Biochemical characterization of selective inhibitors of human group IIA secreted phospholipase A(2) and hyaluronic acid-linked inhibitor conjugates.

We explored the inhibition mode of group IIA secreted phospholipase A(2) (GIIA sPLA(2)) selective inhibitors and tested their ability to inhibit GIIA sPLA(2) activity as chemical conjugates with hyaluronic acid (HA). Analogues of a benzo-fused indole sPLA(2) inhibitor were developed in which the carboxylate group on the inhibitor scaffold, which has been shown to coordinate to a Ca(2+) ligand in the enzyme active site, was replaced with other functionality. Replacing the carboxylate group with amine, amide, or hydroxyl groups had no effect on human GIIA (hGIIA) sPLA(2) inhibition potency but dramatically lowered inhibition potency against hGV and hGX sPLA(2)s. An alkylation protection assay was used to probe active site binding of carboxylate and noncarboxylate inhibitors in the presence and absence of Ca(2+) and/or lipid vesicles. We observed that carboxylate-containing inhibitors bind the hGIIA sPLA(2) active site with low nanomolar affinity, but only when Ca(2+) is present. Noncarboxylate, GIIA sPLA(2) selective inhibitors also bind the hGIIA sPLA(2) active site in the nanomolar range. However, binding for GIIA sPLA(2) selective inhibitors was dependent on the presence of a lipid membrane and not Ca(2+). These results indicate that GIIA sPLA(2) selective inhibitors exert their inhibitory effects by binding to the hGIIA sPLA(2) active site. An HA-linked GIIA inhibitor conjugate was developed using peptide coupling conditions and found to be less potent and selective against hGIIA sPLA(2) than the unconjugated inhibitor. Compounds reported in this study are some of the most potent and selective GIIA sPLA(2) active site binding inhibitors reported to date.