*4.3. MIC Determination*

MICs of the studied compounds on *M. smegmatis* were determined in liquid medium. *M. smegmatis* strains were cultured overnight in 7H9 medium, then diluted in the proportion of 1:200 in fresh medium (to approximately OD600 = 0.05). 196 μl of the diluted culture was poured in sterile nontreated 96-well flat-bottom culture plates (Eppendorf, Germany) and 4 μL of serial two-fold dilutions of the tested compounds in DMSO were added to the wells. The plates were incubated at 37 ◦C and 250 rpm for 48 h. The MIC was determined as the lowest concentration of the compound with no visible bacterial growth.

### *4.4. MSMEG\_1380 Cloning, Expression and Drug-Susceptibility Testing*

*MSMEG\_1380* genes from respective strains were amplified by Phusion High-Fidelity DNA Polymerase (Thermo Scientific, USA) using primers pM\_1380\_f 5-GACACATATGGGAGGAAATGT TGTGAGTGCCCCCGAGACG-3 and pM\_1380\_r 5-TTTTACTAGTTCAGGTGGCGCAGGGCG-3 picked with primer-BLAST [22] and cloned in the pMINDKm- plasmid [15], a modification of pMIND [24] lacking the kanamycin resistance gene, at the *Nde*I and *Spe*I restriction sites, to obtain the following plasmids: pMINDKm-:*msmeg\_1380*, pMINDKm-:*msmeg\_1380-19*, and pMINDKm-:*msmeg\_1380-33*, containing, respectively, the *w.t. msmeg\_1380* gene as well as its mutant variants from strains *atR19* and *atR33.* The resulting plasmids were electroporated in *M. smegmatis mc2 155* cells as described in [23].

*M. smegmatis*transformants were grown inMiddlebrook 7H9 broth supplemented with hygromycin (50 μg/mL) and tetracycline (10 ng/mL) to midexponential phase (OD600 = 1.2). Afterwards the cultures were diluted in the proportion of 1:9:10 (culture:water:M290 medium) and 5 mL were poured as the top layer on Petri dishes with agarized M290 medium. Both top- and bottom-layers were supplemented with hygromycin (50 μg/mL) and tetracycline (10 ng/mL). The plates were allowed to dry for at least 30 min, afterwards sterile paper discs with impregnated imidazo[1,2-*b*][1,2,4,5]tetrazines were plated. The plates were incubated for 2–3 days at 37 ◦C, until the bacterial lawn was fully grown. Growth inhibition halos were measured to the nearest 1 mm. The experiments were carried out as triplicates; the average diameter and standard deviation (SD) were calculated.

### *4.5. Mycobacterial RNA Isolation and Real-Time qPCR*

*M. smegmatis* strains were grown overnight in Middlebrook 7H9 broth to midexponential phase (OD600 = 1.0–1.2); cells from 10 mL culture were harvested by centrifugation for 10 min at 3000× *g* and washed by 1 mL of RNAprotect Bacteria Reagent (Qiagen, USA). Total RNA was extracted by homogenization in Trizol solution (Invitrogen, USA) [25], followed by phenol (pH = 4.5)-chloroform/isoamyl alcohol (25:24:1) purification and precipitation in high salt solution (0.8 M Na citrate, 1.2 M NaCl) with isopropanol. Remaining genomic DNA was removed by DNAse I, Amplification grade (Invitrogen, USA). 50 ng of total RNA was used for cDNA synthesis by iScript Select cDNA Synthesis Kit (Bio-Rad, USA). 1 ng of cDNA was used for real-time qPCR with the qPCRmix-HS SYBR kit (Evrogen, Russia) on a CFX96 Touch machine (Bio-Rad, USA). CFX Manager V 3.1 (Bio-Rad, USA) was used to analyze the qPCR results: relative normalized expression of three biological replicates was calculated as ΔΔCq and genes *sigA* and *ftsZ* were used as reference. The following primers were picked by primer-BLAST [22] for qPCR: q1380-f 5-CTGCTCGACGAACCATGCGAAAC-3 and q1380-r 5-AAGGGTCTTGAGCCGAATCTCAACG-3 (*MSMEG\_1380*), q1382-f 5-ACCACGCAGATCATGAACAACGACT-3 and q1382-r 5-GAAATCGT CGAAGTCCGCCAGATGA-3 (*MSMEG\_1382*), qsigAs-sm-f 5-CGAGCTTGTTGATCACCTCGAC CAT-3 and qsigAs-sm-r 5-CTCGACCTCATCCAGGAAGGCAAC-3 (*sigA*), qftsZs-sm-f 5-AG CAGCTCCTCGATGTCGTCCTT-3 and qftsZs-sm-r 5-GCCTGAAGGGCGTCGAGTTCAT-3 (*ftsZ*).

**Author Contributions:** Conceptualization, D.A.M.; formal analysis, D.A.M., K.V.S., A.A.V.; investigation, D.A.M., K.V.S., A.A.V.; resources, D.A.M., K.V.S., V.N.D.; writing—original draft preparation, D.A.M.; writing review & editing, D.A.M., K.V.S., A.A.V., V.N.D.; visualization, D.A.M.; supervision, V.N.D.; project administration, D.A.M.; funding acquisition, D.A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Russian Science Foundation (RSF), gran<sup>t</sup> number 17-75-20060.

**Acknowledgments:** We would like to thank Acad. V.N. Charushin and G.L. Rusinov of the Laboratory of Heterocyclic Compounds, Postovsky Institute of Organic Synthesis, Ural Branch of RAS, for generously providing compounds for this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
