ABSTRACT
Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with good potency and good in vivo pharmacokinetic properties.
Subject(s)
Amides/chemistry , Complement C1s/antagonists & inhibitors , Drug Design , Polyethylene Glycols/chemistry , Protease Inhibitors/chemical synthesis , Thiophenes/chemistry , Animals , Complement C1s/metabolism , Half-Life , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacokinetics , RatsABSTRACT
We have identified RWJ-671818 (8) as a novel, low molecular weight, orally active inhibitor of human alpha-thrombin (K(i) = 1.3 nM) that is potentially useful for the acute and chronic treatment of venous and arterial thrombosis. In a rat deep venous thrombosis model used to assess antithrombotic efficacy, oral administration of 8 at 30 and 50 mg/kg reduced thrombus weight by 87 and 94%, respectively. In an anesthetized rat antithrombotic model, where electrical stimulation of the carotid artery created a thrombus, 8 prolonged occlusion time 2- and 3-fold at 0.1 and 1.0 mg/kg, i.v., respectively, and more than doubled activated clotting time and activated partial thromboplastin time at the higher dose. This compound had excellent oral bioavailability of 100% in dogs with an estimated half-life of approximately 3 h. On the basis of its noteworthy preclinical data, 8 was advanced into human clinical trials and successfully progressed through phase 1 studies.
Subject(s)
Anticoagulants/chemical synthesis , Fibrinolytic Agents/chemical synthesis , Guanidines/chemical synthesis , Pyrazines/chemical synthesis , Thrombin/antagonists & inhibitors , Amino Acid Motifs , Animals , Anticoagulants/pharmacokinetics , Anticoagulants/pharmacology , Blood Pressure/drug effects , Caco-2 Cells , Crystallography, X-Ray , Cytochrome P-450 CYP3A , Cytochrome P-450 CYP3A Inhibitors , Dogs , Double-Blind Method , Electrocardiography , Female , Fibrinolytic Agents/pharmacokinetics , Fibrinolytic Agents/pharmacology , Guanidines/pharmacokinetics , Guanidines/pharmacology , Guinea Pigs , Heart Rate/drug effects , Hemodynamics/drug effects , Humans , In Vitro Techniques , Male , Microsomes, Liver/metabolism , Models, Molecular , Pyrazines/pharmacokinetics , Pyrazines/pharmacology , Rats , Rats, Sprague-Dawley , Recombinant Proteins/antagonists & inhibitors , Structure-Activity Relationship , Thrombin/chemistry , Venous Thrombosis/blood , Venous Thrombosis/drug therapyABSTRACT
Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement therapy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical pathway of complement is described.
Subject(s)
Complement C1s/antagonists & inhibitors , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Animals , Binding Sites , Half-Life , Models, Molecular , Molecular Structure , Rats , Structure-Activity RelationshipABSTRACT
The pharmacokinetics of TDP4815 was evaluated in rats, rabbits, dogs and monkeys. After intravenous administration, TDP4815 achieved C(O) of 3255 ng/ml in rats at 5 mg/kg, 9066 ng/ml in rabbits and 7858 ng/ml in monkeys at 6 mg/kg, and 4457 ng/ml in dogs at 3 mg/kg. The clearance (C(L)) was 3105, 1692, 835 and 640 ml/h/kg in rats, rabbits, monkeys and dogs, respectively. The volume of distribution (V(Z)) was more than 3861 ml/kg in all species, except 1915 ml/kg in monkeys. The oral bioavailability was rabbit >rat> monkey compared at 100 mg/kg, but it was much higher in dogs (>64%) after oral administrations. The calculated intrinsic clearance data suggested that the clearance of dog and human was restricted by binding to the plasma protein, and the clearance of rat and monkey was dependent on both the free fraction of plasma protein binding and the liver blood flow rate. The unbound hepatic intrinsic clearance of monkey was close to its C(L) suggesting that the hepatic clearance was an important excretion in monkeys. The poor oral bioavailability in the monkey may be related to the extensive glucuronidation. The V(Z).kg and C(L).kg in test species showed good correlation with the animal body weights (R(2)=0.87 and 0.96).
Subject(s)
Anticoagulants/pharmacokinetics , Guanidine/analogs & derivatives , Administration, Oral , Animals , Anticoagulants/administration & dosage , Biological Availability , Body Weight , Dogs , Glucuronides/metabolism , Guanidine/administration & dosage , Guanidine/pharmacokinetics , Humans , In Vitro Techniques , Injections, Intravenous , Liver/blood supply , Liver/metabolism , Macaca fascicularis , Male , Microsomes, Liver/metabolism , Protein Binding , Rabbits , Rats , Rats, Sprague-Dawley , Species Specificity , Tissue DistributionABSTRACT
Inhibiting the classical pathway of complement activation by attenuating the proteolytic activity of the serine protease C1s is a potential strategy for the therapeutic intervention in disease states such as hereditary angioedema, ischemia-reperfusion injury, and acute transplant rejection. A series of arylsulfonylthiophene-2-carboxamidine inhibitors of C1s were synthesized and evaluated for C1s inhibitory activity. The most potent compound had a Ki of 10nM and >1000-fold selectivity over uPA, tPA, FX(a), thrombin, and plasmin.
Subject(s)
Arylsulfonates/chemical synthesis , Complement C1s/antagonists & inhibitors , Serine Proteinase Inhibitors/chemical synthesis , Amidines/chemical synthesis , Amidines/pharmacology , Angioedema/drug therapy , Arylsulfonates/pharmacology , Fibrinolysin/pharmacology , Graft Rejection/drug therapy , Humans , Myocardial Ischemia/drug therapy , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Tetradecanoylphorbol Acetate/pharmacology , Thiophenes/chemical synthesis , Thiophenes/pharmacology , Thrombin/pharmacology , Urokinase-Type Plasminogen Activator/pharmacologyABSTRACT
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
The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.
Subject(s)
Bacterial Proteins/physiology , Genes, Essential/physiology , Nucleoside Diphosphate Sugars/metabolism , Streptococcus pneumoniae/genetics , Transaminases/physiology , Amino Acid Sequence , Benzopyrans/metabolism , Dimerization , Furans/metabolism , Ligands , Molecular Sequence Data , Pyridoxal Phosphate/metabolism , Pyridoxamine/metabolism , Streptococcus pneumoniae/enzymologyABSTRACT
Through structure-based drug design and parallel synthesis, we have discovered a novel series of nonpeptidic phenyl-based thrombin inhibitors using oxyguanidines as guanidine bioisosteres. These compounds have been found to be highly potent, highly selective, and orally bioavailable.
Subject(s)
Anticoagulants/chemical synthesis , Drug Design , Guanidines/chemical synthesis , Thrombin/antagonists & inhibitors , Administration, Oral , Animals , Anticoagulants/pharmacokinetics , Binding Sites , Cell Line , Crystallography, X-Ray , Dogs , Guanidines/pharmacokinetics , Humans , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacokinetics , Structure-Activity RelationshipABSTRACT
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
Although thrombin has been extensively researched with many examples of potent and selective inhibitors, the key characteristics of oral bioavailability and long half-life have been elusive. We report here a novel series non-peptidic phenyl-based, highly potent, highly selective and orally bioavailable thrombin inhibitors using oxyguanidines as guanidine-mimetics.
Subject(s)
Benzene Derivatives/chemistry , Benzene Derivatives/pharmacology , Guanidines/chemistry , Guanidines/pharmacology , Thrombin/antagonists & inhibitors , Administration, Oral , Animals , Benzene Derivatives/pharmacokinetics , Biological Availability , Caco-2 Cells , Dogs , Guanidines/pharmacokinetics , Humans , Mice , Microsomes/drug effects , Rats , Serine Endopeptidases/drug effects , Serine Proteinase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
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.