Biochemical Characterization of Selective Inhibitors of Human Group IIA Secreted Phospholipase A2 and Hyaluronic Acid-Linked Inhibitor Conjugates.

We explored the inhibition mode of group IIA secreted phospholipase A2 (GIIA sPLA2) selective inhibitors and tested their ability to inhibit GIIA sPLA2 activity as chemical conjugates with hyaluronic acid (HA). Analogues of a benzo-fused indole sPLA2 inhibitor were developed in which the carboxylate group on the inhibitor scaffold, which has been shown to coordinate to a Ca2+ 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) sPLA2 inhibition potency but dramatically lowered inhibition potency against hGV and hGX sPLA2s. An alkylation protection assay was used to probe active site binding of carboxylate and noncarboxylate inhibitors in the presence and absence of Ca2+ and/or lipid vesicles. We observed that carboxylate-containing inhibitors bind the hGIIA sPLA2 active site with low nanomolar affinity, but only when Ca2+ is present. Noncarboxylate, GIIA sPLA2 selective inhibitors also bind the hGIIA sPLA2 active site in the nanomolar range. However, binding for GIIA sPLA2 selective inhibitors was dependent on the presence of a lipid membrane and not Ca2+. These results indicate that GIIA sPLA2 selective inhibitors exert their inhibitory effects by binding to the hGIIA sPLA2 active site. An HA-linked GIIA inhibitor conjugate was developed using peptide coupling conditions and found to be less potent and selective against hGIIA sPLA2 than the unconjugated inhibitor. Compounds reported in this study are some of the most potent and selective GIIA sPLA2 active site binding inhibitors reported to date. [ABSTRACT FROM AUTHOR]