James W. Janc

Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets

BACKGROUND Involved or implicated in a wide spectrum of diseases, trypsin-like serine proteases comprise well studied drug targets and anti-targets that can be subdivided into two major classes. In one class there is a serine at position 190 at the S1 site, as in urokinase type plasminogen activator (urokinase or uPA) and factor VIIa, and in the other there is an alanine at 190, as in tissue type plasminogen activator (tPA) and factor Xa. A hydrogen bond unique to Ser190 protease-arylamidine complexes between O gamma(Ser190) and the inhibitor amidine confers an intrinsic preference for such inhibitors toward Ser190 proteases over Ala190 counterparts. RESULTS Based on the structural differences between the S1 sites of Ser190 and Ala190 protease-arylamidine complexes, we amplified the selectivity of amidine inhibitors toward uPA and against tPA, by factors as high as 220-fold, by incorporating a halo group ortho to the amidine of a lead inhibitor scaffold. Comparison of K(i) values of such halo-substituted and parent inhibitors toward a panel of Ser190 and Ala190 proteases demonstrates pronounced selectivity of the halo analogs for Ser190 proteases over Ala190 counterparts. Crystal structures of Ser190 proteases, uPA and trypsin, and of an Ala190 counterpart, thrombin, bound by a set of ortho (halo, amidino) aryl inhibitors and of non-halo parents reveal the structural basis of the exquisite selectivity and validate the design principle. CONCLUSIONS Remarkable selectivity enhancements of exceptionally small inhibitors are achieved toward the uPA target over the highly similar tPA anti-target through a single atom substitution on an otherwise relatively non-selective scaffold. Overall selectivities for uPA over tPA as high as 980-fold at physiological pH were realized. The increase in selectivity results from the displacement of a single bound water molecule common to the S1 site of both the uPA target and the tPA anti-target because of the ensuing deficit in hydrogen bonding of the arylamidine inhibitor when bound in the Ala190 protease anti-target.

Structure-activity relationship studies of a bisbenzimidazole-based, Zn2+-dependent inhibitor of HCV NS3 serine protease

A survey of isosteric replacements of the phosphonoalanine side chain coupled with a process of conformational constraint of a bisbenzimidazole-based, Zn(2+)-dependent inhibitor of hepatitis C virus (HCV) NS3 serine protease resulted in the identification of novel series of active compounds with extended side chains. However, Zn(2+)-dependent HCV NS3 inhibition was relatively insensitive to the structural variations examined but dependent on the presence of negatively charged functionality. This result was interpreted in the context of an initial electrostatic interaction between protease and inhibitor that is subsequently consolidated by Zn(2+), with binding facilitated by the featureless active site and proximal regions of the HCV NS3 protein.

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.

Synthesis of a statistically exhaustive fluorescent peptide substrate library for profiling protease specificity

A statistically exhaustive, 8800 compound tripeptidal amidomethylcoumarin library was synthesized as discreet compounds using solid-phase combinatorial chemistry. A subset of the compounds was purified by HPLC and tested in a high-throughput fluorometric assay against several known serine and cysteine proteases to demonstrate the utility of this library for profiling protease substrate specificity.

Specificity of Induction of the vanA and vanB Operons in Vancomycin-Resistant Enterococci by Telavancin

ABSTRACT Telavancin is a bactericidal, semisynthetic lipoglycopeptide indicated in the United States for the treatment of complicated skin and skin structure infections caused by susceptible Gram-positive bacteria and is under investigation as a once-daily treatment for nosocomial pneumonia. The related vanA and vanB gene clusters mediate acquired resistance to glycopeptides in enterococci by remodeling the dipeptide termini of peptidoglycan precursors from d-alanyl-d-alanine (d-Ala-d-Ala) to d-alanyl-d-lactate (d-Ala-d-Lac). In this study, we assessed the ability of telavancin to induce the expression of van genes in VanA- and VanB-type strains of vancomycin-resistant enterococci. Vancomycin, teicoplanin, and telavancin efficiently induced VanX activity in VanA-type strains, while VanX activity in VanB-type isolates was inducible by vancomycin but not by teicoplanin or telavancin. In VanA-type strains treated with vancomycin or telavancin, high levels of d-Ala-d-Lac-containing pentadepsipeptide were measured, while d-Ala-d-Ala pentapeptide was present at very low levels or not detected at all. In VanB-type strains, vancomycin but not telavancin induced high levels of pentadepsipeptide, while pentapeptide was not detected. Although vancomycin, teicoplanin, and telavancin induced similar levels of VanX activity in VanA-type strains, these organisms were more sensitive to telavancin, which displayed MIC values that were 32- and 128-fold lower than those of vancomycin and teicoplanin, respectively.

Emerging evidence for a shared biosynthetic pathway among clavulanic acid and the structurally diverse clavam metabolites

Abstract [1- 13 C]- l -Valine and [2,3,- 13 C 2 - d,l -proclavaminic acid were administered to Streptomyces antibioticus (Tu 1718). 13 C-NMR spectroscopic analyses demonstrated intact incorporation of the later into valclavam and 2-hydroxyethyl clavam and the former into valclavam. These results, in conjunction with earlier findings, provide evidence for a common biosynthetic origin among all clavam metabolites.

Practical robust fit of enzyme inhibition data.

Publisher Summary This chapter describes the practical robust fit of enzyme inhibition data. The analysis of enzyme inhibition data in the context of preclinical drug screening presents unique challenges to the data analyst. Outliers are data points that are affected by gross errors caused by malfunctioning volumetric equipment, by a human error in data entry, or by countless other possible mishaps. It is shown that Hubers Minimax approach to robust statistical estimation is particularly preferable over the conventional least-squares analysis. The ordinary least square (OLS) estimate of the model parameters is sensitive to the presence of outliers, which has led to the design of various alternatives. All good data points are assigned the same weight in the iteratively reweighted series of LS estimations, exactly as they are in OLS. The practical success of Hubers method applied even to relatively small data sets, such as those arising in preclinical screening, is due to the fact that the method behaves as OLS does if the data are good, but at the same time it gives the least absolute deviation treatment to suspected outliers, while maintaining 95% asymptotic efficiency. It is found that both standardized residuals and leverages play a role in the Hubers method of robust regression analysis, implemented as iteratively re-weighted least squares.

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.