Invited reviewHistone deacetylase inhibitors
Introduction
All of the human genome is packaged in the nucleus into chromatin, a dynamic macromolecular complex made up of repetitive units, the nucleosomes. A single nucleosomal core particle is composed of a fragment of DNA (146 bp) wrapped around a histone octamer formed by four histone partners, an H3–H4 tetramer and two H2A–H2B dimers [1].
Histones are small basic proteins rich in amino-acids, lysine and arginine. The four nucleosomic histones involve two domains: the C-terminal domain, which is located inside the nucleosome core, and the N-terminal domain with lysine residues extending out of the nucleosome. Among the four histone tails, H-3 and H-4 are targeted for various post-translational modifications, including acetylation, phosphorylation and methylation. Histones can be methylated on their lysine (K4 and 27 of H-3 and K-20 of H-4) and also on their arginine residues [2] but, so far, this modification seems to be less dynamic than those caused by the acetylation process.
Thus, histone can be in one of the two antagonist forms, acetylated or deacetylated. Enzymes responsible for this conversion are histone acetyl transferases (HTAs) producing the acetylation, and histone deacetylases (HDACs) which reverse this process. Deacetylation led to the removal of an acetyl group from the ε-amino groups of the lysine side-chains.
Therefore, the N-terminal domain of histones plays a major role towards the transcription regulation. Whereas acetylation correlates with nucleosome remodeling and transcriptional activation, deacetylation of histone tails induces transcriptional repression through chromatin condensation. This may be explained by the fact that neutralization of the positive charge of lysine residues in the N-terminal tail by this acetylation process leads to loosening histone–DNA contacts. This relaxation of the chromatin structure facilitates the accessibility of a variety of factors to DNA [3].
The turnover of the acetyl groups on histone molecules occurs rapidly in the cells and the level of acetylation is controlled by the equilibrium of both activities, acetylation and deacetylation.
Besides this change at the nucleosome scale, acetylation or deacetylation of histones could also interfere [4] with the formation of the chromatin itself by modulating the interactions between the nucleosomes. Moreover, N-terminal amino-acids may also act as signals for interactions [5] with other proteins directly involved in transcription, or via the modification of the chromatin environment. The opposite functions of HTAs and HDACs, in both activating and repressing transcription, show the intricate regulatory processes that are involved in turning genes on or off.
Aberrant gene expression that results in functional inactivation of HAT activity or overexpression of HDACs can mediate tumor cell proliferation [6]. Deregulation of HDAC recruitment to promoters appears to be one of the mechanisms by which these enzymes contribute to tumorigenesis. For example, chromosomal translocations between the retinoic receptor-α (RARα) and either the promyelocytic leukemia zinc finger (PLZF) or the promyelocytic leukemia protein (PML) leading to acute promyelocytic leukemia (APL), recruit a co-repressor and, in turn, HDACs [7]. HDAC-mediated APL pathogenesis serves as a model for lymphoblastic acute leukemia and non-Hodgkin’s lymphoma. These data and others show that inappropriate transcriptional repression mediated by HDACs is a common molecular mechanism used by oncoproteins [8].
For all these reasons, HDAC inhibition has been regarded as a promising anticancer drug target and it is now well established that HDAC inhibitors display ability to affect several cellular processes which are dysregulated in neoplastic cells. They are potent inducers of differentiation with arrest of the cells in the G1 but sometimes also in the G2 phase. They activate transcription of the cyclin-dependent kinase (CDK) inhibitor WAF1 and down-regulate cyclins A and D. They can induce apoptosis in vitro and in vivo by activating both the death-receptor and intrinsic apoptotic pathway. In addition, HDAC inhibitors might lead [8], [9] to activation of the host immune response and inhibition of tumor angiogenesis by multifactorial processes.
So far, 17 human genes that encode proven or putative HDACs have been identified, which belong to two classes identified in eukaryotes and the Sir2-like deacetylases as sirtuins [10].
The class I HDACs (size 42–45 kDa) correspond to the analogues of the Rpd3 yeast protein and include HDAC1, HDAC2, HDAC3 and HDAC8 human enzymes. Contrary to the class I, exclusively expressed in the nucleus, HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10 belonging to the class II (size 120–130 kDa) are shuttled between the cytoplasm and the nucleus. They correspond to the Hda1 yeast protein.
The second family has been identified on the basis of sequence similarity with Sir2, a yeast transcriptional repressor of yeast that requires the co-factor NAD for its deacetylase activity [11], [12]. Human sirtuin SIRT1 has been reported to deacetylate p53 and thus represents the first characterized human sirtuin [13]. The crystal structure of the catalytic domain of human SIRT2 has been reported as well [14].
The first encountered HDAC1 was sodium N-butyrate [15], a potent growth inhibitor and differentiating agent for many tumor cell lines, including breast cancer. The level of Bcl-2 expression involved in the regulation of apoptosis in mammalian cells, regulates [16] the butyrate-induced apoptosis in such a cancer. During the first clinical trials, limited efficacy has been observed [17], with some toxicities including the central nervous system and fatigue. Phase II clinical trials are going on with intravenous administration.
Phenylacetate and phenylbutyrate approximately gave the same results [18], [19], as well as the anti-epileptic agent, valproic acid, which has been reported [20] to delay the growth of primary breast cancers and to act against metastases found in the lung. However, all these compounds were weak inhibitors, in the high micromolar or low millimolar range, and non-selective. Moreover, when used in in vivo experiments at a high concentration, pleiotropic effects of N-butyrate on other enzymes like those involved in cytoskeleton, cell membranes etc., have been reported [21]. Another disadvantage was their low biodisponibility, although some progress have been made with the use of prodrugs such as tributyrin [22] or pivaloyloxymethyl butyrate, or pivanex® [23] which is in phase II trials.
Section snippets
Natural HDACIs and first generation of synthetic analogues from libraries screening
Trichostatin A (TSA) and trichostatin C were initially isolated as fungistatic antibiotics from Streptomyces hygroscopicus by Tsuji et al. [24]. Ten years were required before Morioka et al. [25], [26] and Yoshida et al. [27], [28] demonstrated the very potent activities of trichostatic acid and of trichostatin C in inducing Friend leukemia cell differentiation and inhibiting the cell cycle of normal fibroblasts in both the G1 and G2 phases, respectively (Fig. 1).
In fact, more complete study
Synthetic hydroxamic acid-containing HDAC inhibitors
Based upon the molecular structure of known HDAC inhibitors such as TSA, oxamflatin, and SAHA, several groups have designed and synthesized new inhibitors consisting of a zinc binding group, a 5- or 6-methylene hydrophobic spacer attached to a hydrophobic group via a connection unit. As depicted on Table 1, these target compounds essentially differ by the nature of the hydrophobic group which binds to the enzyme, the connection unit being usually an amide [61], [62], [63], [64], [65], [66], [67]
Synthetic benzamide-containing HDACIs
Although sharing no structural similarity with the previous HDAC, benzamide derivatives which inhibit HDAC in vitro and in vivo have been reported in 1999 by Suzuki et al. [92] from Mitsui Pharmaceuticals. One of the most active derivatives, MS-275, induces hyperacetylation of nuclear histone in various tumor cell lines, and overexpression of p21WAF1, CIP1 and of gelsolin, such as other HDACIs.
As the structural analogue of MS-275, possessing a 3′-aminophenyl instead of a 2′-aminophenyl group
Miscellaneous
Design and synthesis of SAHA-based non-hydroxamates was recently reported by Suzuki et al. [103]. Among them, a semi-carbazide and two bromoacetamides displayed anti-HDAC activities of IC50 of 150, 14, and 17 μM, respectively (Fig. 14).
Arising from Abbott researches, in light of the metabolic instability profile of the hydroxamic acids, three series have been designed which contain trifluoromethyl ketones, heterocyclic ketones, and α-keto-esters or α-keto-amides, like zinc linker.
Inhibitors of Sir2p-like deacetylases
Small molecules acting as inhibitors of Sir2-like deacetylases were simultaneously discovered by Grozinger et al. [107] and that of Bedalov et al. [108]. Both groups used phenotypic screening on compounds derived from chemical libraries, and the drug screenings were performed in 96-well or 394-well plates.
Among the 1600 unbiased small molecules screened, Grozinger et al. [107] identified Sirtinol, A3, and M15 (Fig. 16) as efficient inhibitors of the yeast Sir2p transcriptional silencing
Conclusion
HDAC inhibitors represent a new class of targeted anticancer agents which mediates the regulation of gene expression and induces growth arrest, cell differentiation and apoptosis of tumor cells (Table 3).
However, they seems to alter the expression of a very limited number of genes, contrary to what may be anticipated by the wide distribution of HDACs in chromatin. The gene the most frequently involved is p21WAF1, CIP1 which is up-regulated. Concerning the apoptosis of tumor cells, many
Notes added in proof
After completion of this review, two general papers dealing with the use of HDACs to treat cancer were reported [109], [110] as well as a paper with findings for the design of new inhibitors [111].
Acknowledgements
I am indebted to Dr. M.-F. Poupon and above all to J.P. Buisson for critical reading of the manuscript.
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