RESUMO
Beginning with a screening hit, unique thienopyrazole-indole inhibitors of Itk (interleukin-2-inducible tyrosine kinase) were designed, synthesized, and crystallized in the target kinase. Although initial compounds were highly active in Itk, they were not selective. Increasing the steric bulk around a tertiary alcohol at the 5-indole position dramatically improved selectivity toward Lyk and Syk, but not Txk. Substitutions at the 3- and 4-indole positions gave less active compounds that remained poorly selective. A difluoromethyl substitution at the 5-position of the thienopyrazole led to a highly potent and selective compound. Phenyl at this position reduced activity and selectivity while pushing the side-chains of Lys-391 and Asp-500 away from the binding pocket. Novel and selective thienopyrazole inhibitors of Itk were designed as a result of combining structure-based design and medicinal chemistry.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Cristalografia por Raios X , Humanos , Relação Estrutura-AtividadeRESUMO
The COPII vesicular coat forms on the endoplasmic reticulum from Sar1-GTP, Sec23/24 and Sec13/31 protein subunits. Here, we define the interaction between Sec23/24.Sar1 and Sec13/31, involving a 40 residue Sec31 fragment. In the crystal structure of the ternary complex, Sec31 binds as an extended polypeptide across a composite surface of the Sec23 and Sar1-GTP molecules, explaining the stepwise character of Sec23/24.Sar1 and Sec13/31 recruitment to the membrane. The Sec31 fragment stimulates GAP activity of Sec23/24, and a convergence of Sec31 and Sec23 residues at the Sar1 GTPase active site explains how GTP hydrolysis is triggered leading to COPII coat disassembly. The Sec31 active fragment is accommodated in a binding groove supported in part by Sec23 residue Phe380. Substitution of the corresponding residue F382L in human Sec23A causes cranio-lenticulo-sutural dysplasia, and we suggest that this mutation disrupts the nucleation of COPII coat proteins at endoplasmic reticulum exit sites.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Ligação Proteica , Conformação Proteica , Proteínas de Transporte Vesicular/genéticaRESUMO
COPII-coated vesicles export newly synthesized proteins from the endoplasmic reticulum. The COPII coat consists of the Sec23/24-Sar1 complex that selects cargo and the Sec13/31 assembly unit that can polymerize into an octahedral cage and deform the membrane into a bud. Crystallographic analysis of the assembly unit reveals a 28 nm long rod comprising a central alpha-solenoid dimer capped by two beta-propeller domains at each end. We construct a molecular model of the COPII cage by fitting Sec13/31 crystal structures into a recently determined electron microscopy density map. The vertex geometry involves four copies of the Sec31 beta-propeller that converge through their axial ends; there is no interdigitation of assembly units of the kind seen in clathrin cages. We also propose that the assembly unit has a central hinge-an arrangement of interlocked alpha-solenoids-about which it can bend to adapt to cages of variable curvature.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/química , Animais , Células Cultivadas , Clatrina/metabolismo , Cristalografia por Raios X , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Modelos Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
COPII-coated vesicles form on the endoplasmic reticulum by the stepwise recruitment of three cytosolic components: Sar1-GTP to initiate coat formation, Sec23/24 heterodimer to select SNARE and cargo molecules, and Sec13/31 to induce coat polymerization and membrane deformation. Crystallographic analysis of the Saccharomyces cerevisiae Sec23/24-Sar1 complex reveals a bow-tie-shaped structure, 15 nm long, with a membrane-proximal surface that is concave and positively charged to conform to the size and acidic-phospholipid composition of the COPII vesicle. Sec23 and Sar1 form a continuous surface stabilized by a non-hydrolysable GTP analogue, and Sar1 has rearranged from the GDP conformation to expose amino-terminal residues that will probably embed in the bilayer. The GTPase-activating protein (GAP) activity of Sec23 involves an arginine side chain inserted into the Sar1 active site. These observations establish the structural basis for GTP-dependent recruitment of a vesicular coat complex, and for uncoating through coat-controlled GTP hydrolysis.