Inhibition of CorA-Dependent Magnesium Homeostasis Is Cidal in Mycobacterium tuberculosis

Abstract

Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.

Drivers and sites of diversity in the DNA adenine methylomes of 93 Mycobacterium tuberculosis complex clinical isolates

“…The functions of genes with promoter motif sites that were hypervariable ( Figures 7 and 8 , Figure 7—figure supplement 1 ) or in stereotyped configurations with −10 promoter elements ( Figures 6 and 8 ; Supplementary file 6 ) implicate clinically important processes as under control of DNA adenine methylation ( Supplementary files 1 and 6 , Figure 7—figure supplement 1 ). These include drug resistance, metal ion homeostasis ( corA ( Park et al, 2019 ), lpqS ( Darwin, 2015 ), mmpS4 ( Jones et al, 2014 ) , higA ( Schuessler et al, 2013 ) , mbtJ ( Chownk et al, 2018 ), and hemN McMahon et al, 2012 ), and the induction and maintenance of dormancy—all functions with examples of modulation by DNA adenine methylation in other human pathogens (Beaulaurier et al; Brunet et al, 2020 ; Cohen et al, 2016 ; Sánchez-Romero and Casadesús, 2020 ). The resistance-implicating genes among these mediate resistance through gene regulation ( whiB7 -controlled expression of eis, tap, and Rv1473 ( Duan et al, 2019 ; Morris et al, 2005 ), and raaS- controlled expression of Rv1218c and Rv1217c Wang et al, 2013 ), drug efflux ( drrA ( Mustyala et al, 2016 ) , iniA ( Colangeli et al, 2005 ), Rv3728 ( Gupta et al, 2010 ), and efflux-targets of whiB7 and raaS ), and other mechanisms ( glf ( Chen and Bishai, 1998 ) , mshC ( Parida et al, 2015 ) , mshD ( Vilchèze et al, 2008 ) , pafA ( Samanovic and Darwin, 2016 ), Rv3050c ( Nieto R et al, 2018 ), and gyrB ( Nosova et al, 2013 ).…”

Development and Optimization of Chromosomally-Integrated Fluorescent Mycobacterium tuberculosis Reporter Constructs

“…For magnesium (Mg 2+ ) ions, however, no tools are currently available to reliably quantify or read out its concentration. The importance of Mg 2+ for mycobacterial growth has been demonstrated in in vitro culture using media with low Mg 2+ concentration ( Supplementary Figure 1B ; Buchmeier et al, 2000 ; Piddington et al, 2000 ; Walters et al, 2006 ; Goodsmith et al, 2015 ) and inhibitors of Mg 2+ homeostasis ( Supplementary Figure 2A ; Lopez Quezada et al, 2019 ; Park et al, 2019 ). In addition, there is accumulating evidence that survival of Mtb within the acidic pH of phagosomes is Mg 2+ dependent ( Buchmeier et al, 2000 ; Piddington et al, 2000 ).…”

Data_Sheet_1.PDF

“…Recently, two groups have identified novel antimycobacterial molecules, whose efficacy was Mg 2+ -dependent. Mutations of resistant strains exclusively mapped to the gene rv1239c, which encodes CorA (Lopez Quezada et al, 2019;Park et al, 2019). We explored one of the compounds from these studies, PAA10 (Park et al, 2019).…”

“…When the reporter was cultured in high (500 µM) Mg 2+ levels, the FI, detected at a PAA10 concentration of 100 nM, exceeded that measured at 10 µM environmental MgSO 4 , confirming the proposed mechanism of action of intracellular Mg 2+ starvation. Our new reporter strain will be used to identify Mg 2+ concentrations of different Mtb lesions within the lungs which may address whether the lack of in vivo efficacy of PAA10 (Park et al, 2019) was due to poor exposure to the compound at the site of infection or due to high concentrations of Mg 2+ in the Mtb microenvironment.…”

“…For magnesium (Mg 2+ ) ions, however, no tools are currently available to reliably quantify or read out its concentration. The importance of Mg 2+ for mycobacterial growth has been demonstrated in in vitro culture using media with low Mg 2+ concentration (Supplementary Figure 1B; Buchmeier et al, 2000;Piddington et al, 2000;Walters et al, 2006;Goodsmith et al, 2015) and inhibitors of Mg 2+ homeostasis ( Supplementary Figure 2A; Lopez Quezada et al, 2019;Park et al, 2019). In addition, there is accumulating evidence that survival of Mtb within the acidic pH of phagosomes is Mg 2+ dependent (Buchmeier et al, 2000;Piddington et al, 2000).…”