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.

Keywords: DNA-encoded X-Chem technology; DNA-encoded libraries; InhA; Mycobacterium tuberculosis; multidrug resistance.

Fig. 1.

InhA inhibitors showing cellular activity in Mtb previously described in the literature. (1) Isoniazid adduct (23). (2) PT70 (24, 25). (3) Pyridomycin (26). (4) Methyl thiazole (15). (5) Pyrazole ELT hit (13). (6) Pyridine dione (27).

Fig. S1.

Chemical structures of biochemically active compounds identified from InhA selections and referenced in Table 2.

Fig. 2.

(A) Crystal structure of compound 1a (green carbon atoms) bound to InhA (yellow carbon atoms) (PDB ID code 5G0S). The inhibitor occupies three distinct subsites, which are depicted by colored ovals; red oval: catalytic site (site I); yellow oval: hydrophobic site (site II); green oval: hydrophilic site (site III). Selected residues are displayed as sticks. NADH is displayed with pink carbon atoms. The active site loop of InhA is displayed in orange. (B) Overlay of all five series [compounds 1a (green carbon atoms) (PDB ID code 5G0S); 2a (yellow) (PDB ID code 5G0T); 4a (orange) (PDB ID code 5G0U); 6a (cyan) (PDB ID code 5G0V); and 8a (red) (PDB ID code 5G0W)]. The proteins from the complexes were superimposed, and only the bound compounds and NADH from the complex of InhA with compound 1a are shown for clarity. The compounds occupy the three sites marked in A, and compounds 6a and 8a occupy an additional subsite above the adenine of NADH marked in cyan. A and B are shown in same orientation.

Fig. 3.

X-ray crystal structure of InhA:NADH in complex with compound 4a (PDB ID code 5G0U) from series 4. Carbon atoms for compounds are shown in green. The protein backbone cartoon is represented in yellow. Selected atoms for the InhA side chains including F97, M98, and Ile202 in the active site loop are shown as sticks. NADH is shown as sticks with magenta carbon atoms. Refined (2f_o-f_c) electron density contoured at 1.0 σ is represented as a wire mesh. Some atoms of the active-site covering loop represented in orange have been removed for clarity.

Fig. 4.

X-ray crystal structure of InhA:NADH in complex with compound 6a (PDB ID code 5G0V) from series 6. Carbon atoms for compounds are shown in green. The protein backbone cartoon is represented in yellow. Selected atoms for the InhA side chains including the catalytic residue Y158, F97, and M98 are shown as sticks. NADH is shown as sticks with magenta carbon atoms. Refined (2f_o-f_c) electron density contoured at 1.0 σ is represented as a wire mesh. Some atoms of the active-site covering loop represented in orange have been removed for clarity.

Fig. 5.

X-ray crystal structure of InhA:NADH in complex with compound 8a (PDB ID code 5G0W) from series 8. Carbon atoms for compounds are shown in green. The protein backbone cartoon is represented in yellow. Selected atoms for the InhA side chains including Phe41, Arg43, F97, and M98 are shown as sticks. NADH is shown as sticks with magenta carbon atoms. Refined (2f_o-f_c) electron density contoured at 1.0 σ is represented as a wire mesh. Some atoms of the active-site covering loop represented in orange have been removed for clarity.