This section is adapted from the article “How the flexibility of human histone deacetylases influences ligand binding - An overview”, published in Drug Discovery Today 2015, 20(6), 736-742.
Nathalie Deschamps1, Claudia A. Simões-Pires1, Pierre-Alain Carrupt1, and Alessandra Nurisso1, ⌂ .
⌂ Corresponding author.
Affiliation
1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne Quai Ernest -Ansermet, 30, CH- 1211, Geneva 4, Switzerland.
Contribution
Article designing, article writing, article proofreading.
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A brief focus on histone deacetylases (HDACs)
HDACs have become interesting as therapeutic targets because of the benefits that their modulation might provide in the impairment of the acetylation status found in many diseases, including aging-related disorders 11. The modulation of gene expression by HDACs via chromatin modification was first highlighted as a potential anticancer target. HDAC catalytic inhibition can promote transcriptional reprogramming, which can be associated with the therapeutic success of inhibitors in cancer therapy that has resulted in positive clinical trials 68. Moreover, we and others recently reviewed the outcomes of a specific type of HDAC modulation in neurodegenerative conditions 69,70. However, there are still unknowns regarding the mechanisms of HDAC modulation, which are dependent upon isoform specificity, tissue and cell particularities, such as the diversity of available partner proteins and cellular signaling pathways 71,72.
The HDAC family is clustered into five evolutionarily classes based on phylogenetic features:
HDAC1–3 and 8 (class I); HDAC4, 5, 7 and 9 (class IIa); HDAC6 and 10 (class IIb); sirtuins SIRT1–7 (class III); and HDAC11 (class IV). Class I, IIa/b and IV enzymes require a divalent zinc ion for catalysis, whereas sirtuins are NAD+-dependent enzymes. The latter, which are structurally different from the other classes, are not discussed here 37,73. Class I HDACs are almost exclusively nuclear, often operating through multiprotein complexes, and are mainly responsible for histone deacetylation. By contrast, class II enzymes either shuttle between the nucleus and cytoplasm or are primarily cytoplasmic 74-78. They are able to deacetylate nonhistone proteins and can be found in complex with several protein partners 79-82. Comparable to class I isoforms, HDAC11, being the sole member of class IV, is also found in the nucleus 83. From a structural point of view, all HDAC enzymes share an approximately 11-Å tube-like catalytic channel, which accommodates a zinc ion that is responsible for their deacetylase activity. With a few exceptions, the residues lining this active pocket in the catalytic domain are widely conserved among different isoforms, making the design of selective inhibitors and/or modulators challenging 33, 38,63,64,84-88. Among class I HDACs, a 14-Å tunnel located perpendicular to the 11-Å channel bottom is also conserved. This water-filled pocket, also called the internal cavity or foot pocket, has been suggested to be the egression route for the acetate product and is potentially an additional target area for selective structure-based inhibitor design 33,36, 38,87,89. This channel has also been observed in HDAC4; however, it differs from class I HDACs in terms of its size and residue composition 85, 90. Many authors agree that the 11-Å channel rim is the best place to find exploitable structural differences for the rational design of selective inhibitors or modulators. For this reason, most of the HDAC inhibitors that have been reported to date obey a common ‘cap–linker–chelator’
pharmacophore model, where the cap moiety is designed to explore selectively the isoform channel rim, whereas the linker mimics the lysine side chains of substrates 32, 91.
Relevance of protein flexibility when exploiting HDACs for drug discovery
Protein flexibility is a property that regulates how certain biological effects are exerted in nature 56, 57. In fact, proteins are found to exist in solution in a dynamic equilibrium of energetically similar conformations 58. Distinct ligands, such as protein-binding partners, physiological compounds, or drug candidates, are hypothesized to modify this structural energy landscape through interactions with a limited number of protein conformations. Conformational selection increases the proportion of specific conformers in the total protein population, leading to an increase in the interaction phenomena that are the basis of biological processes 58. This recently developed mechanistic model overcomes the old but still popular lock-and-key and induced-fit models 59. X-ray crystallography, nuclear magnetic
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resonance (NMR) and molecular modeling strategies, such as molecular dynamics (MD) simulations, have already revealed possible conformer-dependent binding scenarios for several proteins, demonstrating an intimate connection between protein flexibility, complex formation and biological activity 60-62. Flexibility related to HDACs was first detected ten years ago 63,64. Since then, information from crystallography coupled with computational studies has been exploited for not only linking structural information to observed biological functions (mechanistic purposes), but also developing HDAC isoform-selective modulators (drug design purposes). The latter is currently needed to investigate the roles of individual HDAC isoforms in biological pathways as well as for producing efficient drugs with fewer adverse effects compared with the pan-HDAC inhibitors currently on the market as anticancer therapies 65-67. To the best of our knowledge, the dynamic aspects of HDAC isoforms that are the basis of catalytic and protein/protein interaction phenomena have not been previously reviewed. Here, we investigate what is known about HDAC flexibility and how this information can be exploited for the conception of novel potent and selective HDAC modulators.
MD: a powerful tool for detecting the plasticity of HDAC proteins
To date, twenty-nine crystallographic structures of human zinc-dependent HDACs have been deposited in the Protein Data Bank (PDB; http://www.wwpdb.org/). These structural data are a precious resource for the design of new compounds using structure-based techniques. Nevertheless, because crystals are a collection of photos representing particular conformational states of proteins, they lack information concerning their dynamic continuity in a solvated environment. This limitation, together with the expensive and extensive work required to collect crystals, has led to the development of a computational method called MD simulation, which is able to predict protein fluctuations as a function of time in a dynamic solvated environment. This technique is based on Newtonian physics and offers important insights regarding the protein motions that are the basis of ligand recognition and allosteric phenomena 92. Moreover, the recent efforts that have been made to decrease computational time, through the development of MD algorithms supporting Graphics Processing Units (GPU), are having a strong impact on the use of this computational strategy in routine drug design projects 93. To gain insights into the conformational dynamics of HDACs, complementary information from crystallography and MD were taken into account and summarized.
Class I HDACs 1-2 and 8: dynamics of catalytic site might be linked to ligand properties
Dynamic transitions between different conformational states are well-known phenomena in enzymology. Such motions are considered intrinsic enzymatic properties, but they can also be a consequence of external perturbations, such as ligand or protein binding 94.
The reported motions of class I HDACs are in line with the latter concept. Enzymes of this class are mostly nuclear and are studied as relevant targets against cancer and central nervous system (CNS) diseases 11,65. Among them, HDAC8 is the most documented in terms of crystallographic data and, to date, two main conformational changes have been observed: mass movements, which involve entire regions of the proteins, and one-off movements, which are associated with conformational variations in single amino acid side chains. By investigating the superimposition of co-crystals and apo-forms, several studies have shown that the active pocket of HDAC8 can convert from a wide-open state (also called the open state, which displays a single wide-open pocket 95) into a sub-open state, which displays two cavities (one deep tunnel adjacent to the 11 Å channel), and then into a unique cavity (the 11 Å channel), which is the closed conformational state (Fig. 4) 63, 95, 96. These dynamic transitions have all been reported or hypothesized to be in equilibrium and to be a consequence of ligand (substrate or inhibitor)
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binding, ligand release, or product egression through the protein structure 95-97. Two loops have been identified as primarily responsible for such induced-fit motions: the L1 loop, which comprises seven residues from Ser30 to Lys36, and the L2 loop, which comprises residues from Pro91 to Thr105. L1 is capable of moving outward from the catalytic site to widen the cavity opening or inward to occlude any adjacent cavities and create the closed conformational state, in which only the 11 Å tube-like cavity is accessible 50, 63. L2 is organized in complexes but not in the apo-form 63, 95,97. The architecture of the catalytic site and the channel rim of HDAC8 adapt to accommodate ligands according to their size, shape and chemical properties; a large and hydrophobic cap group (such as for CRA-A) would preferably bury into a deep hydrophobic groove (i.e. the open state) to reduce solvent exposure, whereas a small and rather hydrophilic cap group (such as SAHA) would rather stay freely exposed at the protein surface (i.e. binding to the HDAC8 closed state) (Fig. 4) 63,95. Punctual residues have an important role in HDAC8 cavity plasticity. For example, Tyr306 forms the end of the 11 Å channel wall and is involved in ligand recognition, and Phe152 (which is found on the 11 Å channel rim next to Tyr306) can either be packed against the Lys33 side chain from the L1 loop, occluding the second pocket of the HDAC8 sub-open conformation, or be rotated away from Lys33 in a position approximately 6 Å away to create the open state conformation of HDAC8 (Fig. 4) 36, 63, 96, 98, 99. One-off movements also characterize Trp141, which is located at the bottom of the HDAC8 catalytic pocket and is part of a gate-keeping system that controls access to the 14 Å channel (Table 1) 96, 98, 100. The 14-Å channel, which has an entrance that is governed by other gatekeepers (Table 1), is part of the inner machinery of the enzyme.
Interestingly, it has been suggested to be a possible egression route for both acetate product and ligands 89,100-102.
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Figure 4. Transition between histone deacetylase 8 (HDAC8) conformations: wide-open (A) (Protein Data Bank (PDB) code 1VKG), sub-open (B) (PDB code 1T64) and closed (C) (PDB code 1T69) conformational states. Molecular surfaces are drawn 8 Å around the co-crystallized ligands using a lipophilicity color code from magenta to green for hydrophilic and hydrophobic areas, respectively. In (A), a unique wide pocket is clearly visible, whereas in (B), two pockets are present, separated by a hydrophobic wall. In (C), only the 11-Å channel remains. The zinc ion is indicated as a blue sphere. (D) The mass and punctual movements of HDAC8 in L1 and L2 loops. The HDAC8 sub-open (PDB code 1T64) and closed (PDB code 2V5X) states are superimposed to highlight the mass movements of both the L1 and L2 loops and the punctual motions of the residues. L1 loop, Lys33 and Phe152 motions are shown either in pink or light blue, depending on whether they belong to the sub-open or the closed conformational state, respectively. The zinc ion (light-blue sphere) and its chelating triad are shown as reference points. The L2 loop (from Pro91 to Thr105) is shown either in violet or in dark blue, depending on whether it belongs to the sub-open (loop only) or the closed (short pseudo helix) conformation, respectively. Images were generated with MOE 2012.10 103.
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Table 1. Areas of flexibility detected in class I HDACs Isoform Region(s) and/or residue(s)
involved Structural consequences Refs
MASS MOTIONS HDAC1
Loops surrounding active site (Tyr201–Lys220) and terminal
regions Opening and closing of 11 Å channel; 14 Å channel; subchannel involved in the acetate release (unique to HDAC1); ligand accommodation
33, 38, 99, 104
HDAC2
Loops surrounding active site (Val190–Ala210) and terminal regions
HDAC3 Loop L1 (Ala19–Pro25); Helix 6 (Gln78–Asn88)
HDAC3–DAD–IP4 complex stabilization:
catalytic activity allowed
86, 99, 100
HDAC8
L1 or B loop (Ser30–Lys36) Opening and closing of 11 Å channel; 14 Å channel
Ser39 phosphorylation might inactivate HDAC8 through L1 conformational changes
36, 38, 50, 63, 89, 95-102, 105,
106 L2 or A loop (Glu85–Glu106)
Loops lining catalytic site (Val204–
Gln223; Gly272–Met274) Ligand accommodation ONE-OFF MOTIONS
HDAC1
Phe205 Accommodation of ligands with large cap group
33, 38, 99, 104 Met30, Tyr303 Opening of subchannel unique to HDAC1
Tyr22, Tyr24 and Phe109 Closing of 14 Å channel
Phe150 Ligand-release mechanisms through enlargement of zinc-binding area
HDAC2
Tyr27, Tyr29 and Phe144 Closing of 14 Å channel
33, 38, 99, 104 Phe155 Ligand-release mechanism through
enlargement of zinc-binding area
HDAC3
Tyr107 and Leu133 Avoiding accommodation of ligands with bulky zinc-binding groups
86, 99, 100 Tyr298
Assuming an inward orientation when HDAC3–DAD–IP4 complex is formed;
substrate recognition
HDAC8
Phe152 and Tyr306 Interconversion between wide-open, sub-open, or closed state of catalytic site; ligand
accommodation and release 36, 38, 50, 63, 89, 95-102, 105,
106 Tyr111, Trp141 and Tyr154
Leu31, Arg37, Tyr111, Trp141,
Gly139, Gly303 and Gly305 Closing of 14 Å channel
Tyr18, Tyr20 and His42 Regulation of second adjacent pocket exit
In the cases of HDAC1 and 2, several egression routes and gatekeepers have been hypothesized to be important in the design of selective compounds. An aromatic wall formed by face-to-face Phe150/Phe155 and Phe205/210 residues lines the 11-Å channel entrance of HDAC1 and 2, respectively. These residues seem to be involved in both ligand stabilization, such as for N-(2-aminophenyl)benzamide, through favorable hydrophobic contacts, and product release 104. Moreover, the 14-Å channel exit is mediated in HDAC1 and 2 by a transient zip-on zip-off mechanism via the tilting of a Tyr residue toward a Phe residue (Table 1) 104. Another transient subchannel, which is unique
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to HDAC1, also allows ligand egression through the two gatekeepers Met30 and Tyr303, and to the best of our knowledge, to date, this is one of the few differences between the HDAC1 and 2 isoforms 104. MD simulation studies, in contradiction with each other, aimed to provide information about the plasticity of all of the HDAC1–2 catalytic channels. In some studies, no significant flexibility was expected with respect to HDAC8 because of their longer L1 loop (HDAC8 is two amino acids shorter) 38,
50, 63, 96; however, several authors have confirmed the high flexibility of the channels by using root mean square deviation (RMSD) analyses of simulations in line with the high temperature factors reported for L1 in the homologous histone deacetylase-like protein (HDLP from Aquifex aeolicus) crystal 33, 104. Such motions have yet to be confirmed by new X-ray structures.
In summary, isoforms 1, 2 and 8 of the class I HDACs show a different flexibility profile that is intimately linked to ligand properties (polarity, size and shape of each moiety). This should be taken into account as a key factor in the rational design of HDAC-selective catalytic inhibitors.
Class I HDAC3: the dynamics of the catalytic site may be dependent on allosteric mechanisms The dynamics of HDAC3 differs from the isoforms described above in terms of its 11-Å channel, which is flexible in an unbound state. However, HDAC3 appears to assume a more rigid catalytic architecture when recruited into the co-repressor complex N-CoR/SMRT, which is necessary for biologically functional HDAC3 86, 88, 99, 100. This recruitment is dependent on the interaction between SMRT and HDAC3 through the binding of inositol tetraphosphate, IP4, which works as a liaison molecule between the two proteins. This interaction occurs in a region adjacent to the catalytic channel rim of HDAC3 (Table 1) and allows the protein to be catalytically active 86. An allosteric mechanism regulating HDAC3/N-CoR/SMRT complex formation has been suggested 99, 101. Thus, the design of compounds that are able to target the apo-form to prevent complex formation would be an interesting approach for probing the biological functions of HDAC3.
Jiggling and wiggling of an additional zinc-binding module influences the catalytic activity of class IIa HDACs
The work carried out by Bottomley and co-workers provided in-depth structural detail about class IIa HDACs and was the first to describe the crystallographic structure of HDAC4 85. According to that study, the core of the protein exhibits a layered α–β–α-fold, with a central parallel β sheet of eight β strands and a catalytic zinc ion, showing an overall similarity to HDAC isoform 8 (Cα RMSD 2.2 Å) 85. HDAC4, together with all the members of class IIa HDACs, is also characterized by an additional zinc-binding motif in the vicinity of the catalytic site. This module seems to have key roles in both substrate recognition and complex association 84, 85, 90. Characterized by two protein segments held together by a tetra-coordinated structural zinc ion (Table 2), the structural zinc-binding subdomain is able to adopt an open or a closed conformational state, which consequently affects the catalytic pocket size and shape 84,
85. Upon ligand binding, the protein structure is able to adopt (or not) the open conformation depending on the properties of the ligand 84, 85,90. In the open state, the additional zinc-binding motif moves 10–
20 Å away from the active site (Fig. 5). In this conformational state, the 14-Å channel is not formed. By contrast, when the closed state is promoted, the zinc-binding subdomain is packed toward the active site, opening the 14 Å channel, and exposing a groove that some studies have suggested to be the area that mediates the formation of multiprotein complexes, such as the HDAC4/7–HDAC3·N-CoR assembly 84,
85, 107, 108. Interestingly, a zinc-coordination switch coordinates with these motions, highlighting the high dynamism of such systems (Table 2). The debate regarding whether class II HDACs are bona fide deacetylases is still open. Notably, in these HDACs, a His replaces the catalytic Tyr that is found in
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class I HDACs, and this replacement decreases the deacetylase activity of class II HDACs (Table 2) 32,
84, 85, 109. Flexibility related to such a His residue might have a fundamental role in deacetylase activity, but this hypothesis still needs to be proven through additional biological and structural studies.
Figure 5. Transition between the open and closed conformational states of histone deacetylase 4 (HDAC4). The molecular surface is drawn around the zinc-binding subdomain (His665–Arg681;
Leu733–His766). As a reference point, the catalytic zinc ion is shown as a light-blue sphere. (A)The zinc-binding subdomain is open, and its corresponding surface is colored in pink. This conformation is obtained when HDAC4 is complexed to a thiophene derivative, shown as pink balls and sticks (Protein Data Bank (PDB) code 2VQJ). (B) Both open (pink) and closed (dark blue) states are superimposed to highlight the significant differences in terms of spatial arrangement. (C) The zinc-binding subdomain is closed and packed toward the catalytic site, and its corresponding surface is colored in dark blue. This conformation is obtained when HDAC4 is complexed to a carboxamide derivative, shown as blue balls and sticks (PDB code 4CBY). (D) The open and closed conformations of the HDAC6 ubiquitin-binding domain. The open (PDB code 3C5K) and closed (PDB code 3PHD) states of the ubiquitin-binding domain are superimposed to highlight Tyr1156 and Arg1155 one-off movements. A main view of the domain is provided to locate both zinc ions (light -blue spheres) and the involved residues. Tyr1156 and Arg1155 are shown as sticks, either in pink or in light blue depending on whether they belong to the open or the closed conformation, respectively. Images generated with MOE 2012.10 103.
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Table 2. Areas of flexibility detected in class II and IV HDACs Isoform Region(s) and/or residue(s)
involved Structural consequences Refs
MASS MOTIONS
HDAC4
Loops α1–α2 (His665–Glu680) Opening and closing of second zinc-binding subdomain
85, 90 Loops α6–α7–β3–β4 (Leu733–
His766)
Closed state: switch in zinc-chelating residues (from His665 and His678 to Cys669 and His675)
Open state: switch in zinc-chelating residues (from His675 and Cys669 to His665 and His678), with this position locked by Cys669Cys700 disulfide bonds HDAC10 Loops lining active site channel
(Glu302–Tyr305) Ligand accommodation 105, 106
HDAC11 Loops lining active site channel
(Ser301–Tyr304) Ligand accommodation 105, 106
ONE-OFF MOTIONS
HDAC4/7 His976/His843 Deacetylase activity stimulation (?) 32, 84, 85, 90 HDAC6 Arg1155 and Tyr1156 Opening or closing of ubiquitin-binding
groove
110
HDAC10 Glu302–Tyr305 11 Å channel plasticity 105, 106 HDAC11 Phe141, Ser301–Tyr304 11 Å channel plasticity 105, 106
Ideally, the design of a selective compound that is able to interfere with the closed conformation of HDACs would inhibit not only the deacetylase activity, but also all the downstream pathways regulated by these enzymes, providing insights into related biological functions. Nevertheless, the ligand properties that regulate such conformational changes are still unclear (i.e. not all catalytic inhibitors are able to promote an open conformation, which is the case for TSA in HDAC8) 63, 84, 90, 97.
For example, although they have the same zinc-binding group (hydroxamic acid), several inhibitors might or might not promote the open state of HDAC4 or 7; for example, an achiral ligand {N-hydroxy-5[(3-phenyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)carbonyl]thiophene-2-carboxamide}
was co-crystallized within the open conformation of HDAC4, whereas several chiral ligands or more flexible ligands {(1R,2R,3R)-2-[4-(5-fluoranylpyrimidin-2-yl)phenyl]-N-oxidanyl-3phenyl-cyclopropane-1-carboxamide; (1R,2R,3R)-2-[4-(1,3-oxazol-5-yl)phenyl]-N-oxidanyl-3-phenyl-cyclopropane-1-carboxamide; TSA and SAHA, respectively} were crystallized in complex with
was co-crystallized within the open conformation of HDAC4, whereas several chiral ligands or more flexible ligands {(1R,2R,3R)-2-[4-(5-fluoranylpyrimidin-2-yl)phenyl]-N-oxidanyl-3phenyl-cyclopropane-1-carboxamide; (1R,2R,3R)-2-[4-(1,3-oxazol-5-yl)phenyl]-N-oxidanyl-3-phenyl-cyclopropane-1-carboxamide; TSA and SAHA, respectively} were crystallized in complex with