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1.
Proc Natl Acad Sci U S A ; 98(21): 11879-84, 2001 Oct 09.
Article in English | MEDLINE | ID: mdl-11592999

ABSTRACT

The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.


Subject(s)
Morpholines/chemistry , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Antigens, CD/chemistry , Antigens, CD/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Structure , Photochemistry , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Type I , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
2.
Circulation ; 100(18): 1917-22, 1999 Nov 02.
Article in English | MEDLINE | ID: mdl-10545437

ABSTRACT

BACKGROUND: To date, the lack of potent and selective inhibitors has hampered the physiological assessment of modulation of the cardiac slowly activating delayed rectifier current, I(Ks). The present study, using the I(Ks) blocker L-768,673, represents the first in vivo assessment of the cardiac electrophysiological and antiarrhythmic effects of selective I(Ks) blockade. METHODS AND RESULTS: In an anesthetized canine model of recent (8.5+/-0.4 days) anterior myocardial infarction, 0.003 to 0.03 mg/kg L-768,673 IV significantly suppressed electrically induced ventricular tachyarrhythmias and reduced the incidence of lethal arrhythmias precipitated by acute, thrombotically induced posterolateral myocardial ischemia. Antiarrhythmic protection afforded by L-768,673 was accompanied by modest 7% to 10% increases in noninfarct zone ventricular effective refractory period, 3% to 5% increases in infarct zone ventricular effective refractory period, and 4% to 6% increases in QTc interval. In a conscious canine model of healed (3 to 4 weeks) anterior myocardial infarction, ventricular fibrillation was provoked by transient occlusion of the left circumflex coronary artery during submaximal exercise. Pretreatment with 0.03 mg/kg L-768,673 IV elicited a modest 7% increase in QTc, prevented ventricular fibrillation in 5 of 6 animals, and suppressed arrhythmias in 2 additional animals. CONCLUSIONS: The present findings suggest that selective blockade of I(Ks) may be a potentially useful intervention for the prevention of malignant ischemic ventricular arrhythmias.


Subject(s)
Acetamides/therapeutic use , Anti-Arrhythmia Agents/therapeutic use , Arrhythmias, Cardiac/drug therapy , Benzodiazepinones/therapeutic use , Heart Block/therapy , Myocardial Ischemia/drug therapy , Ventricular Dysfunction/drug therapy , Animals , Arrhythmias, Cardiac/etiology , Disease Models, Animal , Dogs , Electrocardiography , Myocardial Ischemia/complications , Sympathetic Nervous System/physiology , Ventricular Dysfunction/etiology
3.
J Biol Chem ; 274(10): 6453-60, 1999 Mar 05.
Article in English | MEDLINE | ID: mdl-10037737

ABSTRACT

Vascular endothelial growth factor is an important physiological regulator of angiogenesis. The function of this endothelial cell selective growth factor is mediated by two homologous tyrosine kinase receptors, fms-like tyrosine kinase 1 (Flt-1) and kinase domain receptor (KDR). Although the functional consequence of vascular endothelial growth factor binding to the Flt-1 receptor is not fully understood, it is well established that mitogenic signaling is mediated by KDR. Upon sequencing several independent cDNA clones spanning the cytoplasmic region of human KDR, we identified and confirmed the identity of a functionally required valine at position 848 in the ATP binding site, rather than the previously reported glutamic acid residue, which corresponds to an inactive tyrosine kinase. The cytoplasmic domain of recombinant native KDR, expressed as a glutathione S-transferase fusion protein, can undergo autophosphorylation in the presence of ATP. In addition, the kinase activity can be substantially increased by autophosphorylation at physiologic ATP concentrations. Mutation analysis indicates that both tyrosine residues 1054 and 1059 are required for activation, which is a consequence of an increased affinity for both ATP and the peptide substrate and has no effect on kcat, the intrinsic catalytic activity of the enzyme. KDR kinase catalyzes phosphotransfer by formation of a ternary complex with ATP and the peptide substrate. We demonstrate that tyrosine kinase antagonists can preferentially inhibit either the unactivated or activated form of the enzyme.


Subject(s)
Endothelial Growth Factors/metabolism , Lymphokines/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Tyrosine/metabolism , DNA, Complementary/analysis , DNA, Complementary/genetics , Enzyme Activation , Humans , Phosphorylation , Protein Conformation , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Growth Factor/chemistry , Receptors, Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
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