**1. Introduction**

Tuberculosis (TB), caused by *Mycobacterium tuberculosis*, is currently the leading killer among the infectious diseases caused by one infectious agent, responsible for an estimate of 1.2 million deaths in 2018 [1]. According to WHO, 1.7 billion people globally are infected with *M. tuberculosis*, and thus are at risk of developing the disease [1]. The emergence and spread of multidrug resistant TB (MDR-TB, defined as TB resistant to rifampicin and isoniazid), extensively drug-resistant TB (defined as MDR-TB with resistance to the fluoroquinolones and second-line injectables) and totally drug-resistant TB (TDR-TB) is a global threat to world-wide TB control [2–5]. Long treatment times (six months for drug-susceptible TB and up to two years for DR-TB) often lead to bad patient compliance, which is one of the causes of drug resistance development and results in worse treatment outcomes. Thus, researchers are forced to search for novel anti-TB drugs and shorter regimens [6].

Eukaryotic-type serine-threonine protein-kinases (ESTPKs) play a key role in *M. tuberculosis* life cycle regulation, controlling some of its vital aspects such as cell division and survival within host macrophages, and, therefore, they represent attractive targets for drug development [7,8]. We have previously described a number of imidazo[1,2-*b*][1,2,4,5]tetrazines with a promising antibacterial activity on *M. tuberculosis* and *M. smegmatis* [9]. Most of these compounds showed activity as potential

ESTPK inhibitors in the original *M. smegmatis aphVIII*+ test-system [10,11], and two of them were able to bind to the *M. tuberculosis* PknB adenine-binding pocket according to docking studies [10]. Despite the predicted activity as ESTPK-inhibitors, both the exact mechanism of action and the mechanism of resistance to these compounds are still unknown.

We were able to obtain spontaneous *M. smegmatis* mutants resistant to four imidazo[1,2-*b*][1,2,4,5] tetrazines (**3a**, **3c**, **3h** and **3n**, Figure 1), which had cross-resistance among them, suggesting a common mechanism of drug-resistance [9]. Whole-genomic sequencing and comparative genomic analysis revealed mutations in *MSMEG\_0641* (binding-protein-dependent transporters inner membrane component) in 1 mutant, in *MSMEG\_1601* (hypothetical protein) in seven mutants, in *MSMEG\_2087* (transporter small conductance mechanosensitive ion channel (MscS) family protein) in one mutant [12], while all the mutants carried different mutations in *MSMEG\_1380* (AcrR/TetR\_N transcriptional regulator) – 1 nonsynonymous SNP, 2 insertions leading to a frameshift, 2 duplications (6 and 501 base pairs-long) and one deletion [13].

**Figure 1.** Chemical structures of imidazo[1,2-*b*][1,2,4,5]tetrazines [9].

In this article we describe the investigation of these mutations' role in mycobacterial drug resistance to imidazo[1,2-*b*][1,2,4,5]tetrazines by different approaches: construction of targeted mutants, overexpression of the wild-type (*w.t.*) and mutant genes, and gene-expression studies.
