Targeting intracellular p-aminobenzoic acid production potentiates the anti-tubercular action of antifolates

1 The ability to revitalize and re-purpose existing drugs offers a powerful approach for novel treatment options against Mycobacterium tuberculosis and other infectious agents. Antifolates are an underutilized drug class in tuberculosis (TB) therapy, capable of disrupting the biosynthesis of tetrahydrofolate, an essential cellular cofactor. Based on the observation that exogenously supplied p-aminobenzoic acid (PABA) can antagonize the action of antifolates that interact with dihydropteroate synthase (DHPS), such as sulfonamides and p-aminosalicylic acid (PAS), we hypothesized that bacterial PABA biosynthesis contributes to intrinsic antifolate resistance. Herein, we demonstrate that disruption of PABA biosynthesis potentiates the anti-tubercular action of DHPS inhibitors and PAS by up to 1000 fold. Disruption of PABA biosynthesis is also demonstrated to lead to loss of viability over time. Further, we demonstrate that this strategy restores the wild type level of PAS susceptibility in a previously characterized PAS resistant strain of M. tuberculosis. Finally, we demonstrate selective inhibition of PABA biosynthesis in M. tuberculosis using the small molecule MAC173979. This study reveals that the M. tuberculosis PABA biosynthetic pathway is responsible for intrinsic resistance to various antifolates and this pathway is a chemically vulnerable target whose disruption could potentiate the tuberculocidal activity of an underutilized class of antimicrobial agents. Tuberculosis (TB) causes over 1.7 million deaths per year and an estimated two billion people latently infected with Mycobacterium tuberculosis provides a large reservoir for ongoing reactivation and transmission of disease 1,2 . Treatment options for TB are longer and more complex than treatments for other bacterial infections. These com-plex therapies have led to patient non-compliance and disrupted treatment, resulting in an increased incidence of multidrug resistant and extensively drug resistant M. tuberculosis infections 1,3 . The search for new therapeutic options for treatment of drug susceptible as well as drug resistant strains has fueled an effort to repurpose existing drugs for TB therapy 4,5 . Sulfonamides are broad spectrum antimicrobials commonly used to treat many bacterial infections 6,7 . These compounds are structurally similar to p-aminobenzoic acid (PABA), an essential precursor for synthe-sis of tetrahydrofolate. This structural similarity enables competitive inhibition of dihydropteroate synthase (DHPS) by sulfonamides thereby leading to disruption of tetrahydrofolate biosynthesis (Fig. 1a) 9 . In addition, it has been shown that sulfonamides can serve as alternative substrates for DHPS leading to the depletion of dihydropterin pools with the synthesis of dead end products that might further impair tetrahydrofolate bio-synthesis 9,10 . Sulfonamides were used in early experimental TB therapy, but were replaced by the more potent anti-tubercular agents streptomycin and p-aminosalicylic acid (PAS) 11–13 . Dapsone, another PABA analog and DHPS inhibitor, is a cornerstone in treatment of infections with the related species Mycobacterium leprae. Yet, due to moderate potency and the extent of adverse drug reactions, this drug is not used for M. tuberculo-sis infections. Despite the limited utility of sulfonamides and dapsone against M. tuberculosis infection, enzy-matic studies have confirmed that these compounds are potent competitive inhibitors for purified recombinant 1