Restriction of telomerase capping by short non-toxic peptides via arresting telomeric G-quadruplex

Abstract

The stabilization of a G-quadruplex structure in human telomeric DNA has become a promising strategy in the development of cancer therapeutics. Here, we report FK13 (a small fragment of human cathelicidin peptide LL37, residues 17-29) and its mutant peptides (KR12A, KR12B and KR12C) inhibiting telomerase activity by stabilizing the telomeric G-quadruplex structures. An array of biophysical studies like fluorescence anisotropy, circular dichroism spectroscopy, circular dichroism melting, isothermal titration calorimetry, and high resolution nuclear magnetic resonance spectroscopy, in conjunction with molecular dynamics simulations, are employed to examine the interaction of peptides with the Gquadruplex structure. Furthermore, the peptide-quadruplex interaction is monitored in ex vivo systems, the telomerase over-expressed MCF7 breast adeno-carcinoma cell line. MTT assay and flow cytometry studies indicate selective antiproliferative activities of the peptides towards cancer cells over normal kidney epithelial cell line. Confocal microscopy evidenced nuclear transport and localisation of the peptides. A telomerase repeat amplification protocol assay further evidences telomere uncapping and abrogation of telomerase catalysing activity upon administration of peptides. Hence, arresting Gquadruplex structures using short peptides brings in a new mechanistic insight for the development of future peptide based therapeutics against cancer.

Understanding the Interactions of Guanine Quadruplexes with Peptides as Novel Strategies for Diagnosis or Tuning Biological Functions

“…A high specificity towards G4 against double-strand DNA has also been observed. [69] NMR spectroscopy has highlighted the interaction of the R7, R3, and R13 with the phosphate backbone of the G4, while MD simulations, coherently with the previous observation on the entire LL37 peptide, have proven that FK13 loses its helicity while interacting with the G4. On the other hand, FK13 treatment on MCF7 breast cancer cells leads to a significative inhibition of their growth, in a dose-dependent manner.…”

“…Indeed, FK13 (FKRIVQRIKDFLR) has been shown to bind to h‐Telo G4, and thus provide additional structural stability. A high specificity towards G4 against double‐strand DNA has also been observed [69] . NMR spectroscopy has highlighted the interaction of the R7, R3, and R13 with the phosphate backbone of the G4, while MD simulations, coherently with the previous observation on the entire LL37 peptide, have proven that FK13 loses its helicity while interacting with the G4.…”

“…In conclusion, it was proposed that this short peptide can provoke cancer cell death through the inhibition of the telomerase activity as well as the binding and stabilization of the telomeric G4. [69] Peptides derived from RNA helicase associated with AU-rich element (RHAU) protein for specific G4 recognition RNA helicase associated with AU-rich element (also known as RHAU) is a protein of the DEAH box family, which has been shown to resolve G4 structures with high specificity. [70] In a recent review, Nguyen and Dang highlight the potential applications of different RHAU peptides which specifically recognize G4s.…”

Site-specific amino acid substitution in dodecameric peptides determines the stability and unfolding ofc-MYCquadruplex promoting apoptosis in cancer cells

“…So far, we observed that KR12C is the best peptide candidate that selectively repressed c-MYC promoter activation in cancer cells. Earlier reports showed its ability to trigger apoptosis in cancer MCF-7 cells ( 89 ). We further validated these results through monitoring the expression profiles of different oncogenes and apoptosis markers in order to deduce the mechanism how G-quadruplex stabilization at c-MYC promoter triggered selective apoptosis in cancer cells.…”

“…Smits et al. showed that the decreased expression of miR-125b by VEGF results in an upregulation of MAZ (Myc-associated zinc finger protein) expression, which specifically binds to a GA box sequence (GGGAGGG) in c-MYC- P 2 promoter to regulate c-MYC transcription ( 88 , 89 ). Since FK13 and KR12B strongly inhibited VEGF-A promoter activity and its transcription, the negative effects of these peptides on c-MYC promoter might be due to their non-specific targeting at VEGF-A quadruplexes.…”