Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis

Yumi Park, Angela Pacitto, Tracy Bayliss, Laura A T Cleghorn, Zhe Wang, Travis Hartman, Kriti Arora, Thomas R Ioerger, Jim Sacchettini, Menico Rizzi, Stefano Donini, Tom L Blundell, David B Ascher, Kyu Rhee, Ardala Breda, Nian Zhou, Veronique Dartois, Surendranadha Reddy Jonnala, Laura E Via, Valerie MizrahiOla Epemolu, Laste Stojanovski, Fred Simeons, Maria Osuna-Cabello, Lucy Ellis, Claire J MacKenzie, Alasdair R C Smith, Susan H Davis, Dinakaran Murugesan, Kirsteen I Buchanan, Penelope A Turner, Margaret Huggett, Fabio Zuccotto, Maria Jose Rebollo-Lopez, Maria Jose Lafuente-Monasterio, Olalla Sanz, Gracia Santos Diaz, Joël Lelièvre, Lluis Ballell, Carolyn Selenski, Matthew Axtman, Sonja Ghidelli-Disse, Hannah Pflaumer, Markus Bösche, Gerard Drewes, Gail M Freiberg, Matthew D Kurnick, Myron Srikumaran, Dale J Kempf, Simon R Green, Peter C Ray, Kevin Read, Paul Wyatt, Clifton E Barry, Helena I Boshoff

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

Original languageEnglish
Pages (from-to)18-33
Number of pages16
JournalACS Infectious Diseases
Volume3
Issue number1
Early online date5 Oct 2016
DOIs
Publication statusPublished - 13 Jan 2017

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Indazoles
Inosine Monophosphate
Sulfonamides
Mycobacterium tuberculosis
Oxidoreductases
Guanine
Tuberculosis
IMP Dehydrogenase
Lung
Gene Amplification
X Ray Crystallography
Microbial Sensitivity Tests
Growth
Green Fluorescent Proteins
Pharmaceutical Preparations
Cell Wall
Pharmacokinetics
Genome
Mutation

Keywords

  • Animals
  • Antitubercular agents
  • Drug design
  • Drug discovery
  • Drug resistance, Bacterial
  • Gene expression regulation, Bacterial
  • Gene expression regulation, Enzymologic
  • Humans
  • IMP Dehydrogenase
  • Mice
  • Mice, Inbred C57BL
  • Molecular structure
  • Mutation
  • Mycobacterium tuberculosis
  • Protein conformation
  • Rabbits
  • Structure-activity relationship
  • Sulfonamides
  • Tuberculosis
  • Journal article

Cite this

Park, Yumi ; Pacitto, Angela ; Bayliss, Tracy ; Cleghorn, Laura A T ; Wang, Zhe ; Hartman, Travis ; Arora, Kriti ; Ioerger, Thomas R ; Sacchettini, Jim ; Rizzi, Menico ; Donini, Stefano ; Blundell, Tom L ; Ascher, David B ; Rhee, Kyu ; Breda, Ardala ; Zhou, Nian ; Dartois, Veronique ; Jonnala, Surendranadha Reddy ; Via, Laura E ; Mizrahi, Valerie ; Epemolu, Ola ; Stojanovski, Laste ; Simeons, Fred ; Osuna-Cabello, Maria ; Ellis, Lucy ; MacKenzie, Claire J ; Smith, Alasdair R C ; Davis, Susan H ; Murugesan, Dinakaran ; Buchanan, Kirsteen I ; Turner, Penelope A ; Huggett, Margaret ; Zuccotto, Fabio ; Rebollo-Lopez, Maria Jose ; Lafuente-Monasterio, Maria Jose ; Sanz, Olalla ; Diaz, Gracia Santos ; Lelièvre, Joël ; Ballell, Lluis ; Selenski, Carolyn ; Axtman, Matthew ; Ghidelli-Disse, Sonja ; Pflaumer, Hannah ; Bösche, Markus ; Drewes, Gerard ; Freiberg, Gail M ; Kurnick, Matthew D ; Srikumaran, Myron ; Kempf, Dale J ; Green, Simon R ; Ray, Peter C ; Read, Kevin ; Wyatt, Paul ; Barry, Clifton E ; Boshoff, Helena I. / Essential but Not Vulnerable : Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis. In: ACS Infectious Diseases. 2017 ; Vol. 3, No. 1. pp. 18-33.
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title = "Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis",
abstract = "A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.",
keywords = "Animals, Antitubercular agents, Drug design, Drug discovery, Drug resistance, Bacterial, Gene expression regulation, Bacterial, Gene expression regulation, Enzymologic, Humans, IMP Dehydrogenase, Mice, Mice, Inbred C57BL, Molecular structure, Mutation, Mycobacterium tuberculosis, Protein conformation, Rabbits, Structure-activity relationship, Sulfonamides, Tuberculosis, Journal article",
author = "Yumi Park and Angela Pacitto and Tracy Bayliss and Cleghorn, {Laura A T} and Zhe Wang and Travis Hartman and Kriti Arora and Ioerger, {Thomas R} and Jim Sacchettini and Menico Rizzi and Stefano Donini and Blundell, {Tom L} and Ascher, {David B} and Kyu Rhee and Ardala Breda and Nian Zhou and Veronique Dartois and Jonnala, {Surendranadha Reddy} and Via, {Laura E} and Valerie Mizrahi and Ola Epemolu and Laste Stojanovski and Fred Simeons and Maria Osuna-Cabello and Lucy Ellis and MacKenzie, {Claire J} and Smith, {Alasdair R C} and Davis, {Susan H} and Dinakaran Murugesan and Buchanan, {Kirsteen I} and Turner, {Penelope A} and Margaret Huggett and Fabio Zuccotto and Rebollo-Lopez, {Maria Jose} and Lafuente-Monasterio, {Maria Jose} and Olalla Sanz and Diaz, {Gracia Santos} and Jo{\"e}l Leli{\`e}vre and Lluis Ballell and Carolyn Selenski and Matthew Axtman and Sonja Ghidelli-Disse and Hannah Pflaumer and Markus B{\"o}sche and Gerard Drewes and Freiberg, {Gail M} and Kurnick, {Matthew D} and Myron Srikumaran and Kempf, {Dale J} and Green, {Simon R} and Ray, {Peter C} and Kevin Read and Paul Wyatt and Barry, {Clifton E} and Boshoff, {Helena I}",
note = "This work was funded, in part, by the Intramural Research Program of NIAID and in part by grants from the Foundation for the National Institutes of Health with support from the Bill & Melinda Gates Foundation (to C.E.B. III, V.M., and P.W.), and the South African Medical Research Council (to V.M.). D.B.A. was supported by an NHMRC C. J. Martin Fellowship (APP1072476). D.B.A. and T.L.B received funding from the Newton Fund RCUK-CONFAP Grant awarded by The Medical Research Council (MRC) and Funda{\cc}{\~a}o de Amparo {\`a} Pesquisa do Estado de Minas Gerais (FAPEMIG) (MR/M026302/1). P.W. received joint funding from the Bill and Melinda Gates Foundation and Wellcome Trust for A Centre of Excellence for Lead Optimisation for Diseases of the Developing World",
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month = "1",
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Park, Y, Pacitto, A, Bayliss, T, Cleghorn, LAT, Wang, Z, Hartman, T, Arora, K, Ioerger, TR, Sacchettini, J, Rizzi, M, Donini, S, Blundell, TL, Ascher, DB, Rhee, K, Breda, A, Zhou, N, Dartois, V, Jonnala, SR, Via, LE, Mizrahi, V, Epemolu, O, Stojanovski, L, Simeons, F, Osuna-Cabello, M, Ellis, L, MacKenzie, CJ, Smith, ARC, Davis, SH, Murugesan, D, Buchanan, KI, Turner, PA, Huggett, M, Zuccotto, F, Rebollo-Lopez, MJ, Lafuente-Monasterio, MJ, Sanz, O, Diaz, GS, Lelièvre, J, Ballell, L, Selenski, C, Axtman, M, Ghidelli-Disse, S, Pflaumer, H, Bösche, M, Drewes, G, Freiberg, GM, Kurnick, MD, Srikumaran, M, Kempf, DJ, Green, SR, Ray, PC, Read, K, Wyatt, P, Barry, CE & Boshoff, HI 2017, 'Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis', ACS Infectious Diseases, vol. 3, no. 1, pp. 18-33. https://doi.org/10.1021/acsinfecdis.6b00103

Essential but Not Vulnerable : Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis. / Park, Yumi; Pacitto, Angela; Bayliss, Tracy; Cleghorn, Laura A T; Wang, Zhe; Hartman, Travis; Arora, Kriti; Ioerger, Thomas R; Sacchettini, Jim; Rizzi, Menico; Donini, Stefano; Blundell, Tom L; Ascher, David B; Rhee, Kyu; Breda, Ardala; Zhou, Nian; Dartois, Veronique; Jonnala, Surendranadha Reddy; Via, Laura E; Mizrahi, Valerie; Epemolu, Ola; Stojanovski, Laste; Simeons, Fred; Osuna-Cabello, Maria; Ellis, Lucy; MacKenzie, Claire J; Smith, Alasdair R C; Davis, Susan H; Murugesan, Dinakaran; Buchanan, Kirsteen I; Turner, Penelope A; Huggett, Margaret; Zuccotto, Fabio; Rebollo-Lopez, Maria Jose; Lafuente-Monasterio, Maria Jose; Sanz, Olalla; Diaz, Gracia Santos; Lelièvre, Joël; Ballell, Lluis; Selenski, Carolyn; Axtman, Matthew; Ghidelli-Disse, Sonja; Pflaumer, Hannah; Bösche, Markus; Drewes, Gerard; Freiberg, Gail M; Kurnick, Matthew D; Srikumaran, Myron; Kempf, Dale J; Green, Simon R; Ray, Peter C; Read, Kevin; Wyatt, Paul; Barry, Clifton E; Boshoff, Helena I (Lead / Corresponding author).

In: ACS Infectious Diseases, Vol. 3, No. 1, 13.01.2017, p. 18-33.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Essential but Not Vulnerable

T2 - Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis

AU - Park, Yumi

AU - Pacitto, Angela

AU - Bayliss, Tracy

AU - Cleghorn, Laura A T

AU - Wang, Zhe

AU - Hartman, Travis

AU - Arora, Kriti

AU - Ioerger, Thomas R

AU - Sacchettini, Jim

AU - Rizzi, Menico

AU - Donini, Stefano

AU - Blundell, Tom L

AU - Ascher, David B

AU - Rhee, Kyu

AU - Breda, Ardala

AU - Zhou, Nian

AU - Dartois, Veronique

AU - Jonnala, Surendranadha Reddy

AU - Via, Laura E

AU - Mizrahi, Valerie

AU - Epemolu, Ola

AU - Stojanovski, Laste

AU - Simeons, Fred

AU - Osuna-Cabello, Maria

AU - Ellis, Lucy

AU - MacKenzie, Claire J

AU - Smith, Alasdair R C

AU - Davis, Susan H

AU - Murugesan, Dinakaran

AU - Buchanan, Kirsteen I

AU - Turner, Penelope A

AU - Huggett, Margaret

AU - Zuccotto, Fabio

AU - Rebollo-Lopez, Maria Jose

AU - Lafuente-Monasterio, Maria Jose

AU - Sanz, Olalla

AU - Diaz, Gracia Santos

AU - Lelièvre, Joël

AU - Ballell, Lluis

AU - Selenski, Carolyn

AU - Axtman, Matthew

AU - Ghidelli-Disse, Sonja

AU - Pflaumer, Hannah

AU - Bösche, Markus

AU - Drewes, Gerard

AU - Freiberg, Gail M

AU - Kurnick, Matthew D

AU - Srikumaran, Myron

AU - Kempf, Dale J

AU - Green, Simon R

AU - Ray, Peter C

AU - Read, Kevin

AU - Wyatt, Paul

AU - Barry, Clifton E

AU - Boshoff, Helena I

N1 - This work was funded, in part, by the Intramural Research Program of NIAID and in part by grants from the Foundation for the National Institutes of Health with support from the Bill & Melinda Gates Foundation (to C.E.B. III, V.M., and P.W.), and the South African Medical Research Council (to V.M.). D.B.A. was supported by an NHMRC C. J. Martin Fellowship (APP1072476). D.B.A. and T.L.B received funding from the Newton Fund RCUK-CONFAP Grant awarded by The Medical Research Council (MRC) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (MR/M026302/1). P.W. received joint funding from the Bill and Melinda Gates Foundation and Wellcome Trust for A Centre of Excellence for Lead Optimisation for Diseases of the Developing World

PY - 2017/1/13

Y1 - 2017/1/13

N2 - A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

AB - A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

KW - Animals

KW - Antitubercular agents

KW - Drug design

KW - Drug discovery

KW - Drug resistance, Bacterial

KW - Gene expression regulation, Bacterial

KW - Gene expression regulation, Enzymologic

KW - Humans

KW - IMP Dehydrogenase

KW - Mice

KW - Mice, Inbred C57BL

KW - Molecular structure

KW - Mutation

KW - Mycobacterium tuberculosis

KW - Protein conformation

KW - Rabbits

KW - Structure-activity relationship

KW - Sulfonamides

KW - Tuberculosis

KW - Journal article

U2 - 10.1021/acsinfecdis.6b00103

DO - 10.1021/acsinfecdis.6b00103

M3 - Article

C2 - 27704782

VL - 3

SP - 18

EP - 33

JO - ACS Infectious Diseases

JF - ACS Infectious Diseases

SN - 2373-8227

IS - 1

ER -