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Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single [alpha]/[beta] domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDACS. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.

Research has been conducted on the serine protease NS3/4A of hepatitis C virus. The development of the peptidic and nonpeptidic inhibitors binding to the NS3/4A prime site is described.

The serine protease NS3 of the hepatitis C virus uses positively charged residues to create local electrostatic potentials that promote the attraction of the ligand for the active site. The interaction energy prevents the formation of a tightly bound complex, which is energetically unfavorable.

Hepatitis C virus (HCS) infection has been studied, and the S'-binding in the context of noncleavable decapeptides in the virus spanning P6-P4' has been optimized. The binding mode of the decapeptides in the study has some of the same features as the binding mode of the natural substrates and novel interactions within the S' subsite. The peptides may be an entry point for a novel class of NS3 inhibitors.

Research has been conducted on the hepatitis C virus replication. Results indicate that hexapeptide, tetrapeptide and tripeptide alpha-ketoacids are slow-binding enzyme inhibitors.

A study was conducted to characterize the design of hepatitis C virus (HCV) inhibitors supporting nanomolar potenties of HCV NS3 proteinase based on eglin c. The eglin c gene was determined using polymerase chain reaction primers while eglin variants were obtained from BL21 Escherichia coli cells altered with pT7-driven vectors. Results indicated that the inhibitors support an accurate evaluation of needs associated with the inhibition of the enzymatic activity of NS3.

Peptide inhibitors of the NS3 protease from human hepatitis C virus (HCV) have been synthesized by product inhibition through single-mutant and combinatorial optimization. These synthetic compounds can be used building blocks for studying the binding sites of NS2 by modeling, nuclear magnetic resonance and crystallography. NS3 is a serine protease needed for the development of the HCV and cleaving at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A and NS5A-NS5B sites.

A study on the product inhibition of the NS3 protease of hepatitis C virus (HCV) and on the role of Lys136 in stabilizing the enzyme-product complex is presented. The NS3 protein of HCV contains a serine protease domain which if inactivated shows potential as a way of dealing with the disease. Results show that N-terminal cleavage products bind to the NS3 protease during product inhibition with low micromolar affinities.

The complex formation between NS3 and its peptide cofactor was studied by monitoring enzymatic activity changes and the enzymes' spectroscopic properties as a physicochemical environment function. Results show that complex formation did not change the protein's secondary structure and depends on the glycerol presence in the buffer. This suggests that NS4A is an important enzyme in the protein complex stability.

A homology model of the chymotrypsin-like serine proteinase domain in the NS3 protein of hepatitis C virus was constructed. The model indicated the existence of a tetradentate metal binding site formed by three cysteines and one histidine. Findings revealed that the hepatitis C virus enzyme has a Zn2+ ion with S3N ligation and that the metal is needed for structural integrity and activity of the enzyme. The residues forming the metal binding site were also conserved in the chymotrypsin-like 2A cysteine proteinases of picornaviruses.

The serine protease and helicase activities of the hepatitis C virus nonstructural 3 protein (NS3) are mediated through an interaction with NS4A. NS3 forms a complex with NS4A, and their dissociation is fully reversible. Expression of the NS3-NS4A precursor in Escherichia coli shows that no helicase activity is exhibited by the purified NS3-NS4A complex. In addition, NS4A tends to uncouple the adenosine triphosphatase/ssRNA binding as well as RNA unwinding activities linked to NS3.

A study on the genetic mechanisms of the hepatitis C virus investigated the interaction of the NS3 protease domain of the virus with its NS4A co-factor peptide to identify factors that influence the binding reaction. It will be shown that complex affinities may differ by nearly three orders of magnitude depending on the physico-chemical conditions under which they are determined. Evidence is also presented for complex stabilization by substrate peptides to the active site of the enzyme.

Zinc-dependent histone deacetylases (HDACs) are a family of hydrolases first identified as components of transcriptional repressor complexes, where they act by deacetylating lysine residues at the N-terminal extensions of core histones, thereby affecting transcription. To get more insight into the biological functions of the individual HDAC family members, we have used RNA interference in combination with microarray analysis in Drosophila S2 cells. Silencing of Drosophila HDAC1 (DHDAC1), but not of the other DHDAC family members, leads to increased histone acetylation. Silencing of either DHDAC1 or DHDAC3 leads to cell growth inhibition and deregulated transcription of both common and distinct groups of genes. Silencing DHDAC2 leads to increased tubulin acetylation levels but was not associated with a deregulation of gene expression. No growth of phenotype and no significant deregulation of gene expression was observed upon silencing of DHDAC4 and DHDACX. Loss of DHDAC1 or exposure of S2 cells to the small molecule HDAC inhibitor trichostatin both lead to a G2 arrest and were associated with significantly overlapping gene expression signatures in which genes involved in nucleobase and lipid metabolism, DNA replication, cell cycle regulation, and signal transduction were over-represented. A large number of these genes were shown to also be deregulated upon loss of the co-repressor SIN3 (Pile, L.A., Spellman, P. T., Katzenberger, R. J., and Wassarman, D. A. (2003) J. Biol. Chem. 278, 37840 -37848). We conclude the following. 1) DHDAC1 and -3 have distinct functions in the control of gene expression. 2) Under the tested conditions, DHDAC2, -4, and X have no detectable transcriptional functions in S2 cells. 3) The anti- proliferative and transcriptional effects of trichostatin are largely recapitulated by the loss of DHDAC1. 4) The deacetylase activity of DHDAC1 significantly contributes to the repressor function of SIN3. [ABSTRACT FROM AUTHOR]