Assay Development and Identification of the First 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase Inhibitors.

In the fight towards eradication of malaria, identifying compounds active against new drug targets constitutes a key approach. 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK) has been advanced as a promising target, as being part of the parasite essential folate biosynthesis pathway while having no orthologue in the human genome. However, no drug discovery efforts have been reported on this enzyme. In this study, we conducted a three-step screening of our in-house antifolate library against HPPK using a newly designed HPPK-GFP protein construct. Combining virtual screening, differential scanning fluorimetry and enzymatic assay, we identified 14 compounds active against HPPK. Compounds' binding modes were investigated by molecular docking, suggesting competitive binding with the HMDP substrate. Cytotoxicity and in vitro ADME properties of hit compounds were also assessed, showing good metabolic stability and low toxicity. The most active compounds displayed low micromolar IC against drug-resistant parasites. The reported hit compounds constitute a good starting point for inhibitor development against HPPK, as an alternative approach to tackle the malaria parasite.