5. The thiol zinc-binding groups of Romidepsin and Largazole are released by reduction or hydrolysis, respectively. The zinc-binding group, side chain linker, and roughly half of the capping group are identical in each inhibitor (red). Reprinted from ref. 57. Copyright 2011 American Chemical Society.
generally ranges ca. 3–7 Å in order to optimize interactions of the capping group for inhibitors shown in Table 1. Aliphatic linkers can also consist of cyclic hydrocarbons such as cyclohexane, cyclohexene, and cyclopentene groups, each of which is capable of binding in the F583-F643 aromatic crevice. The best of these inhibitors, cyclohexenyl hydroxamate, lacks a capping group yet nevertheless exhibits a dissociation constant (Kd) of 3 nM determined by isothermal titration calorimetry and impressive 313-fold selectivity for HDAC6 CD2 compared with the class I deacetylase human HDAC8.6
inhibitors of HDAC10.45 The protection of thiol zinc-binding groups in prodrug form facilitates their use in vivo. Linkers Immediately adjacent to the catalytic zinc ion in the active site of all HDACs is a small aromatic crevice formed by two side chain aromatic rings conserved as Phe-Phe, Trp-Phe, or Tyr-Phe. In the active site of HDAC6 CD2, this aromatic crevice is defined by F583 and F643 (Fig. 6). It is here where the linker segment attached to the inhibitor zincbinding group typically binds. Aromatic or heteroaromatic linkers engage in favorable offset π-π stacking interactions with these aromatic residues (Fig. 6). Binding in the aromatic crevice appears to be entropy driven, even for the simplest aromatic hydroxamate inhibitor, phenylhydroxamate.6 Thus, the binding of aromatic linker segments in the aromatic cleft of HDAC6 CD2 may be driven by desolvation, insofar that this phenomenon dominates enzyme-inhibitor association. In addition to aromatic linkers, saturated and unsaturated aliphatic linkers have been utilized in HDAC6 CD2 inhibitors. However, many of these compounds do not exhibit superior isozyme selectivity, such as SAHA. Even so, there are exceptions, such as the mercaptoacetamidebased inhibitors, some of which exhibit impressive HDAC6 selectivity.54–56 Thus, it appears that an inhibitor bearing an unsaturated aliphatic linker can exhibit appreciable isozyme selectivity if it is paired with an appropriate zinc-binding group. The linker length is clearly important as it is paired with different zinc-binding groups and
Capping groups Inhibitor chemodiversity is manifest in the diverse array of capping groups that interact with the mouth of the HDAC6 CD2 active site. Xray crystal structures of HDAC6 CD2-inhibitor complexes show that capping groups can be designed to target residues in this isozyme that are absent in other isozymes. For example, consider S531, which is unique to the HDAC6 CD2 active site. The side chain of S531 accepts a hydrogen bond from the backbone NH group of the acetyllysine substrate.3 S531 similarly accepts hydrogen bonds from NH groups incorporated into structurally-diverse HDAC6 CD2-selective inhibitors such as ACY-1083 (Fig. 7)5 and others.9,10 Inhibitor capping groups can also make additional interactions at the mouth of the active site, where the three-dimensional contour varies from one HDAC isozyme to another. Specifically, there are two shallow pockets designated L1 and L2. Inhibitor capping groups usually
Fig. 6. Linkers of HDAC6 inhibitors bind in an aromatic crevice defined by F583 and F643. Here, aromatic or heteroaromatic linkers can make favorable offset π-π stacking interactions, as observed for the phenylhydroxamate of DDK-137 (a) (PDB 6DVN) and the oxazolehydroxamate of JS-28 (b) (PDB 6Q0Z). Saturated hydrocarbon linkers, such as that of SAHA (c) (PDB 5EEI), also bind in this crevice. 6
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Fig. 7. (a) Simulated annealing omit map showing the binding of ACY-1083 in the active site of HDAC6 CD2. Hydroxamate-zinc coordination is monodentate, and the hydroxamate carbonyl group accepts a hydrogen bond from the zinc-bound water molecule. An inhibitor NH group donates a hydrogen bond to S531. Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. (b) Cut-away view of the HDAC6 CD2 active site surface showing the binding of ACY1083. This view highlights the zinc coordination polyhedron and the heteroaromatic ring of the inhibitor as it binds in the aromatic crevice defined by F583 and F643. Reprinted with permission from ref. 5. Copyright 2017 National Academy of Sciences.
bind in the L1 pocket; only three examples have been observed to date where inhibitor capping groups bind in the L2 pocket.4,9,13 While Resminostat is one of these examples, its capping group binds in the L2 pocket as a consequence of the binding of two iodide ions present in the crystallization buffer.13 X-ray crystal structures of HDAC6 CD2 complexes with various inhibitors reveal that diverse capping groups can be accommodated in the L1 or L2 pockets. Capping groups can range from polar species containing numerous hydrogen bond donors and acceptors, such as peptides, or nonpolar species such as a monoterpene hydrocarbon.13 While occasional hydrogen bond interactions are observed between capping group atoms and protein atoms, it appears that the majority of such interactions are mediated by intervening water molecules rather than direct enzyme-inhibitor hydrogen bonds. The oftentimes “wet” interface between capping groups and the pockets that accommodate these groups could indicate the potential for further optimization of the steric fit between the enzyme and inhibitor. Interestingly, the recently reported crystal structure of HDAC6 CD2 complexed with Tubastatin A reveals that the inhibitor capping group binds in the L1 pocket, and a number of ordered water molecules are observed to bind near the capping group.60 Some inhibitors contain bifurcated capping groups, such that one substituent binds in the L1 pocket and the other substituent binds in the L2 pocket. For example, consider RTS-V5 (Fig. 8).9 The bifurcated
capping group of this inhibitor simultaneously occupies the L1 and L2 pockets. Large capping groups that are complementary in shape to the contour of the mouth of the active site in the L1 and L2 pockets typically exhibit good affinity and selectivity for HDAC6 CD2, even if no direct enzyme-inhibitor hydrogen bond interactions are made in this region. Enzyme-inhibitor association appears to be driven mainly by steric complementarity between the capping groups and the three-dimensional contours of the L1 and L2 pockets. It is interesting to note that large, bulky capping groups often, but not always, correlate with the alternative monodentate hydroxamate-zinc binding mode. Conclusions Since the first report of human and zebrafish HDAC6 CD2 structures in 2016, more than 50 high-resolution X-ray crystal structures of HDAC6 CD2-inhibitor complexes have been reported to date. Each structure provides important information regarding affinity and isozyme selectivity determinants in the active site. Most inhibitors are tripartite in design, consisting of a zinc-binding group, a linker, and a capping group that can make a variety of interactions at the mouth of the active site cleft. Some inhibitor capping groups are bifurcated (Fig. 1b) and can thus interact with a broader region at the mouth of the HDAC6 CD2 active site (Fig. 8). It is in the outer active site cleft where structural differences among different HDAC isozymes become more pronounced, thereby providing starting points for the structure-based design of isozyme selectivity. Even so, it is striking that simple inhibitors, consisting of only a zinc-binding group and an aromatic linker without a capping group, can bind with impressive nanomolar affinity and better than 300-fold selectivity for HDAC6 CD2 compared with class I HDACs.6 By far, hydroxamate zinc-binding groups are most prevalent among selective HDAC6 CD2 inhibitors, but the susceptibility of the hydroxamate moiety to degradation through the Lossen rearrangement motivates the search for alternative zinc-binding groups. In order to broaden the utility of HDAC6 inhibitors beyond cancer indications, a more stable zinc-binding group is desirable for future inhibitor designs. A thiolate zinc-binding group is one possible alternative. The FDA-approved drug Romidepsin contains a thiol group that is liberated by reduction of an intramolecular disulfide linkage in the drug (Fig. 5). However, Romidepsin is more of a pan-HDAC inhibitor, so the
Fig. 8. The inhibitor RTS-V5 was designed as a dual HDAC6-proteasome inhibitor.9 As an inhibitor of HDAC6 CD2, the bifurcated capping group facilitates binding interactions in the shallow L1 and L2 pockets at the mouth of the active site cleft. Reprinted from ref. 13. 7
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challenge for the future is to incorporate a thiol zinc-binding group into an HDAC6 CD2-selective inhibitor while protecting the thiol group from adventitious oxidation, either by molecular oxygen or by disulfide formation. Promising results in this regard have been acquired with mercaptoacetamide zinc-binding groups.54–56 The X-ray crystal structure of HDAC6 CD2 complexed with a mercaptoacetamide inhibitor reveals a constellation of zinc coordination and hydrogen bond interactions that mimic those of a hydroxamate zinc-binding group (compare Fig. 4b and 4d).8 Finally, it is not clear to what degree the properties of affinity and selectivity are synergistic among different combinations of zinc-binding groups, linkers, and capping groups. As new zinc-binding groups are considered and evaluated in new HDAC6 inhibitors, such data will undoubtedly inform and direct the design of HDAC6 inhibitors with optimized properties.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgments We thank the NIH for grant GM49758 in support of this research. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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Choudhary C, Kumar C, Gnad F, et al. Science. 2009;325:834. López JE, Sullivan ED, Fierke CA. ACS Chem Biol. 2016;11:706. Hai Y, Shinsky SA, Porter NJ, Christianson DW. Nat Commun. 2017;8:15368. Kutil Z, Novakova Z, Meleshin M, Mikesova J, Schutkowski M, Barinka C. ACS Chem Biol. 2018;13:685. Moreno-Yruela C, Galleano I, Madsen AS, Olsen CA. Cell Chem Biol. 2018;25:849. Aramsangtienchai P, Spiegelman NA, He B, et al. ACS Chem Biol. 2016;11:2685. Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E. Nucleic Acids Res. 2015;43:D512. Berger SL, Kouzarides T, Shiekhattar R, Shilatifard A. Genes Dev. 2009;23:781. Delcuve GP, Khan DH, Davie JR. Clin Epigenetics. 2012;4:5. Zhao S, Xu W, Jiang W, et al. Science. 2010;27:1000. Wang Q, Zhang Y, Yang C, et al. Science. 2010;327:1004. Choudhary C, Weinert BT, Nishida Y, Verdin E, Mann M. Nat Rev Mol Cell Biol. 2014;15:536. Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M. Nat Rev Drug Discov. 2012;11:384. Falkenberg KJ, Johnstone RW. Nat Rev Drug Discov. 2014;13:673. Li Y, Seto E. Cold Spring Harb Perspect Med. 2016;6 pii:a026831. Duvic N, Talpur R, Ni X, et al. Blood. 2007;109:31. Piekarz RL, Frye R, Turner M, et al. J Clin Oncol. 2009;27:5410. Molife LR, de Bono JS. Expert Opin Invest Drugs. 2011;20:1723. Libby EN, Becker PS, Burwick N, Green DJ, Holmberg L, Besinger WI. Expert Rev. Hematol. 2015;8:9. Zhang L, Han Y, Jiang Q, et al. Med Res Rev. 2015;35:63. Zhang Y, Gilquin B, Khochbin S, Matthias P. J Biol Chem. 2006;281:2401. Zou H, Wu Y, Navre M, Sang BC. Biochem Biophys Res Commun. 2006;341:45. Kutil Z, Skultetyova L, Rauh D, et al. FASEB J. 2019;33:4035. Osko JD, Christianson DW. Biochemistry. 2019;58:4912. Bottomley MJ, Lo Surdo P, Di Giovine P, et al. J Biol Chem. 2008;283:26694. Porter NJ, Christianson DW. ACS Chem Biol. 2017;12:2281. Herbst-Gervasoni CJ, Christianson DW. Biochemistry. 2019;58:4957. Christianson DW, Lipscomb WN. Proc Natl Acad Sci USA. 1985;82:6840. Christianson DW, David PR, Lipscomb WN. Proc Natl Acad Sci USA. 1987;84:1512. Shoham G, Christianson DW, Oren DA. Proc Natl Acad Sci USA. 1988;85:684. Somoza JR, Skene RJ, Katz BA, et al. Structure. 2004;12:1325. Lee JH, Mahendran A, Yao Y, et al. Proc Natl Acad Sci USA. 2013;112:12005 and correction 2015, 112, E5899. Scolnick LR, Clements AM, Liao J, et al. J Am Chem Soc. 1997;119:850. Kerr JS, Galloway S, Lagrutta A, et al. Int J Toxicol. 2010;29:3. Shen S, Kozikowski AP. ChemMedChem. 2016;11:15. Kozikowski AP, Chen Y, Gaysin A, et al. J Med Chem. 2007;50:3054. Segretti MCF, Vallerini GP, Brochier C, et al. ACS Med Chem Lett. 2015;6:1156. Lv W, Zhang G, Barinka C, Eubanks JH, Kozikowski AP. ACS Med Chem Lett. 2017;8:510. Cole KE, Dowling DP, Boone MA, Phillips AJ, Christianson DW. J Am Chem Soc. 2011;133:12474. Decroos C, Clausen DJ, Haines BE, Wiest O, Williams RM, Christianson DW. Biochemistry. 2015;54:2126. Taori K, Paul VJ, Luesch H. J Am Chem Soc. 2008;130:1806. Shen S, Svoboda M, Zhang G, et al. ACS Med. Chem. Lett. 2020. https://doi.org/10. 1021/acsmedchemlett.9b00560 in press. Senger J, Melesina J, Marek M, et al. J Med Chem. 2016;59:1545. Krukowski K, Ma J, Golonzhka O, et al. Pain. 2017;158:1126. Mandl-Weber S, Meinel FG, Jankowsky R, Oduncu F, Schmidmaier R, Baumann P. Br J Haematol. 2010;149:518.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Digest
Structural determinants of affinity and selectivity in the binding of inhibitors to histone deacetylase 6 Jeremy D. Osko, David W. Christianson
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Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, United States
ARTICLE INFO
ABSTRACT
Keywords: Enzyme Inhibitor X-ray crystallography
Histone deacetylase 6 (HDAC6) is associated with multiple neurological disorders as well as aggressive cancers, making its selective inhibition highly desirable for therapeutic purposes. The basic molecular design of an effective HDAC6 inhibitor consists of a zinc-binding group, a linker, and a capping group capable of making interactions at the mouth of the active site. To date, more than 50 high-resolution X-ray crystal structures of HDAC6-inhibitor complexes have been reported, many of which reveal intermolecular interactions that contribute to isozyme affinity and selectivity. Here, we review the key features of HDAC6 inhibitor design illuminated by these structural studies.
X-ray crystallography is a powerful tool that provides a direct, atomic-resolution view of proteins and their complexes with small molecules. Such structural studies can inform and accelerate the discovery of new chemical entities that regulate protein function, a critical goal of medicinal chemistry. In this Digest, we focus on X-ray crystallographic studies of the tubulin deacetylase domain of histone deacetylase 6 (HDAC6; Fig. 1a)1,2 and its complexes with small molecule inhibitors. Inhibitors of HDAC6 are generally tripartite in design (Fig. 1b), consisting of a zinc-binding group that coordinates to the catalytic zinc ion, a flexible or rigid molecular linker, and a capping group capable of making interactions in the outer active site cleft. Since the crystal structures of human and zebrafish HDAC6 catalytic domains were first reported,3,4 more than 50 X-ray crystal structures of HDAC6inhibitor complexes have been determined and deposited in the Protein Data Bank (www.rcsb.org).5–13 These structures provide a wealth of information regarding the molecular basis of effective inhibition. Four classes of zinc-dependent HDAC isozymes are found in humans: class I (HDACs 1, 2, 3 and 8), class IIa (HDACs 4, 5, 7, and 9), class IIb (HDACs 6 and 10), and class IV (HDAC 11).14 These HDACs adopt the characteristic α/β arginase-deacetylase fold with general conservation of active site residues required for the chemistry of deacetylation.15,16 Most HDAC isozymes catalyze the hydrolysis of acetyllysine-containing protein and peptide substrates to generate products lysine and acetate.17–20 However, there are exceptions: HDAC10 is a polyamine deacetylase,21 and HDAC11 is a lysine fatty acid deacylase,22,23 an activity that has also been observed with HDAC8.24 Focusing solely on the chemistry and biology of lysine
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deacetylation, it is notable that there are approximately 40,000 unique protein acetylation sites identified to date (the “acetylome”) according to the PhosphoSite Plus database.25 Precise regulation of protein acetylation levels is implicated in numerous biological processes such as epigenetics,26,27 metabolism,28,29 and cell signaling.30 Moreover, aberrant regulation of protein acetylation is associated with neurodegenerative disease and cancer.31,32 The HDAC inhibitors Vorinostat (suberanilohydroxamic acid, SAHA), Belinostat, Panobinostat, and Romidepsin have been developed and approved for clinical use in humans.33 However, these inhibitors do not exhibit appreciable selectivity for any particular HDAC isozyme, and as such their use can lead to unwanted off-target effects.34–38 Due to their generally broad activity against all zinc-dependent HDAC isozymes, these inhibitors are often referred to as “pan-HDAC” inhibitors. Structure, function, and inhibition of HDAC6 With 1215 amino acids, human HDAC6 (Uniprot Q9UBN7) is the largest member of the zinc-dependent HDAC family.1,2 HDAC6 is additionally unique in that it contains two catalytic domains, designated CD1 and CD2 (Fig. 1a).39,40 HDAC6 CD2 is the tubulin deacetylase, whereas the biological function of HDAC6 CD1 is less well understood. Enzymological measurements using purified protein constructs corresponding to CD1 and CD2 reveal that CD2 exhibits broad substrate specificity, whereas CD1 exhibits much narrower specificity for peptide substrates bearing C-terminal acetyllysine residues.3 A more recent study utilizing full-length human HDAC6 in acetylome peptide
Corresponding author. E-mail address: [email protected] (D.W. Christianson).
https://doi.org/10.1016/j.bmcl.2020.127023 Received 20 December 2019; Received in revised form 7 February 2020; Accepted 8 February 2020 0960-894X/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Jeremy D. Osko and David W. Christianson, Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2020.127023
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In recent years, X-ray crystallographic studies of HDAC6 CD2 complexed with pan-HDAC inhibitors and isozyme-selective inhibitors have illuminated structural aspects of affinity and isozyme selectivity. The general contributions of the principle components of the tripartite inhibitors – the zinc-binding group, the linker, and the capping group – are now outlined as observed in recent X-ray crystallographic studies of complexes with HDAC6 CD2. All structures discussed below are deposited in the Protein Data Bank (www.rcsb.org) and are publicly available. For reference, molecular structures of selected inhibitors discussed in this Digest are shown in Table 1. Zinc-binding groups X-ray crystallographic studies have yielded key structural insights on the catalytic mechanism of HDAC6 CD2 (Fig. 3), and these insights include the coordination chemistry of the catalytic zinc ion. X-ray crystal structures of Y745F and H574A HDAC6 CD2 complexed with an acetyllysine substrate reveal that the scissile amide carbonyl coordinates to the catalytic zinc ion and accepts a hydrogen bond from Y745.3 Together, these interactions polarize and activate the carbonyl group for nucleophilic attack by a zinc-bound water molecule to yield a tetrahedral intermediate that collapses to yield products lysine and acetate. Tandem histidine residues H573 and H574 function as general base-general acid catalysts in this chemical mechanism, as shown in Fig. 3.3 These structural-mechanistic studies show that the catalytic zinc ion has two possible coordination sites for nonprotein ligands, as represented by the coordination sites of the substrate carbonyl and the zinc-bound water molecule. Ketone-based substrate analogues such as HC Toxin or 7-[(3-aminopropyl)amino]-1,1,1-trifluoroheptan-2-one (AAT) undergo a chemical reaction upon binding to HDAC6 CD2 that mimics the first step of the catalytic mechanism.3 If the ketone carbonyl is isosteric with the scissile carbonyl of the acetyllysine substrate, the ketone undergoes nucleophilic attack by a water molecule to yield a tetrahedral gemdiolate (Fig. 4a) that mimics the actual tetrahedral intermediate in catalysis (Fig. 3c).3 Thus, the binding of these reactive substrate analogues reflects the preferential binding of the tetrahedral transition state-like structure rather than the planar ketone substrate-like structure. This is a common feature observed for the binding of ketone-based inhibitors to HDAC4,43 HDAC8,44 and HDAC10.45 This hydration reaction was first observed in the binding of ketone- and aldehyde-based substrate analogues to the zinc enzyme carboxypeptidase A – regardless of whether or not the carbonyl group is activated by an adjacent trifluoromethyl group, the inhibitor was always observed to bind as the tetrahedral gem-diol(ate).46–48 Since an unactivated ketone exists to less than 0.2% gem-diol in aqueous solution, the measured Kd or IC50 values for the actual inhibiting species can be ~500 fold lower than the measured value, taking into account the low concentration of the inhibiting species in aqueous solution. Hydroxamic acids are the zinc-binding groups most often utilized in HDAC6 CD2 inhibitors. Trichostatin A (TSA) is a natural product bearing a hydroxamic acid moiety that is a pan-HDAC inhibitor. The first HDAC structure determined in complex with TSA was that of HDAC8.49 Suberanilohydroxamic acid (SAHA) is another pan-HDAC inhibitor, currently in use as the cancer chemotherapy drug Vorinostat. The first human HDAC structure determined in complex with SAHA was that of HDAC8.49 The crystal structures of HDAC6 complexes with these pan-HDAC inhibitors were recently reported.3,4 A hydroxamic acid zinc-binding group ionizes to form a negativelycharged hydroxamate upon binding to the enzyme. Hydroxamate-zinc coordination is often bidentate, with zinc chelation by the hydroxamate C]O and NeO– groups (Fig. 4b). This coordination geometry also enables hydrogen bond formation with tandem histidine residues, such that the zinc-bound oxyanion of the hydroxamate NeO– group accepts a hydrogen bond from H573 and the hydroxamate NH group donates a hydrogen bond to H574. The hydroxamate carbonyl group additionally
Fig. 1. (a) Crystal structure of the di-domain construct of zebrafish HDAC6 (PDB 5G0J) in which catalytic domain 1 (CD1, tan) and catalytic domain 2 (CD2, blue) are connected by a 23-residue linker (red). Catalytic zinc ions appear as gray spheres. (b) The general structure of an HDAC inhibitor is tripartite, consisting of a zinc-binding group, a linker, and a capping group that typically occupies the L1 pocket at the mouth of the active site. Inhibitors with bifurcated capping groups can occupy both the L1 and L2 pockets at the mouth of the active site.
microarrays indicates that internal acetyllysine residues in certain peptide sequences can be hydrolyzed by full-length human HDAC6 CD1,41 and recent measurements with zebrafish HDAC6 CD1 reveal weak catalytic activity against peptide substrates containing internal acetyllysine residues flanked by smaller residues.42 The recently-reported X-ray crystal structures of human HDAC6 CD2 from Homo sapiens (human),3 and HDAC6 CD1 and CD2 from Danio rerio (zebrafish),3,4 reveal that the active site contour of HDAC6 CD2 exhibits certain structural differences in comparison with other HDAC isozymes of known structure, indicating significant potential for the structure-based design of HDAC6-selective inhibitors. Comparison of human and zebrafish HDAC6 CD2 structures reveals very similar active sites and similar binding modes for the natural product inhibitor Trichostatin A (Fig. 2).3 Because the zebrafish enzyme is much more amenable to the generation of crystalline enzyme-inhibitor complexes that diffract X-rays to high resolution, zebrafish HDAC6 CD2 is most often used for structural studies aimed at understanding inhibitor structure-affinity and isozyme selectivity relationships. Selectivity for HDAC6 inhibition is most often desired and defined relative to the class I isozymes, HDACs 1–3 and 8. In contrast with pan-HDAC inhibitors, HDAC6 CD2-selective inhibitors can exhibit improved pharmacokinetic properties and fewer off-target effects. The first crystal structures of HDAC6 CD1 complexes with panHDAC inhibitors have only recently been reported.3,42 Slight differences in the active sites of HDAC6 CD1 and CD2 were noted when their crystal structures were first reported,3 suggesting the possibility of selective inhibition of either catalytic domain. A key difference in the active site of HDAC6 CD1 is K330, which corresponds to a leucine residue in HDAC6 CD2. The positively charged side chain of K330 is thought to confer specificity for the negatively charged carboxylate of a C-terminal acetyllysine residue in a peptide substrate.3 Since K330 can interact with bound inhibitors,3,42 interactions with this residue could be exploited by a selective inhibitor. 2
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Fig. 2. (a) The crystal structure of human HDAC6 CD2 (yellow) was determined as a fusion with maltose binding protein (MBP, cyan) (PDB 5EDU). The helix-loop linker (red) at the interface of the two proteins was engineered to optimize their association. Maltose is bound to MBP, and Trichostatin A (TSA) is bound in the active site of human HDAC6 CD2 (Zn2+ = gray sphere, K+ = purple spheres). (b) Superposition of the human HDAC6 CD2-TSA complex (wheat; orange TSA; PDB 5EDU) and the zebrafish HDAC6 CD2-TSA complex (light blue; blue TSA; PDB 5EEK). The Zn2+ ion and K+ ions in the human enzyme are white and purple spheres, respectively; the corresponding metal ions in the zebrafish enzyme are blue spheres. Only two active site residues differ between the two enzymes: N530 and N645 at the mouth of the active site of zebrafish HDAC6 CD2 appear as D567 and M682 in human HDAC6 CD2. (c) Comparison of human HDAC6 CD2 (left) and zebrafish HDAC6 CD2 (right), color-coded by electrostatic surface potential. Bound TSA molecules are represented in ball-and-stick fashion. Reprinted with permission from ref. 3.
Table 1 Selected inhibitors of HDAC6 CD2. (See below-mentioned references for further information.).
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Fig. 3. Snapshots of catalysis by HDAC6 CD2 captured in X-ray crystallographic studies. The active site of the unliganded enzyme in (a) reveals a nucleophilic Zn2+-bound water molecule hydrogen bonded with H573 and H574; a vacant coordination site on the Zn2+ ion is occupied by the scissile carbonyl of acetyllysine in the enzyme-substrate complex in (b). The substrate cannot react in this complex due to the Y745F substitution. In (c), the substrate carbonyl is fully activated and undergoes nucleophilic attack by the Zn2+-bound water molecule to yield a tetrahedral intermediate, but this intermediate cannot collapse due to the loss of the required proton donor through the H574A substitution. The oxyanion of this intermediate is stabilized by Zn2+ and Y745. Collapse of the tetrahedral intermediate yields products lysine and acetate, and the acetate complex in (d) represents a product complex following lysine dissociation. Reprinted with permission from ref. 3.
hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB)3,50 and has since been observed for other inhibitors such as ACY-1083.5 The monodentate coordination mode is only 0.5 kcal/mol less stable than the bidentate coordination mode, based on the 30:70 mixture of binding conformations observed for Trichostatin A in the ultrahigh resolution crystal structure of its complex with HDAC6 CD2.5 Either of the two coordination modes for the hydroxamate zinc-binding group may be favored based on the overall steric demands of the inhibitor in the active site. A third zinc-binding mode is feasible for a hydroxamate moiety, in which metal coordination is achieved by an ionized hydroxamate NH
accepts a hydrogen bond from Y745. This constellation of interactions is well-suited for the hydroxamate moiety and likely contributes to the high affinity of hydroxamate-bearing inhibitors. An alternative hydroxamate-zinc binding mode is sometimes observed that involves monodentate zinc coordination through the hydroxamate NeO– group, which also accepts a hydrogen bond from Y745 (Fig. 4c). The hydroxamate C]O group accepts a hydrogen bond from the zinc-bound water molecule, which is not displaced by inhibitor binding and retains its hydrogen bond interactions with H573 and H574. This monodentate hydroxamate-zinc binding mode was first observed in the HDAC6 CD2 complex with N-hydroxy-4-(2-[(24
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Fig. 4. Overlays of HDAC6 CD2-inhibitor complexes: (a) zinc-bound gem-diolates (PDB accession codes 5EFH, 5EFJ, 5EFN); bidentate hydroxamates (PDB accession codes 5EDU, 5EEI, 5EEK, 5EEM, 5EEN, 5EF8, 5EFB, 5G0H, 5WGL, 6CSP, 6CSQ, 6CSR, 6CSS, 6DVO, 6R0K, 6PZR, 6Q0Z, 6PZO, 6PZU; (c) monodentate hydroxamates (PDB accession codes 5EF7, 5W5K, 5WGK, 5WGM, 6CGP, 6CW8, 6DVL, 6DVM, 6DVN, 6PZO and 6PZS); zinc-bound thiolate (PDB accession code 6MR5).
group as observed for the binding of methylhydroxamic acid and trifluoromethylhydroxamic acid to human carbonic anhydrase II.51 However, this binding mode is not observed for hydroxamate binding to HDAC6. Despite the predominance of hydroxamate zinc-binding groups in HDAC6 inhibitors, the hydroxamate moiety is not particularly ideal for incorporation into a drug molecule due to its susceptibility to degradation. Hydroxamates can undergo Lossen rearrangement to yield a reactive isocyanate that can covalently modify proteins or nucleic acids. This undesirable chemistry underlies the genotoxicity of hydroxamatebased HDAC inhibitors and motivates the search for inhibitors bearing alternative zinc-binding groups with improved safety profiles.52,53 Mercaptoacetamide zinc-binding groups are roughly isosteric with hydroxamates but do not exhibit genotoxicity, and accordingly have been incorporated into HDAC6-selective inhibitors.54–56 The crystal structure of a complex between HDAC6 CD2 and N-(5-(5,6-dichloro-1Hindol-1-yl)pentyl)-2-mercaptoacetamide56 reveals that the zinc-bound
thiolate moiety displaces the zinc-bound water molecule and accepts a hydrogen bond from H573.8 The mercaptoacetamide carbonyl group accepts a hydrogen bond from Y745, and the mercaptoacetamide NH group donates a hydrogen bond to H574 (Fig. 4d). Thus, the mercaptoacetamide group makes the same array of hydrogen bond interactions as observed for a hydroxamate moiety. While this is the only crystal structure of an HDAC6 CD2 complex with a thiol-based inhibitor, the zinc coordination polyhedron is identical to that of HDAC8 and HDAC10 complexes with inhibitors bearing thiolate zinc-binding groups.45,57,58 Notably, thiol zinc-binding groups are also found in pan-HDAC inhibitors, including the FDA-approved drug Romidepsin. Romidepsin is structurally similar to Largazole,59 except that the free thiol of Romidepsin is released by reduction of the internal disulfide group and the free thiol of Largazole is liberated by thioester hydrolysis (Fig. 5). Simple thiols are subject to oxidation and inactivation in aqueous solution, e.g., forming disulfides, as recently observed for thiol-based 5
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Fig. 5. The thiol zinc-binding groups of Romidepsin and Largazole are released by reduction or hydrolysis, respectively. The zinc-binding group, side chain linker, and roughly half of the capping group are identical in each inhibitor (red). Reprinted from ref. 57. Copyright 2011 American Chemical Society.
generally ranges ca. 3–7 Å in order to optimize interactions of the capping group for inhibitors shown in Table 1. Aliphatic linkers can also consist of cyclic hydrocarbons such as cyclohexane, cyclohexene, and cyclopentene groups, each of which is capable of binding in the F583-F643 aromatic crevice. The best of these inhibitors, cyclohexenyl hydroxamate, lacks a capping group yet nevertheless exhibits a dissociation constant (Kd) of 3 nM determined by isothermal titration calorimetry and impressive 313-fold selectivity for HDAC6 CD2 compared with the class I deacetylase human HDAC8.6
inhibitors of HDAC10.45 The protection of thiol zinc-binding groups in prodrug form facilitates their use in vivo. Linkers Immediately adjacent to the catalytic zinc ion in the active site of all HDACs is a small aromatic crevice formed by two side chain aromatic rings conserved as Phe-Phe, Trp-Phe, or Tyr-Phe. In the active site of HDAC6 CD2, this aromatic crevice is defined by F583 and F643 (Fig. 6). It is here where the linker segment attached to the inhibitor zincbinding group typically binds. Aromatic or heteroaromatic linkers engage in favorable offset π-π stacking interactions with these aromatic residues (Fig. 6). Binding in the aromatic crevice appears to be entropy driven, even for the simplest aromatic hydroxamate inhibitor, phenylhydroxamate.6 Thus, the binding of aromatic linker segments in the aromatic cleft of HDAC6 CD2 may be driven by desolvation, insofar that this phenomenon dominates enzyme-inhibitor association. In addition to aromatic linkers, saturated and unsaturated aliphatic linkers have been utilized in HDAC6 CD2 inhibitors. However, many of these compounds do not exhibit superior isozyme selectivity, such as SAHA. Even so, there are exceptions, such as the mercaptoacetamidebased inhibitors, some of which exhibit impressive HDAC6 selectivity.54–56 Thus, it appears that an inhibitor bearing an unsaturated aliphatic linker can exhibit appreciable isozyme selectivity if it is paired with an appropriate zinc-binding group. The linker length is clearly important as it is paired with different zinc-binding groups and
Capping groups Inhibitor chemodiversity is manifest in the diverse array of capping groups that interact with the mouth of the HDAC6 CD2 active site. Xray crystal structures of HDAC6 CD2-inhibitor complexes show that capping groups can be designed to target residues in this isozyme that are absent in other isozymes. For example, consider S531, which is unique to the HDAC6 CD2 active site. The side chain of S531 accepts a hydrogen bond from the backbone NH group of the acetyllysine substrate.3 S531 similarly accepts hydrogen bonds from NH groups incorporated into structurally-diverse HDAC6 CD2-selective inhibitors such as ACY-1083 (Fig. 7)5 and others.9,10 Inhibitor capping groups can also make additional interactions at the mouth of the active site, where the three-dimensional contour varies from one HDAC isozyme to another. Specifically, there are two shallow pockets designated L1 and L2. Inhibitor capping groups usually
Fig. 6. Linkers of HDAC6 inhibitors bind in an aromatic crevice defined by F583 and F643. Here, aromatic or heteroaromatic linkers can make favorable offset π-π stacking interactions, as observed for the phenylhydroxamate of DDK-137 (a) (PDB 6DVN) and the oxazolehydroxamate of JS-28 (b) (PDB 6Q0Z). Saturated hydrocarbon linkers, such as that of SAHA (c) (PDB 5EEI), also bind in this crevice. 6
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Fig. 7. (a) Simulated annealing omit map showing the binding of ACY-1083 in the active site of HDAC6 CD2. Hydroxamate-zinc coordination is monodentate, and the hydroxamate carbonyl group accepts a hydrogen bond from the zinc-bound water molecule. An inhibitor NH group donates a hydrogen bond to S531. Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. (b) Cut-away view of the HDAC6 CD2 active site surface showing the binding of ACY1083. This view highlights the zinc coordination polyhedron and the heteroaromatic ring of the inhibitor as it binds in the aromatic crevice defined by F583 and F643. Reprinted with permission from ref. 5. Copyright 2017 National Academy of Sciences.
bind in the L1 pocket; only three examples have been observed to date where inhibitor capping groups bind in the L2 pocket.4,9,13 While Resminostat is one of these examples, its capping group binds in the L2 pocket as a consequence of the binding of two iodide ions present in the crystallization buffer.13 X-ray crystal structures of HDAC6 CD2 complexes with various inhibitors reveal that diverse capping groups can be accommodated in the L1 or L2 pockets. Capping groups can range from polar species containing numerous hydrogen bond donors and acceptors, such as peptides, or nonpolar species such as a monoterpene hydrocarbon.13 While occasional hydrogen bond interactions are observed between capping group atoms and protein atoms, it appears that the majority of such interactions are mediated by intervening water molecules rather than direct enzyme-inhibitor hydrogen bonds. The oftentimes “wet” interface between capping groups and the pockets that accommodate these groups could indicate the potential for further optimization of the steric fit between the enzyme and inhibitor. Interestingly, the recently reported crystal structure of HDAC6 CD2 complexed with Tubastatin A reveals that the inhibitor capping group binds in the L1 pocket, and a number of ordered water molecules are observed to bind near the capping group.60 Some inhibitors contain bifurcated capping groups, such that one substituent binds in the L1 pocket and the other substituent binds in the L2 pocket. For example, consider RTS-V5 (Fig. 8).9 The bifurcated
capping group of this inhibitor simultaneously occupies the L1 and L2 pockets. Large capping groups that are complementary in shape to the contour of the mouth of the active site in the L1 and L2 pockets typically exhibit good affinity and selectivity for HDAC6 CD2, even if no direct enzyme-inhibitor hydrogen bond interactions are made in this region. Enzyme-inhibitor association appears to be driven mainly by steric complementarity between the capping groups and the three-dimensional contours of the L1 and L2 pockets. It is interesting to note that large, bulky capping groups often, but not always, correlate with the alternative monodentate hydroxamate-zinc binding mode. Conclusions Since the first report of human and zebrafish HDAC6 CD2 structures in 2016, more than 50 high-resolution X-ray crystal structures of HDAC6 CD2-inhibitor complexes have been reported to date. Each structure provides important information regarding affinity and isozyme selectivity determinants in the active site. Most inhibitors are tripartite in design, consisting of a zinc-binding group, a linker, and a capping group that can make a variety of interactions at the mouth of the active site cleft. Some inhibitor capping groups are bifurcated (Fig. 1b) and can thus interact with a broader region at the mouth of the HDAC6 CD2 active site (Fig. 8). It is in the outer active site cleft where structural differences among different HDAC isozymes become more pronounced, thereby providing starting points for the structure-based design of isozyme selectivity. Even so, it is striking that simple inhibitors, consisting of only a zinc-binding group and an aromatic linker without a capping group, can bind with impressive nanomolar affinity and better than 300-fold selectivity for HDAC6 CD2 compared with class I HDACs.6 By far, hydroxamate zinc-binding groups are most prevalent among selective HDAC6 CD2 inhibitors, but the susceptibility of the hydroxamate moiety to degradation through the Lossen rearrangement motivates the search for alternative zinc-binding groups. In order to broaden the utility of HDAC6 inhibitors beyond cancer indications, a more stable zinc-binding group is desirable for future inhibitor designs. A thiolate zinc-binding group is one possible alternative. The FDA-approved drug Romidepsin contains a thiol group that is liberated by reduction of an intramolecular disulfide linkage in the drug (Fig. 5). However, Romidepsin is more of a pan-HDAC inhibitor, so the
Fig. 8. The inhibitor RTS-V5 was designed as a dual HDAC6-proteasome inhibitor.9 As an inhibitor of HDAC6 CD2, the bifurcated capping group facilitates binding interactions in the shallow L1 and L2 pockets at the mouth of the active site cleft. Reprinted from ref. 13. 7
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challenge for the future is to incorporate a thiol zinc-binding group into an HDAC6 CD2-selective inhibitor while protecting the thiol group from adventitious oxidation, either by molecular oxygen or by disulfide formation. Promising results in this regard have been acquired with mercaptoacetamide zinc-binding groups.54–56 The X-ray crystal structure of HDAC6 CD2 complexed with a mercaptoacetamide inhibitor reveals a constellation of zinc coordination and hydrogen bond interactions that mimic those of a hydroxamate zinc-binding group (compare Fig. 4b and 4d).8 Finally, it is not clear to what degree the properties of affinity and selectivity are synergistic among different combinations of zinc-binding groups, linkers, and capping groups. As new zinc-binding groups are considered and evaluated in new HDAC6 inhibitors, such data will undoubtedly inform and direct the design of HDAC6 inhibitors with optimized properties.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgments We thank the NIH for grant GM49758 in support of this research. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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