A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.

Reduced expression of Scaffold/Matrix Attachment Region Binding Protein 1 (SMAR1) is associated with various cancers resulting in poor prognosis of the diseases. However, the precise underlying mechanism elucidating the loss of SMAR1 requires ongoing study. Here, we show that SMAR1 is highly downregulated during aberrant Wnt3a signaling due to proteasomal degradation and predicted poor prognosis of colorectal cancer. However, substitution mutation (Arginine and Lysine to Alanine) in the D-box elements of SMAR1 viz. “RCHL” and “RQRL” completely abrogated its proteasomal degradation despite Wnt3a activity. SMAR1 inhibited Wnt/β-catenin signaling by recruiting Histone deacetylase-5 to β-catenin promoter resulting in reduced cell migration and invasion. Consequently, reduced tumor sizes in in-vivo NOD-SCID mice were observed that strongly associated with suppression of β-catenin. However, loss of SMAR1 led to enriched H3K9 Acetylation in the β-catenin promoter that further increased Wnt/β-catenin signaling activities and enhanced colorectal cancer progression drastically. Using docking and isothermal titration calorimetric studies we show that small microbial peptides viz. AT-01C and AT-01D derived from Mycobacterium tuberculosis mask the D-box elements of SMAR1. These peptides stabilized SMAR1 expression that further inhibited metastatic SW480 colorectal cancer cell migration and invasion. Drastically reduced subcutaneous tumors were observed in in-vivo NOD-SCID mice upon administration of these peptides (25 mg/kg body weight) intraperitoneally. Taken together our structural studies, in-vitro and in-vivo results strongly suggest that the D-box elements of SMAR1 represent novel druggable targets, where the microbial peptides hold promise as novel colorectal cancer therapeutics.

c-MYC proto-oncogene harbours a transcription-inhibitory quadruplex-forming scaffold (Pu27) upstream P1 promoter providing anti-neoplastic therapeutic target. Previous reports showed the binding profile of human Cathelicidin peptide (LL37) and telomeric G-quadruplex. Here, we truncated the quadruplex-binding domain of LL37 to prepare a small library of peptides through site-specific amino acid substitution. We investigated the intracellular selectivity of peptides for Pu27 over other oncogenic quadruplexes and their role in c-MYC promoter repression by dual-luciferase assays. We analysed their thermodynamics of binding reactions with c-MYC quadruplex isomers (Pu27, Myc22, Pu19) by Isothermal Titration Calorimetry. We discussed how amino acid substitutions and peptide helicity enhanced/weakened their affinities for c-MYC quadruplexes and characterized specific non-covalent inter-residual interactions determining their selectivity. Solution NMR structure indicated that KR12C, the best peptide candidate, selectively stabilized the 5′-propeller loop of c-MYC quadruplex by arginine-driven electrostatic-interactions at the sugar-phosphate backbone while KR12A peptide destabilized the quadruplex inducing a single-stranded hairpin-like conformation. Chromatin immunoprecipitations envisaged that KR12C and KR12A depleted and enriched Sp1 and NM23-H2 (Nucleoside diphosphate kinase) occupancy at Pu27 respectively supporting their regulation in stabilizing and unfolding c-MYC quadruplex in MCF-7 cells. We deciphered that selective arresting of c-MYC transcription by KR12C triggered apoptotic-signalling pathway via VEGF-A-BCL-2 axis.

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The nuclease hypersensitive element III1 (NHE III1) upstream c-MYC promoter harbors a transcription-silencing G-quadruplex (Pu27) element. Dynamic turnover of various transcription factors (TFs) across Pu27 to control c-MYC transcription homeostasis is enigmatic. Here, we reveal that native Pu27 evolves truncated G-quadruplex isomers (Pu19, Pu22, Pu24, and Pu25) in cells that are optimal intracellular targets of specific TFs in a sequence- and structure-dependent manner. Nuclear magnetic resonance and isothermal titration calorimetry envisaged that NM23-H2 (nucleoside diphosphate kinase) and nucleolin induce conformational fluctuations in Pu27 to sample specific conformationally restricted conformer(s). Structural investigations revealed that the flanking guanines at 5′-Pu27 control solvent exposure at G-quartets upon NM23-H2 and nucleolin binding driving Pu27 unfolding and folding, respectively. Transient chromatin immunoprecipitations confirmed that NM23-H2 drives the conformation switch to Pu24 that outcompetes nucleolin recruitment. Similarly, nucleolin arrests Pu27 in the Pu22 conformer minimizing NM23-H2 binding at Pu27. hnRNPK (heterogeneous nuclear ribonucleoprotein K) positively regulates NM23-H2 and nucleolin association at Pu27 despite their antagonism. On the basis of these results, we simulated the transcription kinetics in a feed-forward loop in which the transcription output responds to hnRNPK-induced early activation via NM23-H2 association, which favors Pu24 formation at NHE III1 reducing nucleolin occupancy and driving quadruplex unfolding to initiate transcription. NM23-H2 further promotes hnRNPK deposition across NHE III1 altering Pu27 plasticity that finally enriches the nucleolin abundance to drive Pu22 formation and weaken NM23-H2 binding to extinguish transcription. This mechanism involves three positive feedback loops (NM23-H2-hnRNPK, NM23-H2-CNBP, and hnRNPK-nucleolin) and one negative feedback loop (NM23-H2-nucleolin) controlling optimal turnover and residence time of TFs at Pu27 to homeostatically regulate c-MYC transcription.