Mechanism of HDACs

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 

---

Flavone has been shown to be effective at 170 /iM concentration to inhibit HDAC-1 activity, and also to induce histone H3 acetylation in NB4 APL cell lines. Flavone s mechanism of HDAC inhibition is currently unknown, but it has been proposed that similar to the inhibitors of kinases, it might occupy the exit channel or an alternative, shallow binding site found in HDAC with possible effects of causing allosteric modulation. This blockage or allosteric modulation would promote inhibition. ... 

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. 

The use of plants for medicinal purposes is an ancient practice. Nature, with its wealth of traditional knowledge has been the source of inspiration for numerous drugs currently used for the improvement of life as well as treatment for a cure. Considering the beneficial role of many plants and fruits, they were included in the human diets. In many instances, the knowledge of the underlying mechanism of action of a particular natural product is incomplete. Continuous investigation can lead to new mechanisms and new structures, which may open up entirely new windows and perspectives. For instance, before the discovery of apicidin and bispyri-dinium diene, it was believed that unless there is a classical chelator for zinc ion, it cannot be a HDAC inhibitor. SAHA has been approved by FDA, which is inspired from the natural product trichostatin. The natural product, romidepsin has also been approved by FDA and many are on clinical trials. Currently, isozyme-selective inhibition for HDAC is at its nascent stage. The invention of some novel molecules or invention of some novel natural product structures with synthetic modifications will solve the problem. 

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. 

HATs catalyze the post-translational acetylation of amino-terminal lysine tails of core histones, which results in disruption of the repressive chromatin folding and an increased DNA accessibility to regulatory proteins. The level of histone acetylation is highly controlled and balanced by the activity of histone deacetylases (HDACs), the opponents of HATs. Generally, acetylation is correlated with activation and deacetylation with repression of gene expression. Therefore, the dynamic equilibrium of these proteins represents a key mechanism of gene regulation. 

The identification of the first small molecule HDAC inhibitors in the late seventies triggered an exponential growth in medicinal chemistry activity. Three decades and many thousand compounds later, the availabiUty of diverse HDAC inhibitors such as short-chain fatty acids, hydroxamic acids, benzamides and tetracyclic peptides, has not only enabled the elucidation of the catalytic mechanism underlying the deacetylating capacity of HDACs, but has also assisted in the investigation of the biological role of the various HDAC subtypes. Furthermore, HDAC inhibitors are currently being evaluated in the clinic and have shown therapeutic potential in the treatment of cancer. 

In summary, due fo fhe large panel of cell cycle regulatory proteins regulated by HDACs at the level of either their expression or activity, the antiproliferative effect of HDAC inhibitors cannot be linked to a single mechanism of action. The relative importance of the different proteins affected by HDACs varies between tumors. In Fig. 2, a visual overview of the role of HDACs in various hallmark processes in the development of cancer is shown. 

Histone deacetylases are linked to the pathogenesis of malignancy from a mechanistic perspective. The capacity of HDAC inhibitors (HDACi) to interfere with the enzyme fimction has led to the observed prechnical and clinical activity in cancer therapy. Although the exact mechanism of anti-tumor activity is not fully elucidated, various cellular pathways have been shown to be involved. From the first chnical trials involving HDACi with short chain fatty acids to the newer generation hydroxamic acid derivatives and cychc tetrapeptides, a number of structurally diverse compounds have made the transition from the laboratory to the chnical arena. For purposes of this part of the discussion, HDACi are arbitrarily divided into the hydroxamates and nonhydroxamates. 

The mechanism of action of this benzamide compoimd is not entirely understood but it has been shown to inhibit HDAC and cellular proUferation. It displays linear kinetics and is rapidly absorbed after oral administration. The main dose limiting toxicity (DLT) reported was thrombocytopenia with the MTD at 8 mg/m /day, although other toxicities Uke nausea, vomiting, diarrhea and fatigue were seen [136]. One partial response was seen in the 53 patients evaluated. 

From a chemogenomics perspective, the past and present generations of HDAC inhibitors, while providing much insight into the molecular mechan-... 

Inhibition of HDACs is one key mechanism to reactivate the expression of these misregulated genes. The astounding tumor specificity of many HDAC inhibitors relays the potential for many of these new compounds for the treatment of cancer and perhaps other disorders. There are five classes of HDAC inhibitors (reviewed in Refs. 51 and 52) including (i) short-chain fatty adds such as sodium- -butyrate (ii) hydroxyamic acids, such as trichostatin A (TSA), suberoylanilide hydroxamic add (SAHA), m-carboxycinnamic acid bishydroxamic acid (CBHA), azelaic bishydroxamic acid (ABHA), and... 

Figure 11 (a) Lys acetylation catalyzed by acetyltransferases (b) mechanism of Zn-dependent HDACs-catalyzed deacetylation (c) mechanism of... 

Figure 3 Histone deacetylase inhibitors, (a) The mechanism by which many HDAC inhibitors function. A hydrophobic moiety biocks the active site of the HDAC. A fiexibie iinker extends into the cieft, and a poiar group interacts with the active zinc atom, (b) Crystai structure of TSA (ieft) and SAHA (right) in the active site of a HDAC-iike protein HDLP (87). (c) Exampies from four major ciasses of HDAC inhibitors.

Benzamides represent a fourth class of HDAC inhibitors. Unlike the other HDAC inhibitors above, benzamides do not conform to the simple pharmacophore model with an obvious metal-binding group connected to a linear spacer. Whether they work by the same mechanism or target an allosteric site on the enzyme is not fully resolved. Nevertheless, they display nanomolar potency, and more than one compound have reached phase I clinical trials for... 

---