Dynamic closure of the Mycobacterium tuberculosis InhA cofactor site reveals heterogeneous routes to isoniazid resistance
Abstract
Isoniazid remains a cornerstone of tuberculosis therapy, yet resistance-associated mutations in its target, InhA, are not fully explained by reduced NADH binding alone. Here, we combine X-ray crystallography, cryo-electron microscopy, calorimetry, thermal stability profiling, and multi-scale molecular dynamics to dissect ten clinically observed InhA missense variants. The structures show that many substitutions preserve the canonical cofactor-bound architecture, but thermodynamic and simulation analyses reveal divergent routes to resistance. Active-site variants remodel water and hydrophobic networks to weaken NADH binding, whereas I95P and several interface-proximal variants populate a coenzyme closed-loop state that occludes the nicotinamide/adduct-binding pocket. A190S and Y259H instead appear to preserve NADH affinity while rigidifying the Phe149-Asp150 region required to accommodate the larger INH-NAD adduct. These findings establish InhA coding-region resistance as a spectrum of local, allosteric, and adduct-selective mechanisms, providing a framework for interpreting clinical variants and designing next-generation InhA inhibitors.
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BibTeX
@misc{rossini2026dynamic,
title = {{Dynamic closure of the Mycobacterium tuberculosis InhA cofactor site reveals heterogeneous routes to isoniazid resistance}},
author = {Nicolas de Oliveira Rossini and Sair Máximo Chavéz-Pacheco and Gerardo Andrés Libreros-Zúñiga and Stephanie Portelli and Jademilson Celestino dos Santos and Douglas Eduardo Valente Pires and David B Ascher and Pierre-Damien Coureux and Paulo Cesar Telles de Souza and Marcio Vinicius Bertacine Dias},
howpublished = {HAL},
year = {2026},
month = jun,
doi = {10.26434/chemrxiv.15004869/v1},
url = {https://hal.science/hal-05787659v1},
note = {CC BY 4.0},
}
RIS
TY - UNPB
TI - Dynamic closure of the Mycobacterium tuberculosis InhA cofactor site reveals heterogeneous routes to isoniazid resistance
AU - Nicolas de Oliveira Rossini
AU - Sair Máximo Chavéz-Pacheco
AU - Gerardo Andrés Libreros-Zúñiga
AU - Stephanie Portelli
AU - Jademilson Celestino dos Santos
AU - Douglas Eduardo Valente Pires
AU - David B Ascher
AU - Pierre-Damien Coureux
AU - Paulo Cesar Telles de Souza
AU - Marcio Vinicius Bertacine Dias
PY - 2026
DA - 2026/06/17
PB - HAL
DO - 10.26434/chemrxiv.15004869/v1
UR - https://hal.science/hal-05787659v1
AB - Isoniazid remains a cornerstone of tuberculosis therapy, yet resistance-associated mutations in its target, InhA, are not fully explained by reduced NADH binding alone. Here, we combine X-ray crystallography, cryo-electron microscopy, calorimetry, thermal stability profiling, and multi-scale molecular dynamics to dissect ten clinically observed InhA missense variants. The structures show that many substitutions preserve the canonical cofactor-bound architecture, but thermodynamic and simulation analyses reveal divergent routes to resistance. Active-site variants remodel water and hydrophobic networks to weaken NADH binding, whereas I95P and several interface-proximal variants populate a coenzyme closed-loop state that occludes the nicotinamide/adduct-binding pocket. A190S and Y259H instead appear to preserve NADH affinity while rigidifying the Phe149-Asp150 region required to accommodate the larger INH-NAD adduct. These findings establish InhA coding-region resistance as a spectrum of local, allosteric, and adduct-selective mechanisms, providing a framework for interpreting clinical variants and designing next-generation InhA inhibitors.
ER -