Murine mPGES-1 3D Structure Elucidation and Inhibitors Binding Mode Predictions by Homology Modeling and Site-Directed Mutagenesis

Abstract

Microsomal prostaglandin E synthase-1 (mPGES-1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxido-reduction of cyclooxygenase derived PGH₂ into PGE₂. mPGES-1 is an essential enzyme involved in a variety of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain; it is therefore regarded as a primary target for development of next-generation anti-inflammatory drugs. Several compounds targeting human mPGES-1 have been reported in the literature. However, none of them is really specific for mPGES-1, and quite surprisingly, all of these compounds have very low or no activity against murine mPGES-1, making preclinical development hard and very expensive. In order to overcome this unresolved question, the current study focuses on the elucidation of the molecular determinants of murine mPGES-1 ligand binding modes combining protein homology modeling and site-directed mutagenesis approaches. We have developed, for the first time, two murine mPGES-1 models, describing both the closed and the open/active conformation of the enzyme. The 3D structure of human mPGES-1 having been recently disclosed, the main differences between the human and the murine enzyme models are described, emphasizing the smaller dimensions of the rodent substrate binding site, which could account for different activity of a ligand toward the two species. Furthermore, active binding modes are hypothesized, highlighting the most likely important residues for inhibition activity, whose identification is supported by in-house mutagenesis experiments. The results of our work could provide grounds for a rational structure-based drug design aimed to identify new inhibitors active against both human and murine mPGES-1.

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells, and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

“…This could be explained by the fact that the aminothiazoles were identified based on their ability to bind to the active site of the human mPGES‐1 enzyme, and that RAW 264.7 cells are of murine origin. TH‐644 may have a lower binding to the murine orthologue of mPGES‐1 and higher concentrations were therefore required to obtain an inhibitory effect, as it has been reported that there are structural differences between human and rodent mPGES‐1 . However, the higher dose of TH‐644 was also required for PBMCs cultures, as no effect could be observed by 2 μmol/L.…”

“…TH-644 may have a lower binding to the murine orthologue of mPGES-1 and higher concentrations were therefore required to obtain an inhibitory effect, as it has been reported that there are structural differences between human and rodent mPGES-1. 39,40 However, the higher dose of TH-644 was also required for PBMCs cultures, as no effect could be observed by 2 μmol/L. In contrast to TH-644, TH-848 inhibitor was more potent, as it inhibited PGE 2 production and osteoclast formation at much lower concentrations in RAW 264.7 cells, as well as osteoclast formation and bone resorption in human PBMCs, indicating that the potency of this inhibitor seems to be independent of the origin of cells.…”

A Novel Multi‐step Virtual Screening for the Identification ofHumanandMousemPGES‐1 Inhibitors

“… Abstract We present here the development of a novel virtual screening protocol combining Structure‐based and Ligand‐based drug design approaches for the identification of mouse mPGES‐1 inhibitors. We used the existing 3D structural data of the murine enzyme to hypothesize the inhibitors binding mode, which was the starting point for docking simulations, shape screening, and pharmacophore hypothesis screening. The protocol allowed the identification of 16 mouse mPGES‐1 inhibitors with low micromolar activity, which, notably, also inhibit the human enzyme in the same concentration range.…”

“…When experimental 3D data are absent, in silico homology models combined with virtuals creening approaches are powerful techniques in determining the structural features needed for ag iven compoundt oe fficiently interact with the protein.In the last decade, Angelini research in inflammatory disease led to the identificationo fp otent inhibitors of human mPGES-1. [13] Aimed to identify new human/mouse mPGES-1i nhibitors, we built two homology models of mouse mPGES-1 [1] (namely,H M1 and HM2), with HM2 describing the active conformation of the enzyme (Figure 1). Despite the high sequenceh omology between human and mouse mPGES-1 (79 %s equence identity), 12 out of the total 29 differences in the protein sequences are located in the predicted substrate binding site, and, assuming competitiveinhibition,t hey could explain the inactivity of human inhibitors against the murine enzyme.The binding of inhibitors into the human mPGES-1 active site, supported by hydrogen/deuterium exchange kinetics experiments, [14] has beenc onfirmed by x-ray crystallography, [11] indicating that the PGH 2 -competitive human inhibitors bind into ah ydrophobic cleft located between neigh-Abstract:W ep resent here the development of an ovel virtual screening protocol combining Structure-based and Ligand-based drug design approaches for the identification of mouse mPGES-1i nhibitors.…”

“…Despite the high sequenceh omology between human and mouse mPGES-1 (79 %s equence identity), 12 out of the total 29 differences in the protein sequences are located in the predicted substrate binding site, and, assuming competitiveinhibition,t hey could explain the inactivity of human inhibitors against the murine enzyme.The binding of inhibitors into the human mPGES-1 active site, supported by hydrogen/deuterium exchange kinetics experiments, [14] has beenc onfirmed by x-ray crystallography, [11] indicating that the PGH 2 -competitive human inhibitors bind into ah ydrophobic cleft located between neigh-Abstract:W ep resent here the development of an ovel virtual screening protocol combining Structure-based and Ligand-based drug design approaches for the identification of mouse mPGES-1i nhibitors. We used the existing [1] 3D structural data of the murine enzyme to hypothesize the inhibitors binding mode, which was the starting point for docking simulations, shape screening, and pharmacophore hypothesis screening. The protocol allowed the identification of 16 mouse mPGES-1 inhibitors with low micromolar activity,w hich, notably,a lso inhibit the human enzyme in the same concentration range.…”