ABSTRACT
Several P4 domain derivatives of the general d-phenylglycinamide-based scaffold (2) were synthesized and evaluated for their ability to bind to the serine protease factor Xa. Some of the more potent compounds were evaluated for their anticoagulant effects in vitro. A select subset containing various P1 indole constructs was further evaluated for their pharmacokinetic properties after oral administration to rats.
Subject(s)
Antithrombin III/chemical synthesis , Antithrombin III/pharmacology , Glycine/analogs & derivatives , Anticoagulants/chemical synthesis , Anticoagulants/chemistry , Anticoagulants/pharmacology , Antithrombin III/chemistry , Crystallography, X-Ray , Factor Xa/chemistry , Factor Xa/metabolism , Glycine/chemical synthesis , Glycine/chemistry , Glycine/pharmacology , Humans , Models, Molecular , Molecular Structure , Protein Binding , Structure-Activity RelationshipABSTRACT
Analogs to a series of D-phenylglycinamide-derived factor Xa inhibitors were discovered. It was found that the S4 amide linkage can be replaced with an ether linkage to reduce the peptide character of the molecules and that this substitution leads to an increase in binding affinity that is not predicted based on modeling. Inhibitors which incorporate ether, amino, or alkyl S4 linkage motifs exhibit similar levels of binding affinity and also demonstrate potent in vitro functional activity, however, binding affinity in this series is strongly dependent on the nature of the S1 binding element.
Subject(s)
Anticoagulants/pharmacology , Factor Xa Inhibitors , Glycine/analogs & derivatives , Serine Proteinase Inhibitors/pharmacology , Anticoagulants/chemistry , Crystallography, X-Ray , Ethanolamines , Glycine/chemistry , Models, Molecular , Peptides/chemistry , Serine Proteinase Inhibitors/chemistryABSTRACT
The authors show by illustration that procedures used to validate the reliability of single-concentration high-throughput screens such as the signal window and Z' factor do not ensure sufficient reliability in potency estimates from concentration response assays. They develop the minimum significant ratio as a statistical parameter to characterize the fold change between 2 compounds run in the same experiment that can be considered a real difference and use this parameter to characterize the reliability of the assay. They adapt methods described by Bland and Altman to develop a simple set of 2 experiments to estimate the minimum significant ratio and show that this protocol can identify assays that lack reproducibility. The methods are then extended to validate the equivalency of the same assay run by multiple laboratories.
Subject(s)
Models, Statistical , Reproducibility of Results , Factor Xa/metabolism , Factor Xa InhibitorsABSTRACT
Several non-amidino S1 derivatives of the 1,2-diaminobenzene-based scaffold (4) were synthesized and evaluated for their ability to bind to the active site and inhibit the human protease factor Xa. A subset of these compounds were also evaluated for their anticoagulant effects in human plasma as measured by prothrombin time (PT).
Subject(s)
Anticoagulants/chemical synthesis , Benzene Derivatives/chemical synthesis , Factor Xa Inhibitors , Anticoagulants/pharmacology , Benzene Derivatives/pharmacology , Binding Sites , Blood Coagulation/drug effects , Factor Xa/metabolism , Humans , Models, Molecular , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Protein Binding , Prothrombin Time , Structure-Activity RelationshipABSTRACT
Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.
Subject(s)
Anisomycin/antagonists & inhibitors , Anisomycin/chemistry , Cytokines/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Muscle Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Pyrazoles/pharmacology , Quinolines/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Anisomycin/pharmacology , Apoptosis , Blotting, Western , COS Cells , Catalysis , DNA Fragmentation , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Enzyme Inhibitors/pharmacology , Glutathione Transferase/metabolism , Humans , Inhibitory Concentration 50 , Interleukin-1/metabolism , MAP Kinase Kinase 4 , MAP Kinase Kinase Kinases/metabolism , Mice , Models, Chemical , Muscle Proteins/metabolism , Myocardium/metabolism , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Plasmids/metabolism , Protein Serine-Threonine Kinases/metabolism , Pyrazoles/chemistry , Quinolines/chemistry , RNA, Small Interfering/metabolism , Signal Transduction , Time Factors , Transfection , Transgenes , Tumor Necrosis Factor-alpha/metabolism , Ultraviolet RaysABSTRACT
A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group substitution effects on inhibition of the coagulation cascade serine protease factor Xa.
