Jose S. Mendoza

Synthesis and protein kinase C inhibitory activities of balanol analogues with modification of 4-hydroxybenzamido moiety

A series of racemic balanol analogues with modification of the benzamido moiety of balanol have been synthesized and evaluated for their inhibitory activities against human protein kinase C isozymes (PKC-alpha, -beta I, -beta II, -gamma, -delta, -epsilon, and -eta). The structural modification includes replacement of the 4-hydroxyphenyl group with variously substituted phenyl rings, substitution of the amide linkage with a sulfonamide or an ester, and replacement of the 4-hydroxyphenyl substructure with a hydroxyl substituted indole or a hydroxybenzyl group. in general, these analogues were found to be less potent than balanol, but a number of analogues were identified with improved isozyme selectivity. The structure-activity relationship studies of these analogues also indicated that (1) the optimal general PKC inhibition requires a free 4-hydroxyl group in the benzamido portion of the molecule, (2) the amide linkage of the benzamido moiety is important for PKC inhibition, and (3) the conformation associated with the benzamido moiety seems to have a profound effect on PKC inhibition. The requirement of a free 4-hydroxyl group in conjunction with an appropriate conformation of the benzamido moiety for optimal PKC inhibition suggests that the 4-hydroxyphenyl group may be involved in a specific inhibitor-enzyme interaction important for PKC inhibition.

Discovery of Highly Selective Inhibitors of p38α

The p38 MAP kinases are a family of serine/threonine protein kinases that play a key role in cellular pathways leading to pro-inflammatory responses. We have developed and implemented a method for rapidly identifying and optimizing potent and selective p38alpha inhibitors, which is amenable to other targets and target classes. A diverse library of druggable, purified and quantitated molecules was assembled and standardized enzymatic assays were performed in a microfluidic format that provided very accurate and precise inhibition data allowing for development of SAR directly from the primary HTS. All compounds were screened against a collection of more than 60 enzymes (kinases, proteases and phosphatases), allowing for removal of promiscuous and non-selective inhibitors very early in the discovery process. Follow-up enzymological studies included measurement of concentration of compound in buffer, yielding accurate determination of K(i) and IC50 values, as well as mechanism of action. In addition, active compounds were screened against less desirable properties such as inhibition of the enzyme activity by aggregation, irreversible binding, and time-dependence. Screening of an 88,634-compound library through the above-described process led to the rapid identification of multiple scaffolds (>5 active compounds per scaffold) of potential drug leads for p38alpha that are highly selective against all other enzymes tested, including the three other p38 isoforms. Potency and selectivity data allowed prioritization of the identified scaffolds for optimization. Herein we present results around our 3-thio-1,2,4-triazole lead series of p38- selective inhibitors, including identification, SAR, synthesis, selectivity profile, enzymatic and cellular data in their progression towards drug candidates.