Multiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the...

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Autores principales:	Matthew B. McNeil, Theresa O’Malley, Devon Dennison, Catherine D. Shelton, Bjorn Sunde, Tanya Parish
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	cell wall mycobacteria antibiotic resistance drug discovery mycolic acids mechanism of resistance Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/4c02b40a4efc4a7ba9a7328330adaff6
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spelling	oai:doaj.org-article:4c02b40a4efc4a7ba9a7328330adaff62021-11-15T15:30:58ZMultiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors10.1128/mSphere.00985-202379-5042https://doaj.org/article/4c02b40a4efc4a7ba9a7328330adaff62020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00985-20https://doaj.org/toc/2379-5042ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.Matthew B. McNeilTheresa O’MalleyDevon DennisonCatherine D. SheltonBjorn SundeTanya ParishAmerican Society for Microbiologyarticlecell wallmycobacteriaantibiotic resistancedrug discoverymycolic acidsmechanism of resistanceMicrobiologyQR1-502ENmSphere, Vol 5, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	cell wall mycobacteria antibiotic resistance drug discovery mycolic acids mechanism of resistance Microbiology QR1-502
spellingShingle	cell wall mycobacteria antibiotic resistance drug discovery mycolic acids mechanism of resistance Microbiology QR1-502 Matthew B. McNeil Theresa O’Malley Devon Dennison Catherine D. Shelton Bjorn Sunde Tanya Parish Multiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors
description	ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.
format	article
author	Matthew B. McNeil Theresa O’Malley Devon Dennison Catherine D. Shelton Bjorn Sunde Tanya Parish
author_facet	Matthew B. McNeil Theresa O’Malley Devon Dennison Catherine D. Shelton Bjorn Sunde Tanya Parish
author_sort	Matthew B. McNeil
title	Multiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors
title_short	Multiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors
title_full	Multiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors
title_fullStr	Multiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors
title_full_unstemmed	Multiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors
title_sort	multiple mutations in <named-content content-type="genus-species">mycobacterium tuberculosis</named-content> mmpl3 increase resistance to mmpl3 inhibitors
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/4c02b40a4efc4a7ba9a7328330adaff6
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Multiple Mutations in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> MmpL3 Increase Resistance to MmpL3 Inhibitors

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