Evaluation of a Radioiodinated G-quadruplex Binder in Cervical Cancer Models.

We herein describe the radiosynthesis of a 125I-labeled acridine orange derivative ([125I]-C8), acting as a G-quadruplex binder, and its biological evaluation in cervical cancer models, aiming to enlighten its potential as a radioligand for Auger Electron Radiopharmaceutical Therapy (AE-RPT) of cancer. [125I]-C8 was synthesized with a moderate radiochemical yield (ca. 60 %) by a [125I]iodo-destannylation reaction. Its evaluation in cervical cancer HeLa cells demonstrated that the radiocompound has a significant cellular internalization with a notorious accumulation in the cell nucleus. In line with these results, [125I]-C8 strongly compromised the viability of HeLa cells in a dose-dependent manner, inducing non-repairable DNA lesions that are most probably due to the AEs emitted by 125I in close proximity to  the DNA. Biodistribution studies in a murine HeLa xenograft model showed that [125I]-C8 has fast blood clearance and high in vivo stability but poor tumor uptake, after systemic administration. The respective supramolecular conjugate with the AS1411 aptamer ([125I]-C8/AS1411) led to a slower blood clearance in the same animal tumor model, although without improving the tumor uptake. To take advantage of the radiotoxicity of [125I]-C8 against cervical cancer cells other strategies need to be studied, based namely on alternative nanodelivery carriers and/or intratumoral injection approaches.

Effect of molecular crowders on ligand binding kinetics with G-quadruplex DNA probed by fluorescence correlation spectroscopy.

Guanine-rich single-stranded DNA folds into G-quadruplex DNA (GqDNA) structures, which play crucial roles in various biological processes. These structures are also promising targets for ligands, potentially inducing antitumor effects. While thermodynamic parameters of ligand/DNA interactions are well-studied, the kinetics of ligand interaction with GqDNA, particularly in cell-like crowded environments, remain less explored. In this study, we investigate the impact of molecular crowding agents (glucose, sucrose, and ficoll 70) at physiologically relevant concentrations (20% w/v) on the association and dissociation rates of the benzophenoxazine-core based ligand, cresyl violet (CV), with human telomeric antiparallel-GqDNA. We utilized fluorescence correlation spectroscopy (FCS) along with other techniques. Our findings reveal that crowding agents decrease the binding affinity of CV to GqDNA, with the most significant effect-a nearly three-fold decrease-observed with ficoll 70. FCS measurements indicate that this decrease is primarily due to a viscosity-induced slowdown of ligand association in the crowded environment. Interestingly, dissociation rates remain largely unaffected by smaller crowders, with only small effect observed in presence of ficoll 70 due to direct but weak interaction between the ligand and ficoll. These results along with previously reported data provide valuable insights into ligand/GqDNA interactions in cellular contexts, suggesting a conserved mechanism of saccharide crowder influence, regardless of variations in GqDNA structure and ligand binding mode. This underscores the importance of considering crowding effects in the design and development of GqDNA-targeted drugs for potential cancer treatment.

Suppression of CTC1 inhibits hepatocellular carcinoma cell growth and enhances RHPS4 cytotoxicity.

BACKGROUND: Although DNA repair mechanisms function to maintain genomic integrity, in cancer cells these mechanisms may negatively affect treatment efficiency. The strategy of targeting cancer cells via inhibiting DNA damage repair has been successfully used in breast and ovarian cancer using PARP inhibitors. Unfortunately, such strategies have not yet yielded results in liver cancer. Hepatocellular carcinoma (HCC), the most common type of liver cancer, is a treatment-resistant malignancy. Despite the development of guided therapies, treatment regimens for advanced HCC patients still fall short of the current need and significant problems such as cancer relapse with resistance still exist. In this paper, we targeted telomeric replication protein CTC1, which is responsible for telomere maintenance. METHODS: CTC expression was analyzed using tumor and matched-tissue RNA-sequencing data from TCGA and GTEx. In HCC cell lines, q-RT-PCR and Western blotting were used to detect CTC1 expression. The knock-down of CTC1 was achieved using lentiviral plasmids. The effects of CTC1 silencing on HCC cells were analyzed by flow cytometry, MTT, spheroid and colony formation assays. RESULTS: CTC1 is significantly downregulated in HCC tumor samples. However, CTC1 protein levels were higher in sorafenib-resistant cell lines compared to the parental groups. CTC1 inhibition reduced cell proliferation in sorafenib-resistant HCC cell lines and diminished their spheroid and colony forming capacities. Moreover, these cells were more sensitive to single and combined drug treatment with G4 stabilizer RHPS4 and sorafenib. CONCLUSION: Our results suggest that targeting CTC1 might be a viable option for combinational therapies designed for sorafenib resistant HCC patients.

Investigating the preferential interaction between imatinib mesylate and VEGF G-quadruplex DNA as therapeutic strategies for cancer treatment: Biophysical and molecular modelling approaches.

G-Quadruplex DNA (GQ-DNA) is one of the most important non-canonical nucleic acid structures. GQ-DNA forming sequences are present in different crucial genomic regions and are abundant in promoter regions of several oncogenes. Therefore, GQ-DNA is an important target for anticancer drugs and hence binding interactions between GQ-DNA and small molecule ligands are of great importance. Since GQ-DNA is a highly polymorphic structure, it is important to identify ligand molecules which preferentially target a particular quadruplex sequence. In this present study, we have used a FDA approved drug called imatinib mesylate (ligand) which is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia, gastrointestinal stromal tumours. Different spectroscopic techniques as well as molecular docking investigations and molecular simulations have been used to explore the interaction between imatinib mesylate with VEGF GQ DNA structures along with duplex DNA, C-Myc, H-Telo GQ DNA. We found that imatinib mesylate shows preferential interaction towards VEGF GQ DNA compared to C-Myc, H-Telo GQ and duplex DNA. Imatinib mesylate seems to be an efficient ligand for VEGF GQ DNA, suggesting that it might be used to regulate the expression of genes in cancerous cells.

Catalytic and Selective Red Light-Triggered Photodegradation of a G-Quadruplex DNA by a Zinc (II) Phthalocyanine.

Water-soluble phthalocyanine (Pc) derivatives have been regarded as potential G-quadruplex (G4) nucleic acid-targeting ligands for anticancer therapy and have been extensively studied as effective photosensitizers for photodynamic therapy (PDT). Understanding how photosensitizers interact with nucleic acids and the subsequent photolytic reactions is essential for deciphering the initial steps of PDT, thereby aiding in the development of new photosensitizing agents. In this study, we found that red-light irradiation of a mixture of a Zn(II) Pc derivative and an all-parallel G4 DNA leads to catalytic and selective photodegradation of the DNA by reactive oxygen species (ROS) generated from the Zn(II) Pc derivative bound to DNA through a reaction mechanism similar to that of an enzyme reaction. This finding provides a novel insight into the molecular design of a photosensitizer to enhance its PDT efficacy.

Potential protein kinase inhibitors that target G-quadruplex DNA structures in the human telomeric regions.

Telomeric regions contain Guanine-rich sequences arranged in a planar manner and connected by Hoogsteen hydrogen bonds that can fold into G-quadruplex (G4) DNA structures, and can be stabilized by monovalent metal cations. The presence of G4 DNA holds significance in cancer-related processes, especially due to their regulatory potential at transcriptional and translational levels of oncogene and tumor suppressor genes. The objective of this current research is to explore the evolving realm of FDA-approved protein kinase inhibitors, with a specific emphasis on their capacity to stabilize the G4 DNA structures formed at the human telomeric regions. This involves investigating the possibility of repurposing FDA-approved protein kinase inhibitors as a novel approach for targeting multiple cancer types. In this context, we have selected 16 telomeric G4 DNA structures as targets and 71 FDA-approved small-molecule protein kinase inhibitors as ligands. To investigate their binding affinities, molecular docking of human telomeric G4 DNA with nuclear protein kinase inhibitors and their corresponding co-crystalized ligands were performed. We found that Ponatinib and Lapatinib interact with all the selected G4 targets, the binding free energy calculations, and molecular dynamic simulations confirm their binding efficacy and stability. Thus, it is hypothesized that Ponatinib and Lapatinib may stabilize human telomeric G4 DNA in addition to their ability to inhibit BCR-ABL and the other members of the EGFR family. As a result, we also hypothesize that the stabilization of G4 DNA might represent an additional underlying mechanism contributing to their efficacy in exerting anti-cancer effects.

Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

Inhibition of cancer cells by Quinoline-Based compounds: A review with mechanistic insights.

This article includes a thorough examination of the inhibitory potential of quinoline-based drugs on cancer cells, as well as an explanation of their modes of action. Quinoline derivatives, due to their various chemical structures and biological activity, have emerged as interesting candidates in the search for new anticancer drugs. The review paper delves into the numerous effects of quinoline-based chemicals in cancer progression, including apoptosis induction, cell cycle modification, and interference with tumor-growth signaling pathways. Mechanistic insights on quinoline derivative interactions with biological targets enlightens their therapeutic potential. However, obstacles such as poor bioavailability, possible off-target effects, and resistance mechanisms make it difficult to get these molecules from benchside to bedside. Addressing these difficulties might be critical for realizing the full therapeutic potential of quinoline-based drugs in cancer treatment.

FANCJ DNA helicase is recruited to the replisome by AND-1 to ensure genome stability.

FANCJ, a DNA helicase linked to Fanconi anemia and frequently mutated in cancers, counteracts replication stress by dismantling unconventional DNA secondary structures (such as G-quadruplexes) that occur at the DNA replication fork in certain sequence contexts. However, how FANCJ is recruited to the replisome is unknown. Here, we report that FANCJ directly binds to AND-1 (the vertebrate ortholog of budding yeast Ctf4), a homo-trimeric protein adaptor that connects the CDC45/MCM2-7/GINS replicative DNA helicase with DNA polymerase α and several other factors at DNA replication forks. The interaction between FANCJ and AND-1 requires the integrity of an evolutionarily conserved Ctf4-interacting protein (CIP) box located between the FANCJ helicase motifs IV and V. Disruption of the CIP box significantly reduces FANCJ association with the replisome, causing enhanced DNA damage, decreased replication fork recovery and fork asymmetry in cells unchallenged or treated with Pyridostatin, a G-quadruplex-binder, or Mitomycin C, a DNA inter-strand cross-linking agent. Cancer-relevant FANCJ CIP box variants display reduced AND-1-binding and enhanced DNA damage, a finding that suggests their potential role in cancer predisposition.

Computed interactions of berenil with restricted foldamers of c-MYC DNA G-quadruplexes.

G-quadruplexes (G4s) are secondary four-stranded DNA helical structures made up of guanine-rich nucleic acids that can assemble in the promoter regions of multiple genes under the appropriate conditions. Stabilization of G4 structures by small molecules can regulate transcription in non-telomeric regions, including in proto-oncogenes and promoter regions, contributing to anti-proliferative and anti-tumor activities. Because G4s are detectable in cancer cells but not in normal cells, they make excellent drug discovery targets. Diminazene, DMZ (or berenil), has been shown to be an efficient G-quadruplex binder. Due to the stability of the folding topology, G-quadruplex structures are frequently found in the promotor regions of oncogenes and may play a regulatory role in gene activation. Using molecular docking and molecular dynamics simulations on several different binding poses, we have studied DMZ binding toward multiple G4 topologies of the c-MYC G-quadruplex. DMZ binds preferentially to G4s that have extended loops and flanking bases. This preference arises from its interactions with the loops and the flanking nucleotides, which were not found in the structure lacking extended regions. The binding to the G4s with no extended regions instead occurred mostly through end stacking. All binding sites for DMZ were confirmed by 100 ns molecular dynamics simulations and through binding enthalpies calculated using the MM-PBSA method. The primary driving forces were electrostatic, as the cationic DMZ interacts with the anionic phosphate backbone, and through van der Waals interactions, which primarily contributed in end stacking interactions.Communicated by Ramaswamy H. Sarma.

The Influence of Chirality on the ß-Amino-Acid Naphthalenediimides/G-Quadruplex DNA Interaction.

G-quadruplexes (G4s) have been identified as a potential alternative chemotherapy target. A series of eight ß-amino acid derived naphthalenediimides (NDI) were screened against a series of oncogenic G4 sequences: c-KIT1, h-TELO, and TBA. Three sets of enantiomers were investigated to further our understanding of the effect of point chirality on G4 stabilisation. Enantioselective binding behaviour was observed with both c-KIT1 and h-TELO. Docking studies using GNINA and UV-vis titrations were employed to better understand this selective binding behaviour.

The binding of a c-MYC promoter G-quadruplex to neurotransmitters: An analysis of G-quadruplex stabilization using DNA melting, fluorescence spectroscopy, surface-enhanced Raman scattering and molecular docking.

The interactions of several neurotransmitter and neural hormone molecules with the c-MYC G-quadruplex DNA sequence were analyzed using a combination of spectroscopic and computational techniques. The interactions between indole, catecholamine, and amino acid neurotransmitters and DNA sequences could potentially add to the understanding of the role of G-quadruplex structures play in various diseases. Also, the interaction of the DNA sequence derived from the nuclear hypersensitivity element (NHE) III1 region of c-MYC oncogene (Pu22), 5'-TGAGGGTGGGTAGGGTGGGTAA-3', has added significance in that these molecules may promote or inhibit the formation of G-quadruplex DNA which could lead to the development of promising drugs for anticancer therapy. The results showed that these molecules did not disrupt G-quadruplex formation even in the absence of quadruplex-stabilizing cations. There was also evidence of concentration-dependent binding and high binding affinities based on the Stern-Volmer model, and thermodynamically favorable interactions in the form of hydrogen-bonding and interactions involving the π system of the aromatic neurotransmitters.

Development and application of metallo-phthalocyanines as potent G-quadruplex DNA binders and photosensitizers.

Metallo-phthalocyanines (MPc) are common photosensitizers with ideal photophysical and photochemical properties. Also, these molecules have shown to interact with non-canonical nucleic acid structures, such as G-quadruplexes, and modulate oncogenic expression in cancer cells. Herein, we report the synthesis and characterisation of two metallo-phthalocyanines containing either zinc (ZnPc) or nickel (NiPc) in the central aromatic core and four alkyl ammonium lateral chains. The interaction of both molecules with G-quadruplex DNA was assessed by UV-Vis, fluorescence and FRET melting experiments. Both molecules bind strongly to G-quadruplexes and stabilise these structures, being NiPc the most notable G-quadruplex stabiliser. In addition, the photosensitizing ability of both metal complexes was explored by the evaluation of the singlet oxygen generation and their photoactivation in cells. Only ZnPc showed a high singlet oxygen generation either by direct observation or by indirect evaluation using a DPBF dye. The cellular evaluation showed mainly cytoplasmic localization of ZnPc and a decrease of the IC50 values of the cell viability of ZnPc upon light activation of two orders of magnitude. Two metallo-phthalocyanines containing zinc and nickel within the aromatic core have been investigated as G-quadruplex stabilizers and photosensitizers. NiPc shows a high G4 binding but negligible photosensitizing ability while ZnPc exhibits a moderate binding to G-quadruplex together with a high potency to generate singlet oxygen and photocytotoxicity. The interaction with G4s and capacity to be photosensitized is associated with the geometry adopted by the central metal core of the phthalocyanine scaffold.

Electrochemical detection of Oxaliplatin induced DNA damage in G-quadruplex structures.

Oxaliplatin (OXP) is a platinum-based chemotherapeutic agent that induces DNA damage by forming intra- and interstrand crosslinks, mainly at the N7s of adenine (A) and guanine (G) bases. In addition to double-stranded DNA, G-rich G-quadruplex (G4)-forming sequences can also be targeted by OXP. However, high doses of OXP can lead to drug resistance and cause serious adverse effects during treatment. To better understand the targeting of G4 structures by OXP, their interactions as well as the molecular mechanisms underlying OXP resistance and adverse effects, there is a need for a rapid, quantitative, and cost-effective method to detect OXP and the damage it causes. In this study, we successfully fabricated a graphite electrode biosensor modified with gold nanoparticles (AuNPs) to investigate the interactions between OXP and the G4-forming promoter region (Pu22) of Vascular endothelial growth factor (VEGF). The overexpression of VEGF is known to be associated with tumor progression and the stabilization of VEGF G4 by small molecules is shown to suppresses VEGF transcription in different cancer cell lines. Differential pulse voltammetry (DPV) was used to investigate the interactions between OXP and Pu22-G4 DNA by monitoring the decrease in the oxidation signal of guanine with increasing OXP concentration. Under the optimized conditions (37 °C, 1:2 v/v AuNPs/water as electrode surface modifier, and 180 min incubation time) the developed probe showed a linear dynamic range of 1.0-10.0 µM with a detection limit of 0.88 µM and limit of quantification of 2.92 µM. Fluorescence spectroscopy was also used to support the electrochemical studies. We observed a decrease in the fluorescence emission of Thioflavin T in the presence of Pu22 upon addition of OXP. To our knowledge, this is the first electrochemical sensor developed to study OXP-induced damage to G4 DNA structures. Our findings provide new insights into the interactions between VEGF G4 and OXP, which could aid in targeting VEGF G4 structures and the development of new strategies to overcome OXP resistance.

Discovering potential stabilizers for KRAS22RT G-quadruplex DNA: an alternative next generation approach to treat pancreatic cancer.

KRAS is the signature gene responsible for the occurrence of pancreatic cancer, which is a complex, multifactorial and intractable lethal malignancy. Prevention and treatment of the ailment have always been a key motivation behind the search for new therapeutic drug molecules. G-quadruplexes are non-canonical guanine-rich secondary structures, commonly formed at eukaryotic telomeric ends, oncogenic promotors and G-rich regions of the DNA. These G-quadruplexes play a crucial role in the regulation of gene expression and maintenance of genome integrity, therefore, they are considered as emerging potential therapeutic drug targets. The present study is concerned with the discovery of a potential stabilizer for KRAS22RT G-quadruplex DNA, located in the NHE region of the promotor, while inhibiting the upregulation of KRAS proto-oncogene, as an alternative approach for the treatment of pancreatic cancer. Various chemical libraries have been virtually screened against the targeted G4 structure and 143 compounds showed promising results. However, molecular dynamic studies, ADME and toxicity analyses predicted that three compounds belonging to the class of tetra-substituted phenanthrolines (i.e., 7i, 7j and 7k) can not only effectively stabilize KRAS22RT G4 structure but also have least toxic effects in the in vivo system. Therefore, it is highly recommended to further investigate their effectiveness and efficacy through experimental analysis in laboratory.Communicated by Ramaswamy H. Sarma.

Ruthenium(II) Complexes Coupled by Erianin via a Flexible Carbon Chain as a Potential Stabilizer of c-myc G-Quadruplex DNA.

Herein, two novel ruthenium(II) complexes coupled by erianin via a flexible carbon chain, [Ru(phen)2(L1-(CH2)4-erianin)](ClO4)2 (L1 = 2-(2-(tri-fluoromethyphenyl))-imidazo [4,5f][1-10]phenanthroline (1) and [Ru(phen)2(L2-(CH2)4-eria)](ClO4)2 (L2 = 2-(4-(tri-fluoromethyphenyl))-imidazo [4,5f][1,10]phenanthroline (2), have been synthesized and investigated as a potential G-quadruplex(G4) DNA stabilizer. Both complexes, especially 2, can bind to c-myc G4 DNA with high affinity by electronic spectra, and the binding constant calculated for 1 and 2 is about 15.1 and 2.05 × 107 M-1, respectively. This was further confirmed by the increase in fluorescence intensity for both complexes. Moreover, the positive band at 265 nm in the CD spectra of c-myc G4 DNA decreased treated with 2, indicating that 2 may bind to c-myc G4 DNA through extern groove binding mode. Furthermore, fluorescence resonance energy transfer (FRET) assay indicated that the melting point of c-myc G4 DNA treated with 1 and 2 increased 15.5 and 16.5 °C, respectively. Finally, molecular docking showed that 1 can bind to c-myc G4 DNA in the extern groove formed by base pairs G7-G9 and G22-A24, and 2 inserts into the small groove of c-myc G4 DNA formed by base pairs T19-A24. In summary, these ruthenium(II) complexes, especially 2, can be developed as potential c-myc G4 DNA stabilizers and will be exploited as potential anticancer agents in the future.

Roles of G4-DNA and G4-RNA in Class Switch Recombination and Additional Regulations in B-Lymphocytes.

Mature B cells notably diversify immunoglobulin (Ig) production through class switch recombination (CSR), allowing the junction of distant "switch" (S) regions. CSR is initiated by activation-induced deaminase (AID), which targets cytosines adequately exposed within single-stranded DNA of transcribed targeted S regions, with a specific affinity for WRCY motifs. In mammals, G-rich sequences are additionally present in S regions, forming canonical G-quadruplexes (G4s) DNA structures, which favor CSR. Small molecules interacting with G4-DNA (G4 ligands), proved able to regulate CSR in B lymphocytes, either positively (such as for nucleoside diphosphate kinase isoforms) or negatively (such as for RHPS4). G4-DNA is also implicated in the control of transcription, and due to their impact on both CSR and transcriptional regulation, G4-rich sequences likely play a role in the natural history of B cell malignancies. Since G4-DNA stands at multiple locations in the genome, notably within oncogene promoters, it remains to be clarified how it can more specifically promote legitimate CSR in physiology, rather than pathogenic translocation. The specific regulatory role of G4 structures in transcribed DNA and/or in corresponding transcripts and recombination hereby appears as a major issue for understanding immune responses and lymphomagenesis.

Design, synthesis and biological evaluation of novel 9-N-substituted-13-alkylberberine derivatives from Chinese medicine as anti-hepatocellular carcinoma agents.

A series of novel 9-N-substituted-13-alkylberberine derivatives from Chinese medicine were designed and synthesized with improved anti-hepatocellular carcinoma (HCC) activities. The optimal compound 4d showed strong activities against HepG2, Sk-Hep-1, Huh-7 and Hep3B cells with IC50 values of 0.58-1.15 µM, which were superior to positive reference cisplatin. Interestingly, 4d exhibited over 40-fold more potent activity against cisplatin-resistant HepG2/DPP cells while showing lower cytotoxicity in normal LX-2 cells. The mechanism studies revealed 4d greatly stabilized G-quadruplex DNA leading to intracellular c-MYC expression downregulation, blocked G2/M-phase cell cycle by affecting related p-cdc25c, cdc2 and cyclin B1 expressions, and induced apoptosis by a ROS-promoted PI3K/Akt-mitochondrial pathway. Furthermore, 4d possessed good pharmacokinetic properties and significantly inhibited the tumor growth in the H22 liver cancer xenograft mouse model without obvious toxicity. Altogether, the remarkably biological profiles of 4d both in vitro and in vivo would make it a promising candidate for HCC therapy.

Discovery of a potent Guanidine derivative that selectively binds and stabilizes the human BCL-2 G-quadruplex DNA and downregulates the transcription.

G-quadruplex also known as G4 (GQ) structures, are a non-canonical kind of DNA or RNA secondary structure that may develop inside guanine-rich nucleic acid sequences. They may be found in a variety of locations in the human genome, such as gene promoters, 5' untranslated region, and telomeres, among others. Because of their significance in biology, G4 structures are recognized as promising pharmacological targets, particularly for therapeutics against cancer. This has led to the discovery of small molecules that can stabilize G4 structures. Small molecules that interact with quadruplexes offer a wide range of potential applications, including not just as medications but also as sensors for quadruplexes structures. The BCL-2 is a proto-oncogene that often gets mutated in lethal cancer and could be an interesting target for developing an anti-cancer drug. In the present study, we have employed various biophysical techniques such as fluorescence, CD, Isothermal calorimetry, gel retardation, and PCR stop assay, indicating that Guanidine derivatives GD-1 and GD-2 selectively interact with high affinity with BCL-2 GQ over other G-quadruplex DNA and duplex DNA. The most promising small molecule GD-1 increases the thermostability of the BCL-2 GQ structure by 12°C. Our biological experiments such as ROS generation, qRT-PCR, western blot, TFP based reporter assay, show that the GD-1 ligand causes a synthetic lethal interaction by suppressing the expression of BCL-2 genes via interaction and stabilization of its promoter GQ strucure in HeLa cells and act as a potential anti-cancer agent.

G-quadruplex-mediated specific recognition, stabilization and transcriptional repression of bcl-2 by small molecule.

The presence of the G-quadruplex (G4) structure in the promoter region of the human bcl-2 oncogenes makes it a promising target for developing anti-cancer therapeutics. Bcl-2 inhibits apoptosis, and its frequent overexpression in cancer cells contributes to tumor initiation, progression, and resistance to therapy. Small molecules that can specifically bind to bcl-2 G4 with high affinity and selectivity are remaining elusive. Here, we report that small molecule 1,3-bis-) furane-2yl-methylidene-amino) guanidine (BiGh) binds to bcl-2 G4 DNA structure with very high affinity and selectivity over other genomic G4 DNA structures and duplex DNA. BiGh stabilizes folded parallel conformation of bcl-2 G4 via non-covalent and electrostatic interactions and increases the thermal stabilization up to 15 °C. The ligand significantly suppresses the bcl-2 transcription in HeLa cells by a G4-dependent mechanism and induces cell cycle arrest which promotes apoptosis. The in silico ADME profiling confirms the potential 'drug-likeness' of BiGh. Our results showed that BiGh stabilizes the bcl-2 G-quadruplex motif, downregulates the bcl-2 gene transcription as well as translation process in cervical cancer cells, and exhibits potential anti-cancer activity. This work provides a potential platform for the development of lead compound(s) as G4 stabilizers with drug-like properties of BiGh for cancer therapeutics.