3‑Hydroxy-Beta-Lactam Inhibitors of Dihydrofolate Synthetase

The 3-hydroxy-β-lactam (3-HβL) group derived from the natural product tabtoxinine-β-lactam (TβL), an inhibitor of glutamine synthetase, was repurposed to develop an inhibitor of dihydrofolate synthetase (DHFS). We show that replacement of the carboxyl group of p-amino-benzoic acid (PABA) with a 3-HβL moiety on the chemical scaffold of a folate mimic results in a potent inhibitor of DHFS. Using a combination of in vitro steady-state kinetics, enzyme-coupled assays, and molecular modeling, we validate the essential role of the 3-HβL group in DHFS inhibition. We provide an optimized synthesis of the 3-(p-aminophenyl)-3-HβL component via a sequence of the C-C bond-forming Henry reaction and a β-lactam ring-closing Grignard reaction. We demonstrate full elaboration to an antifolate scaffold via chemical or chemoenzymatic conjugation of the PABA analogue 3-(p-aminophenyl)-3-HβL to a pterin mimic. In this proof-of-concept study, we provide the first evidence that the 3-HβL group can be used as a general pharmacophore for inhibitors of enzymes in the ATP-dependent carboxylate-amine ligase superfamily through carboxylate replacement on substrate scaffolds, which could have broad therapeutic applications.