RESUMEN
G-quadruplex (G4) sequences, which can fold into higher-order G4 structures, are abundant in the human genome and are over-represented in the promoter regions of many genes involved in human cancer initiation, progression, and metastasis. They are plausible targets for G4-binding small molecules, which would, in the case of promoter G4s, result in the transcriptional downregulation of these genes. However, structural information is currently available on only a very small number of G4s and their ligand complexes. This limitation, coupled with the currently restricted information on the G4-containing genes involved in most complex human cancers, has led to the development of a phenotypic-led approach to G4 ligand drug discovery. This approach was illustrated by the discovery of several generations of tri- and tetra-substituted naphthalene diimide (ND) ligands that were found to show potent growth inhibition in pancreatic cancer cell lines and are active in in vivo models for this hard-to-treat disease. The cycles of discovery have culminated in a highly potent tetra-substituted ND derivative, QN-302, which is currently being evaluated in a Phase 1 clinical trial. The major genes whose expression has been down-regulated by QN-302 are presented here: all contain G4 propensity and have been found to be up-regulated in human pancreatic cancer. Some of these genes are also upregulated in other human cancers, supporting the hypothesis that QN-302 is a pan-G4 drug of potential utility beyond pancreatic cancer.
Asunto(s)
Antineoplásicos , Descubrimiento de Drogas , G-Cuádruplex , G-Cuádruplex/efectos de los fármacos , Humanos , Descubrimiento de Drogas/métodos , Ligandos , Antineoplásicos/farmacología , Antineoplásicos/química , Fenotipo , Línea Celular Tumoral , Naftalenos/farmacología , Naftalenos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Animales , Imidas/química , Imidas/farmacología , Regiones Promotoras GenéticasRESUMEN
Non-CpG methylation is associated with several cellular processes, especially neuronal development and cancer, while its effect on DNA structure remains unclear. We have determined the crystal structures of DNA duplexes containing -CGCCG- regions as CCG repeat motifs that comprise a non-CpG site with or without cytosine methylation. Crystal structure analyses have revealed that the mC:G base-pair can simultaneously form two alternative conformations arising from non-CpG methylation, including a unique water-mediated cis Watson-Crick/Hoogsteen, (w)cWH, and Watson-Crick (WC) geometries, with partial occupancies of 0.1 and 0.9, respectively. NMR studies showed that an alternative conformation of methylated mC:G base-pair at non-CpG step exhibits characteristics of cWH with a syn-guanosine conformation in solution. DNA duplexes complexed with the DNA binding drug echinomycin result in increased occupancy of the (w)cWH geometry in the methylated base-pair (from 0.1 to 0.3). Our structural results demonstrated that cytosine methylation at a non-CpG step leads to an antiâsyntransition of its complementary guanosine residue toward the (w)cWH geometry as a partial population of WC, in both drug-bound and naked mC:G base pairs. This particular geometry is specific to non-CpG methylated dinucleotide sites in B-form DNA. Overall, the current study provides new insights into DNA conformation during epigenetic regulation.
Asunto(s)
Emparejamiento Base , Citosina , Metilación de ADN , ADN , Conformación de Ácido Nucleico , Agua , ADN/química , Citosina/química , Agua/química , Cristalografía por Rayos X , Modelos MolecularesRESUMEN
The tetrasubstituted naphthalene diimide compound QN-302 binds to G-quadruplex (G4) DNA structures. It shows high potency in pancreatic ductal adenocarcinoma (PDAC) cells and inhibits the transcription of cancer-related genes in these cells and in PDAC animal models. It is currently in Phase 1a clinical evaluation as an anticancer drug. A study of structure-activity relationships of QN-302 and two related analogues (CM03 and SOP1247) is reported here. These have been probed using comparisons of transcriptional profiles from whole-genome RNA-seq analyses, together with molecular modelling and molecular dynamics simulations. Compounds CM03 and SOP1247 differ by the presence of a methoxy substituent in the latter: these two compounds have closely similar transcriptional profiles. Whereas QN-302 (with an additional benzyl-pyrrolidine group), although also showing down-regulatory effects in the same cancer-related pathways, has effects on distinct genes, for example in the hedgehog pathway. This distinctive pattern of genes affected by QN-302 is hypothesized to contribute to its superior potency compared to CM03 and SOP1247. Its enhanced ability to stabilize G4 structures has been attributed to its benzyl-pyrrolidine substituent fitting into and filling most of the space in a G4 groove compared to the hydrogen atom in CM03 or the methoxy group substituent in SOP1247.
Asunto(s)
Carcinoma Ductal Pancreático , G-Cuádruplex , Neoplasias Pancreáticas , Animales , Proteínas Hedgehog , Relación Estructura-Actividad , Simulación de Dinámica Molecular , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Perfilación de la Expresión Génica , Pirrolidinas , LigandosRESUMEN
DNA requires hydration to maintain its structural integrity. Crystallographic analyses have enabled patterns of water arrangements to be visualized. We survey these water motifs in this review, focusing on left- and right-handed duplex and quadruplex DNAs, together with the i-motif. Common patterns of linear spines of water organization in grooves have been identified and are widely prevalent in right-handed duplexes and quadruplexes. By contrast, a left-handed quadruplex has a distinctive wheel of hydration populating the almost completely circular single groove in this structure.
Asunto(s)
ADN de Forma Z , G-Cuádruplex , Agua/química , ADN/química , Fenómenos Químicos , Conformación de Ácido NucleicoRESUMEN
GC-rich sequences can fold into G-quadruplexes and i-motifs and are known to control gene expression in many organisms. The potent G-quadruplex experimental anticancer drug QN-302 down-regulates a number of cancer-related genes, in particular S100P. Here we show this ligand has strong opposing effects with i-motif DNA structures and is one of the most potent i-motif destabilising agents reported to date. QN-302 down-regulates the expression of numerous cancer-related genes by pan-quadruplex targeting. QN-302 exhibits exceptional combined synergistic effects compared to many other G-quadruplex and i-motif interacting compounds. This work further emphasises the importance of considering G-quadruplex and i-motif DNA structures as one dynamic system.
Asunto(s)
G-Cuádruplex , Neoplasias , Humanos , ADN/genética , ADN/química , Regiones Promotoras Genéticas/genética , Neoplasias/genética , Proteínas de Unión al Calcio/genética , Proteínas de NeoplasiasRESUMEN
Combination cancer chemotherapy is one of the most useful treatment methods to achieve a synergistic effect and reduce the toxicity of dosing with a single drug. Here, we use a combination of two well-established anticancer DNA intercalators, actinomycin D (ActD) and echinomycin (Echi), to screen their binding capabilities with DNA duplexes containing different mismatches embedded within Watson-Crick base-pairs. We have found that combining ActD and Echi preferentially stabilised thymine-related T:T mismatches. The enhanced stability of the DNA duplex-drug complexes is mainly due to the cooperative binding of the two drugs to the mismatch duplex, with many stacking interactions between the two different drug molecules. Since the repair of thymine-related mismatches is less efficient in mismatch repair (MMR)-deficient cancer cells, we have also demonstrated that the combination of ActD and Echi exhibits enhanced synergistic effects against MMR-deficient HCT116 cells and synergy is maintained in a MMR-related MLH1 gene knockdown in SW620 cells. We further accessed the clinical potential of the two-drug combination approach with a xenograft mouse model of a colorectal MMR-deficient cancer, which has resulted in a significant synergistic anti-tumour effect. The current study provides a novel approach for the development of combination chemotherapy for the treatment of cancers related to DNA-mismatches.
Asunto(s)
Neoplasias Colorrectales , Equinomicina , Humanos , Animales , Ratones , Dactinomicina/química , Equinomicina/química , Timina , Secuencia de Bases , Sitios de Unión , Conformación de Ácido Nucleico , ADN/químicaRESUMEN
The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302.
Asunto(s)
G-Cuádruplex , Neoplasias Pancreáticas , Humanos , Línea Celular Tumoral , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Expresión Génica , Proteínas de Neoplasias/metabolismo , Neoplasias PancreáticasRESUMEN
Left-handed G quadruplexes (LHG4) have been recently discovered as a new class of G quadruplexes. The biological functions of LHG4s are still unknown, but they share a striking resemblance to Z-DNA in their helicity and jagged phosphate backbone. To further understand structural features of the LHG4s that define their left handedness, we have employed human-interpretable machine-learning methods to classify right- and left-handed G4s purely based on torsional angle analysis. Our results reveal the importance of the α, ß, δ, and χ angles in left-handed structuring across both Z-DNAs and LHG4s. Our analysis may serve as the first step to understanding the conditions of formation for LHG4s and their potential biological relevance.
Asunto(s)
ADN Forma B , G-Cuádruplex , Humanos , ADN/genética , ADN/químicaRESUMEN
The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson-Crick base pair at the two drug-binding interfaces can result in a single-stranded 'chair-shaped' DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a 'curved-shaped' duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs' potential synergetic use.
Asunto(s)
ADN , Emparejamiento Base , ADN/química , ADN/genética , Enlace de Hidrógeno , Estructura Molecular , Conformación de Ácido NucleicoRESUMEN
The use of a small molecule compound to reduce toxic repeat RNA transcripts or their translated aberrant proteins to target repeat-expanded RNA/DNA with a G4C2 motif is a promising strategy to treat C9orf72-linked disorders. In this study, the crystal structures of DNA and RNA-DNA hybrid duplexes with the -GGGCCG- region as a G4C2 repeat motif were solved. Unusual groove widening and sharper bending of the G4C2 DNA duplex A-DNA conformation with B-form characteristics inside was observed. The G4C2 RNA-DNA hybrid duplex adopts a more typical rigid A form structure. Detailed structural analysis revealed that the G4C2 repeat motif of the DNA duplex exhibits a hydration shell and greater flexibility and serves as a 'hot-spot' for binding of the anthracene-based nickel complex, NiII(Chro)2 (Chro = Chromomycin A3). In addition to the original GGCC recognition site, NiII(Chro)2 has extended specificity and binds the flanked G:C base pairs of the GGCC core, resulting in minor groove contraction and straightening of the DNA backbone. We have also shown that Chro-metal complexes inhibit neuronal toxicity and suppresses locomotor deficits in a Drosophila model of C9orf72-associated ALS. The approach represents a new direction for drug discovery against ALS and FTD diseases by targeting G4C2 repeat motif DNA.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Proteína C9orf72/genética , ADN de Forma A/ultraestructura , Demencia Frontotemporal/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Antracenos/química , Antracenos/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , ADN/efectos de los fármacos , ADN/ultraestructura , ADN de Forma A/efectos de los fármacos , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Humanos , Conformación de Ácido Nucleico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The determination of the double helical structure of DNA in 1953 remains the landmark event in the development of modern biological and biomedical science. This structure has also been the starting point for the determination of some 2000 DNA crystal structures in the subsequent 68 years. Their structural diversity has extended to the demonstration of sequence-dependent local structure in duplex DNA, to DNA bending in short and long sequences and in the DNA wound round the nucleosome, and to left-handed duplex DNAs. Beyond the double helix itself, in circumstances where DNA sequences are or can be induced to unwind from being duplex, a wide variety of topologies and forms can exist. Quadruplex structures, based on four-stranded cores of stacked G-quartets, are prevalent though not randomly distributed in the human and other genomes and can play roles in transcription, translation, and replication. Yet more complex folds can result in DNAs with extended tertiary structures and enzymatic/catalytic activity. The Protein Data Bank is the depository of all these structures, and the resource where structures can be critically examined and validated, as well as compared one with another to facilitate analysis of conformational and base morphology features. This review will briefly survey the major structural classes of DNAs and illustrate their significance, together with some examples of how the use of the Protein Data Bank by for example, data mining, has illuminated DNA structural concepts.
Asunto(s)
ADN/química , Bases de Datos de Proteínas , Conformación de Ácido Nucleico , Cristalografía por Rayos X , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
The role of G-quadruplexes in human cancers is increasingly well-defined. Accordingly, G-quadruplexes can be suitable drug targets and many small molecules have been identified to date as G-quadruplex binders, some using computer-based design methods and co-crystal structures. The role of bound water molecules in the crystal structures of G-quadruplex-small molecule complexes has been analyzed in this study, focusing on the water arrangements in several G-quadruplex ligand complexes. One is the complex between the tetrasubstituted naphthalene diimide compound MM41 and a human intramolecular telomeric DNA G-quadruplex, and the others are in substituted acridine bimolecular G-quadruplex complexes. Bridging water molecules form most of the hydrogen-bond contacts between ligands and DNA in the parallel G-quadruplex structures examined here. Clusters of structured water molecules play essential roles in mediating between ligand side chain groups/chromophore core and G-quadruplex. These clusters tend to be conserved between complex and native G-quadruplex structures, suggesting that they more generally serve as platforms for ligand binding, and should be taken into account in docking and in silico studies.
RESUMEN
Quadruplex DNAs can fold into a variety of distinct topologies, depending in part on loop types and orientations of individual strands, as shown by high-resolution crystal and NMR structures. Crystal structures also show associated water molecules. We report here on an analysis of the hydration arrangements around selected folded quadruplex DNAs, which has revealed several prominent features that re-occur in related structures. Many of the primary-sphere water molecules are found in the grooves and loop regions of these structures. At least one groove in anti-parallel and hybrid quadruplex structures is long and narrow and contains an extensive spine of linked primary-sphere water molecules. This spine is analogous to but fundamentally distinct from the well-characterized spine observed in the minor groove of A/T-rich duplex DNA, in that every water molecule in the continuous quadruplex spines makes a direct hydrogen bond contact with groove atoms, principally phosphate oxygen atoms lining groove walls and guanine base nitrogen atoms on the groove floor. By contrast, parallel quadruplexes do not have extended grooves, but primary-sphere water molecules still cluster in them and are especially associated with the loops, helping to stabilize loop conformations.
Asunto(s)
G-Cuádruplex , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , AguaAsunto(s)
Economía del Comportamiento , Aguas del Alcantarillado , Sesgo , Cognición , Responsabilidad SocialRESUMEN
The stabilisation of G-quadruplexes (G4s) by small-molecule compounds is an effective approach for causing cell growth arrest, followed by cell death. Some of these compounds are currently being developed for the treatment of human cancers. We have previously developed a substituted naphthalene diimide G4-binding molecule (CM03) with selective potency for pancreatic cancer cells, including gemcitabine-resistant cells. We report here that CM03 and the histone deacetylase (HDAC) inhibitor SAHA (suberanilohydroxamic acid) have synergistic effects at concentrations close to and below their individual GI50 values, in both gemcitabine-sensitive and resistant pancreatic cancer cell lines. Immunoblot analysis showed elevated levels of γ-H2AX and cleaved PARP proteins upon drug combination treatment, indicating increased levels of DNA damage (double-strand break events: DSBs) and apoptosis induction, respectively. We propose that the mechanism of synergy involves SAHA relaxing condensed chromatin, resulting in higher levels of G4 formation. In turn, CM03 can stabilise a greater number of G4s, leading to the downregulation of more G4-containing genes as well as a higher incidence of DSBs due to torsional strain on DNA and chromatin structure.
Asunto(s)
Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , G-Cuádruplex , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Vorinostat/uso terapéutico , Línea Celular Tumoral , Daño del ADN , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Sinergismo Farmacológico , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Neoplasias Pancreáticas/patología , Vorinostat/química , Vorinostat/farmacología , GemcitabinaRESUMEN
Targeting of genomic quadruplexes is an approach to treating complex human cancers. We describe a series of tetra-substituted naphthalene diimide (ND) derivatives with a phenyl substituent directly attached to the ND core. The lead compound (SOP1812) has 10 times superior cellular and in vivo activity compared with previous ND compounds and nanomolar binding to human quadruplexes. The pharmacological properties of SOP1812 indicate good bioavailability, which is consistent with the in vivo activity in xenograft and genetic models for pancreatic cancer. Transcriptome analysis shows that it down-regulates several cancer gene pathways, including Wnt/ß-catenin signaling.
RESUMEN
Gemcitabine is a drug of choice in the treatment of human pancreatic cancer. Chemo-resistance to this drug is common and has been attributed to a variety of distinct mechanisms, involving > 100 genes. A recently developed small-molecule G-quadruplex ligand, the trisubstituted naphthalene diimide compound CM03, has previously been shown to have equivalent potency to gemcitabine in the pancreatic cancer cell line MIA PaCa-2. We report here on cell lines of increased resistance to gemcitabine that have been generated from this line, with the most resistant having 1,000-fold reduced sensitivity to gemcitabine. These resistant lines retain nM sensitivity to CM03. The molecular basis for the retention of potency by this G-quadruplex ligand has been examined using whole transcriptome data analysis with RNA-seq. This has revealed that the pattern of pathways down regulated by CM03 in the parental MIA PaCa-2 cell line is largely unaffected in the gemcitabine-resistant line. The analysis has also shown that the expression patterns of numerous genes involved in gemcitabine sensitivity are down regulated in the resistant line upon CM03 treatment. These results are supportive of the concept that G-quadruplex small molecules such as CM03 have potential for clinical use in the treatment of gemcitabine-resistant human pancreatic cancer.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , G-Cuádruplex , Imidas/farmacología , Naftalenos/farmacología , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Imidas/química , Ligandos , Naftalenos/química , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Represoras , Regulación hacia Arriba/efectos de los fármacos , Gemcitabina , Neoplasias PancreáticasAsunto(s)
Consumo de Bebidas Alcohólicas , Lactancia Materna , Femenino , Fertilidad , Humanos , Embarazo , Proyectos de InvestigaciónRESUMEN
Interactions are reported of three representative naphthalenediimide derivatives with three quadruplex targets, from the promoter region of the telomerase (hTERT) gene, a human telomeric DNA quadruplex, and a telomeric RNA quadruplex (TERRA). Thermal melting studies showed that these compounds strongly stabilize the quadruplexes, with weak stabilization of a duplex DNA. Binding studies by surface plasmon resonance and fluorescence spectroscopy found that the compounds bind to the quadruplexes with nanomolar equilibrium dissociation constants. Plausible topologies for the quadruplex complexes were deduced from CD spectra, which together with the surface plasmon resonance data indicate that the quadruplexes with parallel quadruplex folds are preferred by two compounds, which was confirmed by qualitative molecular modeling.