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Target engagement and binding mode of an anti-tuberculosis drug to its bacterial target deciphered in whole living cells by NMR Guillaume Bouvier, Catherine Simenel, Jichan Jang, Nitin Pal Kalia, Inhee Choi, Michael Nilges, Kevin Pethe, and Nadia Izadi-Pruneyre Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00975 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018
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Biochemistry
Target engagement and binding mode of an anti-tuberculosis drug to its bacterial target deciphered in whole living cells by NMR. Guillaume Bouvier1, Catherine Simenel2, Jichan Jang3,4, Nitin P Kalia5, Inhee Choi3, Michael Nilges1*, Kevin Pethe3,5& Nadia Izadi-Pruneyre2* 1Institut
Pasteur, Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, CNRS UMR3528, C3BI USR3756, Paris, France; 2Institut Pasteur, NMR of Biomolecules Unit, Department of Structural Biology and Chemistry, CNRS UMR3528, Paris, France ; 3Institut Pasteur Korea, Gyeonggi-do, Korea; 4Division of Life Science, Research Institute of Life Science, Gyeongsang National University, Jinju, Korea; 5Lee Kong Chian School of Medicine and School of Biological Sciences, Nanyang Technological University, Singapore. KEYWORDS. Drug-target interaction, In-cell NMR, Drug Discovery, data driven docking, Tuberculosis ABSTRACT: Detailed information on hit-target interaction is very valuable for drug discovery efforts and indispensable for rational hit to lead optimization. We developed a new approach combining NMR in whole-cells (in-cell NMR) and docking to characterize hit-target interaction at the atomic level. By using in-cell NMR, we validated target engagement of the antituberculosis imidazopyridine amide (IPA) series with the subunit b of the cytochrome bc1:aa3, the major respiratory terminal oxidase in mycobacteria. The most advanced IPA called Q203 is currently in clinical trial. Using its derivative IPA317, we identified the atoms of the drug interacting with the cytochrome b in whole cells. NMR data and the Self-Organising Map algorithm were used to cluster a large set of drug-target complex models. The selected ensemble revealed IPA317 in a transient cavity of the cytochrome b, interacting directly with the residue T313, which is the site of spontaneous mutation conferring resistance to the IPA series. Our approach constitutes a pipeline to obtain atomic information on hit-target interactions in the cellular context.
INTRODUCTION The human pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), killed 1,7 million people in 2016 and one-third of the world’s population is believed to be infected by this bacterium. The number of persons susceptible to TB is increasing because of the emergence of multidrug resistance (MDR) strains and the AIDS pandemic. The treatment of TB is challenging and necessitates up to 24 months of antibiotherapy for MDR forms, with only 52% of success and a significant mortality rate. Therefore TB is a major global health problem and there is an urgent need to develop new therapeutic strategies.(1,2) Despite considerable efforts, only a very small number of drugs have reached clinical trials. Among them, the imidazopyridine amide (IPA) series has recently been identified by phenotype-based high throughput screening.(3) The most advanced IPA is Q203 (IC50 nm range) a clinical candidate currently in phase I clinical trial under a US FDA Investigational New Drug application.(4) By means of whole genome sequencing of spontaneous resistant mutants, the putative target of IPA drugs was identified as the cytochrome b (QcrB subunit) of the cytochrome bc1:aa3 complex, an essential terminal oxidase of the respiratory chain required for energy metabolism. Interestingly, the cytochrome bc1 complex is also the target of several other drugs, such as the antimalarial drug atovaquone and several other preclinical stage anti-TB drugs.(5,2) However, direct experimental
evidence for engagement between IPA drugs, or any other mycobacterial QcrB inhibitors, and their target remains to be demonstrated. Detailed structural information on the binding mode of a drug or a hit to its target is very valuable for drug discovery and lead optimization.(6,7) NMR is a powerful technique to study molecular interactions at atomic resolution under physiological (or near physiological) conditions. The Saturation Transfer Difference (STD) is an NMR ligand-based experiment widely used for fragment screening, epitope mapping and target engagement validation.(8-10) This method is also particularly adapted to obtain interatomic distance information between a ligand and its target. However QcrB, is part of the cytochrome bc1-aa3 membrane supercomplex, making its extraction and purification very challenging. This is also the case of many drug targets, which are often receptors embedded in a membrane that cannot be easily isolated and purified. To overcome this issue, we used the STD approach on entire bacterial cells (in-cell STD) without any extraction and purification of the target. As a drug candidate, we chose IPA317 (Figure 1) from the set of lead compounds for optimal solubility in physiological condition and its antibacterial properties (intracellular activity < 1μm). (11) Using in-cell STD, we studied the interaction of IPA317 with entire Mycobacterium smegmatis cells expressing the M. tuberculosis QcrCAB (QcrCABMtb) complex. This strategy allowed us to demonstrate the target engagement of this drug
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for the first time and to identify its chemical moieties involved in the interaction with the target. The resulting atomic data were then used to obtain a meaningful characterization of drug-target interactions at the atomic level.
Figure 1: IPA317 (a) chemical structure with arbitrary proton numbering; (b) assigned chemical shifts in ppm.
MATERIALS AND METHODS Bacterial strains and plasmids used for target expression. Mycobacteium smegmatis Δ qcrCAB was kindly provided by Valerie Mizrahi and Bavesh Kana. (12) The strain was transformed with a replicative plasmid (pMV262) harboring i) the M. tuberculosis qcrCABMtb operon, to yield the strain M. smegmatis pMV262-MtbqcrCAB, or ii) the M. tuberculosis qcrCAB operon possessing the single-nucleotide polymorphism leading to a T313A mutation in QcrB, to yield the strain M. smegmatis pMV262-MtbqcrCAB-T313A. The cloning strategy is detailed in Supplementary Materials and Methods. Growth kinetics of strains and the relative amount of proteins were analyzed as described in Supplementary Materials and Methods. NMR sample preparation. A stock solution of IPA317 (11) (kind gift from the Chemical Laboratory of Institut Pasteur of Korea) at concentration of 3 mM was prepared by dissolving it in deuterated DMSO (DMSO-d6, 99.96% 2H atoms, Eurisotop, France). An aliquot of this solution was added to a 4 mm Shigemi NMR tube (Shigemi Inc., USA) containing either the buffer alone or with the cell suspension. The final concentration of IPA317 in the NMR sample was 76 μM and the amount of DMSO-d6 at 2.5 % (v/v). The concentration of IPA317 was chosen based on its limit of solubility that was at 78 μM in our buffer. The intracellular antibacterial activity of this compound, called 317 is reported as
