RESUMEN
XIAP, a member of the inhibitor of apoptosis family of proteins, is a critical regulator of apoptosis. Inhibition of the BIR domain-caspase interaction is a promising approach towards treating cancer. Previous work has been directed towards inhibiting the BIR3-caspase-9 interaction, which blocks the intrinsic apoptotic pathway; selectively inhibiting the BIR2-caspase-3 interaction would also block the extrinsic pathway. The BIR2 domain of XIAP has successfully been crystallized; peptides and small-molecule inhibitors can be soaked into these crystals, which diffract to high resolution. Here, the BIR2 apo crystal structure and the structures of five BIR2-tetrapeptide complexes are described. The structural flexibility observed on comparing these structures, along with a comparison with XIAP BIR3, affords an understanding of the structural elements that drive selectivity between BIR2 and BIR3 and which can be used to design BIR2-selective inhibitors.
Asunto(s)
Caspasa 3/química , Caspasa 3/metabolismo , Inhibidores de Caspasas/química , Proteínas Inhibidoras de la Apoptosis/química , Nucleopoliedrovirus/química , Proteínas Virales/química , Proteína Inhibidora de la Apoptosis Ligada a X/química , Secuencia de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Apoptosis/genética , Cristalografía por Rayos X , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Nucleopoliedrovirus/genética , Oligopéptidos/química , Oligopéptidos/genética , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/genética , Proteínas Virales/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.
Asunto(s)
Antineoplásicos/química , Benzamidas/química , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Oxazinas/química , Inhibidores de Proteínas Quinasas/química , Administración Oral , Regulación Alostérica , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Benzamidas/administración & dosificación , Benzamidas/farmacología , Humanos , Modelos Moleculares , Oxazinas/administración & dosificación , Oxazinas/farmacología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
We report crystal structures of the human enzyme phosphoenolpyruvate carboxykinase (PEPCK) with and without bound substrates. These structures are the first to be determined for a GTP-dependent PEPCK, and provide the first view of a novel GTP-binding site unique to the GTP-dependent PEPCK family. Three phenylalanine residues form the walls of the guanine-binding pocket on the enzyme's surface and, most surprisingly, one of the phenylalanine side-chains contributes to the enzyme's specificity for GTP. PEPCK catalyzes the rate-limiting step in the metabolic pathway that produces glucose from lactate and other precursors derived from the citric acid cycle. Because the gluconeogenic pathway contributes to the fasting hyperglycemia of type II diabetes, inhibitors of PEPCK may be useful in the treatment of diabetes.
Asunto(s)
Citosol/enzimología , Guanosina Trifosfato/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/química , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Fenilalanina/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/química , Conformación Proteica , Especificidad por SustratoRESUMEN
Protein-protein interaction (PPI) systems represent a rich potential source of targets for drug discovery, but historically have proven to be difficult, particularly in the lead identification stage. Application of the fragment-based approach may help toward success with this target class. To provide an example toward understanding the potential issues associated with such an application, we have deconstructed one of the best established protein-protein inhibitors, the Nutlin series that inhibits the interaction between MDM2 and p53, into fragments, and surveyed the resulting binding properties using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR), surface plasmon resonance (SPR), and X-ray crystallography. We report the relative contributions toward binding affinity for each of the key substituents of the Nutlin molecule and show that this series could hypothetically have been discovered via a fragment approach. We find that the smallest fragment of Nutlin that retains binding accesses two subpockets of MDM2 and has a molecular weight at the high end of the range that normally defines fragments.
RESUMEN
IL-2-inducible T cell kinase plays an essential role in T cell receptor signaling and is considered a drug target for the treatment of Th2-mediated inflammatory diseases. By applying high-throughput protein engineering and crystallization, we have determined the X-ray crystal structures of IL-2-inducible T cell kinase in complex with its selective inhibitor BMS-509744 and the broad-spectrum kinase inhibitors sunitinib and RO5191614. Sunitinib uniquely stabilizes IL-2-inducible T cell kinase in the helix C-in conformation by inducing side chain conformational changes in the ATP-binding site. This preference of sunitinib to bind to an active kinase conformation is reflective of its broad-spectrum kinase activity. BMS-509744 uniquely stabilizes the activation loop in a substrate-blocking inactive conformation, indicating that structural changes described for Src family kinases are also involved in the regulation of IL-2-inducible T cell kinase activity. The observed BMS-509744 binding mode allows rationalization of structure-activity relationships reported for this inhibitor class and facilitates further structure-based drug design. Sequence-based analysis of this binding mode provides guidance for the rational design of inhibitor selectivity.
Asunto(s)
Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Indoles/química , Indoles/farmacología , Ingeniería de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Pirroles/química , Pirroles/farmacología , Relación Estructura-Actividad , Sunitinib , Familia-src Quinasas/metabolismoRESUMEN
The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2.