ABSTRACT
BACKGROUND: G-quadruplex DNA (G4) is a non-canonical structure forming in guanine-rich regions, which play a vital role in cancer biology and are now being acknowledged in both nuclear and mitochondrial (mt) genome. However, the impact of G4-based targeted therapy on both nuclear and mt genome, affecting mt function and its underlying mechanisms remain largely unexplored. METHODS: The mechanisms of action and therapeutic effects of a G4-binding platinum(II) complex, Pt-ttpy, on mitochondria were conducted through a comprehensive approaches with in vitro and in vivo models, including ICP-MS for platinum measurement, PCR-based genetic analysis, western blotting (WB), confocal microscope for mt morphology study, extracellular flux analyzer, JC1 and Annexin V apoptosis assay, flow cytometry and high content microscope screening with single-cell quantification of both ROS and mt specific ROS, as well as click-chemistry for IF study of mt translation. Decipher Pt-ttpy effects on nuclear-encoded mt related genes expression were undertaken via RNA-seq, Chip-seq and CUT-RUN assays. RESULTS: Pt-ttpy, shows a highest accumulation in the mitochondria of A2780 cancer cells as compared with two other platinum(II) complexes with no/weak G4-binding properties, Pt-tpy and cisplatin. Pt-ttpy induces mtDNA deletion, copy reduction and transcription inhibition, hindering mt protein translation. Functional analysis reveals potent mt dysfunction without reactive oxygen species (ROS) induction. Mechanistic study provided first evidence that most of mt ribosome genes are highly enriched in G4 structures in their promoter regions, notably, Pt-ttpy impairs most nuclear-encoded mt ribosome genes' transcription through dampening the recruiting of transcription initiation and elongation factors of NELFB and TAF1 to their promoter with G4-enriched sequences. In vivo studies show Pt-ttpy's efficient anti-tumor effects, disrupting mt genome function with fewer side effects than cisplatin. CONCLUSION: This study underscores Pt-ttpy as a G4-binding platinum(II) complex, effectively targeting cancer mitochondria through dual action on mt and nuclear G4-enriched genomes without inducing ROS, offering promise for safer and effective platinum-based G4-targeted cancer therapy.
Subject(s)
G-Quadruplexes , Mitochondria , G-Quadruplexes/drug effects , Humans , Mitochondria/metabolism , Mitochondria/drug effects , Cell Line, Tumor , Genome, Mitochondrial , Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Platinum/pharmacology , AnimalsABSTRACT
Photopharmacology is an emerging field that utilizes photo-responsive molecules to enable control over the activity of a drug using light. The aim is to limit the therapeutic action of a drug at the level of diseased tissues and organs. Considering the well-known implications of protein kinases in cancer and the therapeutic issues associated with protein kinase inhibitors, the photopharmacology is seen as an innovative and alternative solution with great potential in oncology. In this context, we developed the first photocaged TAM kinase inhibitors based on UNC2025, a first-in-class small molecule kinase inhibitor. These prodrugs showed good stability in biologically relevant buffer and rapid photorelease of the photoremovable protecting group upon UV-light irradiation (<10â min.). These light-activatable prodrugs led to a 16-fold decrease to a complete loss of kinase inhibition, depending on the protein and the position at which the coumarin-type phototrigger was introduced. The most promising candidate was the N,O-dicaged compound, showing the superiority of having two photolabile protecting groups on UNC2025 for being entirely inactive on TAM kinases. Under UV-light irradiation, the N,O-dicaged compound recovered its inhibitory potency in enzymatic assays and displayed excellent antiproliferative activity in RT112â cell lines.
Subject(s)
Adenine/analogs & derivatives , Antineoplastic Agents , Prodrugs , Urinary Bladder Neoplasms , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Prodrugs/pharmacologyABSTRACT
Apurinic/apyrimidinic (AP) sites, 5-formyluracil (fU) and 5-formylcytosine (fC) are abundant DNA modifications that share aldehyde-type reactivity. Here, we demonstrate that polyamines featuring at least one secondary 1,2-diamine fragment in combination with aromatic units form covalent DNA adducts upon reaction with AP sites (with concomitant cleavage of the AP strand), fU and, to a lesser extent, fC residues. Using small-molecule mimics of AP site and fU, we show that reaction of secondary 1,2-diamines with AP sites leads to the formation of unprecedented 3'-tetrahydrofuro[2,3,4-ef]-1,4-diazepane ('ribodiazepane') scaffold, whereas the reaction with fU produces cationic 2,3-dihydro-1,4-diazepinium adducts via uracil ring opening. The reactivity of polyamines towards AP sites versus fU and fC can be tuned by modulating their chemical structure and pH of the reaction medium, enabling up to 20-fold chemoselectivity for AP sites with respect to fU and fC. This reaction is efficient in near-physiological conditions at low-micromolar concentration of polyamines and tolerant to the presence of a large excess of unmodified DNA. Remarkably, 3'-ribodiazepane adducts are chemically stable and resistant to the action of apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphoesterase 1 (TDP1), two DNA repair enzymes known to cleanse a variety of 3' end-blocking DNA lesions.
Subject(s)
DNA Adducts , Polyamines , DNA/chemistry , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA Damage , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Nucleic Acid Conformation , Polyamines/chemistry , Polyamines/metabolismABSTRACT
Bioactive NHC-transition metal complexes have shown promise as anti-cancer agents, but their potential use as radiosensitizers has been neglected so far. We disclose here a new series of bimetallic platinum(II) complexes displaying NHC-type bridging ligands, (bis-NHC)[trans-Pt(RNH2)I2]2, that have been synthesized via a simple, two-step procedure. They display cytotoxicity in the micromolar range on cancerous cell lines, accumulate in cells, and bind to genomic DNA, by inducing DNA damages. Notably, these bimetallic complexes demonstrate significant radiosensitizing effects on both ovarian cells A2780 and nonsmall lung carcinoma cells H1299. Further investigations revealed that bimetallic species make irradiation-induced DNA damages more persistent by inhibiting repair mechanisms. Indeed, a higher and persistent accumulation of both γ-H2AX and 53BP1 foci post-irradiation was detected, in the presence of the NHC-Pt complexes. Overall, we provide the first in vitro evidence for the radiosensitizing properties of NHC-platinum complexes, which suggests their potential use in combined chemo-radio therapy protocols.
Subject(s)
Ovarian Neoplasms , Radiation-Sensitizing Agents , Humans , Female , Platinum/pharmacology , Amines , Cell Line, Tumor , Radiation-Sensitizing Agents/pharmacologyABSTRACT
Combining the selectivity of G-quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V-shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analyzed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.
Subject(s)
G-Quadruplexes , Indolequinones , Alkylating Agents/chemistry , Humans , LigandsABSTRACT
O6-Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O6-methylguanine residues in DNA. MGMT substrate analogues, such as O6-benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine). Specifically, two series of hybrids, encompassing three compounds each, were obtained by varying the position of the attachment point of BG (N9 of guanine vs. the benzyl group) and the length and nature of the linker. UV/vis absorption and fluorescence data indicate that all six hybrids adopt an intramolecularly stacked conformation in aqueous solutions in a wide range of temperatures. All hybrids interact with double-stranded DNA, as clearly evidenced by spectrophotometric titrations, without intercalation of the acridine ring and do not induce thermal stabilization of the duplex. All hybrids, as well as the reference DNA intercalator (6-chloro-2-methoxy-9-aminoacridine 8), irreversibly inhibit MGMT in vitro with variable efficiency, comparable to that of BG. In a multidrug-resistant glioblastoma cell line T98G, benzyl-linked hybrids 7a-c and the N9-linked hybrid 19b are moderately cytotoxic (GI50 ≥ 15 µM after 96 h), while N9-linked hybrids 19a and 19c are strongly cytotoxic (GI50 = 1-2 µM), similarly to acridine 8 (GI50 = 0.6 µM). Among all compounds, hybrids 19a and 19c, similarly to BG, display synergic cytotoxic effect upon co-treatment with subtoxic doses of TMZ, with combination index (CI) values as low as 0.2-0.3. In agreement with in vitro results, compound 19a inactivates cellular MGMT but, unlike BG, does not induce significant levels of DNA damage, either alone or in combination with TMZ, as indicated by the results of γH2AX immunostaining experiments. Instead, and unlike BG, compound 19a alone induces significant apoptosis of T98G cells, which is not further increased in a combination with TMZ. These results indicate that molecular mechanisms underlying the cytotoxicity of 19a and its combination with TMZ are distinct from that of BG. The strongly synergic properties of this combination represent an interesting therapeutic opportunity in treating TMZ-resistant cancers.
Subject(s)
Acridines/pharmacology , Antineoplastic Agents/pharmacology , DNA Modification Methylases/antagonists & inhibitors , DNA Repair Enzymes/antagonists & inhibitors , DNA/chemistry , Enzyme Inhibitors/pharmacology , Guanine/analogs & derivatives , Tumor Suppressor Proteins/antagonists & inhibitors , Acridines/chemistry , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Binding Sites/drug effects , Cattle , Cell Proliferation/drug effects , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Guanine/chemistry , Guanine/pharmacology , Humans , Molecular Structure , Structure-Activity Relationship , Tumor Suppressor Proteins/metabolismABSTRACT
Pt-ttpy (tolyl terpyridin-Pt complex) covalently binds to G-quadruplex (G4) structures in vitro and to telomeres in cellulo via its Pt moiety. Here, we identified its targets in the human genome, in comparison to Pt-tpy, its derivative without G4 affinity, and cisplatin. Pt-ttpy, but not Pt-tpy, induces the release of the shelterin protein TRF2 from telomeres concomitantly to the formation of DNA damage foci at telomeres but also at other chromosomal locations. γ-H2AX chromatin immunoprecipitation (ChIP-seq) after treatment with Pt-ttpy or cisplatin revealed accumulation in G- and A-rich tandemly repeated sequences, but not particularly in potential G4 forming sequences. Collectively, Pt-ttpy presents dual targeting efficiency on DNA, by inducing telomere dysfunction and genomic DNA damage at specific loci.
Subject(s)
Cisplatin/pharmacology , DNA Damage , G-Quadruplexes , Organoplatinum Compounds/pharmacology , Ovarian Neoplasms/pathology , Telomere/drug effects , Telomeric Repeat Binding Protein 2/metabolism , Antineoplastic Agents/pharmacology , Apoptosis , Cell Proliferation , Female , Humans , Organoplatinum Compounds/chemistry , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Telomeric Repeat Binding Protein 2/genetics , Tumor Cells, CulturedABSTRACT
We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2'-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross-linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL-G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.
Subject(s)
G-Quadruplexes , Minisatellite Repeats/genetics , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Binding Sites , Bromodeoxyuridine/chemistry , Chromatography, High Pressure Liquid , Cross-Linking Reagents/chemistry , Models, Molecular , Nucleic Acid Conformation/drug effects , Phosphoproteins/chemistry , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Proteomics , RNA Recognition Motif , RNA-Binding Proteins/chemistry , Tandem Mass Spectrometry , NucleolinABSTRACT
Mass spectrometry provides exquisite details on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by using a mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By putting more negative charge states on the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows one to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.
Subject(s)
DNA/chemistry , G-Quadruplexes , Potassium/chemistry , Base Sequence , Binding Sites , DNA/genetics , DNA/metabolism , Humans , Ligands , Potassium/metabolism , Pyridines/chemistry , Quinolines/chemistry , Sequence Analysis, DNA/methods , Tandem Mass Spectrometry/methodsABSTRACT
Guanine-rich DNA can form four-stranded structures called G-quadruplexes (G4s) that can regulate many biological processes. Metal complexes have shown high affinity and selectivity toward the quadruplex structure. Here, we report the comparison of a panel of platinum (II) complexes for quadruplex DNA selective recognition by exploring the aromatic core around terpyridine derivatives. Their affinity and selectivity towards G4 structures of various topologies have been evaluated by FRET-melting (Fluorescence Resonance Energy Transfert-melting) and Fluorescent Intercalator Displacement (FID) assays, the latter performed by using three different fluorescent probes (Thiazole Orange (TO), TO-PRO-3, and PhenDV). Their ability to bind covalently to the c-myc G4 structure in vitro and their cytotoxicity potential in two ovarian cancerous cell lines were established. Our results show that the aromatic surface of the metallic ligands governs, in vitro, their affinity, their selectivity for the G4 over the duplex structures, and platination efficiency. However, the structural modifications do not allow significant discrimination among the different G4 topologies. Moreover, all compounds were tested on ovarian cancer cell lines and normal cell lines and were all able to overcome cisplatin resistance highlighting their interest as new anticancer drugs.
Subject(s)
G-Quadruplexes/drug effects , Ovarian Neoplasms/drug therapy , Platinum/chemistry , Proto-Oncogene Proteins c-myc/chemistry , Anticarcinogenic Agents/chemistry , Anticarcinogenic Agents/therapeutic use , Cisplatin/adverse effects , Cisplatin/chemistry , Drug Resistance, Neoplasm/drug effects , Female , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Humans , Ligands , Nucleic Acid Conformation/drug effects , Pyridines/chemistryABSTRACT
Ligands interacting with abasic (AP) sites in DNA may generate roadblocks in base-excision DNA repair (BER) due to indirect inhibition of DNA repair enzymes (e.g., APE1) and/or formation of toxic byproducts, resulting from ligand-induced strand cleavage or covalent cross-links. Herein, a series of 12 putative AP-site ligands, sharing the common naphthalenophane scaffold, but endowed with a variety of substituents, have been prepared and systematically studied. The results demonstrate that most naphthalenophanes bind to AP sites in DNA and inhibit the APE1-induced hydrolysis of the latter in vitro. Remarkably, their APE1 inhibitory activity, as characterized by IC50 and KI values, can be directly related to their affinity and selectivity to AP sites, as assessed by means of fluorescence melting experiments. On the other hand, the molecular design of naphthalenophanes has a crucial influence on their intrinsic AP-site cleavage activity (i.e., ligand-catalyzed ß- and ß,δ-elimination reactions at the AP site), as illustrated by the compounds either having an exceptionally high AP-site cleavage activity (e.g., 2,7-BisNP-S, 125-fold more efficacious than spermine) or being totally devoid of this activity (four compounds). Finally, the unprecedented formation of a stable covalent DNA adduct upon reaction of one ligand (2,7-BisNP-NH) with its own product of the AP-site cleavage is revealed.
Subject(s)
DNA Adducts , DNA Cleavage , DNA/chemistry , Naphthalenes/chemistry , Catalytic Domain , DNA/metabolism , DNA Adducts/chemistry , DNA Repair , Ligands , Naphthalenes/metabolismABSTRACT
We studied photochemical reactions of BrU-substituted G-quadruplex (G4) DNA substrates with two pyrene-substituted polyazamacrocyclic ligands, M-1PY and M-2PY. Both ligands bind to and stabilize G4-DNA structures without altering their folding topology, as demonstrated by FRET-melting experiments, fluorimetric titrations and CD spectroscopy. Notably, the bis-pyrene derivative (M-2PY) behaves as a significantly more affine and selective G4 ligand, compared with its mono-pyrene counterpart (M-1PY) and control compounds. Upon short UVA irradiation (365 nm) both ligands, in particular M-2PY, efficiently sensitize photoreactions at BrU residues incorporated in G4 structures and give rise to two kinds of photoproducts, namely DNA strand cleavage and covalent ligand-DNA photoadducts. Remarkably, the photoinduced strand cleavage is observed exclusively with G4 structures presenting BrU residues in lateral or diagonal loops, but not with parallel G4-DNA structures presenting only propeller loops. In contrast, the formation of fluorescent photoadducts is observed with all BrU-substituted G4-DNA substrates, with M-2PY giving significantly higher yields (up to 27%) than M-1PY. Both ligand-sensitized photoreactions are specific to BrU-modified G4-DNA structures with respect to double-stranded or stem-loop substrates. Thus, ligand-sensitized photoreactions with BrU-substituted G4-DNA may be exploited (i) as a photochemical probe, allowing "photofootprinting" of G4 folding topologies in vitro and (ii) for covalent trapping of G4 structures as photoadducts with pyrene-substituted ligands.
Subject(s)
DNA/chemistry , G-Quadruplexes , Photochemical Processes , Uridine/analogs & derivatives , Bromouracil/analogs & derivatives , DNA Adducts/chemistry , Humans , Kinetics , Ligands , Models, Molecular , Mutation/genetics , Telomere/genetics , Uridine/chemistryABSTRACT
It is suggested that several compounds, including G-quadruplex ligands, can target telomeres, inducing their uncapping and, ultimately, cell death. However, it has never been demonstrated whether such ligands can bind directly and quantitatively to telomeres. Here, we employed the property of platinum and platinum-G-quadruplex complexes to target G-rich sequences to investigate and quantify their covalent binding to telomeres. Using inductively coupled plasma mass spectrometry, surprisingly, we found that, in cellulo, in the presence of cisplatin, a di-functional platinum complex, telomeric DNA was platinated 13-times less than genomic DNA in cellulo, as compared to in vitro data. On the contrary, the amount of mono-functional platinum complexes (Pt-ttpy and Pt-tpy) bound either to telomeric or to genomic DNA was similar and occurred in a G-quadruplex independent-manner. Importantly, the quantification revealed that the low level of cisplatin bound to telomeric DNA could not be the direct physical cause of TRF2 displacement from telomeres. Altogether, our data suggest that platinum complexes can affect telomeres both directly and indirectly.
Subject(s)
Cisplatin/chemistry , G-Quadruplexes , Platinum/chemistry , Molecular Structure , Telomere/chemistryABSTRACT
Telomeres protect the ends of chromosomes against illegitimate recombination and repair. They can be targets for G-quadruplex ligands and platinum complexes due to their repeated G-rich sequences. Protection of telomeres is ensured by a complex of six proteins, including TRF2, which inhibits the DNA damage response pathway. We analyzed telomere modifications induced in cancer cells by the experimental hybrid platinum complex, Pt-MPQ, comprising both an ethylene diamine monofunctional platinum complex and a G-quadruplex recognition moiety (MPQ). Pt-MPQ promotes the displacement of two telomeric proteins (TRF2 and TRF1) from telomeres, as well as the formation of telomere damage and telomere sister losses, whereas the control compound MPQ does not. This suggests that the platinum moiety potentiates the targeting of the G-quadruplex ligand to telomeres, opening a new perspective for telomere biology and anticancer therapy. Interestingly, the chemotherapy drug cisplatin, which has no specific affinity for G-quadruplex structures, partially induces the TRF2 delocalization from telomeres but produces less telomeric DNA damage, suggesting that this TRF2 displacement could be independent of G-quadruplex recognition.
Subject(s)
Coordination Complexes/toxicity , G-Quadruplexes/drug effects , Platinum/chemistry , Telomere/drug effects , Acridines/toxicity , Cell Cycle Checkpoints/drug effects , Cell Line , Cell Proliferation/drug effects , Cisplatin/toxicity , DNA Damage/drug effects , Humans , Ligands , Microscopy, Fluorescence , Organoplatinum Compounds/toxicity , Telomere/metabolism , Telomere Shortening/drug effects , Telomeric Repeat Binding Protein 2/metabolismABSTRACT
Telomerase is an almost universal cancer target that consists minimally of a core protein human telomerase reverse transcriptase (hTERT) and a RNA component human telomerase RNA (hTR). Some inhibitors of this enzyme are thought to function by the covalent binding to one or several cysteine residues; however, this inhibition mechanism has never been investigated because of the difficulty in producing telomerase. In this study, we use a recent method to produce recombinant hTERT to analyze the effect of cysteine-reactive inhibitors on telomerase. Using mass spectrometry and mutagenesis analysis, we identify several targeted residues in separated domains of the hTERT protein and show that cysteine-reactive reagents abolish the interaction with the CR4/5 region of hTR.
Subject(s)
Cysteine/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Inhibitors/metabolism , Telomerase/metabolism , Amino Acid Sequence , Binding Sites/genetics , Cysteine/chemistry , Cysteine/genetics , Humans , Mass Spectrometry/methods , Models, Molecular , Molecular Structure , Mutagenesis , Protein Binding , Protein Domains , Recombinant Proteins/chemistry , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/pharmacology , Sequence Homology, Amino Acid , Structure-Activity Relationship , Telomerase/antagonists & inhibitors , Telomerase/geneticsABSTRACT
G-quadruplex structures (G4) are promising anticancerous targets. A great number of small molecules targeting these structures have already been identified through biophysical methods. In cellulo, some of them are able to target either telomeric DNA and/or some sequences involved in oncogene promotors, both resulting in cancer cell death. However, only a few of them are able to bind to these structures G4 irreversibly. Here we combine within the same molecule the G4-binding agent PDC (pyridodicarboxamide) with a N-heterocyclic carbene-platinum complex NHC-Pt already identified for its antitumor properties. The resulting conjugate platinum complex NHC-Pt-PDC stabilizes strongly G-quadruplex structures in vitro, with affinity slightly affected as compared to PDC. In addition, we show that the new conjugate binds preferentially and irreversibly the quadruplex form of the human telomeric sequence with a profile in a way different from that of NHC-Pt thereby indicating that the platination reaction is oriented by stacking of the PDC moiety onto the G4-structure. In cellulo, NHC-Pt-PDC induces a significant loss of TRF2 from telomeres that is considerably more important than the effect of its two components alone, PDC and NHC-Pt, respectively.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , DNA/chemistry , G-Quadruplexes/drug effects , Organoplatinum Compounds/chemistry , Organoplatinum Compounds/pharmacology , Telomere/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA/metabolism , Humans , Ligands , Protein Transport/drug effects , Stereoisomerism , Telomere/genetics , Telomere/metabolism , Telomeric Repeat Binding Protein 2/metabolismABSTRACT
The structure-activity relationships of chiral 1,2-diaminophenylalkane platinum(II) anticancer derivatives are studied, including interactions with telomeric- and genomic-like DNA sequences, the pKa of their diaqua species, structural properties obtained from DFT calculations and resonant X-ray emission spectroscopy. The binding modes of the compounds to telomeric sequences were elucidated, showing no major differences with conventional cis-platinum(II) complexes like cisplatin, supporting that the cis-square planar geometry governs the binding of small Pt(II) complexes to G4 structures. Double-stranded DNA platination kinetics and acid-base constants of the diaqua species of the compounds were measured and compared, highlighting a strong steric dependence of the DNA-binding kinetics, but independent to stereoisomerism. Structural features of the compounds are discussed on the basis of dispersion-corrected DFT, showing that the most active series presents conformers for which the platinum atom is well devoid of steric hindrance. If reactivity indices derived from conceptual DFT do not show evidences for different reactivity between the compounds, RXES experiments provide new insight into the availability of platinum orbitals for binding to nucleophiles.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , DNA, Neoplasm/drug effects , Hydrocarbons, Chlorinated/pharmacology , Organoplatinum Compounds/chemistry , Organoplatinum Compounds/pharmacology , DNA, Neoplasm/genetics , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , G-Quadruplexes/drug effects , Humans , Hydrocarbons, Chlorinated/chemistry , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Structure , Quantum Theory , Structure-Activity RelationshipABSTRACT
Telomerase is the enzyme that maintains the length of telomeres. It is minimally constituted of two components: a core reverse transcriptase protein (hTERT) and an RNA (hTR). Despite its significance as an almost universal cancer target, the understanding of the structure of telomerase and the optimization of specific inhibitors have been hampered by the limited amount of enzyme available. Here, we present a breakthrough method to produce unprecedented amounts of recombinant hTERT and to reconstitute human telomerase with purified components. This system provides a decisive tool to identify regulators of the assembly of this ribonucleoprotein complex. It also enables the large-scale screening of small-molecules capable to interfere with telomerase assembly. Indeed, it has allowed us to identify a compound that inhibits telomerase activity when added prior to the assembly of the enzyme, while it has no effect on an already assembled telomerase. Therefore, the novel system presented here may accelerate the understanding of human telomerase assembly and facilitate the discovery of potent and mechanistically unique inhibitors.
Subject(s)
Telomerase/biosynthesis , Acridines/chemistry , Acridines/pharmacology , Genetic Techniques , HEK293 Cells , Humans , Niacinamide/analogs & derivatives , RNA/chemistry , RNA/metabolism , RNA Folding , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Small Molecule Libraries , Structure-Activity Relationship , Telomerase/antagonists & inhibitors , Telomerase/chemistry , Telomerase/genetics , Telomerase/metabolism , Thiazoles , Trisaccharides/chemistry , Trisaccharides/pharmacologyABSTRACT
This study provides insights into the interactions of Pt-ttpy, that is, a metallo-organic heterocycle-comprising platinum(II) complex of terpyridine, and G-quadruplexes adopted by G-rich DNA from the transcriptional regulatory element of the c-myc gene, a well-known attractive target for artificial modulation of oncogene expression. A previously noted drug-like potential of Pt-ttpy relies on its antiproliferative activity on cancer cells and its increased selectivity for G-quadruplex binding attributed to the combination of distinct interacting modes. The predominant interaction between the herein used models of a parallel G-quadruplex exhibiting short propeller-type loops and Pt-ttpy occurs through stacking to the outer G-quartets. The presence of adenine versus thymine residue at the 5'-end overhanging region allows the coordinative binding of Pt-ttpy to the G-quadruplex structure. Interestingly, Pt-ttpy triggers the formation of the G-quadruplex even in the absence of cations. Furthermore, NMR-based characterisation revealed common structural features of Pt-ttpy-G-quadruplex complexes in the presence and absence of cations, which indicate that cations may be expelled from the cores of the corresponding structures.
Subject(s)
Antineoplastic Agents/pharmacology , G-Quadruplexes/drug effects , Genes, myc/drug effects , Organoplatinum Compounds/pharmacology , Promoter Regions, Genetic/drug effects , Antineoplastic Agents/chemistry , Base Sequence , DNA/chemistry , Electrophoresis , Humans , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation/drug effects , Organoplatinum Compounds/chemistryABSTRACT
We have developed a straightforward synthetic pathway to a set of six photoactivatable G-quadruplex ligands with a validated G4-binding motif (the bisquinolinium pyridodicarboxamide PDC-360A) tethered through various spacers to two different photo-cross-linking groups: benzophenone and an aryl azide. The high quadruplex-versus-duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well-known G-quadruplexes (human telomeric G4 and oncogene promoter c-myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G-quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c-myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.