5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.

Background: HDAC4, -5, -7, and -9 possess conserved motifs for phosphorylation-dependent 14-3-3 binding. Results: SIK2 and SIK3 phosphorylate the deacetylases at the motifs to stimulate 14-3-3 binding. Conclusion:The tumor suppressor kinase LKB1 activates SIK2 and SIK3 to promote trafficking of class IIa HDACs. Significance: This study indicates that LKB1-dependent SIK activation is an important module upstream from class IIa HDACs.

The in vitro resistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.T he hepatitis C virus (HCV)-encoded NS3 protease is essential for viral replication and has long been considered an attractive target in drug design efforts (3, 5). NS3 protease inhibitors (PIs) can induce substantial reductions in HCV RNA plasma levels, and several candidates have progressed through clinical development to offer improved treatment options (for a review, see reference 27). Two PIs, boceprevir and telaprevir, were recently approved for use in combination with pegylated interferon (Peg-IFN) and ribavirin (1,6,7,19). The selection of drug-resistant variants is commonly observed in patients experiencing virologic rebound during treatment with PIs (16,[20][21][22]24).BI 201335 is a potent HCV NS3/4A PI (15, 28) currently in phase 3 clinical trials in combination with Peg-IFN and ribavirin as well as phase 2 assessment with other HCV direct acting antivirals in IFN-sparing regimens. BI 201335 exhibited a profound reduction in viral load when administered for 14 days as monotherapy in treatment-naïve patients or for 28 days in combination with Peg-IFN and ribavirin in treatment-experienced patients (16). In these studies, viral breakthrough was observed in most patients on monotherapy, whereas breakthrough was less frequent in patients undergoing combination treatment. Distinct resistant NS3 variants R155K and D168V predominated for genotype 1a and 1b (GT 1a and GT 1b), respectively (8,16).This study was designed to evaluate the genotypic and phenotypic profiles of the resistant variants that emerged during in vitro selection in the presence of BI 201335 in the replicon system and to relate these results to clinical observations. Replicons resistant to BI 201335 were selected in GT 1a H77 and GT 1b CON-1 replicon cell lines in the presence of 2 concentrations (100ϫ and 1,000ϫ drug concentration required to reduce HCV RNA or the luciferase reporter levels by 50% [EC 50 ]) of drug for 3 weeks and G-418 as previously described (9). With the lower concentration of BI 201335, resistant variants encoding NS3 changes at residues 155, 156, and 168 were selected with the GT 1b replicon, with D168G as the predominant variant (55%). R155K was the predominant variant (68%) selected with the GT 1a replicon (Table 1) and is consistent with the predominant variant selected in GT 1a HCV-infected patients (16). At the higher concentration of BI 201335, essentially only D168 variants were selected with D168 A and V as the predominant variants in both genotypes.In order to confirm that the mutations ob...

Hepatitis C virus infection, a major cause of liver disease worldwide, is curable, but currently approved therapies have suboptimal efficacy. Supplementing these therapies with directacting antiviral agents has the potential to considerably improve treatment prospects for hepatitis C virus-infected patients. The critical role played by the viral NS3 protease makes it an attractive target, and despite its shallow, solvent-exposed active site, several potent NS3 protease inhibitors are currently in the clinic. BI 201335, which is progressing through Phase IIb trials, contains a unique C-terminal carboxylic acid that binds noncovalently to the active site and a bromo-quinoline substitution on its proline residue that provides significant potency. In this work we have used stopped flow kinetics, x-ray crystallography, and NMR to characterize these distinctive features. Key findings include: slow association and dissociation rates within a singlestep binding mechanism; the critical involvement of water molecules in acid binding; and protein side chain rearrangements, a bromine-oxygen halogen bond, and profound pK a changes within the catalytic triad associated with binding of the bromoquinoline moiety.Worldwide, 130 -170 million people are chronically infected with hepatitis C virus (HCV) 2 (1). Infection can progress to cirrhosis, which can lead to hepatic decompensation or hepatocellular carcinoma. HCV infection can be cured by administering a combination of pegylated interferon and the nonspecific antiviral drug ribavirin for 24 -48 weeks. Unfortunately, this treatment is only 40 -50% successful in clearing genotype 1 infections, the most common type in most regions of the world (2). Clearly there is considerable need for more efficacious therapies.HCV is a small, positive-strand RNA virus with a 9600-nucleotide genome encoding a 3000-amino acid polyprotein that is subsequently processed into individual proteins by cellular and viral proteases. Four of the five cleavage sites between the NS (nonstructural) proteins are cleaved by the viral NS3 serine protease (EC 3.4.21.98). HCV NS3 is a bifunctional protein comprised of an N-terminal 180-amino acid serine protease domain and a C-terminal 420-amino acid helicase domain (3). The central portion of the 54-amino acid NS4A protein is structurally integrated into the NS3 protease domain (4) and is necessary for full protease activity (5).The search for specific, direct-acting, small molecule inhibitors of HCV replication began shortly after the virus was discovered in 1989 (6); the fact that no such drug has yet made it to the market attests to the challenging nature of this research. Throughout this period, the NS3-NS4A protease has remained a major focus of HCV drug discovery efforts. Beginning with the protease inhibitor BILN 2061 in 2002 (7, 8), a number of specific antivirals have been tested in human trials and have shown very promising results (9). The most advanced compounds in the clinic are NS3-NS4A protease inhibitors, with two, telaprevir and boceprevir, cu...

The NS3 protein of hepatitis C virus is unusual because it encodes two unrelated enzymatic activities in linked protease and helicase domains. It has also been intensively studied because inhibitors targeting its protease domain have potential to significantly improve treatment options for those infected with this virus. Many enzymological studies and inhibitor discovery programs have been carried out using the isolated protease domain in complex with a peptide derived from NS4A which stimulates activity. However, some recent publications have suggested that the NS3 helicase domain may influence inhibitor binding and thus suggest work should focus on the full-length NS3-NS4A protein. Here we present the characterization of a single-chain protease in which the NS4A peptide activator is linked to the N-terminus of the NS3 protease domain. This protein behaves well in solution, and its protease activity is very similar to that of full-length NS3-NS4A. We find that this fusion protein, as well as the noncovalent complex of the NS4A peptide with NS3, gives similar Ki values, spanning 3 orders of magnitude, for a set of 25 structurally diverse inhibitors. We also show that simultaneous mutation of three residues on the surface of the helicase domain which has been hypothesized to interact with the protease does not significantly affect enzymatic activity or inhibitor binding. Thus, the protease domain with the NS4A peptide, in a covalent or noncovalent complex, is a good model for the protease activity of native NS3-NS4A.