Inhibiting Mycobacterium tuberculosis DosRST Signaling by Targeting Response Regulator DNA Binding and Sensor Kinase Heme

Abstract

Mycobacterium tuberculosis (Mtb) possesses a two-component regulatory system, DosRST, that enables Mtb to sense host immune cues and establish a state of nonreplicating persistence (NRP). NRP bacteria are tolerant to several antimycobacterial drugs in vitro and are thought to play a role in the long course of tuberculosis therapy. 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 sensor 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 pairwise 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.

The Prospect of Repurposing Immunomodulatory Drugs for Adjunctive Chemotherapy against Tuberculosis: A Critical Review

“…Although originally for Gram-positive bacteria, oxazolidinones—linezolid and its derivatives—are in many clinical trials, with linezolid already approved for use in TB [ 22 ]. In addition, the antiparasitic artemisinin is currently receiving a lot of attention from the research community for its ability to disrupt signalling that regulates persistence in M. tuberculosis [ 23 , 24 , 25 , 26 ].…”

Inhibiting DosRST As a New Approach to Tuberculosis Therapy

“…Targeting TCS has been an active area of research to translate studies of bacterial pathogenesis into new classes of antivirulence antibiotics [ 9 , 10 , 72 ]. A variety of approaches have been employed to discover TCS inhibitors, including whole cell reporter-based high-throughput screens (HTS) [ 45 , 64 , 65 , 73 ]; phenotypic HTS targeting TCS-dependent responses [ 74 ]; target-based biochemical assays focused on inhibition of bacterial histidine kinase activity by small molecules [ 75–77 ] or response regulator mimetic peptides [ 78 , 79 ]; or inhibition of response regulator DNA binding [ 80 , 81 ]. These approaches have generated several new compounds that are in preclinical development as novel antivirulence therapies.…”

“…Mechanism of action studies were initially carried out on three of the inhibitors: artemisinin, HC102A and HC103A ( Figure 2 ). RNAseq transcriptional profiling experiments showed that the compounds inhibit the DosR regulon when compared with a dosR mutant control strain [ 64 , 65 ]. Notably, the triacylglycerol (TAG) synthase gene tgs1 was repressed and treatment with the artemisinin, HC102, HC103 significnatly reduced TAG accumulation.…”

Mechanisms controlling bacterial infection in myeloid cells under hypoxic conditions

“…As the DosR/DosT regulon is important for adaptation and survival of M. tuberculosis under hypoxic conditions, it might be a good target for new therapeutics. One promising inhibitor is artemisinin that disables the heme-base DosT sensor kinase [136]; further inhibitors with multiple distinct mechanisms have been identified [137]. is enhanced, which leads to increased glucose uptake into the cell.…”