Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets

Abstract

Remarkable selectivity enhancements of exceptionally small inhibitors are achieved toward the uPA target over the highly similar tPA anti-target through a single atom substitution on an otherwise relatively non-selective scaffold. Overall selectivities for uPA over tPA as high as 980-fold at physiological pH were realized. The increase in selectivity results from the displacement of a single bound water molecule common to the S1 site of both the uPA target and the tPA anti-target because of the ensuing deficit in hydrogen bonding of the arylamidine inhibitor when bound in the Ala190 protease anti-target.

A Urokinase-type Plasminogen Activator-inhibiting Cyclic Peptide with an Unusual P2 Residue and an Extended Protease Binding Surface Demonstrates New Modalities for Enzyme Inhibition

“…Among other organochemical uPA inhibitors, a 30-fold selectivity for uPA over plasmin, thrombin, fXa, tPA, and/or trypsin was also achieved with a series of (4-aminomethyl)phenylguanidine derivatives targeting the S 1 and the S 1 Ј subsites, displaying K i values for uPA in the micromolar range (15). In other reported studies, selectivity ranging from more than 100-fold up to 2000-fold for uPA inhibition over that of plasmin, trypsin, thrombin, tPA, and fXa has been described for a series of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors with K i values in the nanomolar range (14,(43)(44)(45). In contrast to our results here, measurements of selectivity over plasma kallikrein were reported for only a few of these inhibitors (14), and selectivity over aPC was not investigated.…”

Expression, Crystallization, and Three-dimensional Structure of the Catalytic Domain of Human Plasma Kallikrein

“…Inhibitors-Previously, we developed a large suite of small molecule inhibitors (2-(2-phenol)-benzimidazoles and 2-(2-phenol)-indoles) as serine protease inhibitors (61)(62)(63)(64)(65)(66)(67). Inhibition by such scaffolds is typically mediated by short hydrogen bonds at the active site, often via a multicentered short hydrogen bond array involving the active site Ser 195 , a water in the oxyanion hole, the phenolate of the inhibitor, and the benzimidazole (or indole) nitrogen of the inhibitor (see diagram in TABLE SIX).…”

“…Other hydrogen bonds to benzamidine involve the carbonyl group of Gly 219 and a water molecule. Although the structures of the S1 sites of trypsin-like serine proteases resemble one another, one important difference often occurs at residue 190 (52,62,66 protease-amidine complexes (such as plasma kallikrein-benzamidine; see Fig. 5), the sole hydrogen-bonding partner to one of the amidine protons is a water molecule, and in the corresponding Ser…”

“…Thus, inhibitors designed to displace the water are often highly selective for Ser 190 proteases over Ala 190 counterparts (such as plasma kallikrein) because of the resulting hydrogen-bonding deficit at the S1 site (62,66,67). Thus, viewed as an anti-target, plasma kallikrein offers the S1 site as a potential locus for engineering selectivity against this protease, if the target of interest is a Ser 190 protease, such as urokinase.…”

“…In our other serine protease inhibitor development programs, the S1Ј site has been used extensively for engineering selectivity toward particular protease targets into inhibitors that make short hydrogen bonds at the active site (61)(62)(63)(64)(65)(66). In plasma kallikrein, the side chains of residues Leu 41 , Leu 60B , and Trp 60D form a binding pocket that is readily accessible from the para position of the distal phenyl group of a typical, simple short hydrogen bond-mediated scaffold, like CG-01 (Fig.…”