Inhibition of Mycobacterium tuberculosis DosRST two-component regulatory system signaling by targeting response regulator DNA binding and sensor kinase heme

Mycobacterium tuberculosis (Mtb) possesses a two-component regulatory system, DosRST, that enables Mtb to sense host immune cues and establish a state of non-replicating persistence (NRP). NRP bacteria are tolerant to several anti-mycobacterial drugs and are thought to play a role in the long course of tuberculosis (TB) therapy. Therefore, small molecules that inhibit Mtb from establishing or maintaining NRP could reduce the reservoir of drug tolerant bacteria and function as an adjunct therapy to reduce treatment time. Previously, we reported the discovery of six novel chemical inhibitors of DosRST, named HC101A-106A, from a whole cell, reporter-based phenotypic high throughput screen. Here, we report functional and mechanism of action studies of HC104A and HC106A. RNAseq transcriptional profiling shows that the compounds downregulate genes of the DosRST regulon. Both compounds reduce hypoxia-induced triacylglycerol synthesis by ~50%. HC106A inhibits Mtb survival during hypoxia-induced NRP, however, HC104A did not inhibit survival during NRP. An electrophoretic mobility assay shows that HC104A inhibits DosR DNA binding in a dose-dependent manner, indicating that HC104A may function by directly targeting DosR. In contrast, UV-visible spectroscopy studies suggest HC106A directly targets the histidine kinase heme, via a mechanism that is distinct from the oxidation and alkylation of heme previously observed with artemisinin (HC101A). Synergistic interactions were observed when DosRST inhibitors were examined in pair-wise combinations with the strongest potentiation observed between artemisinin paired with HC102A, HC103A, or HC106A. Our data collectively show that the DosRST pathway can be inhibited by multiple distinct mechanisms.


Introduction
on these findings, we conclude that: 1) HC106A strongly and specifically inhibits the DosRST 142 regulon; and 2) HC104A strongly inhibits a portion of the DosRST regulon, with several notable 143 off-target activities.

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To assess the impact of the inhibitors on the DosRST pathway on intracellular Mtb, 145 murine bone marrow-derived mouse macrophages were infected with Mtb and treated with 40 146 µM HC104A and HC106A for 48 h. Total bacterial RNA was isolated and analyzed by RT-PCR 147 for hspX and tgs1 gene differential expression. The results demonstrate that the induction of 148 hspX and tgs1 were inhibited 185-and 10-fold by HC104A and 6-and 4-fold by HC106A, 149 respectively (Fig. 3a). These finding confirm that HC104A and HC106A can access Mtb inside 150 the macrophage, however, the reduced repression of the pathway by HC106A as compared to 151 broth culture, suggests that the molecule may not be able to efficiently target intracellular Mtb.

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The DosRST pathway is also induced by redox signals such as vitamin C and NO (27).

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To examine whether HC104A and HC106A can repress the induction of DosRST pathway by 154 vitamin C or NO, Mtb cells were pretreated with HC104A or HC106A for 24 h followed by 155 vitamin C or NO induction for 2 h. The expression of DosR-regulated genes (hspX and tgs1) 156 was examined by real time-PCR. Vitamin C and DETA-NONOate (NO donor) strongly induced were isolated and analyzed by thin layer chromatography (TLC). As previously observed, treated with HC104A or HC106A showed a ~50% reduction of TAG accumulation, supporting 179 our hypothesis that the compounds can inhibit TAG biosynthesis.

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DosRST has been previously reported to be required for survival during NRP, where 181 deletion of DosR causes greatly reduced survival during prolonged hypoxic stress (19). The

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HC106A caused the DosS Soret peak to immediately shift to 422 nm, where the peak was 247 stably maintained for 2 h (Fig. 5a). This spectrum shift is different from artemisinin, where under 248 identical conditions, artemisinin causes the DosS Soret peak to gradually shift back to the 249 oxidized state at 403 nm (27). These findings show that HC106A may also interact with sensor 250 kinase heme, but via a mechanism that is distinct from artemisinin-heme interactions.

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The Soret peak at 422 nm is consistent with previously described spectra that are 252 observed when DosS heme interacts with NO or CO (13). To confirm this observation, DosS 253 was treated with 100 µM CORM-2 (a CO donor) which caused a shift of the Soret peak to 422 254 nm, similar to what was observed for HC106A (Fig. 5b). This finding supports a hypothesis that 255 HC106A may also be directly binding to the heme. Notably, CO activates DosS kinase function,

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whereas HC106A functions to inactivate the regulon, demonstrating that the impact of heme 257 binding by CO or HC106A has differing impacts on the sensor kinase switch.   to combinations with artemisinin. Overall, these studies provide the evidence that the inhibitors 294 function by distinct mechanisms and may be combined to improve potency.

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We conducted a catalog search for HC104 and HC106 analogs and obtained 10 298 commercial analogs for each series to define initial structure activity relationships (SAR). For

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HC104A we observed that a bromine in the 5-position is required for activity and that the R2 300 dimethylamine group is not required (Supplementary Table 1). For example, HC104B is 301 not highly potent, its ligand efficiency, cLogD and druglikeness are in the range of what would 305 be considered acceptable to good as a starting point for further manipulation. For HC106A 306 (Table 1), catalog SAR work led to new understandings of the nature of the series. We first 307 found that the simple removal of an ortho chloro on the "A" ring of HC106A leads to ~ 2-fold 308 enhanced activity, with an EC 50 in the whole cell Mtb assay for DosRST inhibition of 1.33 µM 309 (HC106F). It was also found that the use of an alternative isomer of the isoxazole had no 310 detectable activity (HC106C).

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To further understand the SAR of the HC106 series, additional analogs were 313 synthesized to examine the need of the central urea functionality and whether modifications 314 can be tolerated (Table 1)

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To further test the SAR, we conducted a Topliss Tree evaluation of the "A-ring" aniline dichloro (MSU-39452). We found that replacing the 4-chlorophenyl ring with pyridyl analogs

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Kinetic solubility assays were conducted for selected analogs and all exhibited 351 excellent aqueous solubility greater than >100 µM, except for MSU-41443 (Table 2). This 352 finding shows that the urea group present in the HC106A does not have a detrimental impact 353 mouse microsomal stability, including. Overall, the nanomolar whole cell potency, flexible 355 SAR, good microsomal stability and excellent solubility, confirm that the HC106 series is a 356 suitable series for continued optimization to identify a drug-like lead.

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DosRST is a two-component regulatory system required for Mtb environmental sensing, 358 adaptation, and persistence. By using a fluorescent reporter strain CDC1551 (hspX'::GFP), we

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The identification of new antibacterial agents and tuberculosis drugs has been 433 associated with the realization that these compounds can occupy a different region of chemical 434 space relative to drugs in most other therapeutic areas. Series HC106 is easily in the range of 435 the characteristics for compounds currently in use or in development as anti-TB compounds.

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Series HC104 needs further exploration to fully assess its suitability. The physicochemical 437 properties of a drug, such as solubility and permeability, impact its oral bioavailability as these 438 factors influence absorption, distribution, metabolism, and excretion. Series HC106 439 demonstrates excellent aqueous solubility (

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Second, dose response assays were performed with 2 µM DosR treated with different 542 concentrations of HC104A or virstatin from 1 µM to 80 µM. After incubating on ice for 30 min, 543 the reactions were terminated by adding 1 µL 80% glycerol, and loaded on a native 5% 544 Tris/Borate/EDTA (TBE) polyacrylamide gel. The gel was run at 50 V, for 5-6 h at 4°C in 1X TBE 545 buffer, and was imaged using a Typhoon scanner with appropriate filters that can detect 546 florescence at excitation = 495 nm, emission = 520 nm. Binding of the unbound probe was 547 quantified using ImageJ (43). The assay was repeated at least twice with similar results. The

Experimental procedures for urea formation
Formation of acyl chloride 2. To a stirred solution of isoxazole acid 1 (1 eq.) in dry tetrahydrofuran (THF, 0.4 M) under N 2 atmosphere was added oxalyl chloride (1.5 eq.) dropwise over 5-10 min followed by dimethylformamide (DMF) (cat.) and the reaction mixture was continued to stir at room temperature. Upon completion, the reaction mixture was concentrated into a residue in vacuo and the residue was dissolved in THF and concentrated again to ensure the removal of excess oxalyl chloride. The crude acyl chloride 2 was used directly in the next step without further purification.
Formation of acyl azide and rearrangement into isocyanate 3.
The crude acyl chloride 2 was dissolved in THF (0.4 M) and stirred at room temperature under N 2 atmosphere. Trimethylsilyl (TMS) azide (2 eq.) was added dropwise over 5 min and stirring was continued. Upon completion of the reaction, the mixture was diluted with ethyl acetate (0.4 M) and quenched with H 2 O (0.4 M). The two layers were separated, and the organic layer was dried over anhydrous Na 2 SO 4 and filtered. The ethyl acetate solvent was swapped into toluene (0.1 M) by the addition of toluene followed by removal of the ethyl acetate in vacuo. Care was taken not to concentrate the toluene. The toluene acyl azide solution was heated at reflux conditions under N 2 atmosphere for 4 h to give the desired isocyanate 3 which was used as a solution in toluene in the next step.
Formation of urea 4. The crude isocyanate solution in toluene was mixed with different amines (1.5 eq.) and stirred at room temperature overnight. Isolation of the ureas was done by diluting the reaction mixture with hexanes, stirring for few hours and filtration of the formed precipitate.
The solid material was washed with hexanes and dried under high vacuum. The urea products usually do not require further purifications. All products were analyzed by 1 H NMR and highresolution mass spectrometry (HRMS).