RESUMO
Identification and structure-guided optimization of a series of 4-(pyrazol-4-yl)-pyrimidines as selective CDK4/6 inhibitors is reported herein. Several potency and selectivity determinants were established based on the X-ray crystallographic analysis of representative compounds bound to monomeric CDK6. Significant selectivity for CDK4/6 over CDK1 and CDK2 was demonstrated with several compounds in both enzymatic and cellular assays.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Modelos Moleculares , Pirazóis/síntese química , Pirimidinas/síntese química , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 4 Dependente de Ciclina/química , Quinase 6 Dependente de Ciclina/química , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
BODIPY-labeled Soraphen A derivative 4 was synthesized and characterized as an acetyl-CoA carboxylase (ACC) binder. Biophysical measurements indicate that the molecule binds in the biotin carboxylase domain where Soraphen A has been shown to bind. The fluorescent label of the BODIPY can be used to biophysically identify a compound that binds to the Soraphen A site of the biotin carboxylase domain versus the carboxytransferase domain of ACC.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Compostos de Boro/química , Macrolídeos/química , Acetil-CoA Carboxilase/metabolismo , Sítios de Ligação , Compostos de Boro/síntese química , Cristalografia por Raios X , Macrolídeos/síntese química , Estrutura Terciária de ProteínaRESUMO
Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA-dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695-residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical "core," although overall sequence identity is low. Eighty-four expression clones of the BVDV polymerase were designed by fine-sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high-throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96-well format, and two of these proteins were successfully crystallized.
Assuntos
Vírus da Diarreia Viral Bovina/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Cristalização , Cristalografia , Vírus da Diarreia Viral Bovina/genética , Escherichia coli/enzimologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Tripsina/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
The bovine viral diarrhea virus (BVDV) RNA-dependent RNA polymerase can initiate RNA replication by a de novo mechanism without a primer. The structure of BVDV polymerase, determined to 2.9-A resolution, contains a unique N-terminal domain, in addition to the fingers, palm, and thumb domains common to other polymerases. The structure of BVDV polymerase complexed with GTP, which is required for de novo (primer-independent) initiation, shows that GTP binds adjacent to the initiation NTP, suggesting that the GTP mimics a vestigial RNA product. Comparison of five monomers in two different crystal forms showed conformational changes in the fingertip region and in the thumb domain that may help to translocate the RNA template and product strands during elongation. The putative binding sites of previously reported BVDV inhibitors are also discussed.
