Structure of HDACs

The crystal structure of FB188 HDAH (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes strain FB188), a baderial class II HDAC homolog. 

HDAC7 in complex with TSA (PDB code 3C10) and (d) class lla human HDAC4 in complex with an hydroxamic acid inhibitor (PDB code 2vqm). The proteins are colored according to secondary structure elements, the catalytic Zn ions are shown as cyan spheres, the class lla-speciflc structural Zn ions as orange spheres and the structural l ions as magenta spheres. 

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Biochemical and mutagenesis studies and the various structures of HDACs led to the proposal of a catalytic mechanism that would imply the nucleophilic attack by an... 

Figure 3.4 Molecular structures of HDAC inhibitors mentioned in the text.

F. 10 Schematic representation for the photo-affinity-based ABPP strategy. The structure of HDAC photo-crosslinking probe (SAHA-BPyne) is shown in the middle [115]. ABPP activity-based protein profiling, HDAC histone deacetylase, SAHA suberoylanilide hydroxamic acid, CuAAC copper(l)-catalyzed azide-alkyne cycloaddition, LC-MS/MS liquid chromatography-tandem mass spectrometry... 

Before our work [39], only one catalytic mechanism for zinc dependent HDACs has been proposed in the literature, which was originated from the crystallographic study of HDLP [47], a histone-deacetylase-like protein that is widely used as a model for class-I HDACs. In the enzyme active site, the catalytic metal zinc is penta-coordinated by two asp residues, one histidine residues as well as the inhibitor [47], Based on their crystal structures, Finnin et al. [47] postulated a catalytic mechanism for HDACs in which the first reaction step is analogous to the hydroxide mechanism for zinc proteases zinc-bound water is a nucleophile and Zn2+ is five-fold coordinated during the reaction process. However, recent experimental studies by Kapustin et al. suggested that the transition state of HDACs may not be analogous to zinc-proteases [48], which cast some doubts on this mechanism. 

Figure 1 Structures of traditional HDAC inhibitors 1-3 and HDAC inhibitors now in clinical trials (4-8).

Crystal structures of a histone deacetylase-like protein (HDLP) and HDAC8 have confirmed a general pharmacophore model for HDAC inhibitors, comprising a cap joined by a hydrophobic linker to a zinc-binding group (ZBG). This model is exemplified by SAHA and the natural product HDACi Trichostatin A (TSA) 2. 

Figure 4. Therapeutic strategies to counteract CBP loss of function. CBP loss of function leads to a decrease in histone acetylation levels as well as a decrease in CBP-dependent transcription. Two main approaches can be tested to reverse diis process either resetting HAT functionality or resetting global acetylation levels widi die use of HDAC inhibitors. Whereas both strategies would increase histone acetylation levels, HDAC inhibition would act on a broad range of genes, while CBP activation (overexpression or by a pharmacological approach) would specifically target bodi CBP-dependent histone acetylation and transcription. The structure of some of the HDACi that have been tested in different models, such as small fatty acids and hydroxamic acids, are represented in the boxes...

Fig. 4. Domain structure of mammalian DNA methyltransferases. (a) The domain structure of the known DNA methyltransferases, depicting the conserved catalytic domain (dark box) and other identified domains. Conserved aminoacid motifs in the catalytic domain are shown in lighter shade of gray. (b) Schematic representation of the reported protein-protein interactions of Dnmtl with a number of regulatory proteins interactions that modulate Dnmtl methyitransferase activity (darker rectangles) or mediate methylation-independent transcriptional repression mechanisms (lighter rectangles). When Dnmtl represses transcription through its enzymatic activity, it has been described to interact with some proteins PCNA [37] and an oncogenic transcription factor PML-RAR [25]. Note that in the case of the PML-RAR transcription factor, histone deacetylase 1 (HDACl) is also bound to the complex. When Dnmtl represses transcription via methylation-independent pathways, it binds to HDACs either directly [34] or indirectly through other proteins the corepressor DMAPl [33], the retinoblastoma protein, and a gene-specific transcription factor [31].

Fig. 9. The structures of psammaphn HDAC inhibitors. The first inhibitor was originally synthesized and later found to be naturally occurring. Recently discovered derivatives of the plammmaplin A are shown in the right.

Fig. 16. Chemical structures of natural product (a) trapoxin A and (b) trapoxin B. Table 10. IC50 values of trapoxin A and trapoxin B against HDAC isoforms.

Although no structure of class II HDAC has been solved in complex with an acetylated peptide, the structure of FB188 H DAH bound to an acetate molecule, the deacetylation reaction product, showed that the acetate was bound to the Zn ion [48]. A17-A long channel was found in FB188 HDAH, leading from the bottom of the active site cavity to the protein surface, and was proposed to function as an exit tunnel for the acetate, as previously proposed for HDLP [41, 44]. 

R., Gallinari, P. et al. (2004) Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDACS, complexed with a hydroxamic acid inhibitor. Proceedings of the National Academy of Sciences of the United States of America, 101 (42), 15064-15069. 

So far 18 different members of HDACs have been discovered in humans and classified into four classes based on their homology to yeast histone deacetylases [33]. Class I includes four different subtypes (HDACl, 2, 3, 8), class II contains six subtypes tvhich are divided into two subclasses class Ila with subtypes HDAC4, 5, 7, 9 and class Ilb with HDAC6, 10. Class I and class II HDAC share significant structural homology, especially within the highly conserved catalytic domains. HDACs 6 and 10 are unique as they have two catalytic domains. HDACll is referred to as class IV. While the activity of class I, II and IV HDACs depends on a zinc based catalysis mechanism, the class III enzymes, also called sirtuins, require nicotinamide adenine dinucleotide as a cofactor for their catalysis. 

In the crystal structures, the inhibitors coordinate to the active site zinc and make a series of hydrogen bonds via their hydroxamic acid moiety. The hydroxamic acids are linked by a flexible spacer with bulky cap groups. The aromatic or aliphatic spacer participates in van der Waals interactions throughout the long charmel, whereas the terminal part of the inhibitor interacts with residues at the rim of HDAC. In general, the binding mode of the cocrystallized inhibitors TSA and SAHA is conserved among the different species and subtypes [35]. 

The class III deacetylases, named sirtuins, are structurally and functionally different from other HDACs. In contrast to the zinc-dependent deacetylation of classic HDACs, sirtuins depend on NAD" to carry out catalytic reactions. A variety of sirtuin crystal structures have been published over the past few years. The structures of human Sirt2 and SirtS as well as several bacterial Sir2 proteins could be derived, whereas no 3D structure is available for Sirtl and the other subtypes [69]. All solved sirtuin structures contain a conserved 270-amino-acid catalytic domain with variable N- and C-termini. The structure of the catalytic domain consists of a large classic Rossmann fold and a small zinc binding domain. The interface between the large and the small subdomain is commonly subdivided into A, B and C pockets. This division is based on the interaction of adenine (A), ribose (B) and nicotinamide (C) which are parts of the NAD" cofactor. (Figure 3.5) Whereas the interaction of adenine and... 

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