Mycobacterium tuberculosis (Mtb), the principal etiological agent of tuberculosis (TB), infects over one-third of humanity and is now the leading cause of infectious disease mortality by a single pathogen. Mtb requires biotin for survival and synthesizes this essential cofactor de novo. In preliminary studies using a genetic approach, we have shown biotin biosynthesis and ligation are essential for Mtb infection in mice. We have synthesized a selective nanomolar inhibitor of biotin protein ligase termed Bio-AMS that targets the enzyme biotin protein ligase (BPL,) responsible for the ligation of biotin onto biotin-dependent enzymes. We have also identified the natural product acidomycin, which targets the final step of biotin biosynthesis catalyze by BioB. However, Bio- AMS and acidomycin have liabilities in their drug disposition properties leading to rapid clearance, poor volume of distribution, and limited oral bioavailability. There are also gaps in our knowledge regarding their mechanism of resistance and activity when combined with other TB drugs. The objectives of this application are: 1) to develop our lead compounds Bio-AMS and acidomycin through the optimization of their ADME (absorption, distribution, metabolism and elimination) properties and pharmacokinetic parameters into viable preclinical candidates, 2) to more deeply illuminate the mechanism of action and resistance in Mtb, 3) to determine the safety profile and potential drug-drug interactions, and 4) to identify interactive effects with other TB drugs (i.e. synergy). We will accomplish the overall objectives of this application by pursuing three specific aims.
In aim 1, we will carry out an iterative structure-based medicinal chemistry program of Bio-AMS and acidomycin to concurrently optimize pharmacokinetic (PK) parameters and whole-cell activity using a combination of approaches including fluorination, structural simplification, and introduction of conformation constraints.
In aim 2, we will perform biochemical and cellular studies to evaluate enzyme inhibition, target engagement, cellular accumulation, and whole-cell activity against Mtb as well as drug-resistant strains. Generation of resistant strains followed by whole-genome sequencing will be used to characterize potential resistance mechanisms and determine the resistance frequency. Finally, combination studies with various first and second-line TB drugs will be undertaken to assess potential for synergy.
In aim 3, the Bio-AMS and acidomycin analogues will be assessed in vivo to determine their complete pharmacokinetic parameters with a goal to improve on the volume of distribution (Vd), intrinsic clearance (CL), and bioavailability (F). We will evaluate compounds against a panel of assays (hERG, CYP inhibition, Ames mutagenicity) to ensure safety and selectivity. In vivo efficacy studies will be done using murine models of acute and chronic TB infection
SUMMARY Tuberculosis (TB) caused by the slow growing bacillus Mycobacterium tuberculosis (Mtb) is the leading cause of infectious disease mortality in the world by a single pathogen. M. tuberculosis and other atypical mycobacteria are also classified as opportunistic infections of AIDS patients. The proposed research is expected to lead to a new class of antitubercular agents that operate by a unique mechanism of action through disruption of biotin biosynthesis.