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

Abstract

25Mycobacterium tuberculosis (Mtb) possesses a two-component regulatory system, 26DosRST, that enables Mtb to sense host immune cues and establish a state of non-27 replicating persistence (NRP). NRP bacteria are tolerant to several anti-mycobacterial drugs 28 and are thought to play a role in the long course of tuberculosis (TB) therapy. Therefore, 29 small molecules that inhibit Mtb from establishing or maintaining NRP could reduce the 30 reservoir of drug tolerant bacteria and function as an adjunct therapy to reduce treatment 31 time. Previously, we reported the discovery of six novel chemical inhibitors of DosRST, 32 named HC101A-106A, from a whole cell, reporter-based phenotypic high throughput screen. 33Here, we report functional and mechanism of action studies of HC104A and HC106A. 34RNAseq transcriptional profiling shows that the compounds downregulate genes of the 35 DosRST regulon. Both compounds reduce hypoxia-induced triacylglycerol synthesis by ~50%. 36HC106A inhibits Mtb survival during hypoxia-induced NRP, however, HC104A did not inhibit 37 survival during NRP. An electrophoretic mobility assay shows that HC104A inhibits DosR 38 DNA binding in a dose-dependent manner, indicating that HC104A may function by directly 39 targeting DosR. In contrast, UV-visible spectroscopy studies suggest HC106A directly targets 40 the histidine kinase heme, via a mechanism that is distinct from the oxidation and alkylation of 41 heme previously observed with artemisinin (HC101A). Synergistic interactions were observed 42 when DosRST inhibitors were examined in pair-wise combinations with the strongest 43 potentiation observed between artemisinin paired with HC102A, HC103A, or HC106A. Our 44 data collectively show that the DosRST pathway can be inhibited by multiple distinct 45 mechanisms.46 47 Keywords: Chemical biology, two-component regulatory systems, microbial pathogenesis. (NRP) where Mtb modulates its metabolism in response to environmental and host immune 54 cues, such as hypoxia, acidic pH, and nutrient starvation (2, 3). DosRST is a two-component 55 regulatory system that regulates Mtb persistence (4-6). It consists of two sensor histidine 56 kinases, DosS and DosT, and the cognate response regulator DosR, which regulates 57 expression of about 50 genes in the DosRST regulon (6-8). The pathway can be induced by 58 host intracellular stimuli, such as nitric oxide (NO), carbon monoxide (CO) and hypoxia, through 59 DosS and DosT (9-11). DosS is an oxygen and redox sensor, whereas DosT acts an oxygen 60 sensor (12-14). Both kinases sense ligands via the heme group, and are inactive when the 61 heme exists as either the Met (Fe 3+ ) form (DosS) or the oxy (Fe 2+ -O 2 ) form (DosT) in the 62 presence of O 2 (13). However, hypoxic conditions activate the kinases by inducing the 63 conversion of DosS to the ferrous form and DosT to the deoxy form. Therefore, DosS/T play 64 overlapping and distinct roles in sensing the redox status and oxygen level of the environment 65 to turn on the DosR pathway (11, 15). 66Non-re...

Targeting dormant phenotype acquired mycobacteria using natural products by exploring its important targets: In vitro and in silico studies

“…The binding energies of these complexes were calculated over all the frames of the MDS of which the standard ligand HC104A showed the highest binding energy of DG bind = −34.98 (6.11) followed by IC (DG bind = −31.76 (4.31) kcal/mol), UA (DG bind = −22.28 (7.33) kcal/mol), and BA (DG bind = −17.13 (4.81) kcal/mol) (Table 2). These results were expected because ligand HC104A is a known Mtb DosR inhibitor (Zheng et al, 2020); this also strengthens our experimental results about the efficacy of the IC as an inhibitor of DosR in the mycobacteria. The ligand RMSD for compounds bound to the Mtb STPK showed variable RMSDs.…”

Two Component Regulatory Systems and Antibiotic Resistance in Gram-Negative Pathogens

“…The possible sites of intervention for a TCS are to be determined. Studies in the past have targeted the RR DNA binding [273], autophosphorylation sites [274] as well as ATP-binding domains [275]. Suggested sites for targeting include the site for autophosphorylation, site of interaction for HK-RR, facilitating the dephosphorylation of the HK, and inhibiting binding to the downstream genes.…”