Structure-guided design of peptide-based tryptase inhibitors.

Improved peptide-based inhibitors of human beta tryptase were discovered using information gleaned from tripeptide library screening and structure-guided design methods, including fragment screening. Our efforts sought to improve this class of inhibitors by replacing the traditional Lys or Arg P1 element. The optimized compounds display low nanomolar potency against the mast cell target and several hundred-fold selectivity with respect to serine protease off targets.

Discovery of novel heterocyclic factor VIIa inhibitors.

Structure-activity relationships and binding mode of novel heterocyclic factor VIIa inhibitors will be described. In these inhibitors, a highly basic 5-amidinoindole moiety has been successfully replaced with a less basic 5-aminopyrrolo[3,2-b]pyridine scaffold.

Factor VIIa inhibitors: improved pharmacokinetic parameters.

Efforts to improve the potency and pharmacokinetic properties of small molecule factor VIIa inhibitors are described. Small structural modifications to existing leads allow the modulation of half-life and clearance, potentially making these compounds suitable candidates for drug development.

Factor VIIa inhibitors: gaining selectivity within the trypsin family.

Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaffold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.

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.

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.

Expression, crystallization, and three-dimensional structure of the catalytic domain of human plasma kallikrein.

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms.

Dissecting and designing inhibitor selectivity determinants at the S1 site using an artificial Ala190 protease (Ala190 uPA).

A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallography and enzymology involving WT and Ala190 uPA were used to calculate free energy binding contributions of hydrogen bonds involving the Ser190 hydroxyl group (O(gamma)(Ser190)) responsible for the remarkable selectivity of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors toward uPA and against natural Ala190 protease anti-targets. Crystal structures of uPA complexes of novel, active site-directed arylguanidine and 2-aminobenzimidazole inhibitors of WT uPA, together with associated K(i) values for WT and Ala190 uPA, also indicate a significant role of Ser190 in the binding of these classes of uPA inhibitors.

Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases.

Modulation of the acetylation state of histones plays a pivotal role in the regulation of gene expression. Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysines near the N termini of histones. This reaction promotes the condensation of chromatin, leading to repression of transcription.

The structure of the extracellular region of human hepsin reveals a serine protease domain and a novel scavenger receptor cysteine-rich (SRCR) domain.

Hepsin is an integral membrane protein that may participate in cell growth and in maintaining proper cell morphology and is overexpressed in a number of primary tumors. We have determined the 1.75 A resolution structure of the extracellular component of human hepsin.

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).

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.

4-Aminoarylguanidine and 4-aminobenzamidine derivatives as potent and selective urokinase-type plasminogen activator inhibitors.

The structure-based design of potent and selective urokinase-type plasminogen activator (uPA) inhibitors with 4-aminoarylamidine or 4-aminoarylguanidine S1 binding groups, is described.

2-(2-Hydroxy-3-alkoxyphenyl)-1H-benzimidazole-5-carboxamidine derivatives as potent and selective urokinase-type plasminogen activator inhibitors.

The development of potent and selective urokinase-type plasminogen activator (uPA) inhibitors based on the lead molecule 2-(2-hydroxy-3-ethoxyphenyl)-1H-benzimidazole-5-carboxamidine (3a) is described.