Factor VIIa inhibitors: chemical optimization, preclinical pharmacokinetics, pharmacodynamics, and efficacy in an arterial baboon thrombosis model.

Highly selective and potent factor VIIa-tissue factor (fVIIa.TF) complex inhibitors were generated through structure-based design. The pharmacokinetic properties of an optimized analog (9) were characterized in several preclinical species, demonstrating pharmacokinetic characteristics suitable for once-a-day dosing in humans. Analog 9 inhibited platelet and fibrin deposition in a dose-dependent manner after intravenous administration in a baboon thrombosis model, and a pharmacodynamic concentration-response model was developed to describe the platelet deposition data. Results for heparin and enoxaparin (Lovenox) in the baboon model are also presented.

Small molecule inhibitors of plasma kallikrein.

Plasma kallikrein is a serine protease that is involved in pathways of inflammation, complement fixation, coagulation, and fibrinolysis. Herein, we describe the SAR and structural binding modes of a series of inhibitors of plasma kallikrein as well as the pharmacokinetics of a lead analog 11 in rat.

Generation of potent coagulation protease inhibitors utilizing zinc-mediated chelation.

Inhibition of coagulation proteases such as thrombin, fXa, and fVIIa has been a focus of ongoing research to produce safe and effective antithrombotic agents. Herein, we describe a unique zinc-mediated chelation strategy to streamline the discovery of potent inhibitors of fIIa, fXa, and fVIIa. SAR studies that led to the development of selective inhibitors of fXa will also be detailed.

Elaborate manifold of short hydrogen bond arrays mediating binding of active site-directed serine protease inhibitors.

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog. Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.

Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor.

Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.

Expression, purification, crystallization and preliminary X-ray diffraction studies of human cathepsin F complexed with an irreversible vinyl sulfone inhibitor.

Cathepsin F is a cysteine protease believed to be involved in the antigen-presenting process of the class II major histocompatibility complex (MHC-II) in macrophages. It has been expressed, purified and crystallized. A complete data set to a resolution of 2.5 A has been collected at room temperature. The Laue group was determined to be orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 68.9, b = 104.8, c = 68.5 A.

Highly potent non-peptidic inhibitors of the HCV NS3/NS4A serine protease.

Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+)-dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes.

Contribution of multicentered short hydrogen bond arrays to potency of active site-directed serine protease inhibitors.

We describe and compare the pH dependencies of the potencies and of the bound structures of two inhibitor isosteres that form multicentered short hydrogen bond arrays at the active sites of trypsin, thrombin, and urokinase type plasminogen activator (urokinase or uPA) over certain ranges of pH. Depending on the pH, short hydrogen bond arrays at the active site are mediated by two waters, one in the oxyanion hole (H(2)O(oxy)) and one on the other (S2) side of the inhibitor (H(2)O(S2)), by one water (H(2)O(oxy)), or by no water. The dramatic variation in the length of the active site hydrogen bonds as a function of pH, of inhibitor, and of enzyme, along with the involvement or absence of ordered water, produces a large structural manifold of active site hydrogen bond motifs. Diverse examples of multicentered and two-centered short hydrogen bond arrays, both at and away from the active site, recently discovered in several protein crystal systems, suggest that short hydrogen bonds in proteins may be more common than has been recognized. The short hydrogen bond arrays resemble one another with respect to ionic nature, highly polar environment, multitude of associated ordinary hydrogen bonds, and disparate pK(a) values of participating groups. Comparison of structures and K(i) values of trypsin complexes at pH values where the multicentered short hydrogen bond arrays mediating inhibitor binding are present or absent indicate that these arrays have a minor effect on inhibitor potency. These features suggest little covalent nature within the short hydrogen bonds, despite their extraordinary shortness (as short as 2.0 A).