Targeting <i>Mycobacterium tuberculosis</i> Parallel G-Quadruplex Motifs with Aminoglycosides Neomycin and Streptomycin: Spectroscopic and Calorimetric Aspects

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) contains potential G-quadruplex (PGQ) motifs in the genes <i>espK</i> and <i>cyp51</i>, which are crucial for the bacteria's virulence within host cells. Aminoglycoside molecules are commonly used as antibiotics for ribosomal targets. This study provides insight into the interactions between these aminoglycosides and <i>Mtb-PGQ</i> sequences (<i>espK</i> and <i>cyp51</i>), shedding light on the structural and thermodynamic dynamics of their binding. This study demonstrates the stability, affinity, and conformation of <i>Mtb-PGQ</i> in the presence of neomycin and streptomycin. Ultraviolet-visible spectroscopy (UV-vis), circular dichroism spectroscopy (CD), CD thermal melting, isothermal titration calorimetry (ITC), and fluorescence intercalator displacement (FID) assays were used to comprehensively examine these interactions. Our results reveal that neomycin with <i>Mtb-PGQ</i>exhibits hypochromism accompanied by a 4-5 nm red shift in the UV-visible absorption titration, whereas streptomycin exhibits a hypochromic shift without changes in the maximum wavelength. Notably, neomycin shows a nonlinear binding isotherm, suggesting the involvement of more than one binding process in the formation of neomycin.<i>Mtb-PGQ</i> complexes. Scatchard plot analysis indicates higher binding affinity values for neomycin compared with weaker affinity of streptomycin. CD studies reveal that neomycin decreases the ellipticity of <i>Mtb-PGQ</i> with a red shift while retaining the parallel topology, ultimately enhancing the thermal stability of both <i>espK</i> and <i>cyp51</i>. In contrast, streptomycin destabilizes the cells. ITC analysis reveals that neomycin exhibits the strongest binding affinity for <i>cyp51</i>, with the relative order being NEO-<i>cyp51</i> > NEO-<i>espk</i> > STR-<i>cyp51</i> > STR-<i>espk.</i> Moreover, thermodynamic analysis reveals that neomycin possesses a unique dual mode of binding through grooves as well as stacking. FID studies further confirm a lower DC₅₀ value for neomycin than for streptomycin, suggesting that neomycin is a strong displacer of thiazole orange. Thus, the results show that neomycin with amino groups selectively recognizes the grooves of <i>cyp51</i> over <i>espK</i>.