ABSTRACT
The structure activity relationship of the prime region of conformationally restricted hydroxyethylamine (HEA) BACE inhibitors is described. Variation of the P1' region provided selectivity over Cat-D with a series of 2,2-dioxo-isothiochromanes and optimization of the P2' substituent of chromane-HEA(s) with polar substituents provided improvements in the compound's in vitro permeability. Significant potency gains were observed with small aliphatic substituents such as methyl, n-propyl, and cyclopropyl when placed at the C-2 position of the chromane.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Chromans/chemistry , Drug Design , Enzyme Inhibitors/chemical synthesis , Binding Sites , Cells, Cultured , Ethylamines/chemical synthesis , Ethylamines/chemistry , Ethylamines/pharmacology , Inhibitory Concentration 50 , Models, Molecular , Structure-Activity RelationshipABSTRACT
Polo-like kinase-2 (Plk-2) has been implicated as the dominant kinase involved in the phosphorylation of α-synuclein in Lewy bodies, which are one of the hallmarks of Parkinson's disease neuropathology. Potent, selective, brain-penetrant inhibitors of Plk-2 were obtained from a structure-guided drug discovery approach driven by the first reported Plk-2-inhibitor complexes. The best of these compounds showed excellent isoform and kinome-wide selectivity, with physicochemical properties sufficient to interrogate the role of Plk-2 inhibition inâ vivo. One such compound significantly decreased phosphorylation of α-synuclein in rat brain upon oral administration and represents a useful probe for future studies of this therapeutic avenue toward the potential treatment of Parkinson's disease.
Subject(s)
Brain/metabolism , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , alpha-Synuclein/metabolism , Animals , Binding Sites , Blood-Brain Barrier/metabolism , Female , HEK293 Cells , Half-Life , Humans , Male , Mice , Molecular Dynamics Simulation , Phosphorylation/drug effects , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Rats , Rats, Sprague-DawleyABSTRACT
Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aß generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aß generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aß in the CSF of healthy human volunteers.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds, 3-Ring/pharmacology , Pyrazoles/pharmacology , Quinolines/pharmacology , Receptors, Notch/antagonists & inhibitors , Sulfonamides/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/cerebrospinal fluid , Amyloid beta-Peptides/metabolism , Animals , Area Under Curve , Basic Helix-Loop-Helix Transcription Factors/genetics , Dogs , Dose-Response Relationship, Drug , Drug Design , Drug Stability , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Gene Expression/drug effects , Heterocyclic Compounds, 3-Ring/chemistry , Homeodomain Proteins/genetics , Humans , Male , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Models, Chemical , Molecular Structure , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Quinolines/chemical synthesis , Quinolines/pharmacokinetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Notch/metabolism , Structure-Activity Relationship , Sulfonamides/chemistry , Time Factors , Transcription Factor HES-1ABSTRACT
Utilizing a structure based design approach, combined with extensive medicinal chemistry execution, highly selective, potent and novel BACE1 inhibitor 8 (BACE1 Alpha assay IC50=8nM) was made from a weak µM potency hit in an extremely efficient way. The detailed SAR and general design approaches will be discussed.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Drug Design , Isoquinolines/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Thiophenes/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Dose-Response Relationship, Drug , Humans , Isoquinolines/chemistry , Models, Molecular , Molecular Structure , Protease Inhibitors/chemistry , Structure-Activity Relationship , Thiophenes/chemistryABSTRACT
Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familial Parkinson's disease (PD). The kinase activity of this complex protein is increased by pathogenic mutations. Inhibition of LRRK2 kinase activity has therefore emerged as a promising approach for the treatment of PD. Herein we report our findings on a series of 4-alkylamino-7-aryl-3-cyanoquinolines that exhibit kinase inhibitory activity against both wild type and G2019S mutant LRRK2. Activity was determined in both biochemical and cellular assays. Compound 14 was further evaluated in an in vivo pharmacodynamic study and found to significantly inhibit Ser935 phosphorylation after oral dosing.
Subject(s)
Drug Discovery , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinolines/pharmacology , Animals , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice , Mice, Knockout , Mice, Transgenic , Models, Molecular , Molecular Structure , Mutation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Quinolines/chemical synthesis , Quinolines/chemistry , Structure-Activity RelationshipABSTRACT
Polo-like kinase-2 (Plk-2) is a potential therapeutic target for Parkinson's disease and this Letter describes the SAR of a series of dihydropteridinone based Plk-2 inhibitors. By optimizing both the N-8 substituent and the biaryl region of the inhibitors we obtained single digit nanomolar compounds such as 37 with excellent selectivity for Plk-2 over Plk-1. When dosed orally in rats, compound 37 demonstrated a 41-45% reduction of pS129-α-synuclein levels in the cerebral cortex.
Subject(s)
Drug Design , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Administration, Oral , Animals , Brain/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , HEK293 Cells , Half-Life , Humans , Mice , Microsomes, Liver/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Pteridines/chemical synthesis , Pteridines/chemistry , Pteridines/pharmacokinetics , Rats , Structure-Activity Relationship , Polo-Like Kinase 1ABSTRACT
The structure-activity relationship of a series of dihydroisoquinoline BACE-1 inhibitors is described. Application of structure-based design to screening hit 1 yielded sub-micromolar inhibitors. Replacement of the carboxylic acid of 1 was guided by X-ray crystallography, which allowed the replacement of a key water-mediated hydrogen bond. This work culminated in compounds such as 31, which possess good BACE-1 potency, excellent permeability and a low P-gp efflux ratio.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid/chemistry , Drug Design , Isoquinolines/pharmacology , Protease Inhibitors/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Catalysis , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Models, Molecular , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
The SAR of a series of brain penetrant, trisubstituted thiophene based JNK inhibitors with improved pharmacokinetic properties is described. These compounds were designed based on information derived from metabolite identification studies which led to compounds such as 42 with lower clearance, greater brain exposure and longer half life compared to earlier analogs.
Subject(s)
Brain/metabolism , Drug Design , Nerve Degeneration/prevention & control , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Thiophenes/pharmacology , Thiophenes/pharmacokinetics , Animals , Chemistry Techniques, Synthetic , Crystallography, X-Ray , Dose-Response Relationship, Drug , Half-Life , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Stereoisomerism , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/chemistryABSTRACT
In this Letter, we describe the evolution of selective JNK3 inhibitors from 1, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs. Strong SAR was found for substitution of the naphthalene ring, as well as for inhibitors adopting different central scaffolds. Significant potency gains were appreciated by inverting the polarity of the thione of the parent triazolothione 1, resulting in potent compounds with attractive pharmacokinetic profiles.
Subject(s)
Enzyme Inhibitors/pharmacology , Mitogen-Activated Protein Kinase 10/antagonists & inhibitors , Naphthalenes/chemical synthesis , Thiones/chemical synthesis , Animals , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Mice , Microsomes, Liver/enzymology , Molecular Structure , Naphthalenes/chemistry , Naphthalenes/pharmacology , Solubility , Structure-Activity Relationship , Thiones/chemistry , Thiones/pharmacologyABSTRACT
The SAR of a series of tri-substituted thiophene JNK3 inhibitors is described. By optimizing both the N-aryl acetamide region of the inhibitor and the 4-position of the thiophene we obtained single digit nanomolar compounds, such as 47, which demonstrated an in vivo effect on JNK activity when dosed orally in our kainic acid mouse model as measured by phospho-c-jun reduction.
Subject(s)
Brain/metabolism , MAP Kinase Kinase 4/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Administration, Oral , Drug Design , Hydrogen Bonding , Models, Molecular , Protein Kinase Inhibitors/chemical synthesis , Structure-Activity RelationshipABSTRACT
In this Letter, we describe the discovery of selective JNK2 and JNK3 inhibitors, such as 10, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs, p38α and ERK2. Substitution of the naphthalene ring affords an isoform selective JNK3 inhibitor, 30, with approximately 10-fold selectivity over both JNK1 and JNK2. A naphthalene ring penetrates deep into the selectivity pocket accounting for the differentiation amongst the kinases. Interestingly, the gatekeeper Met146 sulfide interacts with the naphthalene ring in a sulfur-π stacking interaction. Compound 38 ameliorates neurotoxicity induced by amyloid-ß in human cortical neurons. Lastly, we demonstrate how to install propitious in vitro CNS-like properties into these selective inhibitors.
Subject(s)
Aminopyridines/chemistry , Mitogen-Activated Protein Kinase 10/antagonists & inhibitors , Mitogen-Activated Protein Kinase 9/antagonists & inhibitors , Neurodegenerative Diseases/drug therapy , Neuroprotective Agents/chemistry , Protein Kinase Inhibitors/chemistry , Triazines/chemistry , Aminopyridines/pharmacokinetics , Aminopyridines/therapeutic use , Animals , Binding Sites , Central Nervous System/metabolism , Computer Simulation , Humans , Mice , Microsomes, Liver/metabolism , Mitogen-Activated Protein Kinase 10/metabolism , Mitogen-Activated Protein Kinase 9/metabolism , Neuroprotective Agents/pharmacokinetics , Neuroprotective Agents/therapeutic use , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Structure-Activity Relationship , Triazines/pharmacokinetics , Triazines/therapeutic useABSTRACT
INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering Aß production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP γ-site cleavage (equivalent to Aß40), and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aß production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aß was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aß reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aß production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aß in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aß was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.
ABSTRACT
In this Letter, we describe our efforts to design HEA BACE-1 inhibitors that are highly permeable coupled with negligible levels of permeability-glycoprotein activity. These efforts culminate in producing 16 which lowers Αß by 28% and 32% in the cortex and CSF, respectively, in the preclinical wild type Hartley guinea pig animal model when dosed orally at 30mpk BID for 2.5days.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Ethylamines/chemical synthesis , Ethylamines/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Alkylation , Alzheimer Disease , Animals , Brain/metabolism , Cell Line , Dogs , Drug Design , Guinea Pigs , Humans , Indicators and Reagents , Protease Inhibitors/pharmacokinetics , Protein Binding , Structure-Activity RelationshipABSTRACT
The structure-activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Ethylamines/chemistry , Ethylamines/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/chemistry , Aspartic Acid Endopeptidases/chemistry , Crystallography, X-Ray , Ethylamines/chemical synthesis , Humans , Models, Molecular , Protease Inhibitors/chemical synthesis , Protein Binding , Structure-Activity RelationshipABSTRACT
Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2' position of the inhibitor with more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic analog 1 but with much higher observed passive permeability in MDCK cellular assay.
Subject(s)
Acetamides/chemistry , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Butanols/chemistry , Cyclohexylamines/chemistry , Protease Inhibitors/chemistry , Acetamides/chemical synthesis , Acetamides/pharmacokinetics , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Binding Sites , Butanols/chemical synthesis , Butanols/pharmacokinetics , Cell Membrane Permeability/drug effects , Crystallography, X-Ray , Cyclohexylamines/chemical synthesis , Cyclohexylamines/pharmacokinetics , Humans , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Using structure-guided design, hydroxyethylamine BACE-1 inhibitors were optimized to nanomolar Abeta cellular inhibition with selectivity against cathepsin-D. X-ray crystallography illuminated the S1' residues critical to this effort, which culminated in compounds 56 and 57 that exhibited potency and selectivity but poor permeability and high P-gp efflux.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Drug Design , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Amyloid Precursor Protein Secretases/chemistry , Amyloid Precursor Protein Secretases/genetics , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Humans , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The bradykinin B(1) receptor plays a critical role in chronic pain and inflammation, although efforts to demonstrate efficacy of receptor antagonists have been hampered by species-dependent potency differences, metabolic instability, and low oral exposure of current agents. The pharmacology, pharmacokinetics, and analgesic efficacy of the novel benzamide B(1) receptor antagonist 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1-one (ELN441958) is described. ELN441958 competitively inhibited the binding of the B(1) agonist ligand [(3)H]desArg(10)-kallidin ([(3)H]DAKD) to IMR-90 human fibroblast membranes with high affinity (K(i) = 0.26 +/- 0.02 nM). ELN441958 potently antagonized DAKD (but not bradykinin)-induced calcium mobilization in IMR-90 cells, indicating that it is highly selective for B(1) over B(2) receptors. Antagonism of agonist-induced calcium responses at B(1) receptors from different species indicated that ELN441958 is selective for primate over rodent B(1) receptors with a rank order potency (K(B), nanomolar) of human (0.12 +/- 0.02) approximately rhesus monkey (0.24 +/- 0.01) > rat (1.5 +/- 0.4) > mouse (14 +/- 4). ELN441958 had good permeability and metabolic stability in vitro consistent with high oral exposure and moderate plasma half-lives in rats and rhesus monkeys. Because ELN441958 is up to 120-fold more potent at primate than at rodent B(1) receptors, it was evaluated in a primate pain model. ELN441958 dose-dependently reduced carrageenan-induced thermal hyperalgesia in a rhesus monkey tail-withdrawal model, with an ED(50) approximately 3 mg/kg s.c. Naltrexone had no effect on the antihyperalgesia produced by ELN441958, indicating a lack of involvement of opioid receptors. ELN441958 is a novel small molecule bradykinin B(1) receptor antagonist exhibiting high oral bioavailability and potent systemic efficacy in rhesus monkey inflammatory pain.
Subject(s)
Analgesics/pharmacology , Bradykinin B1 Receptor Antagonists , Spiro Compounds/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Analgesics/chemistry , Analgesics/pharmacokinetics , Animals , Bradykinin/analogs & derivatives , Bradykinin/pharmacology , Calcium/metabolism , Carrageenan/toxicity , Cell Line , Cell Membrane Permeability , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Kallidin/analogs & derivatives , Kallidin/metabolism , Kallidin/pharmacology , Macaca mulatta , Mice , Mice, Knockout , Microsomes, Liver/metabolism , Molecular Structure , Naltrexone/pharmacology , Naproxen/pharmacology , Naproxen/therapeutic use , Narcotic Antagonists , Rats , Receptor, Bradykinin B1/genetics , Receptor, Bradykinin B1/metabolism , Species Specificity , Spiro Compounds/chemistry , Spiro Compounds/pharmacokinetics , TransfectionABSTRACT
The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the development and progression of several human cancers and are attractive targets for cancer therapy. PHA-665752 was identified as a small molecule, ATP-competitive, active-site inhibitor of the catalytic activity of c-Met kinase (K(i) 4 nM). PHA-665752 also exhibited >50-fold selectivity for c-Met compared with a panel of diverse tyrosine and serine-threonine kinases. In cellular studies, PHA-665752 potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes such as cell growth (proliferation and survival), cell motility, invasion, and/or morphology of a variety of tumor cells. In addition, PHA-665752 inhibited HGF-stimulated or constitutive phosphorylation of mediators of downstream signal transduction of c-Met, including Gab-1, extracellular regulated kinase, Akt, signal transducer and activator of transcription 3, phospholipase C gamma, and focal adhesion kinase, in multiple tumor cell lines in a pattern correlating to the phenotypic response of a given tumor cell. In in vivo studies, a single dose of PHA-665752 inhibited c-Met phosphorylation in tumor xenografts for up to 12 h. Inhibition of c-Met phosphorylation was associated with dose-dependent tumor growth inhibition/growth delay over a repeated administration schedule at well-tolerated doses. Interestingly, potent cytoreductive activity was demonstrated in a gastric carcinoma xenograft model. Collectively, these results demonstrate the feasibility of selectively targeting c-Met with ATP-competitive small-molecules and suggest the therapeutic potential of targeting c-Met in human cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Sulfones/pharmacology , Animals , Cell Line , Cell Line, Tumor , Dogs , Drug Screening Assays, Antitumor , Female , Humans , Kidney/cytology , Kidney/drug effects , Kidney/enzymology , Kinetics , Lung Neoplasms/drug therapy , Lung Neoplasms/enzymology , Mice , Mice, Nude , NIH 3T3 Cells , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/enzymology , Phosphorylation/drug effects , Proto-Oncogene Proteins c-met/metabolism , Proto-Oncogene Proteins c-met/physiology , Rats , Signal Transduction/drug effects , Stomach Neoplasms/drug therapy , Stomach Neoplasms/enzymology , Xenograft Model Antitumor AssaysABSTRACT
Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.