A cell‐based inhibitor screening platform for S‐adenosylmethionine synthetase

Abstract

Infectious diseases are one of the leading causes of death worldwide. The remarkable social and economic burden they cause in society demands new tools, including novel drug approaches, to deal with these diseases. Our present study is based on a growth‐based assay that we have developed in E. coli for screening of inhibitors against S‐adenosylmethionine (AdoMet) synthetase (MAT) from different bacterial, fungal and protozoan species. MAT is the only known enzyme to catalyze the biosynthesis of AdoMet, an essential metabolite for all living organisms. The multitude of metabolic roles of AdoMet makes it an attractive target for development of inhibitors. The sequence and structure of this enzyme is highly conserved particularly in the active site, from bacterial to eukaryotic species. This conservation contributes to the difficulty of finding selective inhibitors. Our in vivo platform for screening inhibitors employs an E. coli metK‐deletion mutant (ΔmetK) that makes its growth dependent upon extracellular AdoMet. Plasmids carrying one of several bacterial, fungal, protozoan or mammalian MAT genes were found to rescue the ΔmetK deficiency without AdoMet supplementation. The growth rates are similar in each complemented strains except for the significant delay in lag phase with the human MAT. An initial proof‐of‐principle experiment with cycloleucine, a known weak inhibitor of MAT, showed different degrees of inhibition in the complemented MATs. Optimization of the inhibitor properties for species selectivity is being guided by determination of the structures of the enzyme from different infectious organisms. Our results highlight the applicability of this screening platform to identify selective inhibitors of bacterial, fungal or protozoan species, with a counter screening to avoid compounds that inhibit human MAT.