ABSTRACT
Medulloblastoma - highly aggressive and heterogeneous tumours of the cerebellum - account for 15-20% of all childhood brain tumours, and are the most common high-grade childhood embryonal tumour of the central nervous system. Herein, potent in vitro anticancer activity against two established medulloblastoma cell lines of the sonic hedgehog subgroup, namely DAOY (p53 mutant) and ONS-76 (p53 wild type), has been achieved. A number of first-generation diarylamides and diarylureas were evaluated and activity is likely to be, in-part, conformation-dependent. The most active compound from this first-generation set of compounds, 1-naphthyl derivative 4b, was selected and a second-generation of compounds were optimised and tested for activity against the medulloblastoma cell lines. This process resulted in drug-like compounds with up to sixty times the activity (sub-micromolar) of the first-generation - thus providing potent new leads for further study.
Subject(s)
Amides/pharmacology , Antineoplastic Agents/pharmacology , Cerebellar Neoplasms/drug therapy , Medulloblastoma/drug therapy , Urea/pharmacology , Amides/chemical synthesis , Amides/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cerebellar Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Medulloblastoma/pathology , Molecular Structure , Structure-Activity Relationship , Urea/analogs & derivatives , Urea/chemistryABSTRACT
Anthocyanins (ACNs) are dietary phytochemicals with an acknowledged therapeutic significance. Pomegranate juice (PJ) is a rich source of ACNs with potential applications in nutraceutical development. Glycogen phosphorylase (GP) catalyzes the first step of glycogenolysis and is a molecular target for the development of antihyperglycemics. The inhibitory potential of the ACN fraction of PJ is assessed through a combination of in vitro assays, ex vivo investigation in hepatic cells, and X-ray crystallography studies. The ACN extract potently inhibits muscle and liver isoforms of GP. Affinity crystallography reveals the structural basis of inhibition through the binding of pelargonidin-3-O-glucoside at the GP inhibitor site. The glucopyranose moiety is revealed as a major determinant of potency as it promotes a structural binding mode different from that observed for other flavonoids. This inhibitory effect of the ACN scaffold and its binding mode at the GP inhibitor binding site may have significant implications for future structure-based drug design endeavors.
Subject(s)
Anthocyanins/chemistry , Enzyme Inhibitors/chemistry , Fruit and Vegetable Juices/analysis , Glycogen Phosphorylase/chemistry , Plant Extracts/chemistry , Pomegranate/chemistry , Amino Acid Motifs , Animals , Binding Sites , Crystallography, X-Ray , Glycogen Phosphorylase/antagonists & inhibitors , Hep G2 Cells , Humans , Kinetics , Protein Binding , RabbitsABSTRACT
Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 angstroms resolution structure of PhK heterotetramer (alphabetagammadelta)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic gamma subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 angstroms resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 angstroms resolution was consistent with a proteolysed activated form of PhK that had lost the alpha subunits and possibly the gamma subunits.
Subject(s)
Glycogen Phosphorylase/metabolism , Phosphorylase Kinase/chemistry , Catalytic Domain , Chromatography, Gel , Cryoelectron Microscopy , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Models, Molecular , Phosphorylase Kinase/metabolism , Protein Conformation , Substrate SpecificityABSTRACT
HupR is a response regulator that controls the synthesis of the membrane-bound [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus. The protein belongs to the NtrC subfamily of response regulators and is the second protein of a two-component system. We have crystallized the full-length protein HupR in the unphosphorylated state in two dimensions using the lipid monolayer technique. The 3D structure of negatively stained HupR was calculated to a resolution of approximately 23 A from tilted electron microscope images. HupR crystallizes as a dimer, and forms an elongated V-shaped structure with extended arms. The dimensions of the dimer are about 80 A length, 40 A width and 85 A thick. The HupR monomer consists of three domains, N-terminal receiver domain, central domain and C-terminal DNA-binding domain. We have fitted the known 3D structure of the central domain from NtrC1 Aquifex aeolicus protein into our 3D model; we propose that contact between the dimers is through the central domain. The N-terminal domain is in contact with the lipid monolayer and is situated on the top of the V-shaped structure. The central domain alone has been expressed and purified; it forms a pentamer in solution and lacks ATPase activity.
