Studying the mechanism of action of new derivatives of quinoxalin-1,4-dioxide on the model organism Mycobacterium smegmatis

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Introduction Multidrug-resistant (MDR) tuberculosis remains a major threat to modern medicine, with 649 000 new cases of rifampicin-resistant tuberculosis (RR-TB) - the most effective first-line drug - occurring between 2018 and 2021, of which 78% were MDR (rifampicinand isoniazid-resistant) [1]. The emergence of MDR strains may result from, among other things, insufficient adherence, interrupted therapy or inappropriate chemotherapy. Russia, along with India and China, is among the countries with the highest prevalence of MDR-tuberculosis (WHO Global Tuberculosis Report 2022). Extensively drug-resistant (XDR) TB strains, resistant to 4-9 drugs, pose the greatest threat among MDR strains [2]. Increasing levels of resistance have also been attributed to the accumulation of antibiotics in nature and the activation of natural drug resistance systems in bacteria. According to recent data, one of the factors accelerating the emergence of DR is the presence of minimal selective concentrations (MSC) of antibiotics in the environment, which can lead to an increase in resistant strains in the population and activation of cell defence mechanisms against antibiotics (release or inactivation of antibiotics) [3-5]. Thus, one of the main current challenges is the search for new antituberculosis drugs (ATTs) that will have fundamentally new mechanisms of action, which will make it possible to overcome the phenomenon of drug resistance. The aim of this study was to investigate the mechanism of action of promising PTP candidates - a new quinoxaline 1,4-dioxide derivative synthesised by us earlier 4 [6]. These compounds were selected due to their high activity against mycobacteria - compounds of this class have been shown to introduce singleand double-strand breaks in DNA, leading to cell death, which makes them promising for further study and modification [7]. Using reverse genetics methods, we have shown that mutations in the MSMEG_4646, MSMEG_5122, and MSMEG_1380 genes confer resistance to compound 4 [6]. In this work, we studied the mechanisms of cross-resistance to quinoxaline 1,4-dioxide derivatives on the model object Mycobacterium smegmatis using genetic constructs with an increased level of gene expression. Materials and methods Bacterial strains and incubation conditions Cells of Mycolicibacterium (Mycobacterium) smegmatis strains (Table 1). were grown in Middlebrook 7H9 liquid medium (Himedia) supplemented with OADC (oleic acid, albumin, dextrose, catalase), 0.05% Tween 80, 0.4% glycerol and in Lemco-Tween liquid medium. Composition of Lemco-Tween medium (per 1 litre): 5 g peptone (Oxoid), 5 g Lab Lemco (Oxoid), 5 g NaCl, 0.05% Tween 80. M290 medium (HiMedia Laboratories Pvt. Ltd) was used to grow M. smegmatis on agarised medium. M. smegmatis was incubated at t = 37°C. Table 1. Bacterial strains used in the study Bacterial strains Title Description Reference M.smegmatis mc2 155 Wild-type strain (w.t.) (8) M. smegmatis qdr Spontaneous mutants of M. smegmatis qdR1, qdR4 and qdR5 resistant to the compound 1 (9) M. smegmatis M. smegmatis strains carrying pMind containing mutant genes: pM4646w, pM4646q314, pM4648w, pM4648q1, pM5122. Present paper Source: compiled by the authors. Cloning of genes containing mutations in the MSMEG_4646, MSMEG_4648, MSMEG_5122 genes into the pMind plasmid vector MSMEG_4646, MSMEG_4648, MSMEG_5122 genes of M. smegmatis were aplified from genomic DNA of mutant strains resistant to quinoxaline 1,4-dioxide-4 derivative and WT strain M. smegmatis mc2 155 using primers selected using NCBI BLAST primer (see Table 2). The optimal annealing temperature of the primers was selected using gradient PCR on a Bio-Rad T100 instrument (USA). Tersus Plus PCR kit (Eurogen) was used for amlification. The amlified fragment was cloned into the shuttle replicative vector pMind... by restriction sites NdeI and SpeI (Fast digest, Thermo Scientific, USA). T4-DNA ligase (Thermo Scientific, USA) was used for ligation. The obtained constructs were transfected into competent E. coli cells according to the standard procedure [10]. The presence of the target insert of the desired length was assessed by PCR screening of colonies. Constructs with target gene inserts were isolated from E. coli cells using a plasmid DNA isolation kit (Eurogen, Russia). The obtained constructs were transformed into competent M. smegmatis mc2 155 cells by electroporation according to the method [11]. Table 2. Primers used in the study Primer’s title Primers for cloning in pMind pM_4646_f NdeI 5′ ttttCATATGggaggaaatgttATGGGTGACAACGGCAACGG 3′ pM_4646_r SpeI 5′ ttttACTAGTTCATGCGTTCGCTCCCACAG 3′ pM_4648_f NdeI 5′ ttttCATATGggaggaaatgttATGGCGCACCGGTACAAGG 3′ pM_4648_r SpeI 5′ ttttACTAGTTCACATCGGCAGGTTGTAGGG 3′ pM_5122_f NdeI 5′ ttttCATATGggaggaaatgttATGACGTACGTCATTGCCGAAC 3′ pM_5122_r SpeI 5′ ttttACTAGTTCAGTCCTCACCCTGAGGC 3′ Source: compiled by the authors. Sensitivity test of M. smegmatis to quinoxaline 1,4-dioxide derivatives Cultures were incubated at 200 rpm and 37 °C overnight until OD600 = 1.2. Sensitivity to quinoxaline 1,4-dioxide derivatives was then determined using the paper disc method (diffusion-disc method): M. smegmatis culture was diluted 1 : 9 : 10 (culture : water : M290 medium (HiMedia Laboratories Pvt. Ltd.)) and seeded on top of the agar base layer on Petri dishes. Petri dishes have been incubated for 2 days at 37 °C until complete growth of the bacterial lawn. Growth inhibition halos were measured to the nearest 1 mm. Experiments were performed in triplicate; mean diameter and standard deviation (SD) were calculated. The criterion for selecting positive results was the presence of a significant difference in the diameter of the growth inhibition zone and the absence of the intersection of standard deviations of the diameters of the growth inhibition zones of the experimental and control samples of M. smegmatis [12]. Results A cross-resistance study of M. smegmatis qdR mutants. To investigate cross-resistance to quinoxaline 1,4-dioxide derivatives, we used spontaneous mutants of M. smegmatis mc2 155 resistant to 4-fold MIC of compound 4 (Table 3), obtained and partially characterized previously [6]. Mutations in four different genes, MSMEG_1380, MSMEG_4646, MSMEG_4648, and in the gene and its promoter region MSMEG_5122, were identified by comparative genomic analysis. Using reverse genetics methods, it was shown that mutations in the MSMEG_1380, MSMEG_4648, and MSMEG_5122 genes lead to resistance to the previously described quinoxaline 1,4-dioxide derivatives [6]. Spontaneous mutants of M. smegmatis resistant to compound 4: M. smegmatis qdR1, M. smegmatis qdR4 and M. smegmatis qdR5 (Table 2) were used to investigate cross-resistance to quinoxaline 1,4-dioxide derivatives, and therefore to understand whether there is a common mechanism of action or resistance of M. smegmatis to different quinoxaline 1,4-dioxides. Possible cross-resistance was tested to the following compounds: 4, 13c, 12c, 13a, 16c, 14a, 15c (compounds shown in Table 3). Table 3. Chemical compounds used in the study No. Title link 4 2-carboethoxy-3-methyl-6-(piperazin-1-yl)-7-chloroquinoxaline 1,4-dioxide hydrochloride (6) 13c 2-acetyl-7-(piperazin-1-yl)-3-trifluoromethyl-6-chloroquinoxaline 1,4-dioxide hydrochloride (9) 12c 7-(piperazin-1-yl)-3-trifluoromethyl-6-chloro-2-ethoxycarbonylquinoxaline 1,4-dioxide hydrochloride 13a 2-acetyl-7-(piperazin-1-yl)-3-trifluoromethylquinoxaline 1,4-dioxide hydrochloride 16c 7-(piperazin-1-yl)-3-trifluoromethyl-2-furanoyl-6-chloroquinoxaline 1,4-dioxide hydrochloride 14a 7-(piperazin-1-yl)-2-propanoyl-3-trifluoromethylquinoxaline 1,4-dioxide hydrochloride 15c 2-benzoyl-7-(piperazin-1-yl)-3-trifluoromethyl-6-chloroquinoxaline 1,4-dioxide hydrochloride Source: compiled by the authors. The study revealed cross-resistance to all tested compounds in almost all cases. The qdR series mutants were resistant to most of the quinoxaline 1,4-dioxide derivatives analysed (Figure 1). The M. smegmatis qdR1 strain was generally more sensitive than the other mutants, although significantly more resistant than the wild-type strain to all compounds. Figure 1. Diameters of growth inhibition zones around discs containing different quinoxaline 1,4-dioxides against spontaneous mutants of M. smegmatis resistant to compound 4. The concentration of compounds is 10 nmol/disc. Error bars reflect standard deviation. Source: compiled by the authors. Investigation of the role of individual genes and mutations in the formation of resistance to quinoxaline 1,4-dioxides Previous full-genome sequencing showed that M. smegmatis, qdR1, M. smegmatis, qdR4, and M. smegmatis, qdR5 mutants have unique nonsynonymous mutations [6], confirming previous reports on the DNA-damaging properties of quinoxaline 1,4-dioxides [7]. The number of unique mutations in each strain correlated with the level of resistance, suggesting that their combination has a synergistic effect. We analysed the possible linkage of all mutant genes and identified several genes related to the oxidation of pyruvate to acetyl-CoA. Overexpression of wild-type target genes and their mutant variants in M. smegmatis. To investigate the role of individual genes and mutations in them in the formation of resistance to quinoxaline 1,4-dioxide, we used the approach of overexpression of target genes of wild type and their mutant variants (Table 4). For this approach, we used the pMIND vector [13], which has origins for replication in E. coli and mycobacterial cells, as well as an inducible tetracycline promoter. Table 4. Unique mutations in the genomes of M. smegmatis qdr1, M. smegmatis qdr4 and M. smegmatis qdr5 mutants Protein ID Locus tag Annotation Codon SNP A.a. Distance to gene M. smegmatis qdr1 YP_888911.1 MSMEG_4648 DNA-binding protein 49 CAG>CCG Q>P - YP_889369.1 MSMEG_5122 ferredoxin - - - 71-72 M. smegmatis qdR4 YP_888909.1 MSMEG_4646 pyruvate synthase 95 AAC>CAC N>H - M. smegmatis qdR5 YP_888909.1 MSMEG_4646 pyruvate synthase 274 CCG>CTG P>L - Source: compiled by the authors. We obtained the following constructs: • pM4646wt: pMIND containing the wild-type MSMEG_4646 gene; • pM4646qdr4: pMIND containing the MSMEG_4646 gene with an AAC > CAC mutation at codon 95 (N→H), corresponding to the M. smegmatis qdR4 mutant; • pM4646qdr5: pMIND containing the MSMEG_4646 gene with a CCG > CTG mutation at codon 274 (P→L), corresponding to the M. smegmatis qdR5 mutant; • pM4648wt: pMIND containing the wild-type MSMEG_4648 gene; • pM4648qdr1: pMIND containing the MSMEG_4648 gene with a CAG > CCG mutation in codon 274 (Q→P), corresponding to the M. smegmatis qdR1 mutant; • pM5122: pMIND containing the wild-type MSMEG_5122 gene. These constructs were used to transform M. smegmatis strain mc2 155. The drug susceptibility phenotype of the obtained M. smegmatis transformants carrying the constructed plasmids was assessed by the disc-diffusion method, rifampicin (rif) was used as a control compound (Figure 2). The results (Figure 2) showed a significant increase in resistance to compounds 4, 12c, 14a and 13c upon overexpression of MSMEG_4646 mutant genes. Overexpression of MSMEG_4648 mutant genes did not result in increased resistance. Overexpression of the wild-type gene MSMEG_5122 consistently resulted in increased sensitivity to compounds 4 and 12c, probably shifting the redox reaction equilibrium due to the presence of more electron donor. Figure 2. Diameters of growth inhibition zones around discs containing different quinoxaline 1,4-dioxides on M. smegmatis cultures. The concentration of compounds is 10 nmol/disc. Error bars reflect standard deviation. Source: compiled by the authors. Conclusion As a result of this research, we were able to establish the putative mechanism of action of quinoxaline 1,4-dioxide derivatives on the model object Mycobacterium smegmatis. It can be assumed that the differences in the level of sensitivity of mutant strains are based on different sets of mutations in different strains. Thus, mutants, qdR4 and qdR5 have mutations in the MSMEG_4646 gene (see Table 4) encoding the alpha subunit of ferredoxin oxidoreductase (pyruvate synthase) involved in pyruvate metabolism. The qdR1 mutant has a mutation in the MSMEG_4648 gene, annotated as a DNA-binding protein, which may act as a regulator of transcription of the adjacent operon encoding the alphaand beta-subunits of the aforementioned pyruvate synthase and MSMEG_5122. In one of the previously studied mutants we could detect a mutation only in the MSMEG_5122 gene encoding ferredoxin directly, and the qdR1 mutant also has two mutations in the putative promoter region of the MSMEG_5122 gene (positions -71-72), and both mutant strains were resistant to the tested compound. Ferredoxin acts as an electron acceptor for pyruvate synthase, in the oxidation of pyruvate to acetyl-CoA. Probably, the mutant subunit competes with the wild-type subunit in the formation of the pyruvate synthase complex, which in total reduces the efficiency of its operation and activation of quinoxaline 1,4-dioxides, leading to reduced sensitivity of the strain to the compound under study.

About the authors

Aleksey A. Vatlin

Vavilov Institute of General Genetics, Russian Academy of Sciences; RUDN University

Author for correspondence.
Email: vatlin_alexey123@mail.ru
ORCID iD: 0000-0002-6673-7633

Candidate of Biological Sciences, Senior Researcher, Laboratory of Microorganism Genetics

3 Gubkin St, Moscow, 119333, Russian Federation

Svetlana G. Frolova

Vavilov Institute of General Genetics, Russian Academy of Sciences

Email: sveta.frolova.1997@bk.ru
Junior Researcher, Laboratory of Microorganism Genetics, Institute of General Genetics 3 Gubkin St, Moscow, 119333, Russian Federation

Olga B. Bekker

Vavilov Institute of General Genetics, Russian Academy of Sciences

Email: obbekker@mail.ru
Candidate of Biological Sciences, Senior Researcher Laboratory of Microorganism Genetics, Institute of General Genetics 3 Gubkin St, Moscow, 119333, Russian Federation

Valeriy N. Danilenko

Vavilov Institute of General Genetics, Russian Academy of Sciences

Email: valerid@vigg.ru
Doctor of Biological Sciences, Professor, Head of the Laboratory of Microorganism Genetics 3 Gubkin St, Moscow, 119333, Russian Federation

References