Hepatitis C virus NS3-4A serine protease inhibitors: SAR of P'2 moiety with improved potency.

We have discovered that introduction of appropriate amino acid derivatives at P'2 position improved the binding potency of P3-capped alpha-ketoamide inhibitors of HCV NS3 serine protease. X-ray crystal structure of one of the inhibitors (43) bound to the protease revealed the importance of the P'2 moiety.

Investigation of the S3 site of thrombin: design, synthesis and biological activity of 4-substituted 3-amino-2-pyridones incorporating P1-argininals.

A novel scaffold for P4-P2 dipeptide mimics containing a rigid pyridone spacer was designed based on a virtual library strategy. Several selected nonpeptidic 4-aralkyl or 4-alkylpyridones incorporating a P1-argininal sequence were prepared. The modeling studies, synthesis and biological activities of these unique pyridone derivatives are reported herein.

Highly selective mechanism-based thrombin inhibitors: structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes.

The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined and refined to R values 0.152-0. 170 at 1.8-2.1 A resolution. Since the selectivity of two of the inhibitors was >1600 with respect to trypsin, the structures of trypsin-inhibited complexes of these inhibitors were also determined (R = 0.142-0.157 at 1.9-2.1 A resolution). The selectivity appears to reside in the inability of a benzenesulfonamide group to bind at the equivalent of the D-enantiomorphic S3 site of thrombin, which may be related to the lack of a 60-insertion loop in trypsin. All the inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. Differences in the binding constants of these inhibitors are correlated with their interactions with thrombin and trypsin. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism. The structures of both thrombin and trypsin complexes show similar well-defined transition states in the S1 site and at the electrophilic carbon atom and Ser195OG. The trypsin structures, however, suggest that the first step in a two-step kinetic mechanism may involve formation of a weak transition-state complex, rather than binding dominated by the P2-P4 positions.

Crystallographic structure of a peptidyl keto acid inhibitor and human alpha-thrombin.

The low molecular weight alpha-keto amide inhibitor CVS-1347, benzyl-SO2-Met(O2)-Pro-Arg(CO)((CONH)CH2)-phenyl, is a slow, tight binding inhibitor of alpha-thrombin amidolytic activity having a Ki = 1.28 x 10(-10) M. A complex between human alpha-thrombin and a hydrolysis product of CVS-1347 has been determined and refined using crystallography. The crystals belong to monoclinic space group C2 with cell dimensions of a = 71.08, b = 72.05 and c = 72.98 A and beta = 100.8 degrees. The structure was solved using isomorphous replacement methods and refined with resolution limits of (8.00-1.76) A to an R-value of 0.162. The Pro-Arg core of the inhibitor binds in the S2 and S1 subsites respectively, as is usually observed for Pro-Arg thrombin inhibitors. The Met(O2) side chain does not make any close contacts with the enzyme but influences the conformation of Glu192; the N-terminal benzylsulfonyl group makes an aromatic-aromatic contact with Trp215 in the hydrophobic part of the active site. The alpha-keto carboxylic acid of the proteolyzed inhibitor binds with the carboxylate group in the oxyanion hole, demonstrating that this region can accommodate an anion in a protease-peptide complex. The alpha-keto carbonyl group interacts closely with the two most important residues in the active site: the carbon atom is within a covalent bond distance of the active site Ser195 O gamma and the carbonyl oxygen is hydrogen bonded to His57.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR structure determination of tick anticoagulant peptide (TAP).

Tick anticoagulant peptide (TAP) is a potent and selective 60-amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo- and heteronuclear two-dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C-labeled, and 15N-labeled TAP provided nearly complete 1H sequence-specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY-derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa.