Inhibiting <i>Mycobacterium tuberculosis</i> DosRST 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 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.