ABSTRACT
Through an internal virtual screen at GlaxoSmithKline a distinct class of 2-phenylimidazo[1,2-a]pyridine-6-carboxamide H-PGDS inhibitors were discovered. Careful evaluation of crystal structures and SAR led to a novel, potent, and orally active imidazopyridine inhibitor of H-PGDS, 20b. Herein, describes the identification of 2 classes of inhibitors, their syntheses, and their challenges.
Subject(s)
Drug Discovery , Enzyme Inhibitors/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Intramolecular Oxidoreductases/antagonists & inhibitors , Intramolecular Oxidoreductases/metabolism , Molecular Structure , Structure-Activity RelationshipABSTRACT
GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.
Subject(s)
Aza Compounds/chemistry , Enzyme Inhibitors/chemistry , Quinolines/chemistry , Animals , Binding Sites , Brain/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Drug Stability , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Intramolecular Oxidoreductases/antagonists & inhibitors , Intramolecular Oxidoreductases/metabolism , Kinetics , Male , Mice , Mice, Inbred C57BL , Molecular Dynamics Simulation , Muscle, Skeletal/chemistry , Muscle, Skeletal/metabolism , Rats , Structure-Activity RelationshipABSTRACT
Biosensor-surface plasmon resonance (SPR) technology is now well established as a quantitative approach for the study of nucleic acid interactions in real time, without the need for labeling any components of the interaction. The method provides real-time equilibrium and kinetic characterization for quadruplex DNA interactions and requires small amounts of materials and no external probe. A detailed protocol for quadruplex-DNA interaction analyses with a variety of binding molecules using biosensor-SPR methods is presented. Explanations of the SPR method with basic fundamentals for use and analysis of results are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples. Details of experimental design, quantitative and qualitative data analyses, and presentation are described. Some specific examples of small molecule-DNA quadruplex interactions are presented with results evaluated by both kinetic and steady-state SPR methods.
Subject(s)
Biosensing Techniques , Nucleic Acids/chemistry , Kinetics , Surface Plasmon ResonanceABSTRACT
With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50â¯=â¯220,000â¯nM, LEâ¯=â¯0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50â¯=â¯3,100â¯nM, LEâ¯=â¯0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50â¯=â¯9.9â¯nM, LEâ¯=â¯0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Intramolecular Oxidoreductases/antagonists & inhibitors , Lipocalins/antagonists & inhibitors , Quinolines/chemistry , Quinolines/pharmacology , Animals , Drug Discovery , Enzyme Inhibitors/pharmacokinetics , Humans , Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/metabolism , Lipocalins/chemistry , Lipocalins/metabolism , Male , Mice, Inbred C57BL , Molecular Docking Simulation , Quinolines/pharmacokineticsABSTRACT
We demonstrate a high-throughput chemoprinting platform that confirms the consistency in the higher-order structure of protein biologics and is sensitive enough to detect single-point mutations. This method addresses the quality and consistency of the tertiary and quaternary structure of biologic drug products, which is arguably the most important, yet rarely examined, parameter. The method described uses specific small-molecule ligands as molecular probes to assess protein structure. Each library of probe molecules provides a "fingerprint" when taken holistically. After proof-of-concept experiments involving enzymes and antibodies, we were able to detect minor conformational perturbations between four 48 kDa protein mutants that only differ by one amino acid residue.
Subject(s)
Biological Products/chemistry , High-Throughput Screening Assays , Proteins/chemistry , Proteins/genetics , Chromatography, Liquid , Mass Spectrometry , Models, Molecular , Molecular StructureABSTRACT
1,10-Phenanthroline (Phen) derivatives are attractive ligands to provide metal complexes that are selective for different DNA secondary structures. Herein, we analyze the binding processes of two bis-Phen analogues and their Ni(II) complexes toward double-stranded DNA and telomeric G-quadruplex DNA by calorimetric and spectroscopic techniques. The free ligands can adapt to both DNA arrangements. Conversely, metal ion coordination produces an increase in ligand affinity for the tetrahelical structure, whereas it dramatically decreases binding to double-stranded DNA as a result of distinct binding modes on the two templates. In fact, Ni(II) complexes effectively stack on the G-quadruplex terminals, with an entropic loss counterbalanced by favorable enthalpy changes, whereas they cause a conformational reshaping of the double-helix form with a substantial decrease in the binding free energy. Consistently, no Ni(II) -DNA ionic pair has ever been identified. These results provide a rationale for the selective recognition of distinct DNA arrangements in view of targeted pharmacological applications.
Subject(s)
DNA/chemistry , DNA/drug effects , G-Quadruplexes/drug effects , Nickel/pharmacology , Organometallic Compounds/pharmacology , Phenanthrolines/pharmacology , Dose-Response Relationship, Drug , Humans , Ligands , Molecular Structure , Nickel/chemistry , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistry , Phenanthrolines/chemistry , Structure-Activity Relationship , ThermodynamicsABSTRACT
Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an "in house" library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters.From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT-dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., α155, HMC1.2 and ROSA cells).Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations.
Subject(s)
G-Quadruplexes , Gene Expression Regulation, Neoplastic/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-kit/genetics , Anthraquinones/chemistry , Anthraquinones/metabolism , Anthraquinones/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Ligands , MCF-7 Cells , Molecular Structure , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Proto-Oncogene Mas , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
Proflavine, a known intercalator of DNA and RNA, promotes duplex formation by nucleic acids with natural and non-natural backbones that otherwise form duplexes with low thermal stability, and even some that show no sign of duplex formation in the absence of proflavine. These findings demonstrate the potential for intercalators to be used as cofactors for the assembly of rationally designed nucleic acid structures, and could provide fundamental insights regarding intercalation of natural nucleic acid duplexes.
Subject(s)
Intercalating Agents/pharmacology , Nucleic Acid Conformation/drug effects , Nucleic Acids/chemistry , Proflavine/pharmacology , Biomimetic Materials/chemistry , DNA/chemistry , Intercalating Agents/chemistry , Models, Molecular , Proflavine/chemistry , RNA/chemistryABSTRACT
Aryl ethynyl anthraquinones have been synthesized by Sonogashira cross-coupling and evaluated as telomeric G-quadruplex ligands, by the FRET melting assay, circular dichroism, the DNA synthesis arrest assay and molecular docking. Both the binding properties and G-quadruplex vs. duplex selectivity are controlled by the structures of the aryl ethynyl moieties.
Subject(s)
Anthraquinones/chemistry , G-Quadruplexes , Telomere/chemistry , Circular Dichroism , DNA/chemistry , Fluorescence , Ligands , Models, Molecular , Taq Polymerase/metabolism , Transition TemperatureABSTRACT
Transition metal complexes allow fine tuning of DNA binding affinity and selectivity. Here we report on the nucleic acid recognition properties of a phenanthroline-based ligand coordinated to Ni(2+) or Cu(2+). The resulting complexes clearly bind to telomeric G-quadruplexes at different sites according to the nature of the bound metal ion.
Subject(s)
Coordination Complexes/chemistry , G-Quadruplexes , Nickel/chemistry , Phenanthrolines/chemistry , Zinc/chemistry , Binding Sites , Circular Dichroism , Ligands , Molecular Structure , Transition Elements/chemistryABSTRACT
The physiological role(s) played by G-quadruplexes renders these 'non-canonical' DNA secondary structures interesting new targets for therapeutic intervention. In particular, the search for ligands for selective recognition and stabilization of G-quadruplex arrangements has led to a number of novel targeted agents. An interesting approach is represented by the use of metal-complexes, their binding to DNA being modulated by ligand and metal ion nature, and by complex stoichiometry. In this work we characterized thermodynamically and stereochemically the interactions of a Ni(II) bis-phenanthroline derivative with telomeric G-quadruplex sequences using calorimetric, chiroptical and NMR techniques. We employed three strictly related sequences based on the human telomeric repeat, namely Tel22, Tel26 and wtTel26, which assume distinct conformations in potassium containing solutions. We were able to monitor specific enthalpy/entropy changes according to the structural features of the target telomeric sequence and to dissect the binding process into distinct events. Interestingly, temperature effects turned out to be prominent both in terms of binding stoichiometry and ΔH/ΔS contributions, while the final G-quadruplex-metal complex architecture tended to merge for the examined sequences. These results underline the critical choice of experimental conditions and DNA sequence for practical use of thermodynamic data in the rational development of effective G-quadruplex binders.
Subject(s)
DNA/chemistry , DNA/metabolism , G-Quadruplexes , Organometallic Compounds/metabolism , Polymorphism, Genetic , Telomere/genetics , Base Sequence , DNA/genetics , G-Quadruplexes/drug effects , Ligands , Organometallic Compounds/pharmacology , ThermodynamicsABSTRACT
The mechanism of action of clinically used Pt-based drugs is through the formation of stable DNA adducts occurring at the nitrogen in position 7 of guanine (N7) and involving one or two spatially closed residues. Nevertheless, proteins can represent alternative targets since in particular sulfur groups, present in cysteine or methionine residues, can efficiently coordinate platinum. Here we have characterized the reactivity profile of cisplatin, transplatin and of two trans-platinum amine derivatives (TPAs) towards three different proteins, bovine α-lactalbumin (α-LA), hen egg lysozyme (LYS) and human serum albumin (HSA). Our results demonstrate that generally the tested metal complexes react with the selected target causing protein oligomerization, likely through a cross-linking reaction. Interestingly, the extent of such a process is largely modulated by the target protein and by the chemical features of the metal complex, TPAs being the most efficient platinating agents. From a structural point of view the resulting reaction products turned out to be depending on the nature of the metal complexes. However, in all instances, a transfer reaction of the metal complex to DNA can also occur, maintaining the relevance of nucleic acids as a biological target. These results can be used to better rationalize the different pharmacological profiles reported for cisplatin and TPAs and can help in designing more predictive SARs within the series.
Subject(s)
Cisplatin/chemistry , DNA Adducts/chemistry , Lactalbumin/chemistry , Platinum/chemistry , Serum Albumin/chemistry , Animals , Cattle , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Humans , Mass Spectrometry , Models, Biological , Molecular Structure , Protein BindingABSTRACT
Nowadays, it has been demonstrated that DNA G-quadruplex arrangements are involved in cellular aging and cancer, thus boosting the discovery of selective binders for these DNA secondary structures. By taking advantage of available structural and biological information on these structures, we performed a high throughput in silico screening of commercially available molecules databases by merging ligand- and structure-based approaches by means of docking experiments. Compounds selected by the virtual screening procedure were then tested for their ability to interact with the human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and photodynamic techniques. Interestingly, our screening succeeded in retrieving a new promising scaffold for G-quadruplex binders characterized by a psoralen moiety.
Subject(s)
G-Quadruplexes , Nucleic Acid Conformation , Biophysics , Circular Dichroism , Fluorescence , Furocoumarins/chemistry , Ligands , Mass SpectrometryABSTRACT
Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets.
Subject(s)
Antineoplastic Agents/chemical synthesis , Apoptosis/drug effects , Cytotoxins/chemical synthesis , Imides/chemical synthesis , Naphthalenes/chemical synthesis , Antineoplastic Agents/pharmacology , Caspases/genetics , Caspases/metabolism , Cell Line, Tumor , Cytotoxins/pharmacology , Drug Screening Assays, Antitumor , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , G-Quadruplexes/drug effects , Gene Expression/drug effects , Humans , Imides/pharmacology , Molecular Docking Simulation , Naphthalenes/pharmacology , Phosphorylation , Signal Transduction/drug effects , Structure-Activity Relationship , Taq Polymerase/antagonists & inhibitors , Taq Polymerase/genetics , Telomerase/antagonists & inhibitors , Telomerase/genetics , ThermodynamicsABSTRACT
Small molecules that can induce and stabilize G-quadruplex DNA structures represent a novel approach for anti-cancer and anti-parasitic therapy and extensive efforts have been directed towards discovering lead compounds that are capable of stabilizing quadruplexes. The purpose of this study is to explore conformational modifications in a series of heterocyclic dications to discover structural motifs that can selectively bind and stabilize specific G-quadruplexes, such as those present in the human telomere. The G-quadruplex has various potential recognition sites for small molecules; however, the primary interaction site of most of these ligands is the terminal tetrads. Similar to duplex-DNA groove recognition, quadruplex groove recognition by small molecules offers the potential for enhanced selectivity that can be developed into a viable therapeutic strategy. The compounds investigated were selected based on preliminary studies with DB832, a bifuryl-phenyl diamidine with a unique telomere interaction. This compound provides a paradigm that can help in understanding the optimum compound-DNA interactions that lead to quadruplex groove recognition. DNA recognition by the DB832 derivatives was investigated by biophysical experiments such as thermal melting, circular dichroism, mass spectrometry and NMR. Biological studies were also performed to complement the biophysical data. The results suggest a complex binding mechanism which involves the recognition of grooves for some ligands as well as stacking at the terminal tetrads of the human telomeric G-quadruplex for most of the ligands. These molecules represent an excellent starting point for further SAR analysis for diverse modes of quadruplex recognition and subsequent structure optimization for drug development.
Subject(s)
G-Quadruplexes , Telomere , Circular Dichroism , DNA/chemistry , Drug Design , Drug Discovery , Furans/pharmacology , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Structure-Activity Relationship , Telomere/chemistryABSTRACT
New trans-platinum planar amines (TPAs) represent a family of platinum-based drugs with cytotoxicity equivalent to that of cisplatin, but with negligible cross-resistance. According to the substitution pattern around the metal center, distinct DNA adducts can be formed which yield various levels of cytotoxicity in cell lines. We compared the effects of leaving groups (Cl(-) versus formate or acetate) and amines (NH(3) versus aromatic heterocyclic planar systems) on the efficiency, kinetics, and mode of DNA platination. We show that the substitution of just a single amino group on the transplatin nucleus is optimal, with major effects on the kinetics of metal complex conversion into the reactive aquo species. Additionally, by monitoring TPA reactivity toward variable DNA structures, a lack of preference for double-stranded DNA in over single-stranded or G-quadruplex DNA was observed which is possibly related to steric effects of the planar amine groups. These properties can lead to a unique distribution of platination sites by TPA relative to the lead compound cisplatin, which may help to explain the unique cytotoxic profile of TPAs.
Subject(s)
Amines/chemistry , DNA/chemistry , Platinum/chemistry , Coordination Complexes/chemistry , DNA Adducts/chemistry , G-Quadruplexes , Transition TemperatureABSTRACT
Transition metal ions can drive the assembly of small planar ligands to produce structures able to efficiently recognize G-quadruplex DNA arrangements.