Invited reviewInside HDAC with HDAC inhibitors
Graphical abstract
This review focuses on the synthetic and biological results obtained by several research groups with HDAC inhibitors designed from structural analyses of the proteins active sites and mechanistic proposals.
Introduction
Eukaryotic DNA is packed in a high level structure called chromatin, resulting from the assembly of an elementary unit, the nucleosome, an octameric structure obtained from eight proteins called histones. About 150 base pairs of DNA are sequentially folded around one nucleosome unit. The interactions between the DNA and the histones terminal tails control the activation or repression of gene transcription and several chemical modifications can change the status of histones with impact on gene transcription. In particular the N-ε-acetylation of lysine residues found in histones is equilibrated by two enzymes: the histone acetyl transferases (HAT) and the histone deacetylases (HDAC). Excessive deacetylated level of these histones has been linked to cancer pathologies by promoting the repression of tumor regulatory genes. HDAC inhibitors (HDACi) activities resulted in an increase of the acetylated level of histones, promoting in turn the re-expression of silenced regulatory genes. These compounds are a promising family of small molecule-based anti-cancer therapies.
Several reviews described the synthetic approaches to such inhibitors, [1] their classification in the more general concept of epigenetic drugs, [2] and their applications in several clinical trials, alone or in combinations with other anti-cancer agents based on dual exploitation of convergent biological pathways [3]. Marmorstein published in 2001 a review describing the HDAC differences in relation to deacetylation mechanisms [4]. A recent review presented a collection of data concerning the isoform selectivity of small molecule HDAC inhibitors based on a biological analysis of preclinical to phase II/III compounds presented in Table 3 [5] completed by additional data. The purpose of this review is to present recent information on the structural features of the HDAC proteins and their impact on the design of their inhibitors by an in depth analysis of X-ray crystallographic data or, when available, molecular modelling. A PDB files viewer program was developed, equivalent to other viewer package, with some new specific graphical functionalities, inspired from structural features of the zinc dependant HDAC family. All graphical representations of HDAC presented in this review were obtained with this software (except Fig. 11a from Pymol). The purpose was to present a clear understanding of the several results published in the literature concerning the molecular analysis of these proteins and their inhibitors. After a short presentation of the structural composition of the HDAC family and their implications in cancers, knowledge on the catalytic core will be presented, with implications on the development of inhibitors.
The eighteen HDACs identified in humans are classified in four classes (Table 1). Classes I, II and IV are zinc dependent metalloproteins and class III is NAD+-dependent. HDAC1, 2, 3 and 8 define the class I group, which is mostly nuclear, smaller in size (350–500 amino acids) and is more importantly implied in cancer progression. Class II has two subgroups. Class IIa is represented by HDAC4, 5, 7 and 9 while class IIb consists of the two HDAC6 and 10. Finally HDAC11 is in its own class IV. Class III HDACs, with some X-ray structures available, are the NAD dependant group of the seven sirtuins SIRT1-7, which are not beyond the scope of this review. In Table 1 are presented the several differences in zinc dependant classes I, II and IV HDACs [6], [6](a), [6](b) according to size (number of amino acids), cellular distribution and known interactions with transcription factors. Table 2 summarizes the implication of HDAC in several types of cancers and the expression level. HDACs are diversely implied in cancer progression and other diseases, [7] and identifying specific biomarkers to maximize patient therapies is an emerging area [8]. In this respect, more selective HDAC inhibitors (HDACi) than the actual panHDAC inhibitors clinically used has emerged as a priority. Chart 1 presents several HDAC inhibitors, classified according to chemical structures, followed by the well accepted pharmacophore for such inhibitors, comprising the zinc binding group chelating the zinc atom in the active site, a linker that accommodate the tubular access of the active site, and a cap group for interactions with the external surface, connected by a small connecting unit to the linker. Table 3 presents data on the selectivity of HDACi for phase II/III molecules. Among the four classes of HDAC, the zinc dependant classes were initially the most studied. Chart 2 summarizes the collection of inhibitors bound to HDLP or HDAC4, 7, 8 and HDAH for which X-ray were obtained, with additional data for HDAC8 variants. Finnin [9] first described the X-ray structure of a histone deacetylase like protein (HDLP, PDB code 1C3P). This description was also accompanied by two models of bound HDLP, with TSA 1 (Trichostatin A, 1C3R, Chart 1) and SAHA 2 (Suberoyl Anilide Hydroxamic Acid, 1C3S, Chart 1) respectively. From these results, a first structure–activity relationship was proposed, with a possible mechanism for the activity of these zinc dependant enzymes.
This pioneering work was followed by the structures obtained by Vannini [10], [11] and Somoza, [12] with inhibitors bound to HDAC8 (PDB 2V5W, 2V5X, 1W22, 1VKG, 1T64, 1T67, 1T69). Min published X-ray crystallographic data for HDAC7 (PDB 3C0Z, 3C10), [13], [13](a), [13](b) with first deposited structure data by Schafer et al. (PDB 2VCG) [14]. The latest published X-ray crystallographic data were obtained by Bottomley for HDAC4 catalytic domain and an example of trifluoromethylketone inhibitor 24 bound (Chart 2) [15]. The catalytic domain of HDAC6 was also described (PDB 3C5K) [16]. Finally, X-ray crystallographic data from a bacterial histone deacetylase homologue (HDAH) were published by Schafer et al. and Nielsen et al. [17] (PDB 2VCG, 2HG6), with the latter one giving another example of a trifluoromethylketone inhibitor 15 bound (Chart 2). Chart 3 presents some inhibitors that were modelled bound to HDACs, either from X-ray HDAC structures (HDLP) or with modelled HDAC (HDAC1 and 6).
Section snippets
On the zinc dependant mechanism of lysine deacetylation
Finnin proposed a first explanation of the catalytic activity of these enzymes (Scheme 1). For HDLP, the zinc atom is surrounded by two histidine–aspartic acid dyads (His131-Asp166 and His132-Asp173), a tyrosine (Tyr297) for proton assistance, and is stably coordinated to two aspartic acids (Asp258, Asp168) and one histidine (His170). This environment is common to all zinc dependant HDAC, with some possible residue changes (Table 4). Class I/II have in common two His–Asp relay system
Background
Applications of the afore mentioned theoretical work can be found in several reviews describing hydroxamate or non-hydroxamate HDAC inhibitors [28], [29]. Based on direct zinc coordination, thiols, disulfides [30] and thioesters [31] were reported with some HDAC selectivity [32], [32](a), [32](b) or alphaketoamides [33], [33](a), [33](b). Inspired from hydroxamates, retrohydroxamates can also be potent HDAC inhibitors. More original compounds were also described like alkyl bispyridinium dienes
Background
The aspects of the zinc atom participation in the mechanism of lysine deacetylation is associated to the way the lysine (or the inhibitors) are able to enter the active site and reach the zinc atom. Thus, the structural impact of the tubular access and its correlation with inhibitor structures was studied by several groups. The acetylated lysine can be accommodated in the active site by a 11 Å deep pocket. The residue composition of the tubular access is almost conserved in HDAC (Table 5). For
Background
In the first described X-ray crystallographic structure of HDAC homologue HDLP, [9] an internal cavity was described. This internal cavity was originally expected to allow the elimination of the resulting acetate ion after lysine hydrolysis, and to help water circulation for the reaction to take place once the lysine is bound to the active site (Fig. 8A). The potential for this secondary cavity for the design of selective HDAC inhibitors was studied later, with the description of other X-ray or
Background
The connection unit in HDAC inhibitors is generally a sp2 hybridized group like ketone, which is found in TSA or SAHA. Only few targeted studies were made on this part of the pharmacophore as it is usually integrated to the cap group.
Modulation of the connection unit
In our work on TSA derivatives, we found that indanone or 2,3-dihydrobenzofuran-2-one 32 (Chart 3) can be good connection units [57]. The furanone ring does not generated specific contact at the pocket entry, but interactions were found more critical compared to
Background
From the X-ray crystallographic structure of TSA bound to HDLP, Richon et al. [9] demonstrated that the aromatic moiety of the TSA is an important feature to obtain higher inhibitory activities. In TSA, the aromatic group interacts with an external shallow pocket resulting from spatial organization of His170, Gln192, Tyr196, Ala197 and Leu265. In a recent work we confirmed the importance of this dimethylamino group that interacted with hydrophobic proline rich regions in HDAC1 and HDAC6 [57].
3-D QSAR studies
The alyklhydroxamate family was particularly investigated with compounds 72–75 as examples (Chart 6).
Study on indole alkylhydroxamic acids was published using CoMFA and CoMSIA [82]. A key parameter in the inhibitory activity of 70 and its specificity for class I may arise from stabilization by a possible H-bond between this Asp99 carboxylate group and an NH group from either the indolylamide or the indole groups (Chart 6), as compared to the interactions found by Vannini for compound 26 (Chart 2
Conclusion
Initially developed as single anti-cancer agents, HDAC inhibitors have recently entered the pool of so called epigenetic modulators, molecules able to modulate the expression of genes by several direct or indirect ways. Many biological targets of HDAC are known today and showed that HDAC are involved in several biologic pathways leading to cancer and other diseases. These finding suggested that HDAC inhibitors may be used with other anti-cancer agents to obtain synergistic results. Thus,
Acknowledgements
Author thanks for providing modelled compounds as PDB files Furumai et al. [36] for compound 30 in HDLP, Suzuki et al. [45] for compound (S)-58 in HDAC1, and Ghadiri et al. [80] for model of compound 68.
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