Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-1*

Abstract

Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E2. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E2 production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substrate PGH2, or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.

Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders

“…Since PGH 2 is produced by COX on the luminal side of the endoplasmic reticulum, PGH 2 apparently diffuses through the membrane before it enters the active site of mPGES-1 near the cytoplasmic side of the endoplasmic reticulum membrane [32]. Thr131, Leu135, Ala138 and potentially Arg52 and His53 act as gate keepers and limit the access of inhibitors to the active site [31,33]. The smaller size of residues in these positions in the human compared to the rat enzyme may explain why several mPGES-1 inhibitors fail to inhibit rat mPGES-1 (see below).…”

Crystal structure of microsomal prostaglandin E2synthase provides insight into diversity in the MAPEG superfamily

“…Based on the low-resolution electron crystallography structure (14) and the assumption that the active site would be analogous to LTC 4 synthase, a series of point mutations were made to rationalize the lack of species cross-over (27). A combination of Thr131, Leu135, and Ala138 mutations were shown to have an effect on altering species selectivity, although the effect did not account for the full difference in inhibition.…”

“…The putative inhibitor binding site is not completely conserved across species and there are reports of compounds displaying good in vitro potency in human mPGES-1 but little or no effect in rat (26,27). Based on the low-resolution electron crystallography structure (14) and the assumption that the active site would be analogous to LTC 4 synthase, a series of point mutations were made to rationalize the lack of species cross-over (27).…”

Dual, Constrictor-to-Dilator, Response of the Mouse Ductus Arteriosus to the Microsomal Prostaglandin E Synthase-1 Inhibitor, 2-(6-Chloro-1H-phenanthro[9,10<i>d</i>]imidazole- 2-yl)isophthalonitrile

“…In each case, the number is given above the columns. susceptibility of the enzyme to inhibitors [7,18,19] . Nevertheless, it was found quite unexpectedly that the action of MF63 also comprises a dilator component, becoming evident with chronic treatment or with acute treatment when a certain dose is exceeded.…”