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. 2013 Aug;9(8):499-506.
doi: 10.1038/nchembio.1277. Epub 2013 Jun 16.

Antituberculosis Thiophenes Define a Requirement for Pks13 in Mycolic Acid Biosynthesis

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Free PMC article

Antituberculosis Thiophenes Define a Requirement for Pks13 in Mycolic Acid Biosynthesis

Regina Wilson et al. Nat Chem Biol. .
Free PMC article

Abstract

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13(F79S) mutant conferred high resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Effect of Thiophene 2 and 4 on mycolic acid biosynthesis in Mtb.
(a, b) Normal-phase TLC analysis of MAMEs/FAMEs from wild-type Mtb (H37Rv) and the TP-resistant Mtb (DRM2) after treatment with increasing concentration of TP2 (a) or isoniazid (b) (INH). Equal counts were loaded and the TLC was developed using hexane/ethyl acetate (19:1, v/v, 2 runs) solvent system. Similar TLCs upon TP4 or Ciprofloxacin treatment are shown in Supplementary Fig. 3. (c, d) Reverse-phase TLCs using acetonitrile/dioxane solvent (1:1, v/v and equal volumes of samples) showing the fatty acid chain length of the FAS-I derived products following treatment with TP2, INH or DA5 from H37Rv (c) or DRM2 (d). Cold methyl esters of fatty acids were used as standards as shown in Supplementary Fig. 4. (e–f) Normal-phase TLCs showing TMM (TM) and TDM (TD) from polar lipids extracted from H37Rv (e) or DRM2 (f) upon treatment with increasing concentrations of TP2, or 5× MIC of INH (H), DA5 (D), or ethambutol (E). Equal volume (5 μl) of each sample was analyzed on a silica gel 60 F254, developed with CHCl3/CH3OH/H2O (62:25:4, v/v/v).
Figure 2
Figure 2. Inhibition of fatty acyl-AMP loading onto purified Pks13 by TP2
The loading of FadD32-activated FL C16 on Pks13_WT (a), Pks13_F79S (b) and MsmPks13 (c) was determined by separating the reaction mixtures on SDS-PAGE gels. Activities were determined by measuring in-gel fluorescence (top panels of a, b and c) and total protein by coomassie blue staining (bottom panels of a, b and c). SeeBlue Plus2 Pre-stained marker (Invitrogen) was used as molecular weight standard and approximate molecular weights in MOPS running buffer are indicated. The loading of FL C16 on Pks13_WT (a), Pks13_F79S (b) and MsmPks13 (c) was quantified using ImageQuant 5.2 (GE healthcare) and quantitation from 5-replicates (mean ± SD) each was used to generate inhibition curve (d). The full length images corresponding to these figures are shown in Supplementary Fig. 14.
Figure 3
Figure 3. Bactericidal Activity of TP2, TP4 and other anti-TB drugs against Mtb.
Killing curves of Mtb strain, H37Rv after incubation with TP2, TP4, isoniazid (INH), rifampicin (RIF) or various combinations of these drugs. Killing activity was monitored using the BACTEC 460TB method at 5× the MIC for all drug compounds (a and b) or by plating for CFU (c) after incubation at 5× and 10× (as indicated) the MIC. “ϕ” indicates out of range growth. The arrows indicate the time when drugs were added to the cultures (a and b); for panel c drugs were added on day zero. The data represented (mean ± SD) is from two independent experiments performed in triplicates.

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