Abstract
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.
Highlights
Since PABA is an essential precursor for tetrahydrofolate biosynthesis, M. tuberculosis must maintain a basal level of intracellular PABA, as was confirmed by a recent metabolomic analysis of M. tuberculosis[17]
To begin assessing the effects of intracellular PABA concentration on potency of DHPS inhibitors, M. tuberculosis transposon mutant strains deficient for PABA production were isolated
One well-known example of this augmentation is in the combined use of β-lactam antibiotics with clavulanic acid in order to neutralize β-lactamases[25]
Summary
Since PABA is an essential precursor for tetrahydrofolate biosynthesis, M. tuberculosis must maintain a basal level of intracellular PABA, as was confirmed by a recent metabolomic analysis of M. tuberculosis[17]. These studies demonstrated a dose dependent increase in intracellular PABA in response to treatment with DHPS inhibitors and PAS17. We reasoned that since internal PABA levels are significantly increased following antifolate treatment, PABA synthesis might play a crucial role in intrinsic resistance to these drugs. We hypothesized that impairing synthesis of PABA would abolish this intrinsic drug resistance and lead to potentiation of antifolate action against M. tuberculosis
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