The small molecule ISRIB rescues the stability and activity of Vanishing White Matter Disease eIF2B mutant complexes.

eIF2B is a dedicated guanine nucleotide exchange factor for eIF2, the GTPase that is essential to initiate mRNA translation. The integrated stress response (ISR) signaling pathway inhibits eIF2B activity, attenuates global protein synthesis and upregulates a set of stress-response proteins. Partial loss-of-function mutations in eIF2B cause a neurodegenerative disorder called Vanishing White Matter Disease (VWMD). Previously, we showed that the small molecule ISRIB is a specific activator of eIF2B (Sidrauski et al., 2015). Here, we report that various VWMD mutations destabilize the decameric eIF2B holoenzyme and impair its enzymatic activity. ISRIB stabilizes VWMD mutant eIF2B in the decameric form and restores the residual catalytic activity to wild-type levels. Moreover, ISRIB blocks activation of the ISR in cells carrying these mutations. As such, ISRIB promises to be an invaluable tool in proof-of-concept studies aiming to ameliorate defects resulting from inappropriate or pathological activation of the ISR.

Relative replication capacity and selective advantage profiles of protease inhibitor-resistant hepatitis C virus (HCV) NS3 protease mutants in the HCV genotype 1b replicon system.

We characterized the selective advantage profiles of a panel of hepatitis C virus (HCV) NS3 protease mutants with three HCV protease inhibitors (PIs), BILN-2061, ITMN-191, and VX-950, using a genotype 1b HCV replicon system. Selective advantage curves were generated by a novel mathematical method that factors in the degree of drug susceptibility provided by the mutation, the base-level replication capacity of the mutant in the absence of drugs, and the overall viral replication levels as a function of drug concentration. Most of the mutants showed significantly increased selective advantages over the wild-type species upon drug treatment. Each drug is associated with unique selective advantage profiles that reflect its antiviral activity and mutant susceptibility. Five mutants (R155K/Q, A156T, and D168A/V) showed significant levels of selective advantage after treatment with >10 nM ( approximately 7 times the wild-type 50% effective concentration [EC50]) of BILN-2061. R155K displayed dominant levels of selective advantage over the other mutants upon treatment with ITMN-191 over a broad range of concentrations. Upon VX-950 treatment, various mutants (A156T, A156S, R155K, T54A, V170A, V36M/R155K, and R155Q) exhibited high levels of selective advantage in different drug concentration ranges, with A156T and A156S being the dominant mutants at >3 microM ( approximately 10 times the wild-type EC50) of VX-950. This method provides more accurate estimates of the behavior of various mutants under drug pressure than replication capacity analysis. We noted that the R155K mutant shows reduced susceptibility to all three PIs and significant selective advantage, raising concern over the potential emergence of R155K as a multidrug-resistant, highly fit mutant in HCV patients treated with PIs.

Activity of a potent hepatitis C virus polymerase inhibitor in the chimpanzee model.

A-837093 is a potent and specific nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase. It possesses nanomolar potencies in both enzymatic and replicon-based cell culture assays. In rats and dogs this compound demonstrated an oral plasma half-life of greater than 7 h, and its bioavailability was >60%. In monkeys it had a half-life of 1.9 h and 15% bioavailability. Its antiviral efficacy was evaluated in two chimpanzees infected with HCV in a proof-of-concept study. The design included oral dosing of 30 mg per kg of body weight twice a day for 14 days, followed by a 14-day posttreatment observation. Maximum viral load reductions of 1.4 and 2.5 log(10) copies RNA/ml for genotype 1a- and 1b-infected chimpanzees, respectively, were observed within 2 days after the initiation of treatment. After this initial drop in the viral load, a rebound of plasma HCV RNA was observed in the genotype 1b-infected chimpanzee, while the genotype 1a-infected chimpanzee experienced a partial rebound that lasted throughout the treatment period. Clonal analysis of NS5B gene sequences derived from the plasma of A-837093-treated chimpanzees revealed the presence of several mutations associated with resistance to A-837093, including Y448H, G554D, and D559G in the genotype 1a-infected chimpanzee and C316Y and G554D in the genotype 1b-infected chimpanzee. The identification of resistance-associated mutations in both chimpanzees is consistent with the findings of in vitro selection studies, in which many of the same mutations were selected. These findings validate the antiviral efficacy and resistance development of benzothiadiazine HCV polymerase inhibitors in vivo.

Microarray compound screening (microARCS) to identify inhibitors of HIV integrase.

A novel high-throughput strand transfer assay has been developed, using Microarray Compound Screening (microARCS) technology, to identify inhibitors of human immunodeficiency virus (HIV) integrase. This technology utilizes agarose matrices to introduce a majority of the reagents throughout the assay. Integration of biotinylated donor DNA with fluorescein isothiocyanate (FITC)-labeled target DNA occurs on a SAM membrane in the presence of integrase. An anti-FITC antibody conjugated to alkaline phosphatase (AP) was used to do an enzyme-linked immunosorbent assay with the SAM. An agarose gel containing AttoPhos, a substrate of AP, was used for detection of the integrase reactions on the SAM. For detection, the AttoPhos gel was separated from the SAM after incubation and then the gel was imaged using an Eagle Eye II closed-circuit device camera system. Potential integrase inhibitors appear as dark spots on the gel image. A library of approximately 250,000 compounds was screened using this HIV integrase strand transfer assay in microARCS format. Compounds from different structural classes were identified in this assay as novel integrase inhibitors.