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.

Tuberculosis, caused by the intracellular pathogen , is a deadly disease that requires a long course of treatment. The emergence of drug-resistant strains has driven efforts to discover new small molecules that can kill the bacterium. Here, we report characterizations of the compound HC2091, which kills in a time- and dose-dependent manner and inhibits growth in macrophages. Whole-genome sequencing of spontaneous HC2091-resistant mutants identified single-nucleotide variants in the mycolic acid transporter gene. HC2091-resistant mutants do not exhibit cross-resistance with the well-characterized membrane protein large 3 (MmpL3) inhibitor SQ109, suggesting a distinct mechanism of interaction with MmpL3. Additionally, HC2091 does not modulate bacterial membrane potential or kill nonreplicating , thus acting differently from other known MmpL3 inhibitors. RNA sequencing (RNA-seq) transcriptional profiling and lipid profiling of treated with HC2091 or SQ109 show that the two compounds target a similar pathway. HC2091 has a chemical structure dissimilar to those of previously described MmpL3 inhibitors, supporting the notion that HC2091 is a new class of MmpL3 inhibitor.

Summary Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here we report that the compound AC2P36 selectively kills Mtb at acidic pH and potentiates the bactericidal activity of isoniazid, clofazimine, and diamide. We show that AC2P36 activity is associated with thiol stress and causes an enhanced accumulation of intracellular ROS at acidic pH. Mechanism of action studies demonstrate that AC2P36 directly depletes Mtb thiol pools, with enhanced depletion of free thiols at acidic pH. These findings support that Mtb is especially vulnerable to thiol stress at acidic pH and that chemical depletion of thiol pools is a promising target to promote Mtb killing and potentiation of antimicrobials.

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.

Progress against tuberculosis (TB) requires faster-acting drugs. Mycobacterium tuberculosis (Mtb) is the leading cause of death by an infectious disease and its treatment is challenging and lengthy. Mtb is remarkably successful, in part, due to its ability to become dormant in response to host immune pressures. The DosRST two-component regulatory system is induced by hypoxia, nitric oxide and carbon monoxide and remodels Mtb physiology to promote nonreplicating persistence (NRP). NRP bacteria are thought to play a role in the long course of TB treatment. Therefore, inhibitors of DosRST-dependent adaptation may function to kill this reservoir of persisters and potentially shorten therapy. This review examines the function of DosRST, newly discovered compounds that inhibit DosRST signaling and considers future development of DosRST inhibitors as adjunct therapies.

No abstract

With the operation of six cascade reservoirs, the flow regime and sediment discharge of the Lancang River have changed greatly. The changes of runoff and suspended load have attracted extensive attention. The hydrological data of Gajiu and Yunjinghong stations in Lancang River from 1964 to 2019 were analyzed by using wavelet analysis, double mass curve and abrupt change analysis. The temporal trends in runoff and suspended load were evaluated. Results revealed that the reduction of suspended load was much more profound than the change of runoff. There was a slight downward trend in annual runoff due to climate change. After the completion of Xiaowan and Nuozhadu reservoirs, the proportion of runoff in flood season decreased by 22.64 and 30.75%, respectively. Wavelet analysis was used to reveal the characteristics of runoff evolution. With the operation of reservoirs, suspended load appeared abrupt changes in 1993 and 2008. The amount of suspended load during 2009–2019 decreased by 95.47–98.78% compared with that before the reservoir construction. This paper presents the latest quantitative study on the temporal variation of runoff and suspended load since the completion of Xiaowan and Nuozhadu reservoirs, which is of great importance for guiding the operation of reservoirs and maximizing the value of the whole Lancang-Mekong River basin.

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...