RESUMEN
The screening of fragments is an alternative approach to high-throughput screening for the identification of leads for therapeutic targets. Fragment hits have been discovered using X-ray crystallographic screening of protein crystals of the serine protease enzyme thrombin. The fragment library was designed to avoid any well-precedented, strongly basic functionality. Screening hits included a novel ligand (3), which binds exclusively to the S2-S4 pocket, in addition to smaller fragments which bind to the S1 pocket. The structure of these protein-ligand complexes are presented. A chemistry strategy to link two such fragments together and to synthesize larger drug-sized compounds resulted in the efficient identification of hybrid inhibitors with nanomolar potency (e.g., 7, IC50 = 3.7 nM). These potent ligands occupy the same area of the active site as previously described peptidic inhibitors, while having very different chemical architecture.
Asunto(s)
Modelos Moleculares , Trombina/antagonistas & inhibidores , Trombina/química , Carbamatos/síntesis química , Carbamatos/química , Cristalografía por Rayos X , Bases de Datos Factuales , Humanos , Conformación Proteica , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Tetrazoles/síntesis química , Tetrazoles/químicaRESUMEN
Foot-and-mouth disease viruses (FMDVs) target epithelial cells via integrin receptors, but can acquire the capacity to bind cell-surface heparan sulphate (or alternative receptors) on passage in cell culture. Vaccine viruses must be propagated in cell culture and, hence, some rationale for the selection of variants in this process is important. Crystal structures are available for type O, A and C viruses and also for a complex of type O strain O(1)BFS with heparin. The structure of FMDV A10(61) (a cell culture-adapted strain) complexed with heparin has now been determined. This virus has an RGSD motif in place of the otherwise conserved RGD integrin-binding motif and the potential to bind heparan sulphate (suggested by sequence analyses). FMDV A10(61) was closely similar in structure to other serotypes, deviating most in antigenic sites. The VP1 GH loop comprising the integrin-binding motif was disordered. Heparin bound at a similar site and in a similar conformation to that seen in the analogous complex with O(1)BFS, although the binding had a lower affinity and was more ionic.
Asunto(s)
Virus de la Fiebre Aftosa/metabolismo , Virus de la Fiebre Aftosa/ultraestructura , Oligosacáridos/metabolismo , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Animales , Variación Antigénica , Sitios de Unión , Células CHO , Cricetinae , Cristalografía por Rayos X , Virus de la Fiebre Aftosa/química , Heparitina Sulfato/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Receptores Virales/química , Serotipificación , Resonancia por Plasmón de SuperficieRESUMEN
Foot-and-mouth disease virus (FMDV) capsids are inherently labile under mildly acidic conditions, dissociating to pentamers at pH values in the region of 6.5, with the release of protein 1A and the viral RNA. This acid-induced disassembly is thought to be required for the entry of the virus genome into the host cell. Previous work has highlighted a histidine-alpha-helix charge-dipole interaction at the twofold axes of symmetry between pentamers and has suggested that this interaction plays a role in acid-induced disassembly. The validity of this theory has now been tested by converting the implicated residue, His-142 of protein 1C, to Arg, Phe and Asp. The effects of such changes were studied by using a previously described vaccinia virus expression system, in which synthesis and processing of FMDV capsid proteins results in the self-assembly of capsids. In agreement with the histidine-alpha-helix charge-dipole theory, assembly in the arginine mutant was found to be greatly reduced, while capsids of the aspartic acid mutant were considerably more stable under acidic conditions than the wild-type. Aberrant but acid-stable complexes were obtained in the phenylalanine mutant.