SUMMARY
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