Prostaglandins function as signaling molecules involved in pain, fever, and many diseases associated with chronic inflammation. The most common therapeutic treatment of inflammation involves the inhibition of the COX enzymes by nonsteroidal anti-inflammatory drugs (NSAIDs) or COX-2 selective inhibitors (coxibs). The inhibition of the COX enzymes prevents the catalytic formation of the intermediate endoperoxide compound, PGH2. With the inhibition of PGH2, the production of other downstream prostaglandins is also inhibited. Therefore, COX inhibition can result in adverse gastrointestinal and cardiovascular side effects because of the subsequent low level of various prostanoids. Microsomal Prostaglandin E2 synthase 1 (MPGES1), a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily, is the most prominent PGE synthase expressed during inflammation and is the terminal enzyme in the PGE2 synthesis pathway. It is a promising therapeutic target for the treatment of chronic inflammation and potential drug candidates have been pursued by the pharmaceutical industry in recent years. However, little is known about the actual chemical mechanism of the enzyme. Glutathione (GSH) appears to participate as a cofactor in the reaction, though there hasn’t been any evidence to support this. In order to investigate GSH as a cofactor in the reaction, it is necessary to detect the thiolate of GSH in the active site and determine its exchange rate off of the enzyme surface. One equivalent of the thiolate is observable by difference spectroscopy. However, simple difference spectroscopy isn’t possible since the enzyme is unstable in the absence of GSH. Therefore, a solution of the enzyme in complex with GSH can be rapidly mixed with the serine analogue of GSH, γ-L-glutamyl-L-serylglycine (GOH), or the alanine analogue γ-L-glutamyl-L-alanylglycine (GH) and the kinetics of the loss in absorbance from the replacement of GS- with GO- or GH would confirm the presence of the thiolate in the complex and its rate of dissociation from the enzyme can be determined. A technique known as Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) can also be utilized to investigate the differences in conformational dynamics between MPGES1 and its natural cofactor, GSH and MPGES1 that is in complex with one of the aforementioned analogues, GOH or GH.

Investigation of the chemical mechanism and inhibition of microsomal prostaglandin E2 synthase 1 (MPGES1)

250th National Meeting (2015)

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