RESUMEN
The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.
Asunto(s)
Bradiquinina/antagonistas & inhibidores , Calcio/metabolismo , Íleon/metabolismo , Receptores de Bradiquinina/metabolismo , Dihidromorfina/farmacología , Humanos , Íleon/fisiología , Técnicas In Vitro , Transducción de SeñalRESUMEN
Molecular modeling studies of potent decapeptide bradykinin antagonists suggested the de novo design of peptide mimetics based on a 1,2,3,4-tetrasubstituted 1,4-piperazin-6-one scaffold. These de novo-designed antagonists exhibited only modest potency (IC50 approximately 55 microM) on a cloned human B2 receptor and antagonist activity in an in vitro human-cell functional assay. The success of these structures led to the creation of prototype libraries based on variously substituted 1,4-piperazine scaffolds, which allowed a rapid and general search of pharmacophores attached to a piperazine scaffold. The parent piperazinedione structures and fully reduced piperazine libraries differ from recently reported diketopiperazine libraries in the use of diverse nonnatural amino acids, on-resin-submonomer synthesis to provide more diverse N-substituted structures, and the adaptation of simultaneous ring closure and resin cleavage to drive the formation of highly hindered amide bonds. Using this chemistry, a rationally directed non-peptide library of approximately 2500 N,N'-disubstituted piperazines and piperazinediones was synthesized and screened for ligand affinity on bradykinin, neurokinin, and opioid receptors. A number of lead structures were identified. Notably, a bradykinin antagonist lead, CP-2458, with good receptor selectivity and antagonist activity in human-cell assays was identified and is undergoing optimization by traditional and combinatorial methods.
Asunto(s)
Bradiquinina/antagonistas & inhibidores , Diseño de Fármacos , Proteínas de Unión al GTP/química , Biblioteca de Péptidos , Piperazinas/síntesis química , Receptores de Superficie Celular/química , Acetilación , Compuestos de Aluminio , Aminas/química , Cromatografía Líquida de Alta Presión , Humanos , Ligandos , Compuestos de Litio , Modelos Moleculares , Piperazinas/química , Piperazinas/farmacología , Sustancias Reductoras , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónAsunto(s)
Bradiquinina/antagonistas & inhibidores , Neuroquinina A/antagonistas & inhibidores , Oligopéptidos/farmacología , Sustancia P/antagonistas & inhibidores , Animales , Antagonistas de los Receptores de Bradiquinina , Cobayas , Humanos , Íleon/efectos de los fármacos , Técnicas In Vitro , Estructura Molecular , Antagonistas del Receptor de Neuroquinina-1 , Oligopéptidos/química , Arteria Pulmonar/efectos de los fármacos , Conejos , Receptor de Bradiquinina B2 , Receptores de Neuroquinina-2/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
We report a systematic probing of the structural requirements of the bradykinin (BK) type 2 (B(2)) receptor for antagonist activity by incorporating N-alkyl-amino acid residues at positions 7 and 8 of a potent antagonist sequence. Compound 1 (D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-N-Chg (8)-Arg(9), CP-0597)(1,2) is a potent (pA(2) = 9.3, rat uterus; pK(i) = 9.62, binding, human receptor clone) B(2) receptor antagonist devoid of in vitro B(1) antagonist activity (rabbit aorta). Compound 1 exhibits high potency (ED(50) = 29.2 pmol/kg/min, iv, rabbit) and duration of action when tested in models for in vivo B(2) antagonist activity. Although devoid of activity in a classic B(1) isolated tissue assay, B(1) antagonist activity for 1 was demonstrated in vivo, in a LPS-treated, inducible BK(1) receptor rabbit blood pressure model (ED(50) = 1.7 nmol/kg/min). D-Arg(0) of 1 can be formally replaced by an achiral arginine surrogate, without significant loss in antagonist potency on rat uterus (compound 11, B(2) pA(2) = 9.1). Antagonist 13 (Hyp(2), Nchg(8)), pK(i) = 10.2, and agonist 4 (N-methylcyclohexyl-Gly(8)), pK(i) = 10.1, also exhibited substantial binding to guinea pig ileum membrane receptors as well as a human B(2) receptor clone. Very minor structural changes in the N-alkyl amino acid residues in positions 7 and 8 can modify the activity of this class of compounds from being extremely potent antagonists to tight binding partial or full agonists. These studies have resulted in a series of compounds containing inexpensive amino acid residues but which produce broad spectrum BK receptor blocking potency and exceptional in vivo duration of action.
Asunto(s)
Antagonistas de los Receptores de Bradiquinina , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Aorta/efectos de los fármacos , Unión Competitiva , Presión Sanguínea/efectos de los fármacos , Femenino , Cobayas , Humanos , Íleon/efectos de los fármacos , Íleon/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Unión Proteica , Conejos , Ratas , Receptor de Bradiquinina B1 , Receptor de Bradiquinina B2 , Receptores de Bradiquinina/metabolismo , Relación Estructura-Actividad , Útero/efectos de los fármacosRESUMEN
p-Nitrophenyl 3-diazopyruvate (DAPpNP) has been developed as a heterobifunctional cross-linking agent for synthesis of photoaffinity probes and photoactivatable cross-linking agents that are nucleophile specific. p-Nitrophenyl chloroglyoxylate is formed in high yield from oxalyl chloride and p-nitrophenol. Subsequent reaction with diazomethane produces DAPpNP in 50-60% overall yield. DAPpNP acylates primary and secondary amines to form 3-diazopyruvamides in high yields. 3-Diazopyruvamide derivatives have been formed from a wide variety of amines including aromatic amines, amino acids, and peptides. 3-Diazopyruvamides undergo photolysis and Wolff rearrangement at 300 nm to produce a ketene amide, which efficiently acylates nucleophilic species to form malonic acid amide derivatives. A family of photoactivatable 3-diazopyruvamide cross-linking agents was synthesized from amino acids. A cleavable, thiol-specific photoactivatable cross-linking agent was synthesized from cystamine. These reagents were caused to react with rabbit muscle aldolase to form mainly dimeric cross-linked species.