ABSTRACT
Based on X-ray crystal structure information, mono charged phosphinate isosteres of phosphotyrosine have been designed and incorporated in a short inhibitory peptide sequence of the Grb2-SH2 domain. The resulting compounds, by exploiting additional interactions, inhibit binding to the Grb2-SH2 domain as potently as the corresponding doubly charged (phosphonomethyl)phenylalanine analogue.
Subject(s)
Adaptor Proteins, Signal Transducing , Oligopeptides/chemical synthesis , Phosphinic Acids/chemical synthesis , Phosphotyrosine/analogs & derivatives , Phosphotyrosine/chemical synthesis , Proteins/antagonists & inhibitors , Proteins/chemistry , Binding Sites , Crystallography, X-Ray , Drug Design , GRB2 Adaptor Protein , Hydrogen Bonding , Ligands , Oligopeptides/chemistry , Oligopeptides/pharmacology , Phosphinic Acids/chemistry , Phosphinic Acids/pharmacology , Phosphotyrosine/chemistry , Structure-Activity Relationship , src Homology DomainsABSTRACT
A series of novel phosphinates, derived from 4-phosphonomethylphenylalanine, are described as isosteres of phosphotyrosine. Benzyl (or alkyl) phosphinomethylphenylalanine derivatives were prepared by alkylation of an amino acid P-H phosphinate.
Subject(s)
Adaptor Proteins, Signal Transducing , Phenylalanine/analogs & derivatives , Phosphinic Acids/chemical synthesis , Phosphotyrosine/analogs & derivatives , Proteins/antagonists & inhibitors , Proteins/chemistry , Amino Acids , Drug Design , GRB2 Adaptor Protein , Molecular Structure , Phosphinic Acids/chemistry , Phosphinic Acids/pharmacology , Phosphotyrosine/chemical synthesis , Phosphotyrosine/chemistry , Structure-Activity Relationship , src Homology DomainsABSTRACT
A series of monocyclic and bicyclic amino acids have been synthesised and incorporated into thrombin inhibitors based on CGH728, an analogue of the Mitsubishi compound MD805. Benzthiazolylalanine (Bta) was found to be a good non-polar substitute for arginine at the P1 position, yielding compounds with low nanomolar potency and good selectivity for thrombin.
Subject(s)
Antithrombins/chemical synthesis , Arginine , Pipecolic Acids/chemistry , Pipecolic Acids/chemical synthesis , Serine Proteinase Inhibitors/chemical synthesis , Alanine , Antithrombins/chemistry , Antithrombins/pharmacology , Chymotrypsin/antagonists & inhibitors , Drug Design , Factor Xa Inhibitors , Fibrinolysin/antagonists & inhibitors , Kallikreins/antagonists & inhibitors , Models, Molecular , Molecular Structure , Pipecolic Acids/pharmacology , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Sulfonamides , Thiazoles , Thrombin/metabolism , Trypsin/metabolismABSTRACT
Mitsubishi's MD-805, a potent and selective inhibitor of thrombin which contains four stereogenic centers, has been the starting point for an optimization program. A systematic synthetic study resulted in thrombin inhibitors achiral at P2 and P3 but with a 10-fold increase in potency over the original lead. A number of 4-substituted piperidines were synthesized and examined as replacements for 2-carboxy-4-methylpiperidine at P2; 4-fluoroethylpiperidine (FEP) among others provided inhibitors (e.g. 45g) of increased potency. An enantioselective route was developed to 3(R)-methyl-1,2,3,4-tetrahydroquinolinesulfonyl chloride. Inhibitors containing this enantiomerically pure P3 (42d) had similar potency to the racemic material and provided support, with modeling studies, for the preparation of the gem 3,3-disubstituted compounds. A series of inhibitors containing the novel 3, 3-dimethyl-1,2,3,4-tetrahydroquinolinesulfonyl (DMTHQS) P3 (Table 5) were synthesized and showed a similar activity profile as the monomethyl series. The combination of P3-DMTHQS, P2-FEP, and P1-arginine (45g) had a K(i) of 6 nM (MD-805 K(i) = 85 nM). In animal models of both venous and arterial thrombosis, one inhibitor (42e) was shown to produce a dose-dependent inhibition of thrombus formation that in some situations was superior to that of MD-805.
Subject(s)
Antithrombins/chemical synthesis , Pipecolic Acids/chemistry , Piperidines/chemical synthesis , Thrombin/antagonists & inhibitors , Animals , Antithrombins/administration & dosage , Antithrombins/chemistry , Antithrombins/pharmacology , Arginine/analogs & derivatives , Cattle , Drug Design , Humans , Injections, Intravenous , Injections, Subcutaneous , Models, Molecular , Pipecolic Acids/pharmacology , Piperidines/administration & dosage , Piperidines/chemistry , Piperidines/pharmacology , Rats , Stereoisomerism , Structure-Activity Relationship , Sulfonamides , Thrombosis/drug therapyABSTRACT
The optimisation of the P2 pharmacophore in a series of thrombin inhibitors is described. The interaction of a number of piperidine P2 functionalities with lysine 60G of thrombin is explored with reference to the crystal structure of inhibitor enzyme complexes. A primary ion-dipole interaction between the terminal P2 side chain group and lysine 60G is evoked to explain the SAR in this series.
Subject(s)
Polyglutamic Acid/analogs & derivatives , Polylysine/analogs & derivatives , Thrombin/antagonists & inhibitors , Thrombin/chemical synthesis , Crystallography, X-Ray , Humans , Kinetics , Models, Molecular , Polyglutamic Acid/metabolism , Polylysine/metabolism , Thrombin/pharmacokineticsABSTRACT
Cardiac myocytes were exposed to concentrations of potassium antimonyl tartrate (PAT) ranging from 1 to 1000 microM for 1 to 24 hr. Toxicity was assessed by measuring lactate dehydrogenase (LDH) release and by monitoring chronotropic depression. Lipid peroxidation was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). PAT produced a concentration- and time-dependent depression in chronotropy and an increase in the release of LDH and TBARS. A 4-hr exposure to 100 microM PAT stopped beating and induced significant increases in TBARS and LDH release in the myocyte cultures. The lipid peroxidation and LDH release induced by 100-200 microM PAT at 4 hr could be prevented by pretreatment of the cardiac myocytes with vitamin E or by the simultaneous addition of other antioxidants. Vitamin E continued to protect against lipid peroxidation up to 18 hr after the addition of 100 microM PAT, but failed to provide significant protection against LDH release at this time-point. Both 50 and 100 microM PAT decreased cardiac myocyte glutathione (GSH) levels after a 4-hr exposure. A series of thiol-containing compounds was evaluated for their effects on PAT toxicity. The addition of dithiothreitol, GSH, and 2-mercaptoethanol afforded some degree of protection against lipid peroxidation and LDH release up to 18 hr after the addition of 100 microM PAT. These results suggest that PAT induces lipid peroxidation in cultured cardiac myocytes but that other mechanisms may contribute to cell death with long-term exposures to PAT. Our results also suggest that PAT interacts with thiol-containing compounds.