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. Thus, replacement of Lys/Arg at P1 in a peptide-like scaffold does not need to be accompanied by a loss in target affinity.

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.

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.

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. 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.

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.

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. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 A for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 A for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.

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. Structures and associated K(i) values for a lead inhibitor (CA-11) bound to uPA and to five other proteases, as well as for other leads bound to multiple proteases, help reveal the features responsible for the potency (K(i)=11nM) and selectivity of the remarkably small inhibitor, CA-11. The 6-fluoro-5-amidinobenzimidzole, CA-11, is more than 1000-fold selective against natural Ala190 protease anti-targets, and more than 100-fold selective against other Ser190 anti-targets.

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. HDAC deregulation has been linked to several types of cancer, suggesting a potential use for HDAC inhibitors in oncology. Here we describe the first crystal structures of a human HDAC: the structures of human HDAC8 complexed with four structurally diverse hydroxamate inhibitors. This work sheds light on the catalytic mechanism of the HDACs, and on differences in substrate specificity across the HDAC family. The structure also suggests how phosphorylation of Ser39 affects HDAC8 activity.

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. This structure includes a 255-residue trypsin-like serine protease domain and a 109-residue region that forms a novel, poorly conserved, scavenger receptor cysteine-rich (SRCR) domain. The two domains are associated with each other through a single disulfide bond and an extensive network of noncovalent interactions. The structure suggests how the extracellular region of hepsin may be positioned with respect to the plasma membrane.

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.

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).

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.

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.