Identification, Validation, and Structural Studies of Multitargeting Inhibitors Against Mycobacterium Tuberculosis Mycolic Acid Methyltransferases

Abstract

Combating the persistent threat and low therapeutic success of treating multidrug-resistant Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, demands a worldwide effort to discover and develop new antitubercular drugs. M. tuberculosis harbors a lipid-rich cell wall that poses a formidable barrier to small molecules including antibiotics. Mycolic acids are the predominant very long chain extensively-modified fatty acids found in the cell wall of Mtb that are determinant of cell wall permeability, intrinsic drug resistance, and virulence. Mycolic acids are modified by a family of mycolic acid methyltransferases (MA-MTs) that have been shown to be simultaneously inhibited by a single small molecule, dioctylamine. As cyclopropane mycolic acid synthases (CMAS) were the primary MA-MT targets of the compound, we believe targeting this family of synthases is a promising antibiotic strategy, as their simultaneous inhibition results in enhanced drug penetration and synergistic killing when combined with established antimycobacterial drugs. Due to the lack of suitable in vitro assays for drug screening, no drug-like molecules have been identified to establish whether simultaneous inhibition of this family of enzymes is a viable therapeutic strategy for the treatment of tuberculosis. We first combined differential scanning fluorimetry (DSF) screening and organic synthesis to design a set of nitrobenzoxadiazole fluorescence enhancement probes suitable for in vitro CMAS multitarget assessment. Furthermore, we show that the CmaA2 displacement assay was suitable for HTS. Small molecule screening revealed that CMAS bind amine-based amphipathic ligands of broad structural diversity. We then tested the power of our assays for identifying multitarget CMAS inhibitors in vivo by incubating mycobacteria with a selected collection of ligands with diverse chemical scaffolds and looked for changes in mycolic acid modification using radio two-dimensional thin layer chromatography. We found that the majority of these molecules inhibited multiple pathways of mycolic acid modification. Using X-ray crystallography to examine the molecular basis of inhibition, we found that these drug-like molecules mimic the carbocation reaction intermediate. The studies provide a structural foundation for further structure-based inhibitor design. Finally, we tested compound lethality using Mtb kill-curve assays where we found that simultaneous inhibition of mycolic acid modification is unlikely to be bactericidal to Mtb in planktonic culture. In sum, we have developed an unprecedented CmaA2 HTS assay platform that enables the identification of drug-like small molecules that bind to and inhibit multiple CMAS in vitro and in vivo. We characterized the chemical space of CMAS ligands, that provides an experimental framework for multi-target drug development against Mtb