Identification and Optimization of Novel Inhibitors of the Polyketide Synthase 13 Thioesterase Domain with Antitubercular Activity.

There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.

Toolkit of Approaches To Support Target-Focused Drug Discovery for Plasmodium falciparum Lysyl tRNA Synthetase.

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.

Targeting Mycobacterium tuberculosis CoaBC through Chemical Inhibition of 4'-Phosphopantothenoyl-l-cysteine Synthetase (CoaB) Activity.

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4'-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4'-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.

Discovery of an Allosteric Binding Site in Kinetoplastid Methionyl-tRNA Synthetase.

Methionyl-tRNA synthetase (MetRS) is a chemically validated drug target in kinetoplastid parasites Trypanosoma brucei and Leishmania donovani. To date, all kinetoplastid MetRS inhibitors described bind in a similar way to an expanded methionine pocket and an adjacent, auxiliary pocket. In the current study, we have identified a structurally novel class of inhibitors containing a 4,6-diamino-substituted pyrazolopyrimidine core (the MetRS02 series). Crystallographic studies revealed that MetRS02 compounds bind to an allosteric pocket in L. major MetRS not previously described, and enzymatic studies demonstrated a noncompetitive mode of inhibition. Homology modeling of the Trypanosoma cruzi MetRS enzyme revealed key differences in the allosteric pocket between the T. cruzi and Leishmania enzymes. These provide a likely explanation for the lower MetRS02 potencies that we observed for the T. cruzi enzyme compared to the Leishmania enzyme. The identification of a new series of MetRS inhibitors and the discovery of a new binding site in kinetoplastid MetRS enzymes provide a novel strategy in the search for new therapeutics for kinetoplastid diseases.