Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.The level of histone acetylation is regulated by the action of two classes of enzymes, histone acetyltransferases and histone deacetylases (HDACs).3 Histone acetyltransferases and HDACs are found in large multiprotein complexes, and recruitment of histone acetylase or deacetylase complexes by coactivators or corepressors is thought to cause a local change in the chromatin structure, resulting in either activation or repression of gene transcription (1). Humans have 18 HDACs and, based on their sequence similarity to yeast factors, they are grouped into four classes (class I-IV). Class II HDACs are homologous to yeast histone deacetylase HDA1 and have been implicated as global regulators of gene expression during cell differentiation and development (2). In humans, class II HDACs are subdivided into classes IIa (HDAC4, HDAC5, HDAC7, and HDAC9) and IIb (HDAC6 and HDAC10). Class IIa HDACs contain two functionally important regions, a highly conserved C-terminal catalytic domain and an N-terminal extension that has no similarity with other proteins, mediates the signal-dependent shuttling between the nucleus and the cytoplasm, and harbors binding sites for transcriptional regulators (2, 3). Class IIa HDACs interact with corepressors such as N-CoR (nuclear receptor corepressor) and the MEF2 (myocyte enhancer factor 2) family of transcription factors that is not only important for controlling gene expression in normal cellular programs like muscle differentiation, T-cell apoptosis, neuronal survival, and synaptic differentiation but has also been linked to cardiac hypertrophy, asthma, atherosclerosis, hypertension, and other pathological conditions (3-5). To date all four class IIa HDACs have been knocked out in mice, and the resulting abnormal phenotypes have been extensively characterized (6 -9). HDAC7 for example, plays an important role in cardiova...

A series of conformationally restricted analogues of the hallucinogenic phenethylamine 1 (2,5-dimethoxy-4-bromophenethylamine, 2C-B) was synthesized to test several hypotheses concerning the bioactive conformation of phenethylamine ligands upon binding to the 5-HT(2A) receptor. These benzocycloalkane analogues were assayed for their receptor binding affinity and ability to activate downstream signaling pathways, and one exceptional compound was selected for testing in an in vivo drug discrimination model of hallucinogenesis. All compounds were examined in silico by virtual docking into a homology model of the 5-HT(2A) receptor. On the basis of these docking experiments, it was predicted that the R enantiomer of benzocyclobutene analogue 2 would be the most potent. Subsequent chemical resolution and X-ray crystallography confirmed this prediction, as (R)-2 proved to be equipotent to LSD in rats trained to discriminate LSD from saline. Thus, we propose that the conformation of 2 mimics the active binding conformation of the more flexible phenethylamine type hallucinogens. In addition, (R)-2 is one of the most potent and selective compounds yet discovered in the in vivo drug discrimination assay. Further, 2 was found to be a functionally selective agonist at the 5-HT(2A) receptor, having 65-fold greater potency in stimulating phosphoinositide turnover than in producing arachidonic acid release. If hallucinogenic effects are correlated with arachidonic acid production, such functionally selective 5-HT(2A) receptor agonists may lack the intoxicating properties of hallucinogens such as LSD.

A series of phenethylamine derivatives with various ring substituents and with or without N-methyl and/or C-alpha methyl or ethyl groups was synthesized and assayed for their ability reversibly to inhibit monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B). Several compounds showed potent and selective MAO-A inhibitory activity (IC(50) in the submicromolar range) but none showed appreciable activity toward MAO-B. A three-dimensional quantitative structure-activity relationship study for MAO-A inhibition was performed on the series using comparative molecular field analysis (CoMFA). The resulting model gave a cross-validated q(2) of 0.72 and showed that in this series of compounds steric properties of the substituents were more important than electrostatic effects. Molecular modeling based on the recently published crystal structure of inhibitor-bound MAO-A provided detailed evidence for specific interactions of the ligands with the enzyme, supported by previous references and consistent with results from the CoMFA. On the basis of these results, structural determinants for selectivity of substituted amphetamines for MAO-A are discussed.

A conformationally restricted analogue of mescaline, C-(4,5,6-trimethoxyindan-1-yl)-methanamine, was designed using a 5-HT(2A) receptor homology model. The compound possessed 3-fold higher affinity and potency than and efficacy equal to that of mescaline at the 5-HT(2A) receptor. The new analogue substituted fully for LSD in drug discrimination studies and was 5-fold more potent than mescaline. Resolution of this analogue into its enantiomers corroborated the docking experiments, showing the R-(+) isomer to have higher affinity and potency and to have efficacy similar to that of mescaline at the 5-HT(2A) receptor.