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Histone deacetylases: structural determinants of inhibitor selectivity Q1

Carmina Micelli and Giulio Rastelli Life Sciences Department, University of Modena and Reggio Emilia, Via Campi 183, 41125 Modena, Italy

Histone deacetylases (HDACs) are epigenetic targets with an important role in cancer, neurodegeneration, inflammation, and metabolic disorders. Although clinically effective HDAC inhibitors have been developed, the design of inhibitors with the desired isoform(s) selectivity remains a challenge. Selective inhibitors could help clarify the function of each isoform, and provide therapeutic agents having potentially fewer adverse effects. Crystal structures of several HDACs have been reported, enabling structure-based drug design and providing important information to understand enzyme function. Here, we provide a comprehensive review of the structural information available on HDACs, discussing both conserved and isoform-specific structural and mechanistic features. We focus on distinctive aspects that help rationalize inhibitor selectivity, and provide structure-based recommendations for achieving the desired selectivity.

Introduction Q2 HDACs are enzymes that are commonly deregulated in a variety of tumors [1] because of their

importance in the regulation of gene expression through modification of histone and nonhistone substrates [2,3]. Histone acetylation and/or deacetylation are reversible processes controlled by the action of two classes of enzyme: histone acetyl-transferases (HAT) and histone deacetylases (HDAC). The former catalyzes the acetylation of histones, leading to chromatin relaxation and gene expression, whereas the latter catalyzes the removal of acetyl groups from N-terminal lysine e-amino groups in nuclear histones, causing chromatin condensation and, therefore, transcriptional repression [4,5]. Although histones remain the first and best-characterized substrates of HDAC enzymes, hundreds of nonhistone proteins modified by acetylation have now been identified, thanks to recent efforts in investigating the so-called ‘acetylome’ [6–8]. Changes in protein acetylation can affect many vital regulatory processes, including gene expression, mRNA stability, protein interactions, protein stability, and enzymatic activity [9,10]. Nonhistone substrates of HDAC include heat shock protein 90 (Hsp90), tubulin, and other cytoplasmic proteins. In fact, HDAC activity in cellular processes appears to be influenced by mechanisms different from regulation of transcription, thus extending the potential effects of HDAC

Carmina Micelli is a research fellow in the Molecular Modeling & Drug Design Laboratory of the University of Modena and Reggio Emilia under the supervision of Giulio Rastelli. After her postgraduate studies, she was a research fellow in the Structural Biology of Protein & Nucleic acid Complexes and Molecular Machines Laboratory of the Institute for Research in Biomedicine in Barcelona. Her research interests focus on structural biology, molecular modeling, and drug design. Giulio Rastelli is a professor of medicinal chemistry and head of the Molecular Modeling & Drug Design Laboratory of the University of Modena and Reggio Emilia. He received his PhD in medicinal chemistry from the University of Modena and Reggio Emilia and has been a research fellow at the University of California San Francisco under the supervision of Daniel Santi and Peter Kollman. His research interests focus on the development and application of computational drug design methodologies. He collaborates with academic and private institutions for the discovery and development of small-molecule inhibitors of relevant drug targets, with a special focus on cancer.

Corresponding author: Rastelli, G. ([email protected]) 1359-6446/ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2015.01.007

www.drugdiscoverytoday.com 1 Please cite this article in press as: Micelli, C., Rastelli, G. Histone deacetylases: structural determinants of inhibitor selectivity, Drug Discov Today (2015), http://dx.doi.org/10.1016/ j.drudis.2015.01.007

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Teaser The discovery of isoform-selective histone deacetylase inhibitors is a highly desirable but challenging goal. In this review, we collect and examine the extensive structural information available on histone deacetylases, discussing both conserved and isoform-specific features. Structure-based recommendations for achieving the desired selectivity are put forward.

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inhibitors (HDACIs) to nonhistone substrates [11], and HDACIs have shown synergistic effects when combined with drugs such as proteasome inhibitors [12]. Therefore, HDACs are involved in a variety of biological processes, including transcription, protein degradation, cellular proliferation, and apoptosis. Given the high number and variability of substrates that characterize this class of enzymes, perturbations of the balance between HAT and HDAC are often associated with a variety of disorders, including cancer, cardiovascular and neurological diseases, inflammation, and metabolic diseases [3,13–16]. HDACs are commonly deregulated in a variety of tumors as a consequence of either an altered expression of the HDAC genes or somatic mutations, the latter being generally rare [1,17,18]. Recently, HDACs have also been found to be involved in developmental disorders, such as Cornelia de Lange Syndrome, in which inactivating mutations cause a loss of deacetylase activity [19]. HDACIs are primarily under development as anticancer drugs, but they have potential medical applications for the treatment of other diseases. In cell-based studies, HDACIs have been shown to induce cell cycle arrest, cell differentiation, and apoptosis. Furthermore, HDACIs were reported to intensify the host immune response and decrease angiogenesis [20–24]. For these reasons, several HDACIs are currently at various stages of clinical trials, individually or in combination with radiotherapy and/or chemotherapy for cancer treatment in patients with hematological and solid malignancies [25]. So far, three HDACIs have been approved by the US Food and Drug Administration (FDA) for the treatment of advanced cutaneous T cell lymphoma, namely SAHA (vorinostat), FK228 (romidepsin), and PXD101 (belinostat) [26–29]. HDACIs currently in clinical trials belong to four different chemical classes: hydroxamic acids, cyclic peptides, benzamides, and shortchain fatty acids. SAHA and PXD101 are hydroxamic acids, whereas FK228 is a member of the cyclic peptide class. Similar to most of the other clinically tested molecules, they do not

exhibit HDAC isoform selectivity. Isoform selectivity is desirable to clarify the biological functions of each isoform and to develop therapeutic agents with potentially fewer adverse effects. However, the design of isoform-selective HDACIs is limited by the absence of structural data of several isoforms, and remains particularly challenging in light of the high structural similarity and sequence conservation of the various HDAC isoforms belonging to the same class. So far, isoform-selective HDACIs reported in the literature are few, whereas class-selective or pan-HDACIs represent the majority [30,31]. Currently, major efforts are focused on developing selective inhibitors and studying combination therapies, with the aim of increasing potency against specific cancer types and overcome drug resistance [24,32]. Here, we collect and examine the extensive structural information available on HDACs, discussing both conserved and isoform-specific structural and mechanistic features. We focus on distinctive aspects that help rationalize inhibitor selectivity, and provide structure-based recommendations for achieving the desired selectivity.

HDAC classification Eighteen different HDAC isoforms, grouped into four classes based on phylogenetic analysis and sequence similarity to yeast factors, have been described in humans (Table 1) [33–35]. Classes I, II, and IV HDACs require zinc ions as cofactors, whereas class III HDACs are NAD+-dependent enzymes known as sirtuins (SIRT1-7) [36]. Class I HDACs (HDAC 1, 2, 3, and 8) are homologous to the Rpd3 yeast protein and are located mainly in the nucleus. They contain an N-terminal catalytic domain, and comprise approximately 400 amino acids. Class II HDACs, homologous to the Hda1 yeast protein, are subdivided into class IIa (HDAC 4, 5, 7, and 9) and class IIb (HDAC 6 and 10), and can shuttle between cytoplasm and nucleus. Class II enzymes are 600–1200 residues long and characterized by the presence of either an N-terminal extension with

TABLE 1

Members of class I, II, and IV HDACs along with their catalytic domain length and composition, size (number of amino acids), and cellular localizationa.

a

HDAC

Catalytic domain

Class I HDAC1 HDAC2 HDAC3

N N N

aa. 9-322 aa. 3-316

HDAC8

N

aa. 14-324

Class IIa HDAC4 HDAC5 HDAC7 HDAC9

N N N N

Class IIb HDAC6 HDAC10

N N

aa. 1-323

Class IV HDAC11

N

aa. 14-326

aa. 9-321

C C C C aa. 655-1084 aa. 684-1028

C C

aa. 518-865 C aa. 631-978 C aa. 87-404

aa. 482-800 482-666 C

C

C

Size

Localization

482 488 428

Nuclear Nuclear Nuclear/cytoplasmic

377

Nuclear

1084 1122 952 1011

Nuclear/cytoplasmic Nuclear/cytoplasmic Nuclear/cytoplasmic Nuclear/cytoplasmic

1215 669

Cytoplasmic Cytoplasmic

347

Nuclear

Key: blue box, functional zinc dependent catalytic domain; gray box catalytically inactive domain.

2

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DRUDIS 1570 1–18

regulatory functions (class IIa) or two catalytic domains (class IIb). Within class IIb, HDAC6 is particularly important in light of its involvement in several malignant cellular processes. HDAC6 is a large protein of 1215 amino acids that contains two functional catalytic domains and a ubiquitin-binding zinc finger domain at the C-terminal region. The crystal structure of HDAC6 has not yet been solved. Therefore, structure-based investigations performed on this isoform rely on homology models based on crystal structure templates of other HDAC isoforms [37]. HDAC11, which is predominantly nuclear, has been classified in a different class (class IV), because its overall sequence identity with other HDACs is limited. Differences among zinc-dependent classes I, II, and IV HDACs are not limited to protein size and subcellular localization, but also involve substrate specificity, enzymatic activity, and tissue expression pattern [10,31].

Catalytic mechanism The active site of zinc ion-dependent HDACs exhibits features of both metallo- and serine proteases [38,39] and the detailed mechanism leading to lysine deacetylation is still unclear. Class I and II HDACs have a largely conserved catalytic core and, therefore, it is assumed that they also share the same catalytic mechanism. Finnin et al. [40] proposed a catalytic mechanism on the basis of crystal structures of histone deacetylase-like protein (HDLP; from Aquifex aeolicus) co-crystallized with the HDAC inhibitors trichostatin A [TSA, Protein Data Bank (PDB) code 1C3R] and suberoylanilide hydroxamic acid (SAHA, PDB code 1C3S). According to the mechanism hypothesized by Finnin (Fig. 1a), the carbonyl oxygen of the acetylated lysine binds the zinc ion, which in turn binds the catalytic water molecule. Given these Q3 interactions, the zinc atom would either polarize the carbonyl group of the substrate, causing an increase in electrophilicity of the carbon, and help orient the water molecule, which nucleoQ4 philicity might be further increased by hydrogen bonding with the histidine side chain of the buried Asp166–His131 charge relay system (numbering based on HDLP). His132, which is part of the partially solvent exposed Asp173–His132 charge relay system, is assumed to be protonated in the initial step of the reaction. The water molecule would carry out a nucleophilic attack on the carbonyl carbon of the acetylated lysine, resulting in the formation of a tetrahedral oxyanion intermediate. This oxyanion could be stabilized by interaction between its two oxygens and the zinc ion and, in addition, by a possible hydrogen bond with the hydroxyl group of the Tyr297 residue. In the last step, breakage of the carbon–nitrogen bond would occur and a proton would be transferred from the Asp173-His132 charge relay system to the nitrogen atom, with a final release of the acetate and lysine products. In 2005, Vanommeslaeghe et al. [41] proposed an alternative mechanism (Fig. 1b) based on a density functional theory (DFT) study of the catalytic site of HDLP. In the initial step of the reaction, the substrate is assumed to be coordinated to the zinc ion through the nitrogen and oxygen atoms of the amide group. Moreover, a hydroxide ion derived from deprotonation of the water molecule by means of His131 (which coordinates the catalytic zinc ion and accepts a hydrogen bond from Tyr297 in the final geometry), and a neutral His132 were assumed. Once the acetylated lysine contacts the zinc atom, the hydroxide would

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attack the amide carbon resulting in a tetrahedral intermediate, and a proton would be transferred from His131 to His132. Subsequently, because of the higher acidity of His132 with respect to His131 as predicted by proton affinity calculations, deprotonation of His132 by the lysine e-nitrogen and breakage of the carbon– nitrogen bond of the tetrahedral intermediate would occur, leading to acetate ion and protonated lysine products, as shown in the last step of Fig. 1b. More recently, Corminboeuf et al. [42] suggested a different catalytic mechanism (Fig. 1c), which deviates from the hydroxide mechanism typical of zinc proteases, based on the difference in total charge observed in zinc protease and histone deacetylase active sites. Indeed, whereas in zinc proteases residues coordinating the catalytic zinc ion are one Glu/Asp and two His residues, in HDACs these are replaced by two Asp and one His residues. DFT quantum mechanics/molecular mechanics (QM/MM) studies predicted that His131 and His132 are not protonated in the initial step of the reaction. Upon binding of the acetyl-lysine, the water molecule would be deprotonated by His132, allowing the nucleophilic attack that leads to the tetrahedral intermediate. At this stage, His132 would transfer a proton to the amide nitrogen, resulting in breakage of the amide bond and product release. The availability of crystallographic data at subatomic resolution, when achievable, could help determine the protonation states of catalytic residues at a fine level of structural detail. Moreover, the integration of NMR studies and computational modeling could provide useful information to better understand enzyme mechanism.

Analysis of HDAC crystal structures The rational design of isoform-selective HDACIs entails accurate evaluation of the available protein structures of the various isoforms, both individually and together with ligands, to highlight structural differences and exploit them to create a network of specific interactions that might confer selectivity. To date, 39 crystal structures of human HDACs (isoforms 1, 2, 3, 4, 7, and 8) and eight for HDAC homologs from bacteria (HDLP and HDAH) have been solved [19,40,43–60] (Tables 2 and 3).

HDAC catalytic pocket The overall fold of zinc-dependent HDACs comprises a single compact a/b domain composed of a central eight-stranded parallel b-sheet flanked by several a-helices on both sides (Fig. 2a,b). Secondary structure elements are partially conserved across the entire family of HDACs, whereas dramatic conformational variations are observed for most of the loops emerging from the protein core. The analysis of the available structures reveal a narrow hydrophobic pocket characterized by a tube-like shape, with a depth of ˚ , that leads to a cavity containing the catalytic approximately 11 A machinery. Given the high sequence similarity of residues constituting the catalytic pocket (Figure S1 in the supplementary material online), the binding site architecture is almost the same across the entire family of HDACs. The walls of the channel are lined mainly by hydrophobic residues, including Pro542, Gly678, Phe679, Phe738, and Leu810 (HDAC7 numbering, PDB code 3C10), whereas catalytic residues coordinating the zinc ion are polar. The pocket reaches the narrowest point approximately

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(a)

H132 D173 NH

H N

Lysine

O H O

Y297

O

OH

H

H131 N

H170

NH

D168 D258

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D166

O H132

H132 D173

D173 NH H N

Lysine

NH

Lysine O

O

N

OH O

H Y297

H131

H

Y297

H131

OH

O H170

NH

D168 D258

H170 D166

NH

D168 D258

D166

O

(b)

O H132

H132 D173

D173 NH

NH H N

Lysine

O

N

H N

Lysine

O

N

H O

O H Y297

H

H

H131 Y297

O H170

H131

H

N

D168 D258

N

OH H170

D166

D168 D258

D166

O

O

H132

H132 D173

Lysine

D173

NH

NH2

O

N

H N

Lysine

O

Y297

HO

H

H131

O N

H H170

Y297 NH

D168 D258

O

N H

O

H131 N

OH H170

D166

NH

D168 D258

D166

O

(c)

O

H132 D173 NH H N

Lysine

O

N H O

O

H Y297

H131

H N

O H170

NH

D168 D258

D166

O H132

H132 D173

D173

H NH

NH Lysine

H N

Lysine

O

N

O

N

H O

O H

H Y297

H131

Y297

H

OH

H131

N

O H170

NH

D168 D258

D166

O

H170

D166

D168 D258 O

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FIGURE 1

Mechanism of hydrolysis of acetylated lysine according to Finnin [40] (a), Vanommeslaeghe [41] (b), and Corminboeuf [42] (c).

4

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TABLE 2

Crystal structures of human and bacterial HDACs available in the PDB (accessed December 15, 2014). Ligand structure and IUPAC name

Resolution Mutation Cofactor Metal ions Organism ˚) (A

HDAC1

4BKX

(1) Acetate

3.00

Zn2+

2 K+

HDAC2

3MAX

(2) N-(2-amino-5-phenylphenyl)benzamide

2.05

Zn2+

Ca2+, Na+

1.85

2+

4LXZ

(3) Octanedioic acid hydroxyamide phenylamide (SAHA)

4LY1

(4) N-[2-amino-5-(thiolan-2-yl)phenyl]-4 acetamidobenzamide 1.57

Refs

Homo sapiens

[43]

H. sapiens

[44]

Zn

+

Ca , Na

H. sapiens

[45]

Zn2+

Ca2+, Na+

H. sapiens

[45]

+

2+

2+

HDAC3

4A69

(1) Acetate

2.06

Zn

2K

H. sapiens

[46]

HDAC4

2VQJ

(5) 2,2,2-trifluoro-1-(5-{3-phenyl-5H,6H,7H,8H-imidazo[1,2a]pyrazine-7-carbonyl}thiophen-2-yl)ethane-1,1-diol (TFMK)

2.10

Zn2+

2 K+

H. sapiens

[47]

2VQM

(6) N-hydroxy-5-{3-phenyl-5H,6H,7H,8H-imidazo[1,2a]pyrazine-7-carbonyl}thiophene-2-carboxamide (HA3)

1.80

Zn2+

2 K+

H. sapiens

[47]

2VQW



3.00

H332Y

Zn2+

2 K+

2+

HDAC7

HDAC8

H. sapiens

[47]

2VQO

(5) TFMK

2.15

C25A; C56A; H332Y

Zn

2 K+

H. sapiens

[47]

2VQQ

(5) TFMK

1.90

C25A; C56A

Zn2+

2 K+

H. sapiens

[47]

2VQV

(6) HA3

3.30

C25A; C56A; H332Y

Zn2+

2 K+

H. sapiens

[47]

4CBT

(7) (1R,2R,3R)-2-[4-(5-fluoropyrimidin-2-yl)phenyl]-N-hydroxy3-phenylcyclopropane-1-carboxamide

3.03

Zn2+



H. sapiens

[48]

4CBY

(8) (1R,2R,3R)-N-hydroxy-2-[4-(1,3-oxazol-5-yl)phenyl]-3phenylcyclopropane-1-carboxamide

2.72

Zn2+

2 Na+

H. sapiens

[48]

3C0Y



2.10

Zn2+

2 K+

2+

H. sapiens

[49]

3C0Z

(3) SAHA

2.10

Zn

2 K+

H. sapiens

[49]

3C10

(9) (2E,4E,6R)-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6dimethyl-7-oxohepta-2,4-dienamide (TSA)

2.00

Zn2+

2 K+

H. sapiens

[49]

3ZNR

(10) N-{[4-(4-phenyl-1,3-thiazol-2-yl)oxan-4-yl]methyl}-3-[5(trifluoromethyl)-1,2,4-oxadiazol-3-yl]benzamide

2.40

Zn2+

2 K+

H. sapiens

[50]

3ZNS

(11) N-{[1-methyl-4-(4-phenyl-1,3-thiazol-2-yl)piperidin-4yl]methyl}-3-[5-(trifluoromethyl)-1,2,4-oxadiazol-3yl]benzamide

2.45

Zn2+

2 K+

H. sapiens

[50]

2V5W

Substrate

2.00

Zn2+

2 K+

H. sapiens

[51]

2+

Zn

2 K+

H. sapiens

[51]



H. sapiens

[52]

Y306F

2V5X

(12) (2R)-N-hydroxy-2-[2-(5-methoxy-2-methyl-1H-indol-3yl)acetamido]-N’-[2-(2-phenyl-1H-indol-3yl)ethyl]octanediamide

2.25

1T69

(3) SAHA

2.91

Zn2+ 2+

S39D

+

1T64

(9) TSA

1.90

Zn

2 Na

H. sapiens

[52]

1VKG

(13) 3-[4-(hydroxycarbamoyl)phenyl]-N,N-dimethyl-5-(4methylbenzamido)benzamide (CRA-19156)

2.20

Zn2+

Na+

H. sapiens

[52]

1T67

(14) 7-{[4-(dimethylamino)phenyl]formamido}-Nhydroxyheptanamide (MS-344)

2.31

Zn2+

2 Na+

H. sapiens

[52]

1W22

(15) N-hydroxy-4-[N-methyl5-(pyridin-2-yl)thiophene-2sulfonamido]benzamide

2.50

Zn2+

2 K+

H. sapiens

[53]

3SFF

(16) (2R)-2-amino-3-(3-chlorophenyl)-1-[4-(2,5 difluorobenzoyl)piperazin-1-yl]propan-1-one

2.00

Zn2+

2 K+

H. sapiens

[54]

3SFH

(17) (2R)-2-amino-3-(2,4-dichlorophenyl)-1-(2,3-dihydro-1Hisoindol-2-yl)propan-1-one

2.70

Zn2+

2 K+

H. sapiens

[54]

3MZ3

(14) MS-344

3.20

Co2+

2 K+

2+

H. sapiens

[55]

3EZT

(14) MS-344

2.85

D101E

Zn

2 K+

H. sapiens

[56]

3F06

(14) MS-344

2.55

D101A

Zn2+

2 K+

H. sapiens

[56]

+

H. sapiens

[55]

3MZ4

(14) MS-344

1.85

D101L

2+

Mn

2K

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Enzyme PDB code

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Drug Discovery Today  Volume 00, Number 00  February 2015

TABLE 2 (Continued ) Enzyme PDB code 3MZ6

Ligand structure and IUPAC name

Resolution Mutation Cofactor Metal ions Organism ˚) (A

(14) MS-344

2.00

Refs

D101L

Fe2+

2 K+

H. sapiens

[55]

2+

+

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3MZ7

(14) MS-344

1.90

D101L

Co

2K

H. sapiens

[55]

3EW8

(14) MS-344

1.80

D101L

Zn2+

2 K+

H. sapiens

[56]

3EZP

(14) MS-344

2.65

D101N

Zn2+

2 K+

H. sapiens

[56]

2+

Zn

+

2K

H. sapiens

[56]

Zn2+

2 K+

2+

3F07

(18) (2E)-N-hydroxy-3-[1-methyl-4-(2-phenylacetyl)-1H-pyrrol2-yl]prop-2-enamide

3.30

3EWF

Substrate

2.50

H143A

H. sapiens

[56]

3F0R

(9) TSA

2.54

Zn

2 K+

H. sapiens

[56]

3RQD

(19) S-((E)-4-((5R,8R,11S)-8-isopropyl-5-methyl-6,9,13-trioxo10-oxa-3,17-dithia-7,14,19,20tetraazatricyclo[14.2.1.12,5]icosa-1(18),2(20),16(19)-trien-11yl)but-3-en-1-yl) octanethioate (Largazole)

2.14

Zn2+

2 K+

H. sapiens

[57]

4QA0

(3) SAHA

2.24

C153F

Zn2+

2 K+

H. sapiens

[19]

2+

+

4QA1

(14) MS-344

1.92

A188T

Zn

2K

H. sapiens

[19]

4QA2

(3) SAHA

2.38

I243N

Zn2+

2 K+

H. sapiens

[19]

2+

+

4QA3

(9) TSA

2.88

T311M

Zn

2K

H. sapiens

[19]

4QA4

(14) MS-344

1.98

H334R

Zn2+

2 K+

H. sapiens

[19]

4QA5

Substrate

1.76

A188T; Y306F

Zn2+

2 K+

H. sapiens

[19]

4QA6

Substrate 2.05

I243N; Y306F

Zn2+

2 K+

H. sapiens

[19]

4QA7

Substrate 2.31

H334R; Y306F

Zn2+

2 K+

H. sapiens

[19]

HDLP

1C3P

1.80





Aquifex aeolicus

[40]

HDAH

1C3S

(3) SAHA

2.50

S75C; S77C

Zn2+



A. aeolicus

[40]

1C3R

(9) TSA

2.00

S75C; S77C

Zn2+



A. aeolicus

[40]

2VCG

(20) Methyl (2S)-3-(4-bromophenyl)-2-[7(hydroxycarbamoyl)heptanamido] propanoate

1.90

Zn2+

2 K+

Alcaligenes sp.

[58]

2GH6

(21) 9,9,9-trifluoro-8-oxo-N-phenylnonanamide

2.20

Zn2+

2 K+

Alcaligenaceae [59] bacterium FB188

1ZZ0

(1) Acetate

1.60

Zn2+

2 K+

[60] Alcaligenaceae bacterium FB188

1ZZ1

(3) SAHA

1.57

Zn2+

2 K+

Alcaligenaceae [60] bacterium FB188

1ZZ3

(22) 3-cyclopentyl-N-hydroxypropanamide

1.76

Zn2+

2 K+

Alcaligenaceae [60] bacterium FB188

halfway down the channel and becomes wider at the bottom, where the zinc ion is present (Fig. 2c). The catalytic zinc is ˚ ), His709 (Nd1, 2.14 A ˚ ), coordinated by Asp707 (Od1, 1.86 A ˚ ), and a water molecule (Wat1, 2.36 A ˚ ), which Asp801 (Od2, 2.02 A constitute the polar catalytic core (Fig. 2d). The water molecule is likely to be responsible for the nucleophilic attack on the carbonyl carbon of the acetyl-lysine, and has additional interactions with His669 and His670. His669 is part of the buried charge relay system (His669–Asp705) that is mostly conserved in all HDACs, whereas His670 is involved in the partially solvent exposed His670–Asn712 charge relay system, in which the asparagine is present in class II HDACs and HDAH but is replaced by an aspartic acid in class I HDACs and HDLP (Figure S1 in the supplementary material online). 6

This His–Asp arrangement is typical of serine proteases, where the aspartic acid carboxylate oxygen accepts a hydrogen bond from Nd1 and polarizes the imidazole Ne2, increasing its basicity [39]. Mutagenesis studies involving human HDACs and the Rpd3 yeast protein homologous to the class I subfamily demonstrate that histidine and aspartic acid residues of the buried charge-relay system are necessary to achieve an effective enzymatic activity. Indeed, the H150A mutation in Rpd3 (corresponding to His669 in HDAC7, Fig. 2d) abolishes HDAC activity [61] and the D174N mutation in HDAC1 (corresponding to Asp705 in HDAC7, Fig. 2d) leads to an approximately 12-times drop in HDAC1 activity compared with wild type [62]. Also, the histidine residue of the partially solvent-exposed charge relay system seems to have a crucial role in catalysis, because the H151A mutation in Rpd3

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TABLE 3

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Chemical structures of HDAC inhibitors discussed in this review.

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Drug Discovery Today  Volume 00, Number 00  February 2015

(a)

(b)

α3 α1

α8

β1 β3

α6

α5

α9 α4

α7

α15

β8

β1

β4

α9

F738

β10

α2

α15

α16

α5

α14

α17

β5

β3

α6

(c)

β2

α8

α16

α2

α3

α4

α1

β2

α18

β9

β7 β4

α7

Reviews  KEYNOTE REVIEW

α10

G678 β6

α13

α12

α11

β7

α10

β5 β6

P542

L810

α19

α13 α12

β8

α11

F679

α14

α21

α17

α20

(d)

(e)

(f)

α11

N712

α7 α9

β7 H670

H709

H669

H709

β4

F718 Wat1

Y308

Wat

H843

H669

D721 Wat2

D801

D705

Site 2 Site 1

D705

Wat V724

β3 α8

D801 D707

α14

S728

α1

D707 F755

(g)

L729

(h)

β3

α6 H709

β3

D101

α3

(i)

α7 D626 α14

α2

α15

β4 D101

C533

H541 α1 α3

β4

C618 D801 D707

α2

α8

F208 α2

C535

F152 H180

Wat1 α7

D178

α3

α8

D267 α1

Drug Discovery Today

FIGURE 2

Ribbon representation of human histone deacetylase (HDAC) 2 (a) representative of class I [Protein Data Bank (PDB) code 3MAX], and human HDAC7 (b) representative of class IIa (PDB code 3C10). The inhibitor is drawn as a stick. (c) Surface representation of the HDAC7 catalytic pocket (PDB code 3C10). Hydrophobic residues lining the walls of the channel are drawn as sticks. The zinc ion is shown as a gray sphere, and the inhibitor Trichostatin A is drawn as a stick and ball. (d) Ribbon representation of a superimposition of HDAC7 (PDB code 3C0Y) and HDAC2 active sites (PDB code 3MAX). HDAC7 is shown in magenta and HDAC2 in light-blue. Water molecules are shown as red spheres, and the potassium ion is yellow. (e) Structure of the two additional metal-binding sites in HDAC7 (PDB code 3C10). Residues coordinating the metal ions are drawn as sticks. Potassium ions, zinc ion, and water molecules are shown as yellow, gray and red spheres, respectively. (f) Close-up view of the molecular surface of HDAC7 (PDB code 3C0Y) at the active site entrance. (g) Superimposition of the secondary structures of HDAC7 (PDB code 3C0Y, magenta), HDAC2 (PDB code 3MAX, light blue) and HDAC8 (PDB code 2V5W, green). The conserved aspartic acid residue located on the rim of the channel is shown (D626 and D101 for HDAC7 and HDAC8, respectively). (h) Residues of the catalytic binding site of the HDAC8–substrate complex (PDB code 2V5W). Coordination bonds are displayed as black lines, hydrogen bonds between the aspartic acid residue and the peptide substrate are shown as broken lines. (i) CCHC zinc-binding motif in the inhibitor-free HDAC7 structure (PDB code 3C0Y). Amino acids coordinating the zinc ions are shown as sticks.

and H143A in HDAC8 (corresponding to His670 in HDAC7, Fig. 2d) make the enzyme completely deficient in HDAC activity [56,61]; in addition, mutation of the same residue in HDAC1 (H141A) [62] drastically reduces deacetylase activity. Another residue involved in catalysis is a tyrosine (Y308 in HDAC2 as shown in Fig. 2d, corresponding to Y297 in HDLP as shown in Fig. 1a–c), which is conserved among bacterial and human class I and IIb HDACs, and positioned adjacent to the zinc ion on the opposite side of the two dyads. The hydroxyl group of this tyrosine is oriented toward the active site and is thought to stabilize the tetrahedral oxyanion intermediate via hydrogen bonding [40]. The tyrosine is replaced by a histidine in class IIa HDACs (Figure S1 in the supplementary material online) and the 8

side chain of this histidine is rotated away from the active site, as shown in HDAC4 and HDAC7 crystal structures (H843 in Fig. 2d). In these structures, a water molecule is found in place of the tyrosine hydroxyl (Wat2, Fig. 2d). The ‘outward’ orientation of the histidine in HDAC4 and HDAC7 is thought to be the cause of the decreased deacetylation activity of class IIa HDACs, which possibly arises from a limited stabilization of the transition state [47,49]. In addition, mutagenesis studies support the crucial role of the tyrosine in catalysis. For example, mutation of the tyrosine into phenylalanine (Y297F in HDLP and Y306F in HDAC8) [40,51] or histidine (Y298H in HDAC3) abolished enzymatic activity [63]. Likewise, mutation of the class IIa (HDAC 4, 5, and 7) histidine into tyrosine (corresponding to H843 in HDAC7, Fig. 2d) restored

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the canonical deacetylase activity, further supporting the important role of this residue for catalytic activity and transition state stabilization [49,63]. Overall, analysis of sequence and structural data suggest that residues coordinating the zinc ion, residues constituting the two dyads, and the catalytic tyrosine have a key role in enzymatic activity.

Additional metal-binding sites Apart from the pocket containing the catalytic metal ion described above, which in vivo can differ from zinc [55,64], class I and IIa HDACs structures have two additional metal-binding sites. These sites, designated as site 1 and site 2, can house potassium, calcium, or sodium ions depending on the salt that was included during ˚ away from crystallization [53]. Whereas site 1 is approximately 7 A the catalytic zinc ion and, therefore, is still in proximity of the ˚ away and so is closer catalytic pocket, site 2 is approximately 21 A to the protein surface. In site 1, a potassium ion has been found in all HDAC7 crystal structures solved so far. The ion is hexacoordinated by the carboxylate of Asp705 (residue of the buried charge relay system H669–D705), the hydroxyl of Ser728, and the backbone carbonyl oxygens of residues Asp707, His709, Asp705, and Leu729 (numbering based on PDB code 3C10) (Fig. 2e). Residues at site 1 are conserved in almost all human zinc-dependent HDACs, with the exception of Leu729 and Ser728 (Figure S1 in the supplementary material online). Site 2 also features a potassium ion in all available HDAC7 crystal structures. The potassium is hexacoordinated by the backbone carbonyl oxygens of residues Phe718, Asp721, Val724, and Phe755, and by two water molecules (numbering based on PDB code 3C10) (Fig. 2e). These residues are less conserved than those of site 1 (Figure S1 in the supplementary material online). The role of these two metal binding sites was investigated in HDAC8 [65] and found to be associated with enzyme activation or inhibition. Kinetic studies demonstrated that HDAC8 is inactive in the absence of added KCl or NaCl and that the enzyme shows higher activity when bound to K+ rather than to Na+, the former ion being generally more abundant than the latter in the cellular cytoplasm. Moreover, HDAC8 is activated by low concentrations of both KCl and NaCl, whereas it is partially inhibited by high concentrations of both salts, suggesting activating or inhibitory effects that were assigned to site 2 and site 1 occupation, respectively. Briefly, an ion bound to site 2 has been proposed to stabilize the active conformation of HDAC8 by acting as an allosteric effector, whereas an ion bound to site 1 has been suggested to reduce the catalytic activity by decreasing the pKa of His142 (corresponding to H669 in HDAC7, numbering based on HDAC8). A recent computational study also supported the finding that catalytic activity is inhibited by the presence of K+ at site 1 [66]. The role of His142 in K+ inhibition is further supported by the finding that the H142A mutant was not inhibited by K+ [65]. Interestingly, a dependence of the SAHA inhibition constant for HDAC8 on KCl concentration was noted, higher affinity being exhibited under saturating potassium conditions. These findings lead to some interesting considerations. First, the dependence of HDAC8 activity on the occupation of site 1 and 2 suggests that a similar regulation exists in other homologous HDAC isoforms. Second, deprotonation of His142 (corresponding to H131 of HDLP, Fig. 1) as a consequence of site 1 occupation suggests that the histidine is protonated in the catalytically active

REVIEWS

form of HDAC8, so questioning the proposed role for this residue as a general base [40,41]. Third, the dependence of SAHA affinity on KCl concentration suggests that HDAC enzymatic activity assays should be carried out under the same experimental conditions [67], so limiting the variability observed for a same HDAC inhibitor. It will be interesting to further investigate the role of these two metal-binding sites in other HDAC isoforms, as well as their connection with the histidine of the buried charge relay system. These studies could contribute to a better understanding of HDAC activity regulation and to the development of HDACIs.

External surface of the catalytic zinc-binding site The entrance to the channel of the catalytic site comprises portions of loop regions and, in some cases, short helices whose structure is highly variable between class I and II HDACs because of differences in length and amino acid composition. This feature leads to different entry shapes in the various HDAC isoform structures (Fig. 2f,g). In fact, these structural elements undergo significant conformational changes also within the same HDAC isoform. This feature was observed by comparing unliganded and liganded structures and complexes of the same isoform with different ligands, and from molecular dynamics simulations [68–70]. The high degree of structural variability of these amino acid sequences has been linked with their ability to recognize specific interacting partners or to detect substrates, so conferring substrate selectivity. In almost all HDAC7 structures, the protein region a7–a8 is characterized by high mobility and poor electron density, except for the inhibitor-free structure (PDB code 3C0Y, Fig. 2g). It includes an aspartic acid (residue D626 in HDAC7 and D101 in HDAC8) located in the b4–a8 loop that is strictly conserved in all class I and II HDACs (Figure S1 in the supplementary material online). The side chain of this residue is located at the entry of the catalytic pocket and has been thought to have a role in anchoring the substrate, as evidenced by interactions established with the peptide backbone in the HDAC8–substrate complex (PDB code 2V5W, Fig. 2h). The D101A, D101L, D101N, and D101E HDAC8 mutants [56], as well as the D99A HDAC1 mutant [71] and the D759A HDAC4 mutant [47] (corresponding to D626 in HDAC7, Fig. 2g) exhibit a loss of enzymatic activity compared with the wild type enzymes, indicating an involvement of this aspartic acid residue in substrate binding. Protein region a7–a8 is also rarely conserved among HDAC isoforms (Fig. 2g), and only in class IIa HDACs does it include a long insertion that participates in formation of an unexpected zinc-binding motif located superficially but close to the catalytic pocket entry (Fig. 2i). In HDAC7, this motif is formed by amino acids of the class IIa-specific insertion contained in region a7–a8 and by amino acids contained in region a1–a3. Specifically, the zinc ion is tetrahedrally coordinated by the side chains of Cys533, Cys535, His541, and Cys618, which are strictly conserved in all class IIa isoforms (Figure S1 in the supplementary material online; Fig. 2i). The so-called ‘CCHC zinc-binding motif’ is clearly visible both in HDAC7 and HDAC4 crystal structures, and hypotheses about its role in protein function have been put forward. This motif is positioned in a solvent-accessible area and is in close proximity to the deep catalytic pocket, thus forming a groove alongside the entry of the catalytic site. This location would enable it to interact with the peptide bearing the

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acetyl-lysine, so suggesting a role as substrate recruitment site. Moreover, given that class IIa HDACs are recruited in a multiprotein complex with the N-Cor/HDAC3 co-repressor [72], a role as a binding site for protein partners has also been proposed for the CCHC zinc-binding motif [47,49]. Mutagenesis data would support this hypothesis. For instance, the inactivity of the C669A/ H675A HDAC4 double mutant (C669 and H675 being equivalent to C535 and H541 in HDAC7) [47] suggests that the CCHC zincbinding motif is important in substrate binding. Moreover, this double mutation interferes with the binding of N-Cor/HDAC3 corepressor, suggesting a key role in the formation of a stable ternary complex. However, so far, biological substrates of class IIa HDACs are unknown, and the level of detected deacetylase activity is mainly the result of the HDAC3-associated partner, given that class IIa HDACs have low enzymatic activity against acetylated lysine-containing peptides. Therefore, it is generally thought that class IIa HDACs act as a bridge between SMRT/N-Cor/HDAC3 complex and transcription factors, rather than having a primary role as deacetylase enzymes. Class IIa isoforms have been shown to be highly active when tested on trifluoroacetyl-lysine that, by contrast, is a poor substrate for class I and IIb HDACs [63], thus suggesting the need to vary substrates to evaluate the activity of different HDAC isoforms. Interestingly, the exclusive presence of an accessible CCHC zinc-binding motif in all class IIa HDACs, which is adjacent to the entrance of the catalytic zinc-binding pocket and, thus, potentially able to contact the inhibitor surface, makes it a potential target site to be exploited for the design of class IIa-selective inhibitors. However, as mentioned above, in most of the HDAC7 crystal structures, the loop a7–a8 is not solved, thus making this task challenging. The region between helices a1 and a3 is 15-residues long in class II HDAC7 (H531–A545, Fig. 2i), 11 residues long in class I HDAC1– 3, and six residues long in HDAC8 (Figure S1 in the supplementary material online, Fig. 2g). This region was also proposed to mediate regulatory protein interactions in class I HDAC1–3, because these isoforms are found in large corepressor complexes. Specifically, HDAC1 and HDAC2 are recruited to NuRD, CoREST, and Sin3A complexes, whereas HDAC3 appears to be exclusively recruited from the SMRT complex or the homologous NCoR complex [73,74]. These hypotheses were confirmed by crystal structures of HDAC3 in complex with SMRT-DAD [46] (PDB code 4A69) and HDAC1 with MTA1 from the NuRD complex [43] (PDB code 4BKX), which also revealed the essential role of D-myo-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4] in class I HDAC repression complex assembly. Interestingly, Ins(1,4,5,6)P4 acts as a joint by enabling two highly basic regions from HDAC and ELM2-SANT or equivalent domains in corepressor proteins to indirectly interact, so allowing a protein assembly that would otherwise be impaired by charge repulsion. Moreover, association with corepressor complexes has been shown to cause a dramatic increase in HDAC activity and the available structural data suggest that interactions with Ins(1,4,5,6)P4 would cause conformational changes, particularly of the relatively mobile loop a12–a13 (PDB code 4A69, Fig. 3a). The loop a12–a13, including Leu266 that forms one wall of the tunnel and Arg265 that establishes a key interaction with Ins(1,4,5,6)P4 (numbering based on HDAC3), could be oriented away from the active site to provide better access 10

Drug Discovery Today  Volume 00, Number 00  February 2015

to the catalytic core. Based on sequence conservation, a similar HDAC activity regulation depending on inositol phosphate concentration could also be postulated for HDAC2. HDAC8 does not appear to be associated with corepressor complexes, and it is fully functional [75]. These insights into the assembly and activation of large multicomplexes in which HDAC1–3 take part suggest alternative ways to modulate HDAC activity, such as by targeting the Ins(1,4,5,6)P4-binding site or enzymes responsible for its synthesis. One structural feature that differentiates HDAC8 from other class I isoforms is the remarkable flexibility of its a1–a2 loop (Fig. 3b). Comparison of several HDAC8–inhibitor complexes reveals large structural differences in the active site topology, primarily mediated by the a1–a2 loop, which depend on the bound inhibitor. Interestingly, the complex between HDAC8 and ligand 9 (PDB code 1T64, Table 3, Fig. 3c) shows a second pocket adjacent to the active site, which is connected with the putative acetate release channel described below and is filled by an additional molecule of inhibitor [52]. The opening of this secondary pocket is caused by a movement of loop a1–a2 and by conformational changes of the Phe152 side chain, which, together with Tyr306 and Trp141, separate the active site from the secondary cavity (numbering based on HDAC8). The complex between HDAC8 and ligand 13 (PDB code 1VKG, Table 3, Fig. 3d) shows a deep binding groove immediately adjacent to the acetyl-lysine binding site [52]. Similarly to the complex with ligand 9, the a1–a2 loop is located away from the protein core, but the loop containing Phe152 is more distant from the zinc ion and from the Tyr306 side chain. Consequently, the wall that separates the two pockets is not present and a deep groove is formed. The structures of HDAC8 in complex with molecule 3 (PDB code 1T69, Table 3) and molecule 17 (PDB code 3SFH, Table 3 and Fig. 3e) show that loop a1–a2 moves toward the active site and the Lys33 side chain packs against the Phe152 side chain, resulting in the occlusion of the second pocket [52,54]. Moreover, Tyr306 and Trp141 are flexible and their position can affect the shape of the active site depending on the bound inhibitor. Such conformational flexibility near the catalytic pocket suggests that HDAC8 is able to recognize acetyl-lysines of structurally different substrates. Additionally, molecular dynamics simulations showed that HDAC8 can evolve among structures with one, two, or a single wide pocket in the protein surface at a relatively low energy cost [68,69]. Based on this evidence, HDAC8 selective inhibitors could be designed by trapping HDAC8 in unique conformations, such as in the case of CRA19156 (ligand 13, Table 3) or PCI34051 (ligand 23, Table 3) bearing bulky aryl linkers able to fit the wide pocket observed in the HDAC8– CRA19156 complex but not that of other HDAC isoforms [76]. Overall, although the peculiar flexibility of the HDAC8 active site can provide opportunities to design HDAC8 selective inhibitors, other HDACs exhibit a greater sequence and structural active site similarity within each class, making the design of selective inhibitors more challenging. One possible way to increase selectivity could be to exploit the structural diversity at the entrance of the active sites by rationally designing molecules bearing surface-binding motifs that complement the surface of the desired isoform.

Structural features important for binding and selectivity of hydroxamic acid inhibitors HDACIs are characterized by a common pharmacophore comprising a zinc-binding group (ZBG) coordinating the active

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(b)

(a)

(c)

L266 K33

K33

F152 R265

α12

F152

β7 W141 W141

Y306

α15

α1

α13

Y306

α1

β1

Reviews  KEYNOTE REVIEW

β8

α14 α2

(e)

(d)

(f)

F152

F152 Cap

K33

ZBG

O K33

W141

H N N H

OH

W141

O Y306 Linker

Y306

(h)

(g) D183

(i) H670 H146

H143 H180

H183

Wat2

H669

H145

Wat1 Y306

H142

D181

D267

D176 D178

H843

H709

D269 D707

Y308

D801

Drug Discovery Today

FIGURE 3

Q5 Title. (a) Electrostatic surface representation of the Ins(1,4,5,6)P4 binding site in histone deacetylase 3 (HDAC3)–SMRT–DAD complex (red and blue indicate negative and positive potential, respectively) [Protein Data Bank (PDB) code 4A69]. HDAC3 is shown in orange, and residues Arg265 and Leu266 are displayed as a stick. SMRT-DAD is shown in green and the D-myo-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4] ligand is drawn as a ball and stick. Hydrogen bonds are shown as dashed lines. (b) Conformational changes that determine the presence of two adjacent pockets (yellow, PDB code 1T64), a deep binding groove adjacent to the acetyl-lysine binding site (green, PDB code 1VKG), or a single pocket (cyan and pink, PDB codes 1T69 and 3SFH, respectively). (c–e) Surface representations of the active site entrance of HDAC8 in complex with inhibitors 9 (PDB code 1T64) (c), 13 (PDB code 1VKG) (D), and 17 (PDB code 3SFH) (E). (f) General pharmacophore of HDAC inhibitors (HDACIs) mapped onto the inhibitor SAHA. (g) Active site residues of HDAC8 bound to the peptidic substrate (light green, PDB code 2V5W) and to the hydroxamic acid Thricostatin A (yellow, PDB code 1T64). Only residues of the binding pocket are shown. (h) Zinc coordination in the HDAC7–TSA complex (PDB code 3C10). (i) Zinc coordination by the benzamide moiety in the HDAC2–2 complex (PDB code 3MAX). Only zinc ligands and catalytic residues are shown.

site zinc ion, a surface recognition motif (CAP) interacting with amino acids at the entrance of the N-acetylated lysine binding channel, and a linker domain connecting the CAP and the ZBG and fitting into the narrow hydrophobic pocket (Fig. 3f). Hydroxamic acid is among the most effective and widely present ZBG in HDAC inhibitors currently under development [23,25]. However, this group generally confers poor isoform selectivity. In addition, it displays some disadvantages, such as poor oral absorption, metabolic and pharmacokinetic problems because of glucuronidation, sulfation, and enzymatic hydrolysis that lead to a short in vivo half-life [77,78]. Moreover, hydroxamates can give rise to multiple off-target effects resulting from the coordination of other metalloenzymes, leading to undesirable adverse effects, such as nausea, thrombocytopenia, anemia, and other metabolic issues

[79]. Therefore, there is growing interest in replacing the hydroxamate group with a weaker ZBG, with the double goal of reducing adverse effects and improving HDAC isoform selectivity. The high efficacy of the hydroxamic acid group has been proposed to derive from both its strong metal binding ability and from its pKa value, which is around 9–9.5. Such a pKa would guarantee that the hydroxamate is neutral in solution at physiological pH, facilitating cell membrane permeation. However, upon coordination of the zinc ion, the pKa of hydroxamic acids would be reduced by approximately three units, the acidic proton being transferred to the adjacent conserved histidine (H669 in HDAC7) [80]. Interestingly, computational studies [41,66,81–83] indicate that the anionic hydroxamate chelates the zinc ion in a bidentate fashion with geometries close to the experimental ones. However,

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whether the hydroxamic acids bind HDAC by adopting a neutral or anionic form is still matter of debate, and further investigations will be necessary to draw more definitive conclusions. Most hydroxamates are pan-inhibitors, because they are unable to discriminate isoforms belonging to the same or different HDAC classes. For instance, vorinostat (SAHA) and trichostatin A (TSA) are potent inhibitors of class I and class IIb isoforms, whereas relatively weak inhibitory activity was observed for class IIa isoforms (Tables 3 and 4). A comparison of crystal structures of different HDAC isoforms helps rationalize the different potency trend displayed by the hydroxamic acids. The crystal structure of the HDAC8–TSA complex (PDB code 1T64) is a representative binding mode of hydroxamic acids in class I and class IIb human HDACs, given the high conservation of residues of the catalytic site. The hydroxamate coordinates the zinc ion in a bidentate fashion by using its ˚ and carbonyl and hydroxyl oxygen atoms (at distances of 2.2 A ˚ 2 A from the zinc, respectively), the latter group replacing the active site water molecule (Wat1, Fig. 3g). Moreover, the hydroxamate hydrogen bonds to residues His142, His143, and Tyr306 proposed to be involved in catalysis (Fig. 3g). A similar situation is observed in the HDAC2–SAHA complex (PDB code 4LXZ) as well as in the inhibited bacterial structures of HDLP and HDAH in complex with hydroxamic acids. In the HDAC8–TSA complex, the fivecarbon-long aliphatic chain connecting the hydroxamate with the cap group fits into the hydrophobic channel, making van der Waals interactions with Phe152, Phe208, and Gly151. At the other end of the aliphatic chain, the aromatic dimethylamino-phenyl group contacts residues at the rim of the pocket, giving p-p stacking interactions with Tyr100. Among class I HDACs, TSA and SAHA display lower inhibitory activity toward HDAC8 (Table 4). By analyzing structural differences between HDAC1–3 and HDAC8, Vannini et al. [53] provided an explanation based on the conformational variability of the loop connecting a1 and a2 (see above). In particular, in HDAC8, the loop is shorter than in HDAC1–3 and unable to interact with the cap group of the inhibitor. Instead, TSA is able to contact the corresponding loop

of HDLP (PDB code 1C3R) and probably also that of HDAC1–3, because their shape is similar to that of HDLP. Another possible explanation based on molecular dynamics simulation results is that the HDAC8 active site was found to be surprisingly malleable [68,69] and in dynamic equilibrium among different conformations, characterized by either one, two, or a single wide pocket, as discussed in detail above. Given these conformational changes, small hydroxamates such as TSA and SAHA might lose several hydrophobic contacts in the linker region [71]. By contrast, computational studies predicted that PCI34051 (ligand 23, Table 3) binds to the conformation characterized by a single wide pocket that is unique to HDAC8 [68], placing an aromatic group in the deep groove adjacent to the catalytic site, thus resulting in higher potency and selectivity (Table 4). Interestingly, another study [64] showed that the affinity of HDAC8 for SAHA depends on the type of bivalent catalytic metal ion, affinity being in the order Co(II) > Fe(II) > Zn(II). The study suggested that, in vivo, HDAC8 requires Fe(II), whose intracellular concentration is higher than that of Co(II) [64]. TSA and SAHA are weaker inhibitors of class IIa HDACs compared with class I and IIb HDACs (Table 4). As previously described above, class IIa HDACs have a histidine residue (H843 in HDAC7) in place of the catalytic tyrosine, and the histidine is rotated away from the catalytic site. Zinc coordination is monodentate, the ˚ from hydroxyl and carbonyl oxygens of TSA being 2.1 and 2.6 A the zinc, respectively. The hydroxyl group, similarly to hydroxamates bound to class I HDAC structures, replaces the catalytic water molecule (Wat1, Fig. 3g) and gives hydrogen bonds with the side chains of His669 and His670 (numbering based on HDAC7; Fig. 3h). In addition, TSA hydrogen bonds with the water molecule Wat2 (Fig. 3h).

Class I selective inhibitors: exploration of the acetate release channel Benzamide-containing HDACIs Compared with hydroxamates, HDACIs with a benzamide ZBG proved to be more selective for class I HDACs [67,84] (e.g., ligands

TABLE 4

Potency of selected inhibitors on different HDAC isoformsa. Inhibitor

Class I, IC50 (mM) HDAC1

HDAC2

Class IIa, IC50 (mM) HDAC3

HDAC8

Molecule 9 (TSA)

0.0049

0.0123

0.00141

0.213

Molecule 3 (SAHA)

0.0607

0.251

0.0186

0.827

Molecule 23 (PCI34051)

4

Molecule 24 (MS–275)

0.243

Molecule 4 Molecule 16 Molecule 17

0.0101 >30 1.7

>50 0.453 0.0563 >30 3.9

>50 0.248

0.01

HDAC4 2.4 >10

HDAC5 0.871 >10

Class IIb, IC50 (mM) HDAC7 0.663 >10

HDAC9 3.7 >10

HDAC6

HDAC10

0.000721

0.0116

0.00944

0.0291









>10

>10

>10

>10

>10

>10

2.9

>10

13 >10

>10

>10

>10

>10

>10

3.4



0.2









>30





0.09









>30



9.4

1.4

0.05

0.03

0.11

0.19

1.9



0.36

0.02

0.004

0.03

0.04





>10

Molecule 7

21



Molecule 8

32

43

12

Molecule 10

>100

>100

>100

4.2

0.157

0.097

0.043

0.023

8.2

>100

Molecule 11

>100

>100

>100

2.02

0.124

0.095

0.046

0.005

9.32

>100



0.32







0.31



Molecule 5 a

5.5



44% inh at 5 mM

Based on [35,38,40,44,66,74].

12

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4 and 24 in Table 4). To date, only HDAC2 has been co-crystallized with benzamide-containing inhibitors (PDB codes 3MAX and 4LY1). In the crystal structure of the complex between HDAC2 and ligand 2 (PDB code 3MAX, Table 3) the zinc ion is coordinated by the amino nitrogen and by the amide oxygen of the inhibitor (with distances of 2.1 and 2.5 A˚ from the zinc, respectively). Furthermore, the amino nitrogen hydrogen bonds with the side chains of His145 and His146, and the amide oxygen hydrogen bonds with the hydroxyl of Tyr308 (Fig. 3i). Interestingly, before zinc chelation occurs, an intramolecular hydrogen bond between the aniline amine and the amide carbonyl must be broken to permit successful zinc chelation, and a desolvation penalty is paid for this rearrangement. These data, which might help explain the slow binding properties of the benzamides as opposed to the rapid kinetic (a)

rates of hydroxamates, should be considered when therapeutic utility is being evaluated [45]. The aromatic linker of the ligand 2 packs in the lipophilic portion of the tube-like pocket interacting with the hydrophobic residues Phe155, Phe210, and Leu276 (numbering based on HDAC2). The p-phenyl substituent fits into an internal cavity located adjacent to the catalytic site. This internal cavity, first described in HDLP structures and identified as a 14-A˚ cavity [40] and subsequently named the ‘foot pocket’ in HDAC2 structures, connects the active site with a solvent-exposed surface and is lined primarily by hydrophobic residues, such as Tyr29, Met35, Phe114, and Leu144 (HDAC2 numbering based on PDB code 3MAX, Fig. 4a) [44]. This internal cavity was observed in all class I HDACs but not in class IIa. Docking calculations of small molecular probes into the

(b)

(c) Y28 α2

Y29 M35 L144 R16

Y107

R39 F114

R27

α1

S113

W141

R39 α1

α2 L144

α3

(d)

(e)

(f)

H143

L31 α1

I34 H180

R37

R37 Y306

H142

Wat2

D267

D178

S138

α2

W141

W141

(g)

(h)

(i)

K33 α1

α1

M35

W141 α2 L144

W141

α2 W141 R37

R37

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FIGURE 4

Q6 Title. (a) Surface representation of the foot pocket in histone deacetylase 2 (HDAC2) [Protein Data Bank (PDB) code 3MAX]. Some of the residues lining the foot pocket are shown in stick. (b) Ribbon superimposition of HDAC2 (PDB code 3MAX) and HDLP (PDB code 1C3R) colored light blue and red, respectively. The HDAC2 foot pocket is shown as mesh surface. Arg27 and Arg16 in HDLP, and Arg39 and Tyr28 in HDAC2 are displayed in stick. (c) Ribbon superimposition of HDAC1 (PDB code 4BKX), HDAC2 (PDB code 4LY1), HDAC3 (PDB code 4A69), and HDAC8 (PDB code 1T64) in green, light blue, orange, and yellow, respectively. Ser113 in HDAC1, Leu144 in HDAC2, Tyr107 in HDAC3, and Trp141 in HDAC8 are drawn as sticks. Molecule 4 is shown as a ball and stick. (d) Zinc coordination by a-amino-ketone moiety in the complex between HDAC8 and ligand 17 (PDB code 3SFH). Only residues of the catalytic machinery are shown in stick. Inhibitor is displayed as a ball and stick. (e) Surface representation of the internal cavity of HDAC8 (PDB code 3SFH). Residues dividing the internal cavity in two subchannels are shown as a stick. (f) Surface of the internal cavity of HDAC8 (PDB code 1T69) in the closed state with Trp141 in the ‘in’ conformation (shown as solid surface) superimposed to the surface corresponding to the open state with Trp141 in the ‘out’ conformation (shown as mesh surface). (g) Ribbon superimposition of HDAC1 (PDB code 4BKX), HDAC2 (PDB code 4LY1), HDAC3 (PDB code 4A69), and HDAC8 (PDB code 3SFH) in green, light blue, orange, and pink, respectively. Met35 and Leu144 in HDAC2, Lys33, and Trp141 in HDAC8 are drawn as a stick. Molecule 17 is shown as a ball and stick. (h) Surface representation of the internal cavity of HDAC8 (PDB code 1T64). Arg37 and Trp141 are drawn as a stick; inhibitors are shown as a ball and stick. (i) Internal cavity of HDAC8 in another crystal structure (PDB code 1VKG). www.drugdiscoverytoday.com 13 Please cite this article in press as: Micelli, C., Rastelli, G. Histone deacetylases: structural determinants of inhibitor selectivity, Drug Discov Today (2015), http://dx.doi.org/10.1016/ j.drudis.2015.01.007

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internal cavity of HDLP suggested that, after lysine deacetylation, the protonated lysine side chain would leave the catalytic site via ˚ tube-shaped hydrophobic channel, whereas the acetate the 11-A ˚ cavity [85]. In HDLP, acetate would be evacuated through the 14-A exit would be accomplished by rearranging a loop between helices a1–a3 (corresponding to loop a1–a2 in HDAC2) (Fig. 4b). In addition, residues Arg27 and Arg16 (corresponding to Arg39 and Tyr28 in HDAC2) were proposed to have a role in guiding the acetate outside of HDLP by forming hydrogen-bonding interactions [85]. Class I HDACs have an internal cavity with the same structure of HDLP. Sequence and structural similarity between ˚ cavity has a similar HDLP and class I HDACs suggests that the 14-A function in these enzymes. A subsequent molecular dynamics investigation on HDAC1 and HDAC2 suggested that three residues (Y22, Y24, and F109 in HDAC1, corresponding to Y27, Y29, and F114 in HDAC2) were involved in an ‘aromatic gating’ mechanism that controls water exchange [86], a finding that supports the previous hypothesis formulated for HDLP. Recent mutagenesis studies performed on HDAC1 [87] confirmed the proposed role of the cavity in acetate binding and release. Moreover, they supported a crucial role of the amino acids lining this cavity, as evidenced by the loss of catalytic activity of HDAC1 after mutation of residues located in the internal cavity. Among class I HDACs, HDAC8 is the least conserved and its internal cavity shows some structural variations compared with HDAC1-3. These structural changes could explain the lower inhibitory activity of MS-275 (molecule 24, Table 4) toward HDAC8 with respect to HDAC1–3 (Table 4). Specifically, the lower HDAC8 activity has been attributed to the replacement of a leucine in HDAC1–3 (Leu144 in HDAC2) with a tryptophan (Trp141 in HDAC8) residue. On the one hand, the bulkier tryptophan side chain would restrict the space available to accommodate the aniline ring, thus precluding an effective zinc coordination (Fig. 4c) [88]. On the other hand, in some HDAC8 crystal structures (e.g., PDB codes 1T69 and 3SFH), the tryptophan adopts an ‘out’ conformation in which the indole ring is oriented away of the zinc ion, suggesting that this residue is more flexible than was previously thought (Fig. 3b). Therefore, the weaker inhibition of HDAC8 by compound MS-275 cannot be explained exclusively on account of the Leu to Trp replacement. For example, at variance with MS-275, molecule 4 (Fig. 4c) carrying a 2-thienyl substituent is inactive on both HDAC8 and HDAC3. The lack of activity on HDAC3 has been attributed to the replacement of a serine (Ser118 in HDAC2 and Ser113 in HDAC1) with a tyrosine (Tyr107 in HDAC3) residue, whose bulkier side chain would restrict the space accessible to substituents (Fig. 4c) [67]. Thus, as a result of sequence variation, the shape and physicochemical nature of the internal cavity can undergo important remodeling, which in turn affects selectivity.

a-Amino-ketone-containing HDACIs Compounds featuring an a-amino-ketone ZBG were recently discovered by means of high-throughput screening, and showed higher selectivity for class I HDACs [54]. Two inhibitors with this functional group, compounds 16 and 17 (Tables 3 and 4), were cocrystallized with HDAC8 (PDB codes 3SFF and 3SFH, respectively). The zinc ion is coordinated by the amino nitrogen and the adjacent carbonyl oxygen, with distances of 2.3 and 3 A˚, respectively. In addition, hydrogen bonds between the amino group and the His142 side 14

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chain, and between the carbonyl oxygen and water molecule Wat2, are formed (Fig. 4d). In both crystal structures, inhibitors establish van der Waals contacts with His143, Gly151, Phe152, His180, and Phe208 residues of the 11-A˚ channel, and hydrophobic interactions with Ile34, Trp141, Gly303, Gly304, and Tyr306 residues of the internal cavity (numbering based on HDAC8). The HDAC8 internal cavity differs in size and shape from that of HDAC1–3 and HDLP described in above. Specifically, it is characterized by two subchannels, a 12-A˚ ‘release’ channel and a 14-A˚ ‘disposal’ channel, delimited by residues Leu31, Ile34, Arg37, Ser138, and Trp141 (Fig. 4e). Based on this structural information, a gating mechanism for the control of acetate release has been proposed [54], according to which residues Arg37 (Arg39 in HDAC2) and Trp141 (Leu144 in HDAC2) would guide acetate release by means of a concerted mechanism. Specifically, Arg37 would reposition the indole of Trp141, which in turn would close the 12-A˚ release channel when acetate moves into the 14-A˚ disposal channel. The release of acetate outside of HDAC8 would be mediated by rearrangement of the Tyr18, Tyr20, Ala38, Val41, and His42 side chains, which are close to the external surface and separate the 14 A˚ channel from solvent. Phosphorylation of Ser39 by PKA was found to inhibit HDAC8 enzymatic activity [89]. This effect was attributed to a possible impairing of the acetate release because of charge repulsion, and further supports the hypothesis that this internal cavity serves for acetate release. Water molecules could reach the active site either via the 11-A˚ channel, as suggested by Whitehead [54], or through an exchange with the acetate ion via the internal cavity, as suggested by Vannini et al. [53]. The role of Arg37 in HDAC8 was experimentally validated with mutagenesis studies, which demonstrated that an arginine side chain is crucial for catalytic activity and acetate binding affinity [90]. These results are consistent with the mutagenesis experiments performed on HDAC1 [87], which confirm the crucial role of this residue in catalysis. Superimposition of different HDAC8 crystal structures reveals that Trp141 is flexible (Fig. 3b). Notably, in some HDAC8 structures (e.g., PDB codes 2V5X, 1T64, 1VKG, and 1T67), Trp141 adopts exclusively the ‘in’ conformation, characterized by an indole ring oriented toward the catalytic zinc ion. In other structures (e.g., PDB codes 3MZ4, 3MZ7, 3EW8, and 3EZP), Trp141 adopts the ‘out’ conformation, in which the indole is rotated outwards, whereas in other structures (e.g., PDB codes 1T69, 3F06, and 3MZ6), Trp141 adopts both the ‘out’ and ‘in’ conformations. The acetate channel is open only when Trp141 is in the ‘out’ conformation (Fig. 4f). The higher inhibitory activity of a-amino-ketone ligands toward HDAC8 compared with other class I HDACs can be rationalized by analyzing amino acid differences in the acetate release channel. Among these, replacement of tryptophan 141 with a leucine in HDAC1-3 (Leu144 in HDAC2) results in a loss of aromatic–aromatic interactions. In addition, a methionine side chain (Met35 in HDAC2) of HDAC1–3, which corresponds to a lysine (Lys33) in HDAC8, protrudes inside the internal cavity and impairs the effective fit of the benzene ring, thus explaining the observed loss of binding affinity (Fig. 4g). Notably, in the complexes between HDAC8 and ligands 9 or 13 (PDB codes 1T64 and 1VKG, respectively), the acetate release channel is not directly connected to the catalytic site as observed in the complexes with molecules 17 or SAHA (PDB codes 3SFH and 1T69, respectively), but is more solvent exposed (Fig. 4h,i). This difference is a

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consequence of the structural variations of the enzyme surface primarily mediated by the loop a1–a2, as discussed above. Overall, HDAC8 exhibits significant conformational variability in the active site, which probably enables the binding of acetyl-lysines from different protein substrates. Given that these structural changes affect the acetate release channel, whether these different HDAC8 conformations are catalytically competent remains to be established. Notwithstanding, such conformational variability makes HDAC8 a promising target for achieving isoform selectivity.

Selective class IIa inhibitors: exploration of the lower pocket Trisubstituted cyclopropane-containing HDACIs Structure-based drug design strategies identified trisubstituted cyclopropane as a suitable scaffold to increase selectivity for class (a)

IIa HDACs [48]. HDAC4 has been co-crystallized with two cyclopropane-based inhibitors (ligand 7, PDB code 4CBT and ligand 8, PDB code 4CBY, Table 3) [48]. The hydroxamate moiety of these inhibitors displays bidentate coordination with the zinc ion (the carbonyl and hydroxyl oxygens being 2 and 2.2 A˚ distant from the zinc ion, Fig. 5a), at variance with other HDAC4 and HDAC7 crystal structures in which coordination is monodentate (Fig. 3h) [47,49]. Hydrogen bonds between the hydroxamate and His802 and His803 (HDAC4 numbering) are formed as in class I isoforms. The selectivity of compounds 7 and 8 for class IIa isoforms (Table 4) has been attributed to the unique presence of a lower pocket in class IIa isoforms [48]. In HDAC4, this pocket is constituted by portions of loops a5–a9, a10–a17, and a2–a3, including Pro676, Glu677, Pro800, Phe812, and His976 (numbering based on PDB code 4CBY) (Fig. 5a,b). Whereas class I HDACs have an acetate release channel immediately adjacent to

(b)

(c) α7

L943 F812

α2

P676

α8

F679 L810

H976

H803

α1

β10

H843

β5

E677 H802

R681 α17

α3 P667

P800

(d)

(e)

(f)

H159 R37 H158 L139 Y303

P156

P155

H332

R154 W141

(g)

L31

Y111

W141

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FIGURE 5

Q7 Title. (a) Surface representation of the lower pocket of histone deacetylase 4 (HDAC4) [Protein Data Bank (PDB) code 4CBY]. Residues constituting the lower pocket are drawn as a stick. Inhibitor is shown as a ball and stick. Hydrogen bonds are shown as dashed lined, coordination bonds as black lines. (b) Ribbon superimposition of two HDAC4 structures (PDB codes 4CBY and 2VQJ), in blue and yellow, respectively. Secondary structure elements participating in lower pocket formation are labeled. (c) Surface representation of HDAC7 lower pocket (PDB code 3ZNR). Residues contacting the inhibitor are shown as a stick. Inhibitor is drawn as a ball and stick. (d) Surface representation of HDAC4 lower pocket (PDB code 2VQJ). HDAC4 is in yellow and residues 154–156, 158–159, and 332 are labeled. The HDAC1 ribbon is in green and residues 139 and 303 are labeled. (e) Surface of the acetate release cavity (mesh) and the lateral internal channel (solid) in HDAC8 (PDB code 1T69). Inhibitor is shown as a ball and stick, the zinc ion is displayed as a gray sphere. (f) Surface of the lateral internal channel when Trp is in the ‘out’ conformation (mesh) and in the ‘in’ conformation (solid) in HDAC8 (PDB code 1T69). Arg 37 and Trp141 are drawn as sticks. (g) Surface of the lateral internal channel in the open state (solid, PDB code 3MZ4) and in the closed state (mesh, PDB code 3EZT). Tyr111 and Leu31 are in magenta for the open state and in light blue for the closed state. www.drugdiscoverytoday.com 15 Please cite this article in press as: Micelli, C., Rastelli, G. Histone deacetylases: structural determinants of inhibitor selectivity, Drug Discov Today (2015), http://dx.doi.org/10.1016/ j.drudis.2015.01.007

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the catalytic site (Fig. 4a,e) [44,54], the channel is absent in class IIa HDAC structures. This difference is probably the result of conformational changes arising from the presence of two consecutive proline residues (Pro799 and Pro800) in HDAC4 instead of two glycine residues in class I HDACs (Figure S1 in the supplementary material online). Moreover, the tyrosine to histidine replacement observed in class IIa HDACs can be exploited to increase selectivity for this class by designing ligands that fill the empty space left by the outwardly rotated histidine (Fig. 5a). These differences are responsible for formation of the lower pocket, which has a completely different size and shape compared with the acetate release channel. The two co-crystallized cyclopropane-based inhibitors occupy the lower pocket with a phenyl ring that displaces wat2, establishes favorable edge-to-face p-stacking interactions with Arg681 and Phe812, and makes hydrophobic interactions with Pro676, Pro800, and Leu943. In contrast to the other reported HDAC4 structures, regions a1–a3 and a7–a8 of the HDAC4– 8 complex (PDB code 4CBY) adopt a closed conformation (Fig. 5b) as in the inhibitor-free HDAC4 (PDB code 2VQW) and HDAC7 crystals structures. This closed conformation is considered to be competent for catalytic activity [47,48].

Trifluoromethyloxadiazolyl-containing HDACIs High-throughput screening identified HDACIs with a trifluoromethyloxadiazolyl (TFMO) moiety preferentially inhibiting class IIa isoforms (molecules 10 and 11, Tables 3 and 4) [50]. Crystal structures of HDAC7 in complex with inhibitors 10 and 11 (PDB code 3ZNR and 3ZNS, respectively) revealed an unexpected binding mode for this class of compounds. The fluorine and the oxadiazole oxygen atoms of the TFMO scaffold bound weakly the catalytic zinc ion (with distances of 3 and 2.7 A˚, respectively), whereas the remainder of the molecule adopted a U-shaped conformation and placed the phenyl ring in the lower pocket typical of class IIa HDACs (Fig. 5c). The U-shaped conformation is stabilized by edge-to-face stacking interactions with Phe679 and hydrophobic interactions with Leu810 (Fig. 5c). Overall, these data suggest that a strong metal chelating group is not required to achieve high potency, provided that additional ligand–protein interactions are formed.

Trifluoromethylketone-containing HDACIs HDACIs containing a trifluoromethylketone (TFMK) moiety have been demonstrated to inhibit preferentially class II isoforms (e.g., molecule 5, Tables 3 and 4) [91–93]. So far, these inhibitors have been co-crystallized only with human HDAC4 (PDB code 2VQJ) and bacterial HDAH (PDB code 2GH6). In the complex between HDAC4 and compound 5, the inhibitor coordinates the zinc ion bidentately by using the hydroxyl oxygens originating from the hydrated form of the trifluoromethyl ketone (with distances to the zinc of 2.1 and 2.4 A˚, Fig. 5d). The trifluoromethyl group occupies the back of the lower pocket similarly to the trifluoromethyl group of molecules 10 and 11 described previously. The greater potency on class II HDACs can be ascribed to more favorable interactions between the trifluoromethyl group and proline 156 (HDAC4 numbering), which is replaced by a glycine in class I HDACs (Figure S1 in the supplementary material online).

Definition of an additional internal channel in HDAC8 Comparison of available crystal structures suggests the presence of an additional internal cavity that appears to be present only in 16

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HDAC8. This channel originates at the bottom of the active site and develops laterally to the acetate release cavity (Fig. 5e). This lateral internal channel is formed by portions of loops a1–a2, a5– a6, and b3–a7, and part of helices a1 and a6. It includes residues Met27, Ser30, Leu31, Ala32, Lys33, Ile 34, Asp101, Pro103, Thr105, Gly107, Ile108, Tyr111, Trp141, Phe152, and Tyr154. The channel is mainly hydrophobic and is in communication with the acetate release cavity described above. Importantly, the lateral internal channel is open only when Trp141 adopts the ‘out’ conformation. Therefore, this conformation appears to determine the opening of both the lateral internal channel (Fig. 5e,f) and of the acetate release cavity (Fig. 4f). In addition to the role of Trp141, the presence of the lateral internal channel is also dependent on the conformation of residues Tyr111 and Leu31. In particular, the channel is open when Tyr111 adopts a solvent-exposed conformation (e.g., as in PDB code 3MZ4; Fig. 5g). Although its function is unknown, it can be hypothesized that the channel serves as an alternative way for acetate release or as a preferential way for water uptake, being connected to the catalytic active site. Overall, the existence of this lateral internal channel might provide mechanistic insights into the deacetylation reaction as well as an additional opportunity to design inhibitors selective for HDAC8.

Concluding remarks HDACs are validated drug targets in oncology. Most of the current research efforts in this field are focused on developing isoformselective compounds, to improve toxicity profiles. The availability of crystal structures of several HDAC isoforms has provided a major contribution to understand isoform selectivity. HDAC8 has proven to be the most promising target to achieve selectivity, although it is so far unclear what disease indication such inhibitors are going to be useful for. Selectivity is mainly the result of the high plasticity of its catalytic channel that enables the binding of molecules otherwise unable to fit the more rigid channel of other HDAC isoforms. Moreover, HDAC8 exhibits an acetate release channel that is structurally different from that of HDAC1–3, and a lateral internal channel that appears to be unique to this isoform. Based on the available structural data, isoform selectivity could be achieved by designing ZBGs bearing substituents able to make favorable and specific interactions into the foot pocket (HDAC1–3) or into the lower pocket (class IIa HDACs) of a given isoform. For instance, replacement of serine 107 by tyrosine in HDAC3 results in a spatially restricted foot pocket that can be exploited to design compounds selective for class I HDACs. Moreover, the choice of surface-binding motifs that make specific contacts with the external characteristic grooves of the desired isoform might be useful to gain selectivity. Additional opportunities could arise from targeting specific complexes between HDACs and other interacting partners that are necessary for efficient deacetylase activity [e.g., the Ins(1,4,5,6)P4 binding site for HDAC1–3, or the CCHC zincbinding motif for class IIa HDACs]. So far, structures showing the location of the acetate ion produced by the deacetylation reaction into the putative acetate release channel have not been solved. The same holds true for structures showing the exit of this channel. Isoform-selective inhibitors could be obtained by targeting the exit of the acetate

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release channel, thus interfering with the catalytic cycle. Finally, structural data for some HDAC isoforms are still missing. Such information, when available, will greatly facilitate the design of isoform-selective inhibitors.

REVIEWS

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.drudis.2015.01. 007.

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Histone deacetylases: structural determinants of inhibitor selectivity.

Histone deacetylases (HDACs) are epigenetic targets with an important role in cancer, neurodegeneration, inflammation, and metabolic disorders. Althou...
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