Thienopyrimidine amide analogs target MmpL3 in Mycobacterium tuberculosis

10

The identification of novel agents with mechanisms of action distinct from those 11 currently utilized in tuberculosis treatment remains a significant challenge. The mycobacterial 12 protein MmpL3 has emerged as a promising drug target due to its essential role in the synthesis 13 of the cell wall of Mycobacterium tuberculosis. We previously identified novel thienopyrimidine 14 amides with good anti-tubercular activity. 15

We profiled a subset of thienopyrimidine amides determining activity against 16 intracellular bacteria and bactericidal activity against replicating bacteria. We ran assays to 17 determine mode of action by measuring cell wall stress, ATP production, and bacterial 18 cytological profiling. We determined activity against a strain of M. tuberculosis with mutations in 19 MmpL3. We isolated and sequenced resistant mutants. 20

We tested five analogs against a strain of M. tuberculosis with mutations in MmpL3 and 21 determined that they lost potency. Analogs induced PiniBAC, a reporter for cell wall stress, and led 22 to an ATP boost characteristic of cell wall inhibitors. Bacterial cytological profiling of a 23 representative compound revealed a morphological profile consistent with other MmpL3 24 inhibitors. 25

Together, our data support MmpL3 as the most probable drug target for the TPA 26 analogs and add to the growing list of scaffolds that can inhibit this vulnerable transporter. 27 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint

28 Despite the availability of drug treatment regimens, tuberculosis remains a major global health 29 concern, with an estimated 8.2 million new cases reported in 2023, including 400,000 cases of 30 drug resistant tuberculosis (1) . Currently, no vaccine provides effective protection against 31 pulmonary TB. Additionally, the bacteria can enter a latent phase, where they display no 32 symptoms, or cause symptoms that mimic other pathologies (2) . Together, these factors 33 highlight the urgent need for intensified efforts in TB control and the development of novel 34 therapies, particularly those targeting drug-resistant bacilli and aiming to shorten the duration of 35 treatment. 36 Mycobacterium tuberculosis is the causative agent of TB in humans. M. tuberculosis has a thick 37 cell wall, which is composed by a highly hydrophobic bilayer of mycolic acids linked to 38 arabinogalactan in turn linked to peptidoglycan . This structure forms a unique barrier against 39 drugs and the host immune defenses (3–5). Hence, cell wall biosynthesis represents an 40 attractive target for the development of new inhibitors. 41 MmpL3 (Rv0206c) is an essential transporter which is highly conserved across the 42 Mycobacterium genus (4, 6, 7) and is the only member of the MmpL family that is essential in M. 43 tuberculosis (2, 4, 7). Its essentiality is attributed to its role in the translocation of trehalose 44 monomycolate (TMM) to the periplasmic space, where it serves as a precursor for the synthesis 45 of trehalose dimycolate (TDM), a critical component of the cell envelope (4, 8) . Given the 46 important role of MmpL3 in constructing the mycobacterial cell wall, it has emerged as a high -47 value, druggable target, and at the moment, one of the most studied for anti- TB drug 48 development (6). There are numerous MmpL3 inhibitors with diverse chemical structures which 49 have potent antibacterial activity (5, 9). 50 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint We previously identified and characterized novel thienopyrimidine amide (TPA) analogs that 51 inhibit M. tuberculosis growth and h ad low cytotoxicity . From this series, we identified two 52 subsets. One subset (TPA -L) had increas ed activity against a LepB (signal peptidase) 53 hypomorph, indicating that the signal peptidase or protein secretion is the target or mode of 54 action (10, 11) . The second subset of molecules (TPA -M) had excellent potency but were 55 equipotent against wild-type and hypomorph strains of M. tuberculosis suggesting that the target 56 was not protein secretion. In this paper we explored the biological profile of the TPA-M series of 57 molecules and identified MmpL3 as the most likely intracellular target. 58

59 Bacterial strains and culture conditions 60 M. tuberculosis H37Rv-LP ATCC 25618 (wild-type) (12) and M. tuberculosis LP-0497754-61 RM301 (MmpL3 F255L, V646M, F644I) (13) were cultured in Middlebrook 7H9 broth medium 62 supplemented with 10% oleic acid, albumin, dextrose, and catalase (OADC) enrichment 63 (BBL/Beckton and Dickson) and 0.05% w/v Tween 80 (7H9- Tw-OADC). Nutrient-starved cells 64 were generated by resuspending cells in phosphate -buffered saline (PBS) with 0.05% w/v 65 Tyloxapol (PBS-Tyl) at OD 1.0 and incubating for 7 days at 37°C without agitation. Avirulent M. 66 tuberculosis mc26206 (ΔpanCD ΔleuCD) was grown in 7H9-Tw-OADC supplemented with 0.5% 67 wt/vol glycerol, 0.2% wt/vol Casamino Acids, 48 µg/mL pantothenate, and 50 µg/mL leucine at 68 30°C. 69 Determination of minimum inhibitory concentration (MIC) 70 TPA molecules ( Figure 1) were synthesized as previously described (10). Compound stocks 71 were resuspended in DMSO and stored at -20°C. MICs were determined in 96 - or 384 -well 72 microplates as described (14) . Briefly, M. tuberculosis was grown to mid -log phase ; cultures 73 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint were dispensed into plates containing test compounds to a final OD 590 of 0.02 and incubated at 74 37°C for 5 days (H37Rv) or 7 days ( mc26206). The OD590 was measured and IC 90 determined 75 as the concentration at which 90% of growth was inhibited as compared to controls (DMSO 76 only). 77 Determination of activity against intracellular bacilli 78 THP-1 cells were cultivated in RPMI -1640 medium supplemented with 10% FBS and incubated 79 at 37°C, 5% CO2. Cells were treated with 80 nM PMA for 24 h prior to infection, harvested using 80 Accumax™ solution, and resuspended in fresh cRPMI at a final density of 9×10 ⁵ cells/mL. Cells 81 were infected overnight at a multiplicity of 1:1 with M. tuberculosis expressing LuxABCDE and 82 exposed to compounds in 96 -well plates for 72 h at 37°C and 5% CO 2. Bacterial viability was 83 assessed by luminescence; IC50 was determined as the concentration at which 50% of growth 84 was inhibited as compared to controls (DMSO only). 85 Determination of bactericidal activity 86 M. tuberculosis H37Rv-LP was cultured in 7H9 -Tw-OADC or starved for 7 days in PBS-Tyl, 87 adjusted to a theoretical OD 590 of 0.02 and exposed to compounds in 96 -well plates. Cultures 88 were spotted onto 7H10 agar supplemented with v/v 10% OADC (7H10-OADC) on day 0, 7 and 89 14. The MBC was defined as the lowest concentration at which no visible growth was observed. 90 Niclosamide was included as a control. 91 Induction of cell wall stress 92 M. tuberculosis strain carrying the P iniBAC-lux reporter plasmid (15) was grown to mid -log in 93 GAST/Fe protein -free medium containing 0.3 g/L Bacto Casitone, 0.05 g/L ferric ammonium 94 citrate, 4 g/L dibasic anhydrous potassium phosphate, 2 g/L citric acid, 1 g/L L -alanine, 1.2 g/L 95 magnesium chloride, 0.6 g/L potassium sulfate, 2 g/L ammonium chloride, 1.8 mL of 10 M 96 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint sodium hydroxide, 10 mL glycerol, 5 mL 10% Tween 80, plus 15 µg/mL kanamycin. Cultures 97 were adjusted to a theoretical OD 590 of 0.02 and used to inoculate 96 -well plates containing 98 compounds. After 72 hours of incubation at 37°C, 1 vol of 10 mg/mL luciferin in of 1M HEPES 99 buffer pH 7.8, 1M DTT was added and RLU was read after 25 min of incubation at RT. 100 Ethambutol was included as a positive control. 101 Determination of ATP levels 102 ATP was measured using the BacTiter-Glo Assay kit (Promega) according to the manufacturer’s 103 instructions. Log phase M. tuberculosis was exposed to compounds for 24 h, .50 µL BacTiter-104 Glo™ was added, incubated at RT for 10 min and RLU read. Growth was measured after 120 h 105 by OD. Q203, was included as a positive control. 106 Isolation of resistant mutants 107 Log phase M. tuberculosis was plated on 7H10 -OADC plates with 5X solid MIC and 10X solid 108 MIC. Plates were incubated at 37°C until isolated colonies appeared. Potential resistant mutants 109 were picked and streaked onto 5X MIC. MIC in liquid medium was measured to confirm 110 resistance. Genomic DNA from three isolates was extracted from cultured cells by heat 111 inactivation for 10 min at 100C followed by 0.22 µm filtration. PCR amplification was performed 112 using 10 µL of extract DNA in a final volume of 50 µL containing 1 µL of Pfu polymerase 113 (Agilent), 5 µL of 10X Pfu amplification buffer (Agilent), 2.5 µL of primers MmpL3_seq_1_fwd 114 (5’-gattcgctacctgagcag-3’) and MmpL3_seq_11_rev (0.5 µM) (5’-catttactgcagccgctg-3’) and 4 µL 115 of 10 mM dNTP mix. PCR amplification was conducted, products were purified using the Qiagen 116 PCR purification kit, and sequencing was performed by Plasmidsaurus using Oxford Nanopore 117 Technology with custom analysis and annotation. 118 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint Bacterial Cytological Profiling (BCP) 119 M. tuberculosis mc26206 for bacterial cytological profiling was prepared as described (16) . 120 Briefly, cultures were adjusted to an OD600 of ~0.06-0.08 and incubated at 30°C for 18 -20 hours 121 before exposure to compounds at 1X and 5X MIC for 48h and 120 hours. Cells were fixed using 122 a mixture of 100 µL of 16% paraformaldehyde, 3 µL of 8% glutaraldehyde, and 20 µL of 0.4 M 123 phosphate buffer pH 7.5 , washed twice with 200 µL of warm medium, concentrated to 124 approximately 30 µL, and stained for 30 min. Full -field fluorescence microscopy images of the 125 samples were preprocessed from their original proprietary microscope file format into a common 126 image format (TIFF). The original image dimensions (3×2048×2048) were cropped by trimming 127 124 pixels from each edge to avoid optical artifacts, resulting in dimensions of 3×1800×1800. A 128 600-pixel square sliding window was passed over this image with a step size of 60 pixels to 129 produce 400 sub -images, which were each passed to a trained convolutional neural network 130 (CNN). Each full-field image, consisting of 400 sub-images, generated a vector that describes a 131 point in latent space. Similarity scores were calculated by comparing the location of unknown 132 compound-treated cells in this space to control compound -treated cells using a scaled version 133 of the average minimum distance. 134

and Discussion 135 TPA analogs are active against intracellular mycobacteria 136 We previously demonstrated that the TPA series of compounds are active against aerobically -137 grown M. tuberculosis (10). A subset of analogs had potent anti -tubercular activity. Since these 138 analogs had equipotent activity against wild-type and LepB hypomorph strains, we hypothesized 139 that they did not target protein secretion. We wanted to determine the mode of action and target 140 of these potent molecules. 141 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint We had already noted that compounds were active against replicating bacteria, so we expanded 142 our work to look at activity against intracellular bacteria and non -replicating bacteria. We 143 selected several active analogs (Figure 1) . Molecules were tested for activity against 144 intracellular M. tuberculosis in THP -1 cells. All compounds were active against intracellular M. 145 tuberculosis, with activity similar to the MICs (Table 1). 146 147 TPA analogs are bactericidal against replicating bacilli 148 We assessed the bactericidal activity of the TPA analogs against M. tuberculosis wild type 149 following a 14 -day exposure under replicating conditions as well as against non-replicating 150 bacteria generated by nutrient starvation. All analogs were bactericidal against replicating M. 151 tuberculosis with MBC/MIC ratio of 100 µM) ( Table 1). These data are in contrast to what we previously noted with 153 inhibitors of LepB which showed bactericidal activity against non -replicating bacteria further 154 supporting a different target or mode of action (17). 155 TPA analogs induce cell wall stress in M. tuberculosis 156 We were interested in determining the mode of action of our molecules. As a first step, we 157 determined whether molecules induced cell wall stress. We used a reporter strain of M. 158 tuberculosis expressing luciferase under the control of P iniBAC, since Induction of iniBAC 159 expression is a marker of cell envelope stress (18). We saw induction of PiniBAC for all 160 analogs, which predominantly occurred at concentrations close to the MIC. These data suggest 161 that TPA analogs cause cell wall stress (Figure 2 and Figure S1). 162 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint TPA analogs boost ATP in M. tuberculosis 163 A link between induction of iniBAC and a burst in ATP production has previously been proposed 164 (19). To determine if our molecules also affect ATP levels, we measured intracellular ATP levels 165 following treatment with TPA analogs. As anticipated, increasing compound concentrations led 166 to a boost in ATP levels (Figure 3 and Figure S2 ). Again we saw the boost occurring at 167 concentrations around the MIC. These data are consistent with the response seen with other 168 MmpL3 inhibitors (3, 20). 169 MmpL3 mutation leads to resistance to TPA analogs 170 We hypothesized that our molecules could be targeting MmpL3, since this is a highly 171 promiscuous target and cell wall stress and elevated intracellular ATP levels are features 172 associated with MmpL3 inhibition in M. tuberculosis (3). We compared TPA activity against the 173 wild-type M. tuberculosis and strain with mutations in MmpL3 which is resistant to a wide range 174 of MmpL3 inhibitors (13, 20) . The MmpL3 mutant strain demonstrated increased resistance to 175 all analogs, with at least a 4 -fold increase in IC90 (Table 1). Interestingly, this strain was highly 176 resistant to three of the five analogs which lost all activity (IC 90 >50-100 M). Thus our data are 177 consistent with MmpL3 being the drug target for this compound series. 178 Resistant strains have a non-synonymous mutation in mmpL3 179 In order to determine whether there might be additional targets, we isolated resistant mutants 180 using TPN -0089300. Three strains were selected with an IC 90 of >100 M. Since MmpL3 181 mutations lead to resistance, we sequenced mmpL3 in three resistant isolates (RM3, RM4, and 182 RM6). All isolates had the same non-synonymous mutation in MmpL3 of F644L. This mutation 183 has been associated with resistance to other MmpL3 inhibitors and is similar to one of the 184 mutations in the MmpL3 mutant strain we used to test for resistance (F644I) (4, 8, 13, 21). 185 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint Cytological profiling 186 We determined the phenotypic effect of a representative TPA analog using bacterial cytological 187 profiling (16). We confirmed that TPN -0089300 was active against the avirulent M. tuberculosis 188 strain mc26206 (MIC of 1 µg/mL). Bacterial cells were exposed to TPN-0089300 for 48 and 120 189 hours at 2X and 5X MIC (2 µg/mL and 5 µg/mL respectively) . Morphological profiling 190 demonstrated that bacterial morphology changed with cells becoming more rounded and losing 191 membrane integrity after 120h (Figure 4) . Based on the similarity score, this profile was 192 identified as a strong match to other MmpL3 inhibitors (Figure 5) (16, 20). 193

194 We provide strong evidence that a subset of TPA analogs, which we now term TPA -M, target 195 MmpL3 in M. tuberculosis. TPA-M compounds were bactericidal for replicating bacilli, induced 196 cell walls stress and boosted ATP production. Mutations in MmpL3 conferred resistance and 197 bacterial cytological profiling identified MmpL3 as the most likely mode of action. We 198 demonstrated that the TPA -M analogs do not kill starved, non -replicating bacilli. This is 199 consistent with other MmpL3 inhibitors that have little effect against non-replicating M. 200 tuberculosis (3, 22). Cells treated with MmpL3 inhibitors develop dimples at the dividing pole, 201 where the protein localizes during active cell division and the lack of activity against non -202 replicating cells has been attributed to the absence of MmpL3 localization at the pole in the non-203 replicating state (7, 23). Taken together our data suggest that MmpL3 is the primary target of the 204 TPA-M series. 205 Acknowledgments 206 The authors kindly thank Grace Liu, Renee Allen, and Lauren Ames for technical assistance. 207 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint Funding Sources 208 This work was supported by the Department of Defense office of the Congressionally Directed 209 Medical Research Programs under award number under award number PR180794 and by 210 NIAID of the National Institutes of Health under award number R01AI182006. The content is 211 solely the responsibility of the authors and does not necessarily represent the official views of 212 the National Institutes of Health. 213 Conflict of interest 214 The authors declare no competing interests. 215

216 1. World Health Organization. 2024. Global tuberculosis report 2024. 217 https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-tuberculosis-218 report-2024 219 2. Diab A, Dickerson H, Al Musaimi O. 2025. Targeting the Heart of Mycobacterium: Advances 220 in Anti-Tubercular Agents Disrupting Cell Wall Biosynthesis. Pharmaceuticals 18:70. 221 3. Ames L, Allen R, Boshoff HI, Cleghorn L, Engelhart C, Schnappinger D, Parish T. Common 222 biological features of Mycobacterium tuberculosis MmpL3 inhibitors. 223 4. Kapp E, Calitz H, Streicher EM, Dippenaar A, Egieyeh S, Jordaan A, Warner DF, Joubert J, 224 Malan SF, Sampson SL. 2023. Discovery and biological evaluation of an adamantyl -amide 225 derivative with likely MmpL3 inhibitory activity. Tuberculosis 141:102350. 226 5. North EJ, Schwartz CP, Zgurskaya HI, Jackson M. 2023. Recent advances in mycobacterial 227 membrane protein large 3 inhibitor drug design for mycobacterial infections. Expert Opin 228 Drug Discov 18:707–724. 229 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint 6. Adams O, Deme JC, Parker JL, Fowler PW, Lea SM, Newstead S. 2021. Cryo-EM structure 230 and resistance landscape of M. tuberculosis MmpL3: An emergent therapeutic target. 231 Structure 29:1182-1191.e4. 232 7. Williams JT, Abramovitch RB. 2023. Molecular Mechanisms of MmpL3 Function and 233 Inhibition. Microb Drug Resist 29:190–212. 234 8. Williams JT, Haiderer ER, Coulson GB, Conner KN, Ellsworth E, Chen C, Alvarez -Cabrera 235 N, Li W, Jackson M, Dick T, Abramovitch RB. Identification of New MmpL3 Inhibitors by 236 Untargeted and Targeted Mutant Scr eens Defines MmpL3 Domains with Differential 237 Resistance. 238 9. Chauhan M, Barot R, Yadav R, Joshi K, Mirza S, Chikhale R, Srivastava VK, Yadav MR, 239 Murumkar PR. 2024. The Mycobacterium tuberculosis Cell Wall: An Alluring Drug Target for 240 Developing Newer Anti‐ TB Drugs—A Perspective. Chem Biol Drug Des 104:e14612. 241 10. Lynde BE, Chemaly DM, Baldin VP, Greve E, Harding CL, Graner JM, Hardy M, Chowdhury 242 S, Parish T. 2025. Novel 3 -Aminothieno[2,3- b ]pyridine-2-carboxamides with Activity 243 against Mycobacterium tuberculosis. ACS Med Chem Lett 16:241–249. 244 11. Bonnett SA, Ollinger J, Chandrasekera S, Florio S, O’Malley T, Files M, Jee J -A, Ahn J, 245 Casey A, Ovechkina Y, Roberts D, Korkegian A, Parish T. 2016. A Target-Based Whole Cell 246 Screen Approach To Identify Potentia l Inhibitors of Mycobacterium tuberculosis Signal 247 Peptidase. ACS Infect Dis 2:893–902. 248 12. Ioerger TR, Feng Y, Ganesula K, Chen X, Dobos KM, Fortune S, Jacobs WR, Mizrahi V, 249 Parish T, Rubin E, Sassetti C, Sacchettini JC. 2010. Variation among genome sequences of 250 H37Rv strains of Mycobacterium tuberculosis from multiple laboratories. J Bacteriol 251 192:3645–3653. 252 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint 13. McNeil MB, O’Malley T, Dennison D, Shelton CD, Sunde B, Parish T. 2020. Multiple 253 Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors. 254 mSphere 5:e00985-20. 255 14. Ollinger J, Bailey MA, Moraski GC, Casey A, Florio S, Alling T, Miller MJ, Parish T. 2013. A 256 Dual Read- Out Assay to Evaluate the Potency of Compounds Active against 257 Mycobacterium tuberculosis. PLoS ONE 8:e60531. 258 15. Naran K, Moosa A, Barry CE, Boshoff HIM, Mizrahi V, Warner DF. 2016. Bioluminescent 259 Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery. 260 Antimicrob Agents Chemother 60:6748–6757. 261 16. Quach D, Sharp M, Ahmed S, Ames L, Bhagwat A, Deshpande A, Parish T, Pogliano J, 262 Sugie J. 2025. Deep learning–driven bacterial cytological profiling to determine antimicrobial 263 mechanisms in Mycobacterium tuberculosis. Proc Natl Acad Sci 122:e2419813122. 264 17. Bonnett SA, Dennison D, Files M, Bajpai A, Parish T. 2018. A class of hydrazones are 265 active against non-replicating Mycobacterium tuberculosis. PloS One 13:e0198059. 266 18. Alland D, Steyn AJ, Weisbrod T, Aldrich K, Jacobs WR. 2000. Characterization of the 267 Mycobacterium tubercu losis iniBAC Promoter, a Promoter That Responds to Cell Wall 268 Biosynthesis Inhibition. J Bacteriol 182:1802–1811. 269 19. Shetty A, Dick T. 2018. Mycobacterial Cell Wall Synthesis Inhibitors Cause Lethal ATP 270 Burst. Front Microbiol 9:1898. 271 20. Allen R, Ames L, Baldin VP, Butts A, Henry KJ, Durst G, Quach D, Sugie J, Pogliano J, 272 Parish T. 2024. An arylsulfonamide that targets cell wall biosynthesis in Mycobacterium 273 tuberculosis. Antimicrob Agents Chemother 68:e01037-24. 274 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint 21. Ioerger TR, O’Malley T, Liao R, Guinn KM, Hickey MJ, Mohaideen N, Murphy KC, Boshoff 275 HIM, Mizrahi V, Rubin EJ, Sassetti CM, Barry CE, Sherman DR, Parish T, Sacchettini JC. 276 2013. Identification of New Drug Targets and Resistance Mechanisms in Mycobacterium 277 tuberculosis. PLoS ONE 8:e75245. 278 22. Rao SPS, Lakshminarayana SB, Kondreddi RR, Herve M, Camacho LR, Bifani P, Kalapala 279 SK, Jiricek J, Ma NL, Tan BH, Ng SH, Nanjundappa M, Ravindran S, Seah PG, Thayalan P, 280 Lim SH, Lee BH, Goh A, Barnes WS, Chen Z, Gagaring K, Chatterjee AK, Pethe K, Kuhen 281 K, Walker J, Feng G, Babu S, Zhang L, Blasco F, Beer D, Weaver M, Dartois V, Glynne R, 282 Dick T, Smith PW, Diagana TT, Manjunatha UH. 2013. Indolcarboxamide Is a Preclinical 283 Candidate for Treating Multidrug-Resistant Tuberculosis. Sci Transl Med 5. 284 23. Lun S, Tasneen R, Chaira T, Stec J, Onajole OK, Yang TJ, Cooper CB, Mdluli K, Converse 285 PJ, Nuermberger EL, Raj VS, Kozikowski A, Bishai WR. 2019. Advancing the Therapeutic 286 Potential of Indoleamides for Tuberculosis. Antimicrob Agents Chemother 63:e00343-19. 287 288 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint 289 290 291 292 293 Table 1. Activity of TPA analogs against M. tuberculosis. 294 Intracellular IC 90 = concentration that leads to 90% inhibition of bacterial replication in THP -1 295 macrophages. Data are the average and standard deviation of two independent replicates. IC90 296 = concentration that leads to 90% inhibition of bacterial growth in aerobic culture. Data are the 297 average and standard deviation of a minimum of 3 replicates. Fold-change = comparison of IC90 298 for wild-type and RM301 (MmpL3 F255L, V646M, F644I ) strains. MBC = minimum bactericidal 299 concentration – concentration at which no viable bacteria were detected after 14 days. Data are 300 the average and standard deviation of three replicates. 301 302 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint 303 304 305 Figure 1. Structure of analogs used in this study. (1) TPN -0102024 (2) TPN -0099994 (3) TPN -306 0099934 (4) TPN-0099730 (5) TPN-0089300 307 308 309 Figure 2. Exposure to TPA analogs induces cell wall stress in M. tuberculosis. 310 M. tuberculosis PiniBAC-Lux was exposed to compounds for 72h and luminescence was read. 311 Data are representative of two independent experiments (see Fig S1). 312 313 314 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint 315 316 Figure 3. TPA analogs boost ATP in M. tuberculosis. 317 M. tuberculosis was exposed to compounds for 24h and ATP was measured using BacTiter-Glo. 318 Growth was measured by OD after 5 d. Data are representative of two independent experiments 319 (see Fig S2). Q203 was used as a control. 320 321 0.1 1 10 100 1000 0.0 0.1 0.2 0.3 1000000 2000000 3000000 TPN-0102024 Concentration (µM) OD RLU Growth (OD) ATP (RLU) 0.1 1 10 100 1000 0.0 0.1 0.2 0.3 1000000 2000000 3000000 TPN-0099994 Concentration (µM) OD RLU ATP (RLU) Growth (OD) 0.1 1 10 100 1000 0.0 0.1 0.2 0.3 1000000 2000000 3000000 TPN-0099934 Concentration (µM) OD RLU ATP (RLU) Growth (OD) 0.1 1 10 100 1000 0.0 0.1 0.2 0.3 1000000 2000000 3000000 TPN-0099730 Concentration (µM) OD RLU Growth (OD) ATP (RLU) 0.1 1 10 100 0.0 0.1 0.2 0.3 1000000 2000000 3000000 Concentration (µM) Growth (OD) ATP (RLU) TPN-0089300 ATP (RLU) Growth (OD) 0.0001 0.001 0.01 0.1 1 0.0 0.1 0.2 0.3 1000000 2000000 3000000 Telacebec, Q203 Concentration (µM) OD RLU Growth (OD) ATP (RLU) .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint 322 Figure 4. Cytological profiling in response to TPN -0089300. M. tuberculosis mc26206 was 323 treated for 48 and 120 h with TPN -0089300 or DMSO (control) and stained. Membranes are 324 shown in red, DNA in blue and green indicates loss of membrane integrity. 325 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint 326 327 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint Figure 5. Similarity scores for cytological profiling in response to TPN-0089300. 328 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 26, 2025. ; https://doi.org/10.1101/2025.06.26.661674doi: bioRxiv preprint