ABSTRACT
The synthesis and characterisation of fluorosulfate covalent inhibitors of the lipid kinase PI4KIIIß is described. The conserved lysine residue located within the ATP binding site was targeted, and optimised compounds based upon reversible inhibitors with good activity and physicochemical profile showed strong reversible interactions and slow onset times for the covalent inhibition, resulting in an excellent selectivity profile for the lipid kinase target. X-Ray crystallography demonstrated a distal tyrosine residue could also be targeted using a fluorosulfate strategy. Combination of this knowledge showed that a dual covalent inhibitor could be developed which reveals potential in addressing the challenges associated with drug resistant mutations.
ABSTRACT
Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.
Subject(s)
Antineoplastic Agents/pharmacology , Peptides/metabolism , Prodrugs/pharmacology , Tissue Inhibitor of Metalloproteinase-3/metabolism , gamma-Glutamyl Hydrolase/metabolism , Antibodies, Monoclonal/pharmacology , Humans , Neoplasms/drug therapy , Neoplasms/metabolism , Nitrogen Mustard Compounds/pharmacology , Recombinant Proteins/pharmacology , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Physicochemical properties, such as solubility, are important when prioritising compounds for progression on a drug discovery project. There is limited literature around the systematic effects of core changes on thermodynamic solubility. This work details the synthesis of nitrogen containing 6,5-bicyclic heterocyclic cores which are common scaffolds in medicinal chemistry and the analysis of their physicochemical properties, particularly, thermodynamic solubility. Crystalline solids were obtained where possible to enable a robust comparison of the thermodynamic solubility. Other parameters such as pKa, melting point and lipophilicity were also measured to determine the key factors affecting the observed solubility.
Subject(s)
Amides/chemistry , Heterocyclic Compounds, 2-Ring/chemistry , Nitrogen/chemistry , Pyrimidines/chemistry , Thermodynamics , Amides/chemical synthesis , Heterocyclic Compounds, 2-Ring/chemical synthesis , Molecular Structure , Pyrimidines/chemical synthesis , SolubilityABSTRACT
Doxorubicin is a cytotoxic anthracycline derivative that has been used as a chemotherapeutic in many different forms of human cancer with some success. However, doxorubicin treatment has several side-effects, the most serious of which is cardiomyopathy, that can be fatal. Doxorubicin encapsulation in PEGylated liposomes (Doxil®) has been shown to increase tumour localisation and decrease cardiotoxicity. Conversely, the stability of such liposomes also leads to increased circulation times and accumulation in the skin, resulting in palmar planter erythrodysesthesia, while also limiting release of the drug at the tumour site. Specific targeting of such liposomes to tumour cells has been attempted using various receptor-specific peptides and antibodies. However, targeting a single epitope limits the likely number of tumour targets and increases the risk of tumour resistance through mutation. In this report, Doxil® was coupled to peptide sequence p700 derived from tissue inhibitor of metalloproteinase 3. This Doxil® -P700 complex results in an approximately 100-fold increase in drug uptake, relative to Doxil® alone, by both mouse and human breast cancer cells and immortalised vascular cells resulting in an increase in cytotoxicity. Using p700 to target liposomes in this way may enable specific delivery of doxorubicin or other drugs to a broad range of cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Doxorubicin/analogs & derivatives , Liposomes/chemistry , Peptides/metabolism , Polyethylene Glycols/chemistry , Tissue Inhibitor of Metalloproteinase-3/metabolism , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Doxorubicin/pharmacology , Female , Humans , MCF-7 Cells , Mice , Mice, Inbred BALB C , Polyethylene Glycols/pharmacologyABSTRACT
The discovery and lead optimisation of a novel series of SYK inhibitors is described. These were optimised for SYK potency and selectivity against Aurora B. Compounds were profiled in a human skin penetration study to identify a suitable candidate molecule for pre-clinical development. Compound 44 (GSK2646264) was selected for progression and is currently in Phase I clinical trials.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Skin/drug effects , Syk Kinase/antagonists & inhibitors , Administration, Topical , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/chemistry , Catalytic Domain , Cell Line, Tumor , Humans , Molecular Docking Simulation , Molecular Structure , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyridines/administration & dosage , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity Relationship , Syk Kinase/chemistryABSTRACT
The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay.
Subject(s)
Naphthyridines/pharmacology , Protein Kinase Inhibitors/pharmacology , Administration, Oral , Animals , Biological Availability , Crystallography, X-Ray , Humans , Mutagenicity Tests , Naphthyridines/administration & dosage , Naphthyridines/pharmacokinetics , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 µM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).
Subject(s)
Enzyme Inhibitors/chemistry , Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Pyridines/chemistry , Amination , Cell Line , Cell Membrane Permeability , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Histone Demethylases/chemistry , Histone Demethylases/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Models, Molecular , Pyridines/pharmacokinetics , Pyridines/pharmacologyABSTRACT
Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Cell Line , Cell Membrane Permeability , Crystallography, X-Ray , Enzyme Inhibitors/pharmacokinetics , Histone Demethylases/chemistry , Histone Demethylases/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Models, Molecular , Molecular Docking Simulation , Pyrimidinones/pharmacokinetics , Structure-Activity RelationshipABSTRACT
PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
Subject(s)
Benzimidazoles/pharmacology , Enzyme Inhibitors/pharmacology , Hydrolases/antagonists & inhibitors , Neutrophils/drug effects , Animals , Benzimidazoles/chemical synthesis , Binding, Competitive , Calcium/metabolism , Citrulline/metabolism , Enzyme Inhibitors/chemical synthesis , HEK293 Cells , Histones/metabolism , Humans , In Vitro Techniques , Mice , Models, Molecular , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Small Molecule Libraries , Substrate SpecificityABSTRACT
The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the surface of vascular endothelial cells stimulates many steps in the angiogenic pathway. Inhibition of this interaction is proving of value in moderating the neovascularization accompanying age-related macular degeneration and in the treatment of cancer. Tissue inhibitor of metalloproteinases-3 (TIMP-3) has been shown to be a natural VEGFR-2 specific antagonist-an activity that is independent of its ability to inhibit metalloproteinases. In this investigation we localize this activity to the C-terminal domain of the TIMP-3 molecule and characterize a short peptide, corresponding to part of this domain, that not only inhibits all three VEGF-family receptors, but also fibroblast growth factor and platelet-derived growth factor receptors. This multiple-receptor inhibition may explain why the peptide was also seen to be a powerful inhibitor of tumour growth and also a partial inhibitor of arthritic joint inflammation in vivo.
Subject(s)
Arthritis/drug therapy , Mammary Neoplasms, Experimental/drug therapy , Peptides/pharmacology , Tissue Inhibitor of Metalloproteinase-3/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Animals , Arthritis/metabolism , Arthritis/pathology , Female , Human Umbilical Vein Endothelial Cells , Humans , Macular Degeneration/drug therapy , Macular Degeneration/metabolism , Macular Degeneration/pathology , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Peptides/chemistry , Tissue Inhibitor of Metalloproteinase-3/chemistry , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent and selective IKK2 inhibitor showing good efficacy in a rat model of neutrophil activation.
Subject(s)
I-kappa B Kinase/antagonists & inhibitors , Indoles/chemistry , Protein Kinase Inhibitors/chemistry , Animals , Biological Availability , Disease Models, Animal , Half-Life , Humans , I-kappa B Kinase/metabolism , Indoles/chemical synthesis , Indoles/pharmacokinetics , Lung/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.
Subject(s)
Aniline Compounds/chemistry , Aniline Compounds/therapeutic use , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Arthus Reaction/drug therapy , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Administration, Oral , Aniline Compounds/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Crystallography, X-Ray , Drug Discovery , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Models, Molecular , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/metabolism , Pyrimidines/pharmacology , Rats , Structure-Activity Relationship , Syk KinaseABSTRACT
Functionalized cyclohexanones are prepared from cyclic enol ethers via a Pd-catalyzed [1,3]-O-to-C rearrangement reaction. α-Arylketones are generated with excellent diastereocontrol when basic phosphine ligands are used. In contrast, a Lewis acid is required to promote the rearrangement of the alkyl-substituted enol ether systems.
Subject(s)
Carbon/chemistry , Cyclohexanones/chemistry , Oxygen/chemistry , Palladium/chemistry , Pyrans/chemistry , Catalysis , Ligands , Molecular StructureABSTRACT
Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the six aggrecanases (ADAMTS-1, -4, -5, -8, -9 and -15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that four of the six aggrecanases are expressed in immortalized chondrocyte cell-lines and can be upregulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knock-down of ADAMTS-5 and -9 expression in these cells and, by culturing them on three-dimensional (3D) scaffolds, show that reduction in expression of ADAMTS-5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue-engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS-5 may be a therapeutic target in the treatment of arthritic disease.
Subject(s)
ADAM Proteins/metabolism , Chondrocytes/enzymology , Extracellular Matrix/metabolism , Gene Knockdown Techniques , Lentivirus/genetics , RNA, Small Interfering/genetics , ADAM Proteins/genetics , ADAMTS5 Protein , ADAMTS9 Protein , Cell Line , Cell Line, Transformed , Cells, Cultured , Gene Expression Regulation, Enzymologic , Glycosaminoglycans/metabolism , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tissue ScaffoldsABSTRACT
Crystallography driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of quinoline-3-carboxamides as highly potent and selective inhibitors of phosphodiesterase 4B. This series has been optimized to GSK256066, a potent PDE4B inhibitor which also inhibits LPS induced production of TNF-alpha from isolated human peripheral blood mononuclear cells with a pIC(50) of 11.1. GSK256066 also has a suitable profile for inhaled dosing.
Subject(s)
Anti-Inflammatory Agents/chemistry , Phosphodiesterase 4 Inhibitors , Phosphodiesterase Inhibitors/chemistry , Quinolines/chemistry , Administration, Inhalation , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacokinetics , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Humans , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/pharmacokinetics , Quinolines/chemical synthesis , Quinolines/pharmacokinetics , Rats , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The synthesis and SAR of a novel series of IKK2 inhibitors are described. Modification around the hinge binding region of the 7-azaindole led to a series of potent and selective inhibitors with good cellular activity.
Subject(s)
Chemistry, Pharmaceutical/methods , I-kappa B Kinase/antagonists & inhibitors , Indoles/chemical synthesis , Indoles/pharmacology , Adenosine Triphosphate/chemistry , Binding Sites , Drug Design , Humans , Inhibitory Concentration 50 , Models, Chemical , Models, Molecular , Molecular Structure , Protein Binding , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 degrees C and 25 degrees C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters/one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.
Subject(s)
Collagen Type III/metabolism , Collagen Type I/metabolism , Matrix Metalloproteinase 9/metabolism , Animals , Collagen Type I/chemistry , Collagen Type III/chemistry , Culture Media, Conditioned/chemistry , DNA, Complementary , Escherichia coli/genetics , Humans , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/isolation & purification , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solubility , Spodoptera/cytology , Spodoptera/metabolism , TemperatureABSTRACT
Sorsby's fundus dystrophy (SFD) is a rare autosomal dominant disorder that results in degeneration of the macular region of the retina, with onset usually in the fourth to fifth decade of life. It leads to the rapid loss of central vision, often followed by further loss of peripheral vision. SFD shares several pathological features commonly found in the 'wet' or exudative form of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in developed countries. These phenotypic similarities have led to SFD being proposed as an acceptable genetic model for AMD. Whereas AMD appears to have a complex aetiology, with both genetic and environmental factors playing a role, SFD has been shown to be a single-gene disorder, linked to mutations in exon 5 of the tissue inhibitor of metalloproteinases 3 (TIMP3) gene on chromosome 22q12-q13. This review confines itself to a discussion of the known biochemical properties of the wild-type and SFD TIMP3 proteins and attempts to relate these to the pathology encountered in SFD patients. We also discuss briefly how, despite the lack of inherited mutations in the structural gene, the TIMP3 protein might play a role in the onset and progression of AMD.
Subject(s)
Macular Degeneration/genetics , Mutation , Tissue Inhibitor of Metalloproteinase-3/genetics , Animals , Humans , Macular Degeneration/pathology , Tissue Inhibitor of Metalloproteinase-3/chemistry , Tissue Inhibitor of Metalloproteinase-3/physiologyABSTRACT
Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the retina, caused by mutations in exon 5 of the gene for tissue inhibitor of metalloproteinases-3 (TIMP-3). The mechanism by which these mutations give rise to the disease phenotype is unknown. In an attempt to identify common properties of these molecules that might underlie the disease phenotype, a range of SFD mutants were expressed from human retinal pigment epithelial (RPE) cells. This showed that resistance to turnover, resulting from intermolecular disulfide bond formation, was a common property of all the SFD mutants examined, providing a possible explanation for the increased deposition of the protein observed in eyes from SFD patients. In contrast, SFD mutants varied in their ability to inhibit cell-surface activation of matrix metalloproteinase-2 (MMP-2), a potent mediator of angiogenesis, ranging from being fully active to totally inactive. These data show that increased deposition of active TIMP-3, rather than dysregulation of metalloproteinase inhibition, is likely to be the primary, initiating event in SFD.