Total Synthesis of Covalent Cysteine Protease Inhibitor N-Desmethyl Thalassospiramide C and Crystallographic Evidence for Its Mode of Action.

A total synthesis of N-desmethyl thalassospiramide C, a unique strained macrocyclic proteobacterial depsipeptide, enabled a detailed crystallographic study of its covalent complex with cathepsin K, a member of a medicinally important family of cysteine proteases. The study provides support for the mechanism of action, and the insight gained can be used for structure-based drug design targeting these calpain proteases.

Preclinical Characterization and Human Microdose Pharmacokinetics of ITMN-8187, a Nonmacrocyclic Inhibitor of the Hepatitis C Virus NS3 Protease.

The current paradigm for the treatment of chronic hepatitis C virus (HCV) infection involves combinations of agents that act directly on steps of the HCV life cycle. Here we report the preclinical characteristics of ITMN-8187, a nonmacrocyclic inhibitor of the NS3/4A HCV protease. X-ray crystallographic studies of ITMN-8187 and simeprevir binding to NS3/4A protease demonstrated good agreement between structures. Low nanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 1, 2b, 4, 5, and 6. In cell-based potency assays, half-maximal reduction of genotype 1a and 1b HCV replicon RNA was afforded by 11 and 4 nM doses of ITMN-8187, respectively. Combinations of ITMN-8187 with other directly acting antiviral agents in vitro displayed additive antiviral efficacy. A 30-mg/kg of body weight dose of ITMN-8187 administered for 4 days yielded significant viral load reductions through day 5 in a chimeric mouse model of HCV. A 3-mg/kg oral dose administered to rats, dogs, or monkeys yielded concentrations in plasma 16 h after dosing that exceeded the half-maximal effective concentration of ITMN-8187. Human microdose pharmacokinetics showed low intersubject variability and prolonged oral absorption with first-order elimination kinetics compatible with once-daily dosing. These preclinical characteristics compare favorably with those of other NS3/4A inhibitors approved for the treatment of chronic HCV infection.

3-heterocyclyl quinolone inhibitors of the HCV NS5B polymerase.

The discovery and optimization of a novel class of quinolone small-molecules that inhibit NS5B polymerase, a key enzyme of the HCV viral life-cycle, is described. Our research led to the replacement of a hydrolytically labile ester functionality with bio-isosteric heterocycles. An X-ray crystal structure of a key analog bound to NS5B facilitated the optimization of this series of compounds to afford increased activity against the target enzyme and in the cell-based replicon assay system.

Quinolones as HCV NS5B polymerase inhibitors.

Hepatitis C virus (HCV) infection is treated with a combination of peginterferon alfa-2a/b and ribavirin. To address the limitations of this therapy, numerous small molecule agents are in development, which act by directly affecting key steps in the viral life-cycle. Herein we describe our discovery of quinolone derivatives, novel small-molecules that inhibit NS5b polymerase, a key enzyme of the viral life-cycle. A crystal structure of a quinoline analog bound to NS5B reveals that this class of compounds binds to allosteric site-II (non-nucleoside inhibitor-site 2, NNI-2) of this protein.

Optimization of the multiple enzymatic activities of the hepatitis C virus NS3 protein.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is known to possess multiple enzymatic activities. In addition to its well-characterized protease activity, HCV NS3 also has ATP hydrolase (ATPase) and nucleic acid unwinding (helicase) activities. We systematically studied the effect of common reagents on all three enzymatic activities with a view to improving assay sensitivity for compound screening and profiling. Inclusion of the detergent lauryl dimethylamine oxide (LDAO) improves protease and helicase activities significantly, allowing robust assays at much lower NS3 concentrations. These conditions enable a particularly sensitive protease assay that uses picomolar concentrations of NS3.

Inhibition and binding kinetics of the hepatitis C virus NS3 protease inhibitor ITMN-191 reveals tight binding and slow dissociative behavior.

The protease activity of hepatitis C virus nonstructural protein 3 (NS3) is essential for viral replication. ITMN-191, a macrocyclic inhibitor of the NS3 protease active site, promotes rapid, multilog viral load reductions in chronic HCV patients. Here, ITMN-191 is shown to be a potent inhibitor of NS3 with a two-step binding mechanism. Progress curves are consistent with the formation of an initial collision complex (EI) that isomerizes to a highly stable complex (EI*) from which ITMN-191 dissociates very slowly. K(i), the dissociation constant of EI, is 100 nM, and the rate constant for conversion of EI to EI* is 6.2 x 10(-2) s(-1). Binding experiments using protein fluorescence confirm this isomerization rate. From progress curve analysis, the rate constant for dissociation of ITMN-191 from the EI* complex is 3.8 x 10(-5) s(-1) with a calculated complex half-life of approximately 5 h and a true biochemical potency (K(i)*) of approximately 62 pM. Surface plasmon resonance studies and assessment of enzyme reactivation following dilution of the EI* complex confirm slow dissociation and suggest that the half-life may be considerably longer. Abrogation of the tight binding and slow dissociative properties of ITMN-191 is observed with proteases that carry the R155K or D168A substitution, each of which is likely in drug resistant mutants. Slow dissociation is not observed with closely related macrocyclic inhibitors of NS3, suggesting that members of this class may display distinct binding kinetics.

MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea.

We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.