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

Various G4 binding ligands have been investigated for their therapeutic potential by affecting the stability of the G4 structure, such as TMPyP4, BRACO-19, etc. 11, 41, 42 TMPyP4 acts as an anti-cancer agent by stabilizing G4 structures present at human telomeres. 43 It also inhibits viral RNA processing inside host cells by stabilizing the G4 structure present in the L gene of the Ebola virus and acts as an anti-Ebola virus (EBOV) agent. 11, 41 Recently, BRACO-19 was shown to inhibit M. tuberculosis growth, but its cellular target remains to be identified. 17 In our study, we assessed the stabilizing activity of TMPyP4 for MTB-PGQs by CD melting curve analysis. Melting curves of MTB-PGQs in the absence and presence of TMPyP4 yielded an increase in melting temperature (T m ) of $8 C, indicative of a stabilizing effect of TMPyP4 on the thermostability of G4 structures formed by MTB-PGQs ( Figure 4A ). Further, we assessed the binding affinity of TMPyP4 for the G4 forming MTB-PGQ oligos by ITC. Changes in enthalpy (DH 1 ) associated with binding to MTB-PGQ1, MTB-PGQ-2, and MTB-PGQ3 were very negative, indicative of an energetically favorable binding of TMPyP4 to MTB-PGQs. 44 The association constants (K A ) for the high-affinity site of MTB-PGQ1, MTB-PGQ2, and MTB-PGQ3 were 1.33 Â 10 6 M À1 , 2.58 Â 10 6 M À1 , and 2.05 Â 10 6 M À1 , respectively. These results confirm a thermodynamically stable interaction between TMPyP4 and MTB-PGQs with affinities in the micromolar range ( Figure 4B) . We also took a non-G4-forming sequence as a negative control to observe selectivity of TMPyP4 for the G4 structure. ITC results indicate 33-, 64-, and 50-fold tighter binding of TMPyP4 to MTB-PGQ1, MTB-PGQ2, and MTB-PGQ3, respectively, than to the non-quadruplexforming sequence. ( Figure 5 ). The observed IC 50 value was 6.25 mM, a value lower than the IC 50 values of the TMPyP4 for both malignant and normal eukaryotic cells. 45 TMPyP4 Stalls the Replication Machinery at G4 Sites Next, we sought to investigate whether TMPyP4 binding to the PGQ motif inhibits DNA replication or not. To accomplish this, we employed two variants of stop assay: (1) PCR stop assay and (2) Taq DNA polymerase stop assay. 46 In PCR stop assay, a G-region overlapping primer was utilized for PCR amplification. In the absence of TMPyP4, a double-stranded product formed because of the primer annealing and extension. 46 The presence of TMPyP4 leads to the stabilization of G4 in the template that results in the unavailability of the template for primer annealing, and thus PCR got inhibited (fully inhibited at higher concentration and partially inhibited at a lower concentration). As shown in Figure S7 , increasing concentrations of TMPyP4 have a strong impact on PCR amplification. On the contrary, when mut-MTB-PGQs were used as a DNA template, TMPyP4 did not inhibit DNA synthesis leading to full product generation.