Development of Trypsin-Like Serine Protease Inhibitors as Therapeutic Agents: Opportunities, Challenges, and their Unique Structure-Based Rationales.

There has been a revolution in the development of effective, small-molecule anticoagulants and antiplatelet agents. Numerous trypsin-like serine proteases have been under active pursuit as therapeutic targets. Important examples include thrombin, factor VIIa, factor Xa, and β-tryptase with indications ranging from thrombosis and inflammation to asthma and chronic obstructive pulmonary disease (COPD).

Dimerization of β-tryptase inhibitors, does it work for both basic and neutral P1 groups?

The tetrameric folding of β-tryptase and the pair-wise distribution of its substrate binding sites offer a unique opportunity for development of inhibitors that span two adjacent binding sites. A series of dimeric inhibitors with two basic P1 moieties was discovered using this design strategy and exhibited tight-binder characteristics. Using the same strategy, an attempt was made to design and synthesize dimeric inhibitors with two neutral-P1 groups in hope to exploit the dimeric binding mode to achieve a starting point for further optimization.

Human kallikrein 6 inhibitors with a para-amidobenzylanmine P1 group identified through virtual screening.

A series of hK6 inhibitors with a para-amidobenzylamine P1 group and a 2-hydroxybenzamide scaffold linker was discovered through virtual screening. The X-ray structure of hK6 complexed with compound 9b was determined to a resolution of 1.68Å.

Virtual Screening and X-ray Crystallography for Human Kallikrein 6 Inhibitors with an Amidinothiophene P1 Group.

A series of compounds with an amidinothiophene P1 group and a pyrrolidinone-sulphonamide scaffold linker was identified as potent inhibitors of human kallikrein 6 by structure-based virtual screening based on the union accessible binding space of serine proteases. As the first series of potent nonmechanism-based hK6 inhibitors, they may be used as tool compounds for target validation. An X-ray structure of a representative compound complexed with hK6, resolved at a resolution of 1.

A β-tryptase inhibitor with a tropanylamide scaffold to improve in vitro stability and to lower hERG channel binding affinity.

Tropanylamide was investigated as a possible scaffold for β-tryptase inhibitors with a basic benzylamine P1 group and a substituted thiophene P4 group. Comparing to piperidinylamide, the tropanylamide scaffold is much more rigid, which presents less opportunity for the inhibitor to bind with off-target proteins, such as cytochrome P450, SSAO, and hERG potassium channel. The proposed binding mode was further confirmed by an in-house X-ray structure through co-crystallization.

Structure-based library design and the discovery of a potent and selective mast cell β-tryptase inhibitor as an oral therapeutic agent.

A solid phase combinatorial library was designed based on X-ray structures and in-silico models to explore an inducible S4+ pocket, which is formed by a simple side-chain rotation of Tyr95. This inducible S4+ pocket is unique to β-tryptase and does not exist for other trypsin-like serine proteases of interest. Therefore, inhibitors utilizing this pocket have inherent advantages for being selective against other proteases in the same family.

A conformationally constrained inhibitor with an enhanced potency for β-tryptase and stability against semicarbazide-sensitive amine oxidase (SSAO).

A novel β-tryptase inhibitor with a basic benzylamine P1 group, a piperidine-amide linker, and a substituted indole P4 group was discovered. A substitution at 4-indole position was introduced to constrain the conformational flexibility of the inhibitor to the bioactive conformation exhibited by X-ray structures so that entropic penalty was decreased. More importantly, this constrained conformation limited the accessibility of this molecule to anti-targets, especially SSAO, so that an enhanced metabolic profile was achieved.

Design, synthesis, and biological activity of potent and selective inhibitors of mast cell tryptase.

A new series of novel mast cell tryptase inhibitors is reported, which features the use of an indole structure as the hydrophobic substituent on a m-benzylaminepiperidine template. The best members of this series display good in vitro activity and excellent selectivity against other serine proteases.

Structure based design of 4-(3-aminomethylphenyl)piperidinyl-1-amides: novel, potent, selective, and orally bioavailable inhibitors of betaII tryptase.

Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests tryptase beta is the most important form released during mast cell activation in allergic diseases.

Design, synthesis and characterization of a novel class of coumarin-based inhibitors of inducible nitric oxide synthase.

Inducible nitric oxide synthase (iNOS) has been implicated in various central and peripheral pathophysiological diseases. Our high throughput screening initially identified a weak inhibitor of iNOS, thiocoumarin 13. From this lead, a number of potent derivatives were prepared that demonstrate favorable potency, selectivity and kinetics.

Novel pyrazinone inhibitors of mast cell tryptase: synthesis and SAR evaluation.

In this manuscript, the synthesis and SAR evaluation of a novel pyrazinone class of tryptase inhibitors is described. Chemical optimization of the P1 and P4 groups led to the identification of 7p (K(i)=93 nM) as a potent inhibitor of mast cell tryptase.

Discovery of an orally efficacious inhibitor of coagulation factor Xa which incorporates a neutral P1 ligand.

The discovery and SAR of ketopiperazino methylazaindole factor Xa inhibitors are described. Structure-activity data suggesting that this class of inhibitors does not bind in the canonical mode were confirmed by an X-ray crystal structure showing the neutral haloaromatic bound in the S(1) subsite. The most potent azaindole, 33 (RPR209685), is selective against related serine proteases and attains higher levels of exposure upon oral dosing than comparable benzamidines and benzamidine isosteres.

Molecular mechanics (MM4) force field development for phosphine and its alkyl derivatives.

Based on the experimental structures and vibrational spectra of eight alkylphosphines, as well as ten ab initio calculated (MP2/6-31+g*) structures and five potential energy profiles, the MM4 force field has been extended to include this important functional group. The results are comparable to experimental values for phosphorus-containing compounds. The addition of various cross-terms significantly improved the MM4 calculated structures relative to its predecessor, MM3.