Biochemical and Structural Studies of LjSK1, a Lotus japonicus GSK3ß/SHAGGY-like Kinase, Reveal Its Functional Role.

The crystal structure of a truncated form of the Lotus japonicus glycogen synthase kinase 3ß (GSK3ß) like kinase (LjSK190-467) has been resolved at 2.9 Å resolution, providing, for the first time, structural data for a plant GKS3ß like kinase. The 3D structure of LjSK190-467 revealed conservation at the structural level for this plant member of the GSK3ß family. However, comparative structural analysis to the human homologue revealed significant differences at the N- and C-termini, supporting the notion for an additional regulatory mechanism in plant GSK3-like kinases. Structural similarities at the catalytic site and the ATP binding site explained the similarity in the function of the human and plant protein. LjSK1 and lupeol are strongly linked to symbiotic bacterial infection and nodulation initiation. An inhibitory capacity of lupeol (IC50 = 0.77 µM) for LjSK1 was discovered, providing a biochemical explanation for the involvement of these two molecules in nodule formation, and constituted LjSK1 as a molecular target for the discovery of small molecule modulators for crop protection and development. Studies on the inhibitory capacity of two phytogenic triterpenoids (betulinic acid and hederacoside C) to LjSK1 provided their structure-activity relationship and showed that hederacoside C can be the starting point for such endeavors.

Novel diarylamides and diarylureas with N-substitution dependent activity against medulloblastoma.

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.

Mutagenesis of a Lotus japonicus GSK3ß/Shaggy-like kinase reveals functionally conserved regulatory residues.

The glycogen synthase kinases 3 family (GSK3s/SKs; serine/threonine protein kinases) is conserved throughout eukaryotic evolution from yeast to plants and mammals. We studied a plant SK kinase from Lotus japonicus (LjSK1), previously implicated in nodule development, by enzyme kinetics and mutagenesis studies to compare it to mammalian homologues. Using a phosphorylated peptide as substrate, LjSK1 displays optimum kinase activity at pH 8.0 and 20 °C following Michaelis-Menten kinetics with Km and Vmax values of 48.2 µM and 111.6 nmol/min/mg, respectively, for ATP. Mutation of critical residues, as inferred by sequence comparison to the human homologue GSK3ß and molecular modeling, showed a conserved role for Lys167, while residues conferring substrate specificity in the human enzyme are not as significant in modulating LjSK1 substrate specificity. Mutagenesis studies also indicate a regulation mechanism for LjSK1 via proteolysis since removal of a 98 residue long N-terminal segment increases its catalytic efficiency by almost two-fold. In addition, we evaluated the alteration of LjSK1 kinase activity in planta, by overexpressing the mutant variants in hairy-roots and a phenotype in nodulation and lateral root development was verified.

Probing the ß-pocket of the active site of human liver glycogen phosphorylase with 3-(C-ß-d-glucopyranosyl)-5-(4-substituted-phenyl)-1, 2, 4-triazole inhibitors.

Human liver glycogen phosphorylase (hlGP), a key enzyme in glycogen metabolism, is a valid pharmaceutical target for the development of new anti-hyperglycaemic agents for type 2 diabetes. Inhibitor discovery studies have focused on the active site and in particular on glucopyranose based compounds with a ß-1 substituent long enough to exploit interactions with a cavity adjacent to the active site, termed the ß-pocket. Recently, C-ß-d-glucopyranosyl imidazoles and 1, 2, 4-triazoles proved to be the best known glucose derived inhibitors of hlGP. Here we probe the ß-pocket by studying the inhibitory effect of six different groups at the para position of 3-(ß-d-glucopyranosyl phenyl)-5-phenyl-, 1, 2, 4-triazoles in hlGP by kinetics and X-ray crystallography. The most bioactive compound was the one with an amine substituent to show a Ki value of 0.43 µM. Structural studies have revealed the physicochemical diversity of the ß-pocket providing information for future rational inhibitor design studies.

Nanomolar Inhibitors of Glycogen Phosphorylase Based on ß-d-Glucosaminyl Heterocycles: A Combined Synthetic, Enzyme Kinetic, and Protein Crystallography Study.

Aryl substituted 1-(ß-d-glucosaminyl)-1,2,3-triazoles as well as C-ß-d-glucosaminyl 1,2,4-triazoles and imidazoles were synthesized and tested as inhibitors against muscle and liver isoforms of glycogen phosphorylase (GP). While the N-ß-d-glucosaminyl 1,2,3-triazoles showed weak or no inhibition, the C-ß-d-glucosaminyl derivatives had potent activity, and the best inhibitor was the 2-(ß-d-glucosaminyl)-4(5)-(2-naphthyl)-imidazole with a Ki value of 143 nM against human liver GPa. An X-ray crystallography study of the rabbit muscle GPb inhibitor complexes revealed structural features of the strong binding and offered an explanation for the differences in inhibitory potency between glucosyl and glucosaminyl derivatives and also for the differences between imidazole and 1,2,4-triazole analogues.

van der Waals interactions govern C-ß-d-glucopyranosyl triazoles' nM inhibitory potency in human liver glycogen phosphorylase.

3-(C-Glucopyranosyl)-5aryl-1,2,4-triazoles with an aryl moiety larger than phenyl have been shown to have strong inhibitory potency (Ki values in the range of upper nM) for human liver glycogen phosphorylase (hlGP), a pharmacologically relevant target for diabetes type 2. In this study we investigate in a comparative manner the inhibitory effect of the above triazoles and their respective imidazoles on hlGPa. Kinetic studies show that the imidazole derivatives are 6-8 times more potent than their corresponding triazoles. We also seek to answer how the type of the aryl moiety affects the potency in hlGPa, and by determination of the crystal structure of rmGPb in complex with the triazole derivatives the structural basis of their inhibitory efficacy is also elucidated. Our studies revealed that the van der Waals interactions between the aryl moiety and residues in a hydrophobic pocket within the active site are mainly responsible for the variations in the potency of these inhibitors.