Article Figures & Data
Figures
- 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
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
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
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
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
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
- TABLE 1
Drug susceptibilities of actively replicating versus oxygen-starved M. tuberculosisa
Drug MIC (μM) for M. tuberculosis Replicating Nonreplicating RIF 0.05 1.41 SQ109 2.37 3.35 AU1235 0.31 >310 THPP-2 6.25 >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
Compound pH outside pH inside ΔpH No drug 6.82 7.09 0.26 Isoniazid 6.79 7.08 0.29 Rifampin 6.81 7.08 0.27 Streptomycin 6.77 7.05 0.28 CCCP (2× MIC) 6.85 6.85 0 Nigericin (20 μM) 6.82 6.82 0 Valinomycin (33 μM) 6.83 7.07 0.24 Ro 48-8071 (2× MIC) 6.83 6.83 0 SQ109 (2× MIC) 6.81 6.81 0 AU1235 (2× MIC) 6.82 6.82 0 BM212 (1× MIC) 6.85 6.85 0 2418 (2× MIC) 6.81 6.81 0 THPP-2 (2× MIC)b 6.82 6.82 0 ↵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: TMM TDM DAT PAT Ac2SL SL-I PDIM MBT SQ109 211 −88 73 −68 145 −20 −47 −16 AU1235 318 −84 70 −44 88 −19 −29 −13 INH −72 −82 −18 −43 −9 −25 −63 NDc ↵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
Isolatea MIC (μg/ml) forb: WTc AU2 AU3 AU4 AU5 AU6 DA5_1 DA5_2 DA8_1 DA8_2 AU1235 0.2–0.4 >1.6 >1.6 >1.6 >1.6 >1.6 >1.6 1.6 >1.6 1.6 BM212 1.25 10 5–10 10 5 5 10 10 10 5 SQ109 1 NDd ND ND ND ND 4 2 2–8 2–4 ↵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.
Supplemental material
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