Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence

Abstract

The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ∼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme-artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.

Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at Acidic pH Kills the Bacterium and Potentiates Antibiotics

“…Two whole-cell phenotypic high throughput screens (HTS) were conducted at neutral and acidic pH for inhibitors of the DosRST and PhoPR regulons, respectively, using an identical chemical library (Johnson, et al, 2015; Zheng, et al, 2017). The ~220,000 compound screening library is composed of small molecules representing broad chemical diversity.…”

“…These compounds may identify novel drug targets or pathways that are essential for growth at acidic pH, which would have been missed in previously reported drug screens performed in rich medium at neutral pH. Our team has completed two, independent high throughput screens searching for inhibitors of acidic pH-inducible or hypoxia-inducible signaling pathways (Johnson, et al, 2015; Zheng, et al, 2017). The screens were performed under near identical conditions, including the same ~220,000 compound library, with the primary difference being the pH of the medium (pH 7.0 or pH 5.7).…”

Small Molecules That Sabotage Bacterial Virulence

“…A DosRST-dependent, hypoxia-inducible fluorescent Mtb reporter strain was generated by fusing the DosR - regulated hspX promoter to GFP [50]. This reporter strain was used in to screen a >540,000 compound library for chemicals that inhibit hypoxia-dependent induction of reporter strain fluorescence and identified six new inhibitors of the DosRST regulon [74]. Mechanism of action studies were carried out on three prioritized inhibitors: artemisinin, HC102A and HC103A (Figure 2).…”

“…Previously, disruption of tgs1 has been shown to reduce Mtb tolerance to the first-line Mtb drug isoniazid (INH) during NRP [76, 77]. In the hypoxic shiftdown model Mtb exhibits 100% survival following 10 days of treatment with 5 μM INH[74]. However, treatment of Mtb with 5 μM INH in combination with 40 μM of artemisinin, HC102A or HC103A, broke Mtb tolerance to INH and caused a ~25% enhancement of Mtb killing by INH.…”

“…Using UV-visible spectroscopy, artemisinin was shown to modulate DosS and DosT sensor kinases by oxidizing and alkylating the heme group carried by DosS/T[74]. Using structural modeling DosS and DosT amino acid substitutions were identified that would interfere with artemisinin docking with heme and it was observed that these substitutions promoted resistance to artemisinin-mediated inhibition of DosS and DosT in biochemical assays.…”