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 ␣͞␤ 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 HDAC8. 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.T he epigenetic control of gene expression is operated through a series of posttranslational modifications of chromatin that influence the electrostatics of DNA-protein interactions and generate docking sites for a large number of chromatininteracting proteins (1, 2). The acetylation status of lysine residues found in the accessible N termini of core histones is one of the posttranslational chromatin modifications that impinge on gene expression. Acetylation and deacetylation of histones are controlled by the enzymatic activity of histone acetyltransferases and histone deacetylases (HDACs) (3, 4). Alterations of gene expression are a hallmark of cancer, and mounting evidence suggests that at least a part of these alterations is mediated by epigenetic mechanisms (5, 6). Importantly, the aberrant recruitment of HDACs has been mechanistically linked to malignancy in leukemias and lymphomas (7,8), and small-molecule HDAC inhibitors show antitumor activity in preclinical models and in clinical trials and have the promise to become effective, new antineoplastic therapeutics (9).At least 18 HDAC subtypes exist, and they are subdivided into three classes (10): class I (HDACs 1-3 and 8), homologous to the yeast Rpd3 deacetylase; class II (HDACs 4-7, 9, and 10), related to the yeast Hda1 deacetylase; and class III proteins (Sirtuins 1-7), which are yeast Sir2 homologs. HDAC11 has homology to both class I and II enzymes but cannot unambiguously be assigned to either class. Class I and II HDACs, as well as HDAC11, are all zinc-dependent hydrolases. The therapeutically relevant HDAC inhibitors are thought to be nonselective or poorly selective inhibitors of all or most of class I and II enzymes but do not inhibit class III HDACs (9). It is not clear whether the antitumor properties of HDAC inhibitors are due to their l...

Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 Å resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/ Tyr306Phe mutants.

The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3-and 2.4-Å resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small ␣-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen. The hepatitis C virus (HCV)1 is a small positive-strand RNA virus responsible for a considerable proportion of acute and chronic hepatitis in humans (1, 2). It is estimated that more than 170 million people worldwide are infected by this virus (3). There is no vaccine available for HCV, and the current therapy, based on interferon and ribavirin, is poorly tolerated and of limited efficacy. Therefore, there are intense research efforts toward the development of new drugs targeting essential HCV enzymes and in particular the polymerase. The HCV nonstructural protein 5B (NS5B) is the RNA-dependent RNA polymerase (RdRp) responsible for replication of the viral genome. NS5B can initiate RNA synthesis by two different mechanisms: primer-independent initiation from the 3Ј terminus of the viral genome, also known as de novo initiation (4, 5), and primer-dependent initiation using either DNA or RNA as primers (6). The de novo synthesis is likely used during virus replication in infected cells (7).The three-dimensional structures of soluble forms of the HCV polymerase genotype 1b, ⌬C21 and ⌬C55, lacking the last 21 and 55 C-terminal residues, respectively, have been reported (8 -10). These structures revealed a classical "right hand" shape formed by the palm, thumb, and fingers domains as initially defined in the Klenow fragment of Escherichia coli DNA polymerase I (11) and showed the presence of an extension in the fingers, the so-called fingertip subdomain, containing two loops, ⌳1 and ⌳2 (secondary structure nomenclature according to Ref. 9), which anchor the fingers to the thumb. As a result, the polymerase has a relatively closed and spherical appearance, and the active site cavity, to which the RNA template and the NTP substrates have access via two positively charged tunnels, is completely encircled. Structural studies have shown that other viral RdRps, such a...

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) has recently emerged as a major regulator of plasma low-density lipoprotein cholesterol (LDLc) and is a promising therapeutic target for treating coronary heart Press, October 19, 2010 DOI 10.1194 Abbreviations: AF, Alexa Fluor; CETP, cholesteryl ester transfer protein; CHD, coronary heart disease; DELFIA, dissociation-enhanced lanthanide fl uorescence immunoassays; HDLc, high-density lipoprotein cholesterol; LDLc, low-density lipoprotein cholesterol; LDLr, lowdensity lipoprotein receptor; MAb, monoclonal antibody; PEG, polyethylene glycol 6000; TCEP, Tris(2-carboxyethyl)phosphine; TG, triglycerides; TR-FRET, time-resolved fl uorescence resonance energy transfer. Published, JLR Papers in1 The atomic coordinates and structure factors have been deposited in the Protein Data Bank, accession code 2xtj.3 Current address: Okairos S.r.l.,

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of ␤-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9⌬C, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) 4 is a key regulator of plasma low density lipoprotein (LDL) cholesterol and has emerged as a promising target for prevention and treatment of coronary heart disease. A strong link between PCSK9, LDL, cholesterol, and coronary heart disease has been established by multiple laboratories. Human genetic studies demonstrated remarkable correlations between several nonsense or missense PCSK9 mutations with plasma LDL cholesterol levels and the risk of coronary heart disease. Thus, putative gain-or loss-of-function mutants were found to correlate with increased or reduced plasma LDL levels and cardiovascular risk, respectively (1-7). A recent genome-wide association study further bolstered the importance of PCSK9 by establishing a linkage between a single nucleotide polymorphism at a locus near PCSK9 with early-onset myocardial infarction (8).There is extensive evidence that plasma PCSK9 raises LDL cholesterol levels by binding to cell surface LDLR and targeting the receptor to lysosomes for degradation (9 -13). Accordingly, inhibition of PCSK9 by recombinant LDLR fragments (14 -16) or by mono-or polyclonal antibodies (17, 18) restored LDL cholesterol uptake in cells. Moreover, either RNAi targeting liver PCSK9 (19) or intravenous injection of a monoclonal antibody disrupting the PCSK9-LDLR inter...