Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

Abstract

Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl–acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening

“…This enzyme is known to require cofactor to bind inhibitors and has an active site loop that plays a critical role in inhibition525354. Recent publications report ELT selections for InhA consistent with the notion that the presence of cofactor improves this target’s tractability3342. In fact, the method described here could be used to test the same target under many conditions or multiple constructs of the same target for optimization.…”

Novel Nucleic Acid Binding Small Molecules Discovered Using DNA-Encoded Chemistry

“…Individual DNA-encoded chemical libraries were synthesized using a DNA-recorded split and pool procedure in which DNA tagging and building block installation events occurred during each split. We have described this general methodology before [3,4,5,6,7,8,27,28]. The synthetic schemes defining each library (A and B) are shown in Figure 1.…”

“…Altering the target concentration, the inclusion of selectivity targets, and the addition of known binders to compete with library compounds are individual variables that can be explored in parallel in a single-selection campaign. We have previously reported the result of multiple parallel selection conditions that successfully identified novel small-molecule inhibitors of therapeutic protein targets, including BTK [3], PAR2 [4], sEH [5], InhA [6], Mcl1 [7], and ATAD2 [8]: Other groups have reported successes with similar platforms [1].…”

Quantitative Comparison of Enrichment from DNA-Encoded Chemical Library Selections

“…10 Similarly, Soutter et al successfully used parallel DEL selections to identify compounds with affinity for specific binding sites to the enoyl-acyl-carrier protein reductase InhA from Mycobacterium tuberculosis . 11 Cuozzo et al used parallel selections of a DEL for Bruton’s tyrosine kinase (BTK) with varying target concentrations and the presence or absence of ATP and dasatinib to elucidate the relative binding affinities and binding mechanisms of novel BTK inhibitors. 12 Other selection parameters, including the washing protocol and incubation time, can be varied to probe binding characteristics like on and off rates.…”