title: para-Aminosalicylic acid is a prodrug targeting dihydrofolate reductase in Mycobacterium tuberculosis.
abstract: para-Aminosalicylic acid (PAS) is one of the antimycobacterial drugs currently used for multidrug-resistant tuberculosis. Although it has been in clinical use for over 60 years, its mechanism(s) of action remains elusive. Here we report that PAS is a prodrug targeting dihydrofolate reductase (DHFR) through an unusual and novel mechanism of action. We provide evidences that PAS is incorporated into the folate pathway by dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS) to generate a hydroxyl dihydrofolate antimetabolite, which in turn inhibits DHFR enzymatic activity. Interestingly, PAS is recognized by DHPS as efficiently as its natural substrate para-amino benzoic acid. Chemical inhibition of DHPS or mutation in DHFS prevents the formation of the antimetabolite, thereby conferring resistance to PAS. In addition, we identified a bifunctional enzyme (riboflavin biosynthesis protein (RibD)), a putative functional analog of DHFR in a knock-out strain. This finding is further supported by the identification of PAS-resistant clinical isolates encoding a RibD overexpression mutation displaying cross-resistance to genuine DHFR inhibitors. Our findings reveal that a metabolite of PAS inhibits DHFR in the folate pathway. RibD was shown to act as a functional analog of DHFR, and as for DHFS, both were shown to be associated in PAS resistance in laboratory strains and clinical isolates.
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore 138670. zhjunyy@hotmail.com
authors.forename: Jun
authors.initials: J
authors.lastname: Zheng
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rubin
authors.forename: Pablo
authors.initials: P
authors.lastname: Bifani
authors.forename: Vanessa
authors.initials: V
authors.lastname: Mathys
authors.forename: Vivian
authors.initials: V
authors.lastname: Lim
authors.forename: Melvin
authors.initials: M
authors.lastname: Au
authors.forename: Jichan
authors.initials: J
authors.lastname: Jang
authors.forename: Jiyoun
authors.initials: J
authors.lastname: Nam
authors.forename: Thomas
authors.initials: T
authors.lastname: Dick
authors.forename: John R
authors.initials: JR
authors.lastname: Walker
authors.forename: Kevin
authors.initials: K
authors.lastname: Pethe
authors.forename: Luis R
authors.initials: LR
authors.lastname: Camacho
mesh_terms: D010131:Aminosalicylic Acid / Q000493:pharmacokinetics / Q000494:pharmacology; D000995:Antitubercular Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D004094:Dihydropteroate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000493:pharmacokinetics / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D009154:Mutation; D011355:Prodrugs / Q000493:pharmacokinetics / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D005493:Folic Acid Antagonists; D011355:Prodrugs; D010131:Aminosalicylic Acid; D013762:Tetrahydrofolate Dehydrogenase; D004094:Dihydropteroate Synthase
keywords: Antibiotic Action; Antibiotic Resistance; Drug Development; Folate Metabolism; Microbiology; Mycobacterium tuberculosis