ABSTRACT
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.
Subject(s)
Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Models, Molecular , Pyrazoles/chemical synthesis , Pyrimidines/chemical synthesis , Cell Line, Tumor , Crystallography, X-Ray , Cyclin-Dependent Kinase 4/chemistry , Cyclin-Dependent Kinase 6/chemistry , High-Throughput Screening Assays , Humans , Kinetics , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Acetyl-CoA Carboxylase/antagonists & inhibitors , Boron Compounds/chemistry , Macrolides/chemistry , Acetyl-CoA Carboxylase/metabolism , Binding Sites , Boron Compounds/chemical synthesis , Crystallography, X-Ray , Macrolides/chemical synthesis , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Diarrhea Viruses, Bovine Viral/enzymology , RNA-Dependent RNA Polymerase/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Animals , Cattle , Cloning, Molecular , Crystallization , Crystallography , Diarrhea Viruses, Bovine Viral/genetics , Escherichia coli/enzymology , Molecular Sequence Data , Protein Structure, Tertiary , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Trypsin/chemistry , Viral Proteins/genetics , Viral Proteins/isolation & purificationABSTRACT
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.