Polyguanine sequences fold into G-quadruplex structures in the presence of monovalent cations. It is accepted that the telomeric DNA region consists of G-quadruplex structure. There are reports that potential G-quadruplex forming motifs are also present in the promoter region of some proto-oncogenes such as c-myc, c-kit, KRAS, etc. Small molecules with the potential to stabilize the telomeric DNA quadruplex have emerged as potential anticancer agents. We have studied the interaction of ellipticine, a putative anticancer agent from a plant source, with a human telomeric DNA sequence (H24). Spectroscopic and calorimetric studies indicate that the association of ellipticine with H24 is an entropically driven phenomenon with a 2:3 (H24:ellipticine) stoichiometry. Though ellipticine binding does not induce any major structural perturbation in H24, the association leads to formation of a complex with enhanced thermal stability. An assay with the telomerase repeat amplification protocol shows that ellipticine inhibits telomerase activity in MDAMB-231 breast cancer cell line extracts. This is the first report of the quadruplex binding ability of ellipticine. Using the results, we propose that along with DNA intercalation and/or topoisomerase II inhibition, interaction with the telomeric DNA region and the resultant inhibition of telomerase activity might be an additional mode of action for its anticancer property.

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A- and B-type lamins are intermediate filament proteins constituting the nuclear lamina underneath the nuclear envelope thereby conferring proper shape and mechanical rigidity to the nucleus. Lamin proteins are also shown to be related diversely to basic nuclear processes. More than 400 mutations in human lamin A protein alone have been reported to produce at least 11 different disease conditions jointly termed as laminopathies. These mutations in lamin A are scattered throughout its helical rod domain, as well as the C-terminal domain containing the conserved Ig-fold region. The commonality of phenotypes in all these diseases is characterized by misshapen nuclei of the affected tissues which might stem from altered rigidity of the supporting lamina hence lamins. Here we have focused on autosomal dominant Emery-Dreifuss Muscular Dystrophy, one such laminopathy where R453W is the causative mutation located in the Ig domain of lamin A. We have investigated by single-molecule force spectroscopy how a stretching mechanical perturbation senses the destabilizing effect of the mutation in the lamin A Ig domain and compared the mechanoelastic properties of the mutant R453W with that of the wild-type in conjunction with steered molecular dynamics. Furthermore, we have shown the interaction of Ig domain with emerin, another key player and interacting partner in the pathogenesis of EDMD, is disrupted in the R453W mutant. This altered mechanoresistance of Ig domain itself and consequent uncoupling of lamin A-emerin interaction might underlie the altered mechanotransduction properties of EDMD affected nuclei.

Small molecules that interact with G-quadruplex structures formed by the human telomeric region and stabilize them have the potential to evolve as anticancer therapeutic agents. Herein we report the interaction of a putative anticancer agent from a plant source, chelerythrine, with the human telomeric DNA sequence. It has telomerase inhibitory potential as demonstrated from telomerase repeat amplification assay in cancer cell line extract. We have attributed this to the quadruplex binding potential of the molecule and characterized the molecular details of the interaction by means of optical spectroscopy such as absorbance and circular dichroism and calorimetric techniques such as isothermal titration calorimetry and differential scanning calorimetry. The results show that chelerythrine binds with micromolar dissociation constant and 2:1 binding stoichiometry to the human telomeric DNA sequence. Chelerythrine association stabilizes the G-quadruplex. Nuclear magnetic resonance spectroscopy ((1)H and (31)P) shows that chelerythrine binds to both G-quartet and phosphate backbone of the quadruplex leading to quadruplex aggregation. Molecular dynamics simulation studies support the above inferences and provide further insight into the mechanism of ligand binding. The specificity toward quartet binding for chelerythrine is higher compared to that of groove binding. MM-PBSA calculation mines out the energy penalty for quartet binding to be -4.7 kcal/mol, whereas that of the groove binding is -1.7 kcal/mol. We propose that the first chelerythrine molecule binds to the quartet followed by a second molecule which binds to the groove. This second molecule might bring about aggregation of the quadruplex structure which is evident from the results of nuclear magnetic resonance.

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