Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence

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1 Supplementary Information Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence uiqing Zheng 1, Christopher J. Colvin 1, Benjamin K. Johnson 1, Paul D. Kirchhoff 2, Michael Wilson 2, Katriana Jorgensen-Muga 3, Scott D. Larsen 2 and Robert B. Abramovitch 1 * 1 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, 48824, United States. 2 Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI Sweet Briar College, Sweet Briar, VA ature Chemical Biology: doi: /nchembio.2259

2 Supplementary Results Supplementary Tables Supplementary Table 1. GFP fluorescence inhibition and eukaryotic cytotoxicity by DosR regulon inhibitors. For reporter fluorescence EC 50 determination, CDC1551(hspX ::GFP) reporter was treated with compounds over an 8 point dose response curve ranging from 400 µm to 0.65 µm. Following 6 days of treatment, GFP florescence and optical density were measured. In all cases, no inhibition of growth was observed at the reported EC 50 for inhibition of GFP fluorescence. For eukaryotic cytotoxicity, macrophage cells including bone marrow derived macrophage (BMDM), TP-1 and J774, were tested with serial dilutions of DosR regulon inhibitors over an 8 point dose response curve ranging from 400 µm to 0.65 µm. Macrophages were incubated for 3 days and viability was determined using CellTiter-Glo (Promega) luminescent cell viability assay. Supplementary Table 2. Small molecule screening data. Supplementary Datasets Supplementary Dataset 1. Differential gene expression data of WT Mtb treated with inhibitors and the DMS treated DosR mutant Supplementary Dataset 2. Differential gene expression data of the DosR mutant treated with the inhibitors. ature Chemical Biology: doi: /nchembio.2259

3 Supplementary Dataset 3. Complete gene expression tables for transcriptional profiling experiments. Supplementary figure legends Supplementary Figure 1. Statistical analysis of TS controls. A. Box plots showing the variation of DMS control wells (negative control) from the screens of the ICCB- Longwood and Molecular Libraries Program (MLP) collections. Boxes show the 25 and 75% quartiles and the whiskers are 1.5x the interquartile range (approximately 3 standard deviations away from the mean). The dots are considered outliers. B. Table with the means and standard deviations from the DMS controls. Along with the Z- factor of 0.9, tight clustering of the control wells around 0% inhibition for both fluorescence and optical density support robustness of the screen. C. FDR p-values plotted vs. the fluorescence inhibition to growth inhibition ratios shows that the chosen 1.5-fold cut-off ratio (red line) is significant (p<0.0003). Supplementary Figure 2. Identification of inhibitors of the DosRST pathway. Dose response curves of GFP fluorescence inhibition of CDC1551(hspX ::GFP) reporter treated with DosRST regulon inhibitor compounds. A. Artemisinin analogs, artesunate and dihydroartemisinin (DA). B. C103B and C104A-C106A. C. C102A was generated by organic synthesis (CCG ) and exhibited expected activity. D. C103A was generated by organic synthesis (CCG ) and exhibited expected activity. Error bars represent the standard deviation and experiments were repeated at least twice with similar results. Supplementary Figure 3. Transcriptional profiling shows Artemisinin, C102A and C103A inhibit the core genes of the DosRST regulon during hypoxia. A. Inhibition ature Chemical Biology: doi: /nchembio.2259

4 of DosR regulon under hypoxic conditions by the DosR regulon inhibitors. Mtb treated with compounds of interest was grown at 37 C without shaking for 6 days, and total RA was extracted for RT-PCR quantification. RT-PCR shows three highly induced DosR regulon genes (dosr, hspx and tgs1) under hypoxia were repressed by the DosR regulon inhibitors. Error bars represent the standard deviation. B. and Vitamin C assays. Mtb cells were pre-treated with compounds of interest for 24 hours, and total RA was extracted after inducing with or vitamin C for 2 hours. C102A and C103A inhibited the induction of DosR regulon by and vitamin C, but DA had a little effect. In all cases, the difference in the drug treated samples compared to DMS treated samples in response to vitamin C or is significant with a p-value <0.001 based on T-test, except those marked as non-significant (n.s.). Error bars represent the standard deviation. Mtb differential gene expression in response to C102A (panel C) and C103A (panel D). Genes in red have a p-value <0.05, and indicated gene names are DosR regulated genes. The transcriptional analysis from different assays collectively support that DosR pathway is the target of artemisinin, C102A and C103A. Experiments were repeated at least twice with similar results. Supplementary Figure 4. Artemisinin, C102A and C103A inhibit TAG synthesis, survival and isoniazid tolerance during RP. A. Quantification of TAG accumulation for Mtb treated with compounds of interest shows that DosRST regulon inhibitors repress TAG synthesis to the level similar to ΔdosR mutant. Error bars represent the standard deviation. B. Dose dependent inhibition of Mtb survival during RP following 15 days of treatment. Percent viability was calculated relative to the viable bacteria in the DMS control at Day 15. Error bars represent the standard deviation of the mean. C. Fifteen days of treatment with artemisinin, C102A or C103A, with or without I, significantly reduces bacterial survival (p-value <0.05 based on a T-test) during RP ature Chemical Biology: doi: /nchembio.2259

5 relative to the respective DMS controls. D. Fifteen days of treatment with artemisinin, C102A and C103A reduces isoniazid tolerance during RP. To quantify I tolerance, percent viability at 1, 5 and 25 µm I was measured relative to the 0 µm I control (DMS control). Significant differences (marked with an asterisks, p<0.05 based on a T-test) were calculated relative to the respective DMS control sample. For example, cells treated with 5 µm I and artemisinin, C102A and C103A are significantly different from the DMS control treated with 5 µm I. Experiments were repeated at least twice with similar results. Supplementary Figure 5. Artemisinin directly modulates DosS and DosT (G85L) heme in a dose-dependent manner. UV-visible spectra of DosS (panel A) and DosT (G85L) (panel B) treated with different concentrations of artemisinin. Artemisinin modulated DosT heme at a lower concentration (50 µm, Figure 5D) than DosS heme (400 µm), supporting that DosT is more sensitive to artemisinin. DosT (G85L) exhibits a similar profile to WT DosT (Figure 5D) Supplementary Figure 6. Substitutions in the DosT provide resistance to artemisinin. Mtb was transformed with a replicating plasmid that overexpresses WT dost, dost (G85L) or dost (G115L). Cells were treated with µm - 20 µm artemisinin, and total RA was extracted after 6 days incubation at 37 C. RT-PCR quantification of DosR-regulated genes (dosr, hspx and tgs1) shows that strains expressing DosT (G115L) exhibited EC 50 for artemisinin-mediated inhibition of DosR regulon genes of µm, which is ~5-fold more resistant than WT DosT or DosT (G85L) with EC 50 of µM. ature Chemical Biology: doi: /nchembio.2259

6 Supplementary Figure 7. C102A and C103A do not modulate DosS redox. DosS treated with C102A (panel A) or C103A (panel B) shows a similar overall spectrum as DMS control. This indicates that C102A and C103A has no effect in modulating redox status of DosS, and may inhibit DosR regulon by distinct mechanism. Supplementary Figure 8. Modulation of DosS and DosT kinase activities by C102A and C103A. Full western blots from which the cropped images in Figure 6 were generated. DosS and DosT protein is marked by the asterisk. ature Chemical Biology: doi: /nchembio.2259

7 Supplementary,Table,1.,EC50,calculation,of,GFP,flourescence,inhition,and,eukaryotic,cytotoxicity,by,DosR,regulon,inhibitors ame Structure, GFP,FI,(EC50,,μM) Eukaryotic,cytotoxicity,(EC50,,μM) BMDM TPM1 J774 3 C C 3 C 3 C101A&(Artemisinin) 1.2 >400 >400 >400 C 3 3 C C 3 C101B&(Artesunate) C 3 3 C C101C& C 3 (Dihydroartemisinin) C C 3 3 C C102A 12.4 >400 >400 >400 S C103A 2.7 > C103B C 3 C 3 C104A Br C S C 3 C105A Cl C106A Cl ature Chemical Biology: doi: /nchembio.2259

8 Supplementary Table 2. Small molecule screening data! Category Parameter Description Assay Type of assay Whole Cell Phenotypic Screen against a M. tuberculosis hypoxia-inducible fluorescent reporter strain Target Inhibition of DosRST regulated reporter fluorescence. The intended targets were DosR (P95193), DosS (P95194) or DosT (53473). Primary measurement Inhibition of hypoxia-inducible reporter GFP fluorescence and with limited impact on reporter growth (measured by optical density) Key reagents CDC1551 (apra ::GFP) reporter strain Assay protocol Described in methods section igh throughput screening and data analysis Additional comments Validation of reporter strain is published in Tan et al., Plos Pathogens 2013, ref 26). Library Library size 540,288 Library composition Small molecules and known bioactives Source arvard Medical Schools Institute of Chemistry and Cell Biology s (ICCB) in house library (211,655 compounds) and the I MLP collection (328,633 compounds) Additional comments Compound libraries described at: Screen Format 384-well optical plate Concentration(s) tested Plate controls Reagent/ compound dispensing system Detection instrument and software Assay validation/qc Correction factors ~10 µm in DMS DMS negative control, 0.3 µm rifampin positive control Compounds were arrayed using arvard s in house robotics platform. Reporter strain was added to the plates using a CyBio Selma Liquid handler. Plates were read using a Perkin-Elmer Enspire plate reader. Z -Factors are calculated for each plate. Z -factor for whole screen averaged 0.9. one ormalization Percent inhibition relative to the positive and negative controls. See equation of page 17 in the methods section. Additional comments The screen was conducted with a clinical strain of M. tuberculosis at Michigan State University in a BSL-3 TS facility. Post-TS analysis it criteria >35% inhibition of reporter GFP fluorescence, and >1.5 fold inhibition of reporter fluorescence relative to optical density it rate Remains to be fully determined, but below 0.01%. Additional assay(s) Confirmation of hit purity and structure Additional comments GFP quenching; eukaryotic cytotoxicity in primary bone marrow macrophages and two macrophage like cell lines; Ec50 determinations; RA-seq; inhibition of Mtb survival during persistence; biochemical assays showing targeting of DosS or DosT using UV-visible spectroscopy or autophosphorylation assays. C101 (artemisinin and analogs) are well characterized and were purchased from Sigma. For C102A and C103A,the commercial compounds were purchased from Enamine and Chembridge, respectively, and verified by chemists (see methods). C102A and C103A were also synthesized by organic chemists and confirmed in the whole cell and biochemical assays. ature Chemical Biology: doi: /nchembio.2259

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16 Supplementary Figure 8 ature Chemical Biology: doi: /nchembio.2259