Targeting Two Late-Stage Enzymes of the Mycobacterium tuberculosis Biotin Biosynthetic Pathway

Abstract

Mycobacterium tuberculosis (Mtb), responsible for both latent and symptomatic tuberculosis (TB), remains the leading cause of mortality among infectious diseases worldwide. The rise and propagation of drug-resistant TB remains a global health crisis and has prompted researchers to investigate novel mechanisms of action for the development of antitubercular agents. Chapter 1 discusses the biotin biosynthetic pathway as a target for the development of antibiotics targeting Mtb, providing both chemical and genetic validation evidence of inhibiting this pathway in Mtb infections. This chapter thoroughly examines each enzyme in the biotin biosynthetic pathway by reviewing: the reaction it catalyzes, its mechanism of action, structural and sequence analysis, and catalogue of inhibitors known for each enzyme. The late-stage biotin synthase (BioB) and biotin protein ligase (BPL) proteins are elaborated on and will be the focus of this thesis. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. Chapter 2 reports the synthesis and evaluation of a systematic series of potent nucleoside-based bisubstrate inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KDs ≤ 2 nM. Additionally, this chapter discusses the structural interactions between the inhibitors and MtBPL using the highly-resolved x-ray co-crystal structures. Despite relatively uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MICs) ranging from 0.78 to >100 uM. Cellular accumulation studies showed a nearly ten-fold enhancement in accumulation of a C-2′-a-fluoro analogue over the corresponding C-2′-b-fluoro analogue, consistent with their differential whole-cell activity. The parent compound, Bio-AMS, was also evaluated for its pharmacokinetic (PK) parameters, and although it shows stability toward plasma and liver microsomes, Bio-AMS is rapidly cleared form CD-1 mice. From chapter 2, the potent compound Bio-AMS was shown to possess selective activity against MtBPL. However, Mtb develops spontaneous resistance to Bio-AMS with a frequency of resistance (FOR) of at least 1 x 10-7 by overexpression of Rv3406, a type II sulfatase that enzymatically inactivates Bio-AMS. In an effort to circumvent this resistance mechanism, chapter 3 describes the strategic modification of the Bio-AMS at the 5’-position to prevent enzymatic inactivation. The new analogues retain subnanomolar potency to MtBPL, and the 5′R-C-methyl derivative exhibited identical antimycobacterial activity toward: Mtb H37Rv, MtBPL overexpression, and an isogenic Rv3406 overexpression strain (MIC = 1.56 uM). Moreover, this compound was not metabolized by recombinant Rv3406 and resistant mutants to this compound could not be isolated (FOR < 1.4 x 10-10) demonstrating it successfully overcame Rv3406-mediated resistance. The natural product acidomycin, discovered in 1952 and isolated from Streptomyces spp., was originally shown to have selective antibiotic activity against Mtb grown in the absence of biotin, implying it is an antimetabolite of the biotin biosynthetic pathway. Chapter 4 fully investigates the mechanism of action and selectivity of acidomycin. Acidomycin was evaluated against an array of drug susceptible and drug resistant Mtb strains, as well as a panel of gram-positive and gram-negative pathogens, and showed remarkable selectivity to Mtb with MICs ranging from 0.096 – 6.2 uM for the Mtb strains and >100 M for the other microorganisms. Acidomycin was also shown to be a reversible, competitive inhibitor of E. coli biotin synthase (EcBioB), with a Ki of 1.5 uM, and a homology model shows substantial sequence alignment in the mycobacterial enzyme (MtBioB). The selectivity of acidomycin against E. coli versus Mtb is due to differential levels of cellular accumulation, with a 30-fold increase in the amount of acidomycin accumulated in Mtb over E. coli. In vivo, acidomycin was shown to be rapidly eliminated from CD-1 mice, is a half-life of 9.6 min, but exhibited remarkable plasma and microsomal stability. A brief series of acidomycin analogues showed a very tight SAR window for modifications, with the primary amide analogue being the best analogue with an MIC less than two-fold of acidomycin.