Bruce A. Malcolm

Discovery of Narlaprevir (SCH 900518): A Potent, Second Generation HCV NS3 Serine Protease Inhibitor

Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (∼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.

Rapid mass spectrometric determination of preferred irreversible proteinase inhibitors in combinatorial libraries

Abstract Optimal N -iodoacetyldipeptide inactivators of hepatitis A virus 3C proteinase were identified directly from equimolar mixtures of these compounds using electrospray ionization mass spectrometry (ESI-MS). Limiting amounts of proteinase were allowed to react with the library of inhibitors and subsequently analyzed by ESI-MS to determine the mass of the adducts formed. N -iodoacetyl-Ser-Phe-NH 2 was found to be the most potent inactivator with a second order rate constant of 840 ± 90 M −1 s −1 . Fragmentation of the complexex by using cyanogen bromide and trypsin followed by liquid chromatography/ESI-MS confirmed the identity of the adduct and allowed inhibitor mass differences of as little as 6 Da to be distinguished in a single experiment. This approach allows the rapid screening and identification of preferred covalent inhibitors or intermediates from combinatorial libraries without deconvolution or resynthesis and should be applicable to irreversible inhibitors of virtually any enzyme that uses a covalent catalysis mechanism.

Synthesis and testing of azaglutamine derivatives as inhibitors of Hepatitis A Virus (HAV) 3C proteinase

Abstract Hepatitis A virus (HAV) 3C proteinase is a picornaviral cysteine proteinase that is essential for cleavage of the initially synthesized viral polyprotein precursor to mature fragments and is therefore required for viral replication in vivo. Since the enzyme generally recognizes peptide substrates with l-glutamine at the P1 site, four types of analogues having an azaglutamine residue were chemically synthesized: hydrazo-o-nitrophenylsulfenamides A (e.g. 16); frame-shifted hydrazo-o-nitrophenylsulfenamides B (e.g. 25–28); the azaglutamine sulfonamides C (e.g. 7, 8, 11, 12); and haloacetyl azaglutamine analogues 2 and 3. Testing of these compounds for inhibition of the HAV 3C proteinase employed a C24S mutant in which the non-essential surface cysteine was replaced with serine and which displays identical catalytic parameters to the wild-type enzyme. Sulfenamide 16 (type A) showed no significant inhibition. Sulfenamide 27 (type B) had an IC50 of ca 100μM and gave time-dependent inactivation of the enzyme due to disulfide bond formation with the active site cysteine thiol, as demonstrated by electrospray mass spectrometry. Sulfonamide 8 (type C) was a weak competitive inhibitor with an IC50 of approximately 75μM. The haloacetyl azaglutamine analogues 2 and 3 were time-dependent irreversible inactivators of HAV 3C proteinase with rate constants k obs/[I] of 680M−1 s−1 and 870M−1 s−1, respectively, and were shown to alkylate the active site thiol. ©

Identification of a decapeptide with the binding reactivity for tumor-associated TAG72 antigen from a phage displayed library.

Peptide ligands for tumor‐associated TAG72 antigen were identified by screening a large, diverse decapeptide library expressed on the surface of filamentous phages. Fifty‐eight clones of phages were selected from the eluates after the third round of biopanning and their DNA inserts were sequenced. A dominant decapeptide HYVSIELPDH (14/58) was found with the binding reactivity for TAG72 antigen in the TAG72‐binding ELISA and Western dot blotting. It also showed a preferential binding to colonic adenocarcinomatous cells expressing the TAG72 antigen in the histochemical study. Therefore, this anti‐TAG72 decapeptide may be useful in serving as the starting point with regard to further designing peptidomimetics for potential pharmaceuticals.