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. 2015 Jan 7;7(269):269ra3.
doi: 10.1126/scitranslmed.3010597.

Direct inhibitors of InhA are active against Mycobacterium tuberculosis

Ujjini H Manjunatha  1 , Srinivasa P S Rao  2 , Ravinder Reddy Kondreddi  2 , Christian G Noble  2 , Luis R Camacho  2 , Bee H Tan  2 , Seow H Ng  2 , Pearly Shuyi Ng  2 , Ng L Ma  2 , Suresh B Lakshminarayana  2 , Maxime Herve  2 , Susan W Barnes  3 , Weixuan Yu  4 , Kelli Kuhen  3 , Francesca Blasco  2 , David Beer  2 , John R Walker  3 , Peter J Tonge  4 , Richard Glynne  3 , Paul W Smith  2 , Thierry T Diagana  1
Affiliations

Direct inhibitors of InhA are active against Mycobacterium tuberculosis

Ujjini H Manjunatha et al. Sci Transl Med. .

Abstract

New chemotherapeutic agents are urgently required to combat the global spread of multidrug-resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase InhA is one of the few clinically validated targets in tuberculosis drug discovery. We report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH (reduced form of nicotinamide adenine dinucleotide)-dependent manner and blocked the enoyl substrate-binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of Mycobacterium tuberculosis infection. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB.

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Figures

Fig. 1
Fig. 1. Chemical structures of 4-hydroxy-2pyridones and in vitro and in vivo antimycobacterial activity of these compounds
(A) Chemical structures of 4-hydroxy-2-pyridone analogs used in this study. MIC50 values against Mtb are given in parentheses (μM). R1-6 numbering is shown in the NITD-529 structure. (B) The concentration dependent bactericidal kill-kinetics activity of NITD-529 and NITD-916 against in vitro replicating Mtb, and compared with isoniazid. (C) Concentration dependent activity of NITD-916, NITD-529 and isoniazid against Mtb in intracellular activated THP-1 macrophages with five days drug exposure. IC90 and IC99 values are indicated by stippled lines. Both kill kinetic and intra-macrophage analysis were performed in biological replicates (n = 2) and results are shown as mean values with standard errors.
Fig. 2
Fig. 2. Mechanism of action of pyridones
Fatty acid (A) and mycolic acid (B) lipid profiles of Mtb after exposure to 4-hydroxy-2-pyridones. Mycolic acid/fatty acid methyl esters prepared after [14C]acetate metabolic labelling and analyzed by thin layer chromatographic and phosphor-imaging. Isothermal titration calorimetry binding of NITD-529 (C) and NITD-564 (D) to apo-InhA (triangle), the InhA-NAD+ complex (circles), and the InhA-NADH complex (squares).
Fig. 3
Fig. 3. Structural analysis of the 2-pyridone binding site in the InhA-NADH complex
(A) Superimposed crystal structures of InhA-NADH-NITD564 (yellow) and InhA-NADH-NITD916 (orange); respective 2-pyridone ligands are shown in green and cyan. Substrate binding loop encompassing residues 196 to 211 is shown in red. (B) Close-up of NITD-916 (cyan) binding pocket in InhA-NADH complex, with protein polar (cyan) and hydrophobic (grey) surfaces shown. The side chains of Y158 and M199 residues are shown. The distance (in Å) between the ligand and side chains of with Y158, M199 and 2′-OH on the ribose sugar of NADH is highlighted by dotted lines. (C) Hydrogen bonding interactions of NITD-916 with critical residues in the active site of InhA. Side chains of amino acid residues responsible for NITD-916 resistance (T17, S94, G96, D148 and M161) are shown. (D) The InhA-NITD916 (green) structure overlaid with the fatty acyl substrate (cyan, 1BVR), along with other direct InhA inhibitors namely, triclosan derivative (orange, 3FNG), alkyl diphenyl ether (grey, 2×23), pyrrolidine carboxamides (pink, 2H7I) and methyl-thiazoles (blue, 4BQP). (E) The InhA-NITD916 structure (red) overlaid with co-crystal structures of fatty acyl substrate (blue, 1BVR) and alkyl diphenyl ether (yellow, 2×23). NITD-916, fatty acyl substrate and alkyl diphenyl ether ligands are colored in green, cyan and grey respectively. The shift in the conformation of the substrate binding loop is shown by an arrow.
Fig. 4
Fig. 4. In vivo efficacy of NITD-916 in Mtb infected mouse models
BALB/c mice were infected intranasally with 103 H37Rv strain of Mtb, and animals were orally treated for 4 weeks following 1 or 4 weeks of infection in acute (A) and established (B) efficacy models, respectively. Efficacy of test compounds was measured by Δlog CFUs compared to untreated controls independently for lung and spleen. Statistical evaluation was done using one way ANOVA and the data was analyzed using Tukey's multiple comparison test. Statistical significance was accepted with P values <0.05. All bars labeled with the same letter of the alphabet do not differ significantly. INH, isoniazid; RIF, rifampicin and EMB, Ethambutol.
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