RESUMEN
The novel phosphotyrosine (pTyr) mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp) has been designed and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60(c-Src) (Src) tyrosine kinase. A 2.2 A X-ray crystal structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure-activity relationships and supports the design concept of this new pTyr mimetic.
Asunto(s)
Benzamidas/química , Benzamidas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Organofosfonatos/química , Organofosfonatos/farmacología , Fosfotirosina/química , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Dominios Homologos src/efectos de los fármacos , Animales , Sitios de Unión , Resorción Ósea , Cristalografía por Rayos X , Dentina/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Espectroscopía de Resonancia Magnética , Imitación Molecular , Mutación , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Conejos , Relación Estructura-ActividadRESUMEN
Using a novel, solid-phase parallel synthetic route and a computational docking program, a series of phosphorylated nonpeptides were generated to determine their structure-activity relationships (SAR) for binding at the SH2 domain of pp60src (Src). A functionalized benzoic acid intermediate was attached to solid support via Rink amide linkage, which upon acid cleavage generated the desired benzamide template-based nonpeptides in a facile manner. Compounds were synthesized using a combination of solid- and solution-phase techniques. Purification using reversed-phase, semipreparative HPLC allowed for quantitative SAR studies. Specifically, this work focused on functional group modifications, in a parallel fashion, designed to explore hydrophobic binding at the pY+3 pocket of the Src SH2 domain.
Asunto(s)
Benzamidas/síntesis química , Sitios de Unión , Proteínas Proto-Oncogénicas pp60(c-src)/química , Dominios Homologos src , Benzamidas/química , Cristalografía por Rayos X , Diseño de Fármacos , Fosforilación , Relación Estructura-ActividadRESUMEN
A series of 1,2,4-oxadiazole analogues has been shown to be potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70, a potential therapeutic target for immune suppression. These compounds typically are 200-400-fold more potent than the native, monophosphorylated tetrapeptide sequences. When compared with the high-affinity zeta-1-ITAM peptide (Ac-NQL-pYNELNLGRREE-pYDVLD-NH(2), wherein pY refers to phosphotyrosine) some of the best 1,2, 4-oxadiazole analogues are approximately 1 order of magnitude less active. This series of compounds displays an unprecedented level of selectivity over the closely related tyrosine kinase Syk, as well as other SH2-containing proteins such as Src and Grb2. Gel shift studies using a protein construct consisting only of C-terminal ZAP-70 SH2 demonstrate that these compounds can effectively engage this particular SH2 domain.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Oxadiazoles/síntesis química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Dominios Homologos src , Inhibidores Enzimáticos/química , Precursores Enzimáticos/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular , Modelos Moleculares , Oxadiazoles/química , Relación Estructura-Actividad , Quinasa Syk , Proteína Tirosina Quinasa ZAP-70RESUMEN
As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.
Asunto(s)
Fármacos Anti-VIH/química , Inhibidores de la Proteasa del VIH/química , Proteasa del VIH/química , Azepinas/química , VIH-1/enzimología , Enlace de Hidrógeno , Conformación Molecular , Urea/análogos & derivados , Urea/química , Urea/farmacologíaRESUMEN
Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.
Asunto(s)
Fármacos Anti-VIH , Guanidinas , Inhibidores de la Proteasa del VIH , Proteasa del VIH/metabolismo , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Línea Celular , Cristalografía por Rayos X , Guanidinas/síntesis química , Guanidinas/química , Guanidinas/metabolismo , Guanidinas/farmacología , Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/enzimología , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Relación Estructura-Actividad , Urea/análogos & derivados , Urea/químicaRESUMEN
High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure-activity relationships of C2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and "unbound" (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2' pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure-activity relationships, and molecular recognition of this series of C2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2' is also discussed.
Asunto(s)
Inhibidores de la Proteasa del VIH/síntesis química , Urea/síntesis química , Animales , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Conformación Molecular , Relación Estructura-Actividad , Urea/química , Urea/farmacologíaRESUMEN
BACKGROUND: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds. RESULTS: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg-1 DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC90 suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response. CONCLUSIONS: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.
Asunto(s)
Azepinas/síntesis química , Azepinas/farmacología , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/enzimología , Urea/análogos & derivados , Administración Oral , Animales , Azepinas/química , Azepinas/farmacocinética , Cristalografía por Rayos X , Farmacorresistencia Microbiana/genética , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacocinética , VIH-1/fisiología , Humanos , Infusiones Intravenosas , Ratones , Microscopía Electrónica , Solubilidad , Urea/síntesis química , Urea/química , Urea/farmacocinética , Urea/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
PURPOSE: The effects of structural modifications on the membrane permeability of angiotensin II (Ang II) receptor antagonists and the usefulness of in vitro and in situ intestinal absorption models in predicting in vivo absorption or bioavailability were investigated. METHODS: Intestinal permeability was determined in vitro using Caco-2 cell monolayers and in situ using a perfused rat intestine method. Several physicochemical parameters were either measured or computed, and correlated with intestinal permeation. RESULTS: Permeation coefficients (Pa) across Caco-2 cell monolayers correlated well with both in situ absorption rate constants (ka) and in vivo bioavailability or % absorption. For these Ang II antagonists, Pa values larger than 3 x 10(-6) cm sec-1 and in situ ka values of 2 x 10(-4) min-1 cm-1 or above were associated with good in vivo absorption. Structural modifications at the R5 position, where a COOH group was substituted with either a CHO or CH2OH group, enhanced the permeability of the Ang II receptor antagonists up to 100-fold. There were good correlations between permeability and log P(octanol/buffer), log PHPLC, charge, solvation/desolvation energy and assigned hydrogen bonding potential. CONCLUSIONS: The correlations obtained in this study indicate that both the Caco-2 cell model and the in situ perfused rat intestine could be used to predict intestinal absorption in vivo. Structural modifications of the Ang II antagonists had a significant impact on the intestinal permeability. Charge, solvation energy, and hydrogen bonding are predominant determinants of intestinal permeability and oral bioavailability of these compounds.
Asunto(s)
Antagonistas de Receptores de Angiotensina , Indoles/farmacocinética , Absorción Intestinal/fisiología , Mesilatos/farmacocinética , Péptidos Cíclicos/farmacocinética , Piridinas/farmacocinética , Tetrazoles/farmacocinética , Animales , Disponibilidad Biológica , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacocinética , Células CACO-2/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión , Humanos , Concentración de Iones de Hidrógeno , Imidazoles/química , Imidazoles/farmacocinética , Técnicas In Vitro , Indoles/química , Losartán , Masculino , Mesilatos/química , Octanoles/química , Péptidos Cíclicos/química , Piridinas/química , Ratas , Ratas Sprague-Dawley , Solubilidad , Relación Estructura-Actividad , Tetrazoles/químicaRESUMEN
Mechanistic information and structure-based design methods have been used to design a series of nonpeptide cyclic ureas that are potent inhibitors of human immunodeficiency virus (HIV) protease and HIV replication. A fundamental feature of these inhibitors is the cyclic urea carbonyl oxygen that mimics the hydrogen-bonding features of a key structural water molecule. The success of the design in both displacing and mimicking the structural water molecule was confirmed by x-ray crystallographic studies. Highly selective, preorganized inhibitors with relatively low molecular weight and high oral bioavailability were synthesized.