Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence

Journal name:
Nature Chemical Biology
Volume:
13,
Pages:
218–225
Year published:
DOI:
doi:10.1038/nchembio.2259
Received
Accepted
Published online

Abstract

The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ~540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme–artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.

At a glance

Figures

  1. Discovery of inhibitors of the DosRST pathway.
    Figure 1: Discovery of inhibitors of the DosRST pathway.

    (a) Scatter plot of primary screening data showing compounds that inhibit CDC1551(hspX′::GFP) reporter GFP fluorescence with limited impact on Mtb growth. Six distinct classes of compounds (HC101–HC106) are highlighted. (b) Structures of compounds confirmed to selectively inhibit CDC1551(hspX′::GFP) reporter fluorescence. (c) Dose–response curves for artemisinin (Art, HC101A), HC102A and HC103A inhibition of GFP fluorescence. Dose–response curves for other characterized molecules are presented in Supplementary Figure 2.

  2. Transcriptional profiling shows artemisinin, HC102A and HC103A inhibit the core genes of the DosRST regulon during hypoxia.
    Figure 2: Transcriptional profiling shows artemisinin, HC102A and HC103A inhibit the core genes of the DosRST regulon during hypoxia.

    (a) Mtb differential gene expression in response to artemisinin. Genes in red have a P value < 0.05. Indicated gene names include characterized DosR-regulated genes. (b) Venn diagram showing genes that are downregulated (>2-fold; P < 0.05) in CDC1551 treated with artemisinin, HC102A or HC103A relative to a DMSO-treated CDC1551 control. Also shown are genes downregulated (>2-fold; P < 0.05) in a DMSO-treated CDC1551(ΔdosR) mutant strain relative to a DMSO-treated CDC1551 control. (c) Venn diagram showing genes that are downregulated (>2-fold; P < 0.05) in a CDC1551(ΔdosR) mutant strain treated with artemisinin, HC102A or HC103A relative to a DMSO-treated CDC1551(ΔdosR) control. The limited genes modulated by HC102A and HC103A support that these compounds are highly specific for the DosR regulon.

  3. Artemisinin, HC102A and HC103A inhibit TAG synthesis, survival and isoniazid (INH) tolerance.
    Figure 3: Artemisinin, HC102A and HC103A inhibit TAG synthesis, survival and isoniazid (INH) tolerance.

    (a) CDC1551 treated with compounds of interest (at 40 μM) exhibits a 60–70% reduction of TAG accumulation, similar to the CDC1551(ΔdosR) mutant control. The position of TAG is indicated by the asterisk. (b) CDC1551 and Erdman strains treated with 40 μM of the compounds of interest exhibit reduced survival following 10 d in the hypoxic shiftdown assay. Colony-forming units (CFUs) were counted and percent survival calculated relative to the WT control at day 0. Differences between treated samples as compared to the DMSO control are significant (*P value < 0.05 based on a t-test). (c) Dose-dependent inhibition of Mtb survival in the hypoxic shiftdown assay. Percent viability was calculated relative to viable bacteria in the DMSO control at day 10. (d) Following 10 d in the hypoxic shiftdown assay, treatment with artemisinin, HC102A or HC103A, with or without INH, significantly reduces bacterial survival relative to the respective DMSO controls (P value < 0.05 based on a t-test). (e) Artemisinin, HC102A and HC103A reduce INH tolerance in the hypoxic shiftdown assay. Percent viability at 1, 5 and 25 μM INH was calculated relative to the 0 μM INH control (using data in d). Significant inhibition of INH tolerance (*P value < 0.05 based on a t-test) is observed relative to the respective DMSO controls. For each panel, error bars represent the s.d. and experiments were repeated at least twice with similar results.

  4. Artemisinin directly inhibits DosS and DosT by targeting sensor-kinase heme.
    Figure 4: Artemisinin directly inhibits DosS and DosT by targeting sensor-kinase heme.

    UV–visible spectra of DosS (a) and DosT (b) showing treatment with dithionite (DTN) reduces the heme (the “on” state for the kinases) and that artemisinin oxidizes or degrades the heme (“off” states of the kinases). (c) Mass spectra showing the presence of peaks at ~898 Da that are present in artemisinin-treated DosS samples, but absent in DMSO-treated samples. This mass is the approximate combined mass of heme (616.487 Da) and artemisinin (282.332 Da) and is consistent with the formation of heme–artemisinin adducts. Experiments were repeated at least twice with similar results.

  5. Amino acid substitutions in DosS or DosT promote resistance to artemisinin.
    Figure 5: Amino acid substitutions in DosS or DosT promote resistance to artemisinin.

    (a) Molecular modeling indicates a channel exists in DosS and DosT through which artemisinin may access the heme. In WT DosT, the heme (yellow) and iron (green ball) are accessible to artemisinin via a channel. G85L and G115L substitutions are predicted to block this channel and access to the heme. (b,c) UV–visible spectra show that DosS (E87L) (b) and DosS (G117L) (c) proteins can be reduced by the addition of dithionite (DTN) but are resistant to oxidation by artemisinin (Art). (d,e) WT DosT exhibits a dose-dependent decrease in the amplitude of the Soret peak at 430 nm (left, d) and a loss of the peak at 560 nm (magnified in right side d). In contrast, DosT (G115L) exhibits resistance to artemisinin because it does not exhibit a dose-dependent decrease in the 430-nm peak (left, e) and the 560 nm peak is maintained at treatments of 50 and 100 μM artemisinin (right side e). DosT (G85L) exhibits spectra similar to WT DosT (Supplementary Fig. 5b). (f) Overexpression of DosT (G115L) in CDC1551 provides artemisinin resistance. Mtb overexpressing WT DosT or DosT (G85L) exhibit strongly inhibited expression of DosR regulated genes (dosR, hspX and tgs1) in the presence of 1 μM artemisinin, while the strain overexpressing DosT (G115L) is resistant. Dose–response curves are presented in Supplementary Figure 6. Experiments were repeated at least twice with similar results.

  6. HC103A inhibits DosS and DosT autophosphorylation.
    Figure 6: HC103A inhibits DosS and DosT autophosphorylation.

    Recombinant DosS or DosT was treated with HC103A (a) or HC102A (b) across a dose–response curve. The autophosphorylation assay was incubated for 1 h, the proteins were analyzed by western blotting and the protein autophosphorylation was assessed by exposure of the blot to a phosphor screen. HC102A and HC103A inhibit DosS autophosphorylation, with IC50 of 1.9 μM and 0.5 μM, respectively. HC102A had limited impact on DosT autophosphorylation, whereas HC103A inhibited DosT autophosphorylation with an IC50 of ~5 μM. Experiments were repeated at least twice with similar results.

Compounds

5 compounds View all compounds
  1. (±)-(5S,9R)-7,7,9- trimethyl-1,3- diazaspiro[4.5]decane-2,4-dione
    Compound 1 (±)-(5S,9R)-7,7,9- trimethyl-1,3- diazaspiro[4.5]decane-2,4-dione
  2. N-[3-[(3-hydroxyphenyl)carbamoyl]phenyl]thiophene-2-carboxamide
    Compound 2 N-[3-[(3-hydroxyphenyl)carbamoyl]phenyl]thiophene-2-carboxamide
  3. 6-bromo-2-[3-(dimethylamino)propyl]benzo[de]isoquinoline-1,3-dione
    Compound 3 6-bromo-2-[3-(dimethylamino)propyl]benzo[de]isoquinoline-1,3-dione
  4. 9-ethyl-3-((4-(propylsulfonyl)piperazin-1-ium-1-yl)methyl)-9H-carbazol-9-ium oxalate
    Compound 4 9-ethyl-3-((4-(propylsulfonyl)piperazin-1-ium-1-yl)methyl)-9H-carbazol-9-ium oxalate
  5. 1-(2,4-dichlorophenyl)-3-(1,2-oxazol-5-yl)urea
    Compound 5 1-(2,4-dichlorophenyl)-3-(1,2-oxazol-5-yl)urea

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Protein Data Bank

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Author information

Affiliations

  1. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.

    • Huiqing Zheng,
    • Christopher J Colvin,
    • Benjamin K Johnson &
    • Robert B Abramovitch
  2. Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan, USA.

    • Paul D Kirchhoff,
    • Michael Wilson &
    • Scott D Larsen
  3. Sweet Briar College, Sweet Briar, Virginia, USA.

    • Katriana Jorgensen-Muga

Contributions

H.Z., C.J.C., B.K.J. and R.B.A. conducted high throughput screen and follow-up experiments. M.W. and S.D.L. synthesized chemical compounds; K.J.-M. contributed reagents. P.D.K. performed structural modeling; H.Z. and R.B.A. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (8,456 KB)

    Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–8

  2. Supplementary Note (160 KB)

    Synthetic procedures.

Excel files

  1. Supplementary Dataset 1 (169 KB)

    Differential gene expression data of WT Mtb treated with inhibitors and the DMSO treated DosR mutant.

  2. Supplementary Dataset 2 (83 KB)

    Differential gene expression data of the DosR mutant treated with the inhibitors.

  3. Supplementary Dataset 3 (3,400 KB)

    Complete gene expression tables for transcriptional profiling experiments.

Additional data