A virtual screen discovers novel, fragment-sized inhibitors of Mycobacterium tuberculosis InhA

Abstract

Isoniazid (INH) is usually administered to treat latent Mycobacterium tuberculosis (Mtb) infections and is used in combination therapy to treat active tuberculosis (TB). Unfortunately, resistance to this drug is hampering its clinical effectiveness. INH is a prodrug that must be activated by Mtb catalase-peroxidase (KatG) before it can inhibit InhA (Mtb enoyl-acyl-carrier-protein reductase). Isoniazid-resistant cases of TB found in clinical settings usually involve mutations in or deletion of katG, which abrogate INH activation. Compounds that inhibit InhA without requiring prior activation by KatG would not be affected by this resistance mechanism and hence would display continued potency against these drug-resistant isolates of Mtb. Virtual screening experiments versus InhA in the GO Fight Against Malaria (GO FAM) project were designed to discover new scaffolds that display base-stacking interactions with the NAD cofactor. GO FAM experiments included targets from other pathogens, including Mtb, when they had structural similarity to a malaria target. Eight of the 16 soluble compounds identified by docking against InhA plus visual inspection were modest inhibitors and did not require prior activation by KatG. The best two inhibitors discovered are both fragment-sized compounds and displayed Ki values of 54 and 59 μM, respectively. Importantly, the novel inhibitors discovered have low structural similarity to known InhA inhibitors and thus help expand the number of chemotypes on which future medicinal chemistry efforts can be focused. These new fragment hits could eventually help advance the fight against INH-resistant Mtb strains, which pose a significant global health threat.

Figure 1. Workflow Used to Discover Novel InhA Inhibitors in the Virtual Screen with the NCI Library on GO FAM

(A) The workflow used energetic and interaction-based filters (e.g., requiring compounds to display an estimated free energy of binding ≤ −8.0 kcal/mol, base stack with the NAD cofactor, and form at least two hydrogen bonds with the active site) to filter the VS results and harvest NCI compounds for visual inspection. Candidates that passed visual inspection were then ordered and tested in InhA inhibition assays. Eight of the sixteen soluble compounds inhibited InhA activity by 27 to 71% at 100 μM. (B) The predicted binding modes for all eight novel InhA inhibitors are displayed as sticks-and-balls with cyan carbons, while the InhA target is shown in magenta. The NAD cofactor is rendered as CPK, and the key residues Gly96, Ser123, Phe149, Tyr158, Thr196, and Met199 are shown as thin sticks.

Figure 2. Predicted binding modes of the two most potent new InhA inhibitors discovered in GO FAM experiment 5

The docked modes produced by AutoDock Vina are displayed as ball-and-sticks with cyan carbon atoms, and the InhA target (2X23.pdb) is displayed as magenta ribbons. The NAD cofactor is displayed in CPK, and the key residues Gly96, Ser123, Phe149, Tyr158, Thr196, and Met199 are shown as thin sticks. A close-up view of the predicted binding mode of the top fragment hit, NCI 99389 (K_i^app = 54.1 ± 5.4 μM), is shown in (A), while the full view is displayed in (B). In (C) the docked mode of NCI 99389 is compared to the experimentally-determined binding mode of PT70, the inhibitor that crystallized with InhA in 2×23.pdb, which is displayed as ball-and-sticks with magenta carbons. In (D) the predicted binding mode of the 2^nd most potent fragment hit, NCI 111591 (K_i^app = 59.2 ± 8.7 μM), is displayed.

Figure 3. Predicted binding modes of the least potent new InhA inhibitors discovered in GO FAM experiment 5

The docked modes produced by AutoDock Vina are displayed as ball-and-sticks with cyan carbon atoms. The InhA target (2×23.pdb) is displayed as magenta ribbons, with the NAD cofactor as CPK. The key residues Gly96, Ser123, Phe149, Tyr158, Thr196, and Met199 are shown as thin sticks. The predicted binding modes of the following new InhA inhibitors are depicted: (A) NSC 112144 (K_i^app = 205.6 ± 46 μM), (B) NSC 111589, (C) NSC 111590, (D) NSC 111588, (E) NSC 135809, and (F) NSC 196166.

Figure 4. Summary of the 2D structures, docking scores, and InhA inhibitory activities of the eight new inhibitors discovered

The most potent new InhA inhibitor discovered is shown in the top-left corner, while the least potent new inhibitor is displayed in the bottom-right corner. These eight new inhibitors correspond to five novel scaffolds versus InhA (i.e., NCI 111588 – 111591 represent analogs of one scaffold, according to a Tanimoto cut-off of 0.7; see Supporting Information Table S1). FEB signifies the estimated free energy of binding value from AutoDock Vina’s scoring function, in kcal/mol. The Lig. Eff. is the calculated ligand efficiency from AutoDock Vina, in kcal/mol/heavy atom. The % inhibition of InhA activity was produced when each compound was present at 100 μM. The region of each compound that was predicted to base stack with the NAD cofactor is highlighted with a red circle.

Figure 5. Comparing the chemical space of the new InhA inhibitors to known InhA inhibitors

A Principal Component Analysis (PCA) was performed on the combination of the 157 known InhA inhibitors in the TB Mobile 2 data set and the 8 novel InhA inhibitors discovered. Three PCs explain 84.8% of the variance observed. The 157 InhA inhibitors in the TB Mobile 2 data set are displayed in magenta. The two most potent new InhA inhibitors discovered are depicted in green, and the other six novel InhA inhibitors identified are in blue. A red circle highlights the location of NCI 111591. The PCA indicates that the 8 new InhA inhibitors have similar chemical properties to known InhA inhibitors, but they are generally not within the main clusters of these previously characterized InhA inhibitors.

Figure 6. Kinetic data on the two most potent InhA inhibitors discovered indicate NCI 99389 is a competitive inhibitor, while 111591 is noncompetitive

IC₅₀ values were measured for the top 2 fragment hits, followed by a detailed mechanistic study to measure the K_i values. NCI 99389 showed a Ki^app of 54.1 ± 5.4 μM and a competitive binding mechanism, indicating that the inhibitor competed with the CoA substrate and bound directly to the enzyme. Conversely, NCI111591 had a Ki^app of 59.2 ± 8.7 μM and a non-competitive binding mechanism, suggesting a more complex scenario where the inhibitor could bind to both the holo enzyme and to the substrate-enzyme complex.