ABSTRACT
Activated factor XI (FXIa) inhibitors are promising novel anticoagulants with low bleeding risk compared with current anticoagulants. The discovery of potent FXIa inhibitors with good oral bioavailability has been challenging. Herein, we describe our discovery effort, utilizing nonclassical interactions to improve potency, cellular permeability, and oral bioavailability by enhancing the binding while reducing polar atoms. Beginning with literature-inspired pyridine N-oxide-based FXIa inhibitor 1, the imidazole linker was first replaced with a pyrazole moiety to establish a polar C-H···water hydrogen-bonding interaction. Then, structure-based drug design was employed to modify lead molecule 2d in the P1' and P2' regions, with substituents interacting with key residues through various nonclassical interactions. As a result, a potent FXIa inhibitor 3f (Ki = 0.17 nM) was discovered. This compound demonstrated oral bioavailability in preclinical species (rat 36.4%, dog 80.5%, and monkey 43.0%) and displayed a dose-dependent antithrombotic effect in a rabbit arteriovenous shunt model of thrombosis.
Subject(s)
Factor XIa , Pyridines , Animals , Anticoagulants/chemistry , Anticoagulants/pharmacology , Dogs , Drug Design , Factor XIa/metabolism , Pyridines/pharmacology , Rabbits , RatsABSTRACT
G-protein coupled receptor kinase 2 (GRK2), which is upregulated in the failing heart, appears to play a critical role in heart failure (HF) progression in part because enhanced GRK2 activity promotes dysfunction of ß-adrenergic signaling and myocyte death. An orally bioavailable GRK2 inhibitor could offer unique therapeutic outcomes that cannot be attained by current heart failure treatments that directly target GPCRs or angiotensin-converting enzyme. Herein, we describe the discovery of a potent, selective, and orally bioavailable GRK2 inhibitor, 8h, through high-throughput screening, hit-to-lead optimization, structure-based design, molecular modelling, synthesis, and biological evaluation. In the cellular target engagement assays, 8h enhances isoproterenol-mediated cyclic adenosine 3',5'-monophosphate (cAMP) production in HEK293 cells overexpressing GRK2. Compound 8h was further evaluated in a human stem cell-derived cardiomyocyte (HSC-CM) contractility assay and potentiated isoproterenol-induced beating rate in HSC-CMs.
Subject(s)
G-Protein-Coupled Receptor Kinase 2/antagonists & inhibitors , Phthalazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Animals , Enzyme Assays , G-Protein-Coupled Receptor Kinase 2/metabolism , HEK293 Cells , Humans , Mice, Inbred C57BL , Molecular Docking Simulation , Molecular Structure , Myocytes, Cardiac/drug effects , Phthalazines/chemical synthesis , Phthalazines/pharmacokinetics , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Quinazolines/chemical synthesis , Quinazolines/metabolism , Quinazolines/pharmacokinetics , Structure-Activity RelationshipABSTRACT
We screen for fragments using X-ray crystallography as the primary screen. There are several unique features in our screening methodology. As a result of using X-ray diffraction as our primary screen, we do not use affinity data to bias our data collection or design in progressing hits toward a lead. Another difference in our methodology is that we choose to group our compounds as shape-similar groups. We also screen in a first pass mode without recollecting failed diffraction experiments. This method of screening results in an average loss of 5-10% of the data sets for the primary screen. The remaining data sets offer enough information to successfully advance three to five scaffolds into the secondary library design. We do not deconvolute the wells which show evidence of fragment binding by repeating the soaks with single compounds. Instead, evaluation of the possible fragments is done by refinement and examination of the resulting electron density difference maps. These methods allow us to complete the initial screen of a primary library of fragments in less than 3 months. A secondary library of fragments is designed using the base structures with electron density envelopes from the successful fragment hits of the primary library. Chemistry is chosen to probe interactions with the target and push the observed binding pocket limits in order to more clearly define the plasticity and range of possible extensions to the scaffolds chosen. The secondary library compounds are also screened in shape-similar groupings of five that are chosen without the knowledge of binding affinity. Our approach is a completely orthogonal one from traditional high-throughput screening in finding novel compounds.
Subject(s)
Crystallography, X-Ray , Drug Evaluation, Preclinical/methods , Proteins/chemistry , Fatty Acid-Binding Proteins/chemistry , Humans , Models, Molecular , Protein Binding , Small Molecule Libraries , SoftwareABSTRACT
2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K(i)=1.2nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.
Subject(s)
Acetamides , Amino Acid Motifs , Anticoagulants/administration & dosage , Drug Design , Nitriles , Thrombin/antagonists & inhibitors , Thrombosis/drug therapy , Acetamides/chemical synthesis , Acetamides/pharmacokinetics , Acetamides/therapeutic use , Administration, Oral , Animals , Anticoagulants/chemical synthesis , Anticoagulants/pharmacokinetics , Binding Sites , Disease Models, Animal , Dogs , Dose-Response Relationship, Drug , Haplorhini , Humans , Hydrogen Bonding , Models, Chemical , Nitriles/chemical synthesis , Nitriles/pharmacokinetics , Nitriles/therapeutic use , Rats , Structure-Activity RelationshipABSTRACT
We have explored a series of spirocyclic piperidine amide derivatives (5) as tryptase inhibitors. Thus, 4 (JNJ-27390467) was identified as a potent, selective tryptase inhibitor with oral efficacy in two animal models of airway inflammation (sheep and guinea pig asthma models). An X-ray co-crystal structure of 4 x tryptase revealed a hydrophobic pocket in the enzyme's active site, which is induced by the phenylethynyl group and is comprised of amino acid residues from two different monomers of the tetrameric protein.
Subject(s)
Asthma/drug therapy , Respiratory Hypersensitivity/drug therapy , Serine Proteinase Inhibitors/pharmacology , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacology , Tryptases/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Cytochrome P-450 Enzyme Inhibitors , Disease Models, Animal , Dogs , Guinea Pigs , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Molecular Structure , Rats , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacokinetics , Sheep , Spectrometry, Mass, Electrospray Ionization , Spiro Compounds/pharmacokinetics , Trypsin/metabolism , Tryptases/metabolismABSTRACT
The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.
Subject(s)
Protein Kinase Inhibitors/chemistry , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/chemistry , Receptor, Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptor, Macrophage Colony-Stimulating Factor/chemistry , Amides/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Humans , Molecular Sequence Data , Mutant Chimeric Proteins/antagonists & inhibitors , Mutant Chimeric Proteins/chemistry , Protein Structure, Tertiary/genetics , Proto-Oncogene Mas , Quinolones/chemistry , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Macrophage Colony-Stimulating Factor/genetics , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Receptor, TIE-2/chemistry , Receptor, TIE-2/genetics , Receptors, Fibroblast Growth Factor/chemistry , Receptors, Fibroblast Growth Factor/geneticsABSTRACT
The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.
Subject(s)
Chemistry, Pharmaceutical/methods , Crystallography, X-Ray/methods , Dipeptidyl Peptidase 4/chemistry , Enzyme Inhibitors/pharmacology , Protease Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacology , Binding Sites , Blood Glucose/metabolism , Caco-2 Cells , Dipeptidyl Peptidase 4/metabolism , Drug Design , Drug Screening Assays, Antitumor , Humans , Kinetics , Mice , Models, Chemical , Models, Molecular , Rats , Structure-Activity RelationshipABSTRACT
HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.
Subject(s)
Benzodiazepines/chemical synthesis , Nuclear Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/agonists , Benzodiazepines/chemistry , Benzodiazepines/pharmacology , Binding Sites , Cell Line, Tumor , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Mimicry , Molecular Structure , Proto-Oncogene Proteins c-mdm2 , Stereoisomerism , Structure-Activity Relationship , Tumor Suppressor Protein p53/biosynthesisABSTRACT
Activation of the classical pathway of complement has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury and acute transplant rejection. The trypsin-like serine protease C1s represents a pivotal upstream point of control in the classical pathway of complement activation and is therefore likely to be a useful target in the therapeutic intervention of these disease states. A series of thiopheneamidine-based inhibitors of C1s has been optimized to give a 70 nM inhibitor that inhibits the classical pathway of complement activation in vitro.
Subject(s)
Complement Inactivator Proteins/chemical synthesis , Complement Pathway, Classical/drug effects , Pyrazoles/chemical synthesis , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemical synthesis , Thiazoles/chemical synthesis , Thiophenes/chemical synthesis , Binding Sites/physiology , Complement C1/metabolism , Complement Inactivator Proteins/pharmacology , Complement Pathway, Classical/physiology , Humans , Myocardial Ischemia/drug therapy , Myocardial Ischemia/enzymology , Pyrazoles/pharmacology , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Thiazoles/pharmacology , Thiophenes/pharmacologyABSTRACT
A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.