RESUMEN
Peptides that inhibit cyclin-dependent kinase 2 by blocking the macromolecular substrate recruitment site of cyclin A were simplified, for example, by replacement of dipeptide units with beta-amino acids. The smallest inhibitor retaining activity was a tripeptide, whose binding mode was confirmed by X-ray crystallography. This result suggests that nonpeptidic cyclin groove inhibitors may be feasible therapeutic agents.The cyclin-dependent kinase 2-cyclin A complex is an important regulator of the DNA-synthesis phase of the mammalian cell cycle, which is frequently deregulated in cancer. Rather than blocking the ATP-binding site of the apparently redundant kinase subunit, targeting the binding site for macromolecular substrates and regulatory proteins of cyclin A represents a promising strategy to enforce tumour-selective apoptosis. The cyclin-binding groove can be blocked with comparatively small synthetic peptides, which indirectly leads to inhibition of kinase function, but these peptides are metabolically labile and membrane impermeable. As part of our ongoing effort to develop more druglike peptidomimetics derived from cyclin-groove-binding peptides, we report the results of our studies aimed at a detailed understanding of the structural determinants required for effective binding. Using a combination of peptide synthesis, biochemical assays and X-ray crystallography, we show that it is possible to simplify peptide structures through the replacement of dipeptide units in which one of the residues is not directly involved in binding, through the introduction of beta-amino acid residues that retain only the dipeptide residue side chain that is important for binding. This approach also allowed us to probe spatial constraints in general, as well as the importance of peptide backbone hydrogen-bonding functions. Our identification of potent beta-homoleucine-containing tetrapeptide inhibitors, as well as the finding that an optimised N-terminally acetylated tripeptide retains some cyclin A-binding affinity, suggest that the pharmacological targeting of the cyclin A binding groove may be feasible.
Asunto(s)
Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Péptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Simulación por Computador , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/metabolismo , Diseño de Fármacos , Humanos , Péptidos/síntesis química , Péptidos/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Estructura Terciaria de Proteína , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Relatively little attention has been devoted to the social validation of potentially effective autism interventions. Thus, it is often difficult to identify and implement evidence-based practices, and programming is often inadequate. The authors identified autism intervention components with reported effectiveness for school settings. The results of a social validation survey completed by parents, teachers, and administrators indicate strong, consistent support for program components falling within five functional areas: (a) individualized programming, (b) data collection, (c) the use of empirically-based strategies, (d) active collaboration, and (e) a focus on long-term outcomes. These socially validated interventions can be used to evaluate existing autism curricula and develop training for professionals, parents, and students in order to improve public school autism programs.
Asunto(s)
Trastorno Autístico , Docentes , Padres , Conducta Social , Encuestas y Cuestionarios , Preescolar , Educación Especial , HumanosRESUMEN
We describe a drug-design strategy termed REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) in which nonpeptidic surrogates for specific determinants of known peptide ligands are identified in silico by using a core peptide-bound protein structure as a design anchor. In the REPLACE application example, we present the effective replacement of two critical binding motifs in a lead protein-protein interaction inhibitor pentapeptide with more druglike phenyltriazole and diphenyl ether groups. These were identified through docking of fragment libraries into the volume of the cyclin-binding groove of CDK2/cyclin A vacated through truncation of the inhibitor peptide-binding determinants. Proof of concept for this strategy was obtained through the generation of potent peptide-small-molecule hybrids and by the confirmation of inhibitor-binding modes in X-ray crystal structures. This method therefore allows nonpeptide fragments to be identified without the requirement for a high-sensitivity binding assay and should be generally applicable in replacing amino acids as individual residues or groups in peptide inhibitors to generate pharmaceutically acceptable lead molecules.
Asunto(s)
Ciclina A/química , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/química , Diseño de Fármacos , Biblioteca de Péptidos , Péptidos/química , Ingeniería de Proteínas/métodos , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Ciclina A/antagonistas & inhibidores , Ciclina A/metabolismo , Estructura Molecular , Péptidos/metabolismo , Péptidos/farmacologíaRESUMEN
Inhibition of cyclin A- and cyclin E-associated cyclin-dependent kinase-2 (CDK2) activities is an effective way of selective induction of apoptotic cell death via the E2F pathway in tumour cells. The cyclin groove recognition motif (CRM) in the natural CDK-inhibitory (CDKI) tumour suppressor protein p27KIP1 was used as the basis for the design and synthesis of a series of cyclic peptides whose biological activity and structural characterisation by NMR and X-ray crystallography is reported. Whereas linear p27KIP1 sequence peptides were comparatively ineffective, introduction of side chain-to-tail constraints was found to be productive. An optimal macrocyclic ring size for the conformational constraint was determined, mimicking the intramolecular H-bonding system of p27. Molecular dynamics calculations of various macrocycles suggested a close correlation between ring flexibility and biological activity. Truncated inhibitor peptide analogues also confirmed the hypothesis that introduction of a cyclic conformational constraint is favourable in terms of affinity and potency. The structural basis for the potency increase in cyclic versus linear peptides was demonstrated through the determination and interpretation of X-ray crystal structures of complexes between CDK2/cylin A (CDK2A) and a constrained pentapeptide.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Complejos Multienzimáticos/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Sitios de Unión/efectos de los fármacos , Biología Computacional , Cristalografía por Rayos X , Ciclina A/química , Quinasa 2 Dependiente de la Ciclina/química , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/química , Evaluación Preclínica de Medicamentos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/efectos de los fármacos , Biblioteca de Péptidos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Conformación ProteicaRESUMEN
Inhibition of CDK2/CA (cyclin-dependent kinase 2/cyclin A complex) activity through blocking of the substrate recognition site in the cyclin A subunit has been demonstrated to be an effective method for inducing apoptosis in tumor cells. We have used the cyclin binding motif (CBM) present in the tumor suppressor proteins p21(WAF1) and p27(KIP1) as a template to optimize the minimal sequence necessary for CDK2/CA inhibition. A series of peptides were prepared, containing nonnatural amino acids, which possess nano- to micromolar CDK2-inhibitory activity. Here we present X-ray structures of the protein complex CDK2/CA, together with the cyclin groove-bound peptides H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH(2) (peptide 1), H-Arg-Arg-Leu-Ile-Phe-NH(2) (peptide 2), Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH(2) (peptide 3), H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH(2) (peptide 4), and H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH(2) (peptide 5). Some of the peptide complexes presented here were obtained through the novel technique of ligand exchange within protein crystals. This method may find general application for obtaining complex structures of proteins with surface-bound ligands.
Asunto(s)
Ciclinas/química , Secuencias de Aminoácidos , Sitios de Unión , Quinasas CDC2-CDC28/antagonistas & inhibidores , Quinasas CDC2-CDC28/química , Cristalografía por Rayos X , Ciclina A/antagonistas & inhibidores , Ciclina A/química , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Humanos , Ligandos , Modelos Moleculares , Péptidos/química , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
CDK2 inhibitors have been proposed as effective anti-cancer therapeutics. We show here that CYC202 (R-roscovitine) is a potent inhibitor of recombinant CDK2/cyclin E kinase activity (IC(50) = 0.10 microM) with an average cytotoxic IC(50) of 15.2 microM in a panel of 19 human tumour cell lines, and we also demonstrate selectivity for rapidly proliferating cells over non-proliferating cells. A study of the cell cycle effects of CYC202 in Lovo colorectal carcinoma cells showed that the major effect was not the predicted arrest in one part of the cycle, but rather an induction of cell death from all compartments of the cell cycle. The maximum tolerated dose given intravenously to mice was in excess of 20 mg/kg. Doses up to 2,000 mg/kg were tolerated when administered orally in mice. Following repeated intraperitoneal administration (3 times daily for 5 days) of 100 mg/kg to nude mice bearing the Lovo human colorectal tumour, CYC202 induced a significant antitumour effect with a 45% reduction in tumour growth compared to controls. A second experiment using the human uterine xenograft MESSA-DX5 treated with orally administered CYC202 (500 mg/kg 3 times daily for 4 days) also exhibited a significant reduction in the rate of growth of the tumour (62%). These data, showing enzyme and cellular potency together with antitumour activity, confirm the potential of CDK2 inhibitors such as CYC202 as anticancer drugs.
Asunto(s)
Antineoplásicos/uso terapéutico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Purinas/farmacología , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Ciclina E , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Recombinantes , Roscovitina , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
A focused series of octapeptides based on the lead compound H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH(2) 1, in which the C-terminal phenylalanine residue was replaced by alpha and/or beta-modified variants, was synthesized using solid-phase chemistry. Both the L-threo-beta-hydroxy-phenylalanine (beta-phenylserine, Pse) and (2S)-phenylalaninol derivatives, as competitive binders at the cyclin-recruitment site, displayed potent inhibitory activity towards the CDK2-cyclin A complex. Unexpectedly, the D-threo-Pse derivatives also showed inhibitory activity.