Characterization of G-Quadruplex Motifs in espB, espK, and cyp51 Genes of Mycobacterium tuberculosis as Potential Drug Targets

Mishra, Subodh Kumar; Shankar, Uma; Jain, Neha; Sikri, Kriti; Tyagi, Jaya Sivaswami; Sharma, Tarun Kumar; Mergny, Jean-Louis; Kumar, Amit

Because these G4s are present in the coding regions of the espK, espB, and cyp51 genes, we probed the presence of GQs and the consequence of stabilizing these motifs using TMPyP4, a wellknown G4 stabilizing ligand. First, the observed increase in T m by >8 C in the presence of TMPyP4 established the presence of PGQ sequences in these genes. Second, the presence of PGQ sequences was confirmed by the observed stalling of DNA replication in the presence of TMPyP4 in a PCR stop assay. The binding interaction between TMPyP4 and MTB-PGQs was determined by ITC analysis to be thermodynamically stable, energetically favorable, and selective. Finally, we evaluated the inhibitory effect of TMPyP4 on M. tuberculosis growth; IC 50 value was calculated to be 6.25 mM. Highly stable G4s in the open reading frame of genes were previously shown to downregulate the expression of genes. 13, 41, 53 The treatment of M. tuberculosis cultures with TMPyP4 led to a significant decrease in the expression of espK and espB relative to 16S rRNA, a housekeeping gene, suggesting that a G4-mediated inhibition mechanism is involved in this process. As a schematic model elaborated in Figure S1B , G4-mediated inhibition of espK and espB genes is expected to increase the innate immune response of host cell. The inhibited expression of the espB and espK proteins would restore the phagosome maturation process and reduce the secretion of CFP-10 and ESAT-6, in turn leading to a rescued antigen presentation to the host immune cells.