Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Antimicrobial Agents and Chemotherapy
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Mechanisms of Action: Physiological Effects

Novel Insights into the Mechanism of Inhibition of MmpL3, a Target of Multiple Pharmacophores in Mycobacterium tuberculosis

Wei Li, Ashutosh Upadhyay, Fabio L. Fontes, E. Jeffrey North, Yuehong Wang, Debbie C. Crans, Anna E. Grzegorzewicz, Victoria Jones, Scott G. Franzblau, Richard E. Lee, Dean C. Crick, Mary Jackson
Wei Li
aMycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ashutosh Upadhyay
aMycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Fabio L. Fontes
aMycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
E. Jeffrey North
bDepartment of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yuehong Wang
cInstitute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Debbie C. Crans
dDepartment of Chemistry, Colorado State University, Fort Collins, Colorado, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Anna E. Grzegorzewicz
aMycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Victoria Jones
aMycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Scott G. Franzblau
cInstitute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Richard E. Lee
bDepartment of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dean C. Crick
aMycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mary Jackson
aMycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AAC.03229-14
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Additional Files
  • FIG 1
    • Open in new tab
    • Download powerpoint
    FIG 1

    Structures of compounds discussed in the text. Shown are the MmpL3 inhibitors SQ109 (an ethylenediamine analog of ethambutol), BM212 (a 1,5-diarylpyrrole), THPP-2 (a tetrahydropyrazolo pyrimidine), 2418 (an indolecarboxamide), AU1235 (an adamantyl urea), and DA5 (a SQ109-related compound). The known menaquinone inhibitor (Ro 48-8071) and PMF uncoupler (CCCP) are boxed. The estimated pKa values are indicated. The pKa values were predicted using the pKa component of Pipeline Pilot (Accelrys, Inc.). The actual pKa for CCCP is 6.09 (48).

  • FIG 2
    • Open in new tab
    • Download powerpoint
    FIG 2

    Drug susceptibilities of actively replicating versus nutrient-starved M. tuberculosis cultures. The effects of SQ109, DA5, INH, RIF, THPP-2, BM212, 2418, and AU1235 on the viability of log-phase and 2- and 6-week nutrient-starved M. tuberculosis H37Rv ATCC 25618 cultures were investigated. Triplicate wells of M. tuberculosis cultures incubated for 7 days in the absence or presence of 5× the MIC (white bars) or 50× the MIC (black bars) of each of the drugs were processed, and mean values and standard deviations of the viable CFU counts are shown. (a) log-phase cultures; (b) 2-week-starved cultures; (c) 6-week-starved cultures. The drug concentrations (5× and 50× the MIC, respectively) were as follows: INH, 0.5 and 5 μg/ml; RIF, 0.5 and 5 μg/ml; DA5, 16 and 160 μg/ml; AU1235, 0.5 and 5 μg/ml; SQ109, 2.5 and 25 μg/ml; THPP-2, 7 and 70 μg/ml; BM212, 7.8 and 78 μg/ml; and 2418, 7.8 and 78 μg/ml.

  • FIG 3
    • Open in new tab
    • Download powerpoint
    FIG 3

    Effect of CCCP on the transfer of mycolic acids onto its cell envelope acceptors. (a) Lipid analysis of untreated, SQ109-treated, and CCCP-treated M. smegmatis and M. Tuberculosis (Mtb) H37Rv mc26206 cells. The same volume of [14C]acetate-labeled lipids from bacterial cells treated with CCCP or SQ109, or untreated cells were analyzed by TLC in the solvent system (CHCl3/CH3OH/H2O, 20:4:0.5) and revealed by phosphorimaging. PE, phosphatidylethanolamine. (b) TLC analysis of cell wall-bound mycolic acid methyl esters (MAMEs) prepared from the same untreated, SQ109-treated, and CCCP-treated M. smegmatis and M. tuberculosis H37Rv mc26206 cells as in panel a. The TLC plates were developed thrice in the solvent system (n-hexane/ethyl acetate, 95:5) and revealed by phosphorimaging. (c) The amount of radioactivity incorporated in TMM, TDM, and cell wall-bound mycolic acids (CW) in the M. smegmatis treated and untreated cells was semiquantified using a PhosphorImager, and the results (expressed as a fold increase or decrease over the value measured in the untreated control arbitrarily set to 1) are presented as histograms. (d) Lipid analysis of untreated and valinomycin-treated M. tuberculosis H37Rv mc26206 cells. The same volume of [14C]acetate-labeled total lipids was analyzed by TLC for each sample as described in panel a. (e) TLC analysis as in panel b of cell wall-bound mycolic acid methyl esters (MAMEs) prepared from the same untreated and valinomycin-treated M. tuberculosis H37Rv mc26206 cells.

  • FIG 4
    • Open in new tab
    • Download powerpoint
    FIG 4

    Effects of SQ109 and AU1235 on the biosynthesis and export of sulfolipids, di- and polyacyltrehaloses, and phthiocerol dimycocerosates in M. tuberculosis. Lipid analysis of untreated, SQ109-treated, and AU1235-treated M. tuberculosis H37Rv mc26206 cells. Surface-exposed (S) and cell-associated (C) [14C]acetate-labeled and [1-14C]propionate-labeled lipids from bacterial cultures treated with SQ109 or AU1235 or untreated were analyzed by TLC in the solvent systems (CHCl3/CH3OH/H2O [20:4:0.5]) ([14C]-acetate-labeled lipids) (a), (CHCl3/CH3OH/H2O [60:30:6]) ([1-14C]propionate-labeled lipids) (b), (petroleum ether [60/80°C]/ethyl acetate [98:2]; three developments) ([1-14C]propionate-labeled lipids) (c), first dimension: (petroleum ether [60/80°C]/acetone (92:8); three developments); second dimension: (acetone-toluene [95:5]) ([1-14C]propionate-labeled lipids), and revealed by autoradiography (d). The same volume of samples was loaded per lane. The amount of radioactivity incorporated in SL-I, the diacylated sulfolipid precursor (Ac2SL), DAT, PAT, and PDIM in the treated and untreated cells was semiquantified using a PhosphorImager, and the results are presented in Table 3. CL, cardiolipin; PE, phosphatidylethanolamine.

  • FIG 5
    • Open in new tab
    • Download powerpoint
    FIG 5

    Effects of inhibitors on ΔΨ dissipation in M. smegmatis cells. DisC3 (5) fluorescence is quenched in the presence of the cells due to intercalation into the membrane. The addition of compounds (at time zero) that dissipate ΔΨ causes an increase in fluorescence due to dissociation of the dye from the membranes. Note: the MIC of THPP-2 against M. smegmatis is >22 μg/ml (the highest concentration tested here).

  • FIG 6
    • Open in new tab
    • Download powerpoint
    FIG 6

    Menaquinone biosynthesis in untreated, Ro 48-8071-treated, and SQ109-treated M. tuberculosis cells. Shown is a TLC analysis of neutral lipids isolated from M. tuberculosis H37Rv mc26206 after metabolic labeling with l-[methyl-14C]methionine in the presence of the indicated concentrations of Ro 48-8071 and SQ109. Menaquinone [MK-9 (II-H2)] was identified by comigration with an authentic standard (27). The MIC of Ro 48-8071 against M. tuberculosis H37Rv mc26206 under the culture conditions used in this experiment is 25 μg/ml; the MIC of SQ109 is 0.8 μg/ml.

Tables

  • Figures
  • Additional Files
  • TABLE 1

    Drug susceptibilities of actively replicating versus oxygen-starved M. tuberculosisa

    DrugMIC (μM) for M. tuberculosis
    ReplicatingNonreplicating
    RIF0.051.41
    SQ1092.373.35
    AU12350.31>310
    THPP-26.25>25
    • ↵a The MICs of RIF, SQ109, and AU1235 determined in the MABA and LORA assays (28, 29) are against virulent M. tuberculosis H37Rv. The MICs of THPP-2 are against M. tuberculosis H37Rv mc26206. The susceptibility of the actively replicating M. tuberculosis cells to this compound was determined in 7H9-OADC-Tween 80 broth using the resazurin microplate reduction assay, while that of the hypoxic nonreplicating bacilli was determined in Dubos Tween albumin medium, as described by Wayne and Hayes (26). The nonreplicating M. tuberculosis bacilli were exposed to THPP-2 for 7 days.

  • TABLE 2

    31P NMR-derived ΔpH collapse in inhibitor and ionophore-treated M. smegmatis cellsa

    CompoundpH outsidepH insideΔpH
    No drug6.827.090.26
    Isoniazid6.797.080.29
    Rifampin6.817.080.27
    Streptomycin6.777.050.28
    CCCP (2× MIC)6.856.850
    Nigericin (20 μM)6.826.820
    Valinomycin (33 μM)6.837.070.24
    Ro 48-8071 (2× MIC)6.836.830
    SQ109 (2× MIC)6.816.810
    AU1235 (2× MIC)6.826.820
    BM212 (1× MIC)6.856.850
    2418 (2× MIC)6.816.810
    THPP-2 (2× MIC)b6.826.820
    • ↵a The average pH values were calculated from the equation pH = 6.75 + log (d − 10.85)/(13.25 − d) (49), where d is the 31P NMR chemical shift difference between peaks and that of the α phosphorus on ATP. The error is estimated to be no more than ±0.02 pH units.

    • ↵b THPP-2 was tested against M. tuberculosis H37Rv mc26206 cells, since this compound did not show any inhibitory effect against M. smegmatis in culture at the highest concentration tested (22 μg/ml).

  • TABLE 3

    Quantitative assessment of the effects of SQ109 on the biosynthesis and export of sulfolipids, di- and polyacyltrehaloses, and phthiocerol dimycocerosates in M. tuberculosis

    Whole-cell treatmenta% increase/decrease radioactivity compared to control forb:
    TMMTDMDATPATAc2SLSL-IPDIMMBT
    SQ109211−8873−68145−20−47−16
    AU1235318−8470−4488−19−29−13
    INH−72−82−18−43−9−25−63NDc
    • ↵a All were at 10× the MIC.

    • ↵b The amount of radioactivity incorporated by whole cells (including cell-associated and surface-exposed materials) in TMM, TDM, SL-I, the diacylated SL precursor (Ac2SL), DAT, PAT, PDIM, and (carboxy)mycobactins (MBT) of the M. tuberculosis SQ109-, AU1235-, and INH-treated cells was analyzed by TLC (Fig. 4 and data not shown) and semiquantified using a PhosphorImager. The results are expressed as percent increases or decreases of the values measured in the untreated control.

    • ↵c ND, not determined.

  • TABLE 4

    Cross-resistance of adamantyl urea and SQ109 spontaneous resistant mutants of M. tuberculosis H37Rv to AU1235, BM212, and SQ109

    IsolateaMIC (μg/ml) forb:
    WTcAU2AU3AU4AU5AU6DA5_1DA5_2DA8_1DA8_2
    AU12350.2–0.4>1.6>1.6>1.6>1.6>1.6>1.61.6>1.61.6
    BM2121.25105–1010551010105
    SQ1091NDdNDNDNDND422–82–4
    • ↵a The mutation for the isolates AU2, AU3, AU4, AU5, and AU6 is G253E, that for DA5_1 and DA5_2 is A700T, that for DA8_1 is L567P, and that for DA8_2 is Q40R. The adamantyl urea (isolates AU2, AU3, AU4, AU5, and AU6) and SQ109 (isolates DA5_1, DA5_2, DA8_1, and DA8_2) spontaneous resistant mutants of M. tuberculosis H37Rv were reported previously (9, 19).

    • ↵b The MICs, defined as the concentration of inhibitors that inhibited 100% of the growth and metabolic activity of the various M. tuberculosis H37Rv isolates, were determined in 96-well microtiter plates using the colorimetric resazurin assay in 7H9-OADC-Tween 80 broth at 37°C.

    • ↵c The wild-type (WT) strain was M. tuberculosis H37Rv TMC102.

    • ↵d ND, not determined.

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 -

      Supplemental Figures S1 to S3

      PDF, 1.5M

PreviousNext
Back to top
Download PDF
Citation Tools
Novel Insights into the Mechanism of Inhibition of MmpL3, a Target of Multiple Pharmacophores in Mycobacterium tuberculosis
Wei Li, Ashutosh Upadhyay, Fabio L. Fontes, E. Jeffrey North, Yuehong Wang, Debbie C. Crans, Anna E. Grzegorzewicz, Victoria Jones, Scott G. Franzblau, Richard E. Lee, Dean C. Crick, Mary Jackson
Antimicrobial Agents and Chemotherapy Oct 2014, 58 (11) 6413-6423; DOI: 10.1128/AAC.03229-14

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Antimicrobial Agents and Chemotherapy article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Novel Insights into the Mechanism of Inhibition of MmpL3, a Target of Multiple Pharmacophores in Mycobacterium tuberculosis
(Your Name) has forwarded a page to you from Antimicrobial Agents and Chemotherapy
(Your Name) thought you would be interested in this article in Antimicrobial Agents and Chemotherapy.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Novel Insights into the Mechanism of Inhibition of MmpL3, a Target of Multiple Pharmacophores in Mycobacterium tuberculosis
Wei Li, Ashutosh Upadhyay, Fabio L. Fontes, E. Jeffrey North, Yuehong Wang, Debbie C. Crans, Anna E. Grzegorzewicz, Victoria Jones, Scott G. Franzblau, Richard E. Lee, Dean C. Crick, Mary Jackson
Antimicrobial Agents and Chemotherapy Oct 2014, 58 (11) 6413-6423; DOI: 10.1128/AAC.03229-14
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

About

  • About AAC
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • AAC Podcast
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AACJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0066-4804; Online ISSN: 1098-6596