Structure and inhibitors of S. aureus HPPK - a folate pathway enzyme

Folate is an essential vitamin (vitamin B9) that is required for many one-carbon transfer reactions and is a critical precursor for the biosynthesis of purines, pyrimidines, and amino acids. Folate is a dietary requirement for man and animals. However bacteria, parasites and plants can synthesize folate de novo using enzymes of the folate biosynthesis pathway. Thus folate biosynthesis remains a key target for antimicrobial therapy as exemplified by the sulfa drugs that date back to the 1940s, acting on the enzyme DHPS. HPPK, like other enzymes of the folate biosynthesis pathway, is absent in man and therefore is a potential drug target for the development of antimicrobial agents. Increasing rates of drug resistance to current antibiotics is a serious problem, due to the rapid adaptation of microorganisms to our chemical interventions. Because HPPK is not the target of any existing antibiotic, it is attractive for the development of new antibiotics against resistant strains. The dearth of substrate-site inhibitors for HPPK observed in the literature reflects a combination of difficult pterin-like chemistry and a paucity of commercially available pterin-like molecules, compounded by a highly specific substrate site. This knowledge gap presents interesting research opportunities. Herein, the first structural and biophysical data on 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK catalyses the pyrophosphoryl transfer from the cofactor (ATP) to the pterin substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). The expression, purification and protein stability was optimised to provide high yields for both screening and crystallisation experiments. A combination of fragment and in silico based methods was adopted to identify small molecule inhibitors of the SaHPPK enzyme