High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-N-acyl-ß-d-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors.

Structure-based design and synthesis of two biphenyl-N-acyl-ß-d-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico-derived models of the binding mode generated during the design process corresponded very well with the crystallographic data.

Kinetic and Structural Studies of the Plastidial Solanum tuberosum Phosphorylase.

Kinetics and structural studies of the plastidial Solanum tuberosum phosphorylase (stPho1) revealed that the most active form of the enzyme (stPho1ΔL78) is composed by two segments generated by proteolytic degradation of an approximately 65-residue-long peptide (L78) approximately in the middle of the stPho1 primary structure. stPho1ΔL78 is 1.5 times more active than the nonproteolyzed enzyme in solution and shows stronger specificity for glycogen, α-d-glucose, caffeine, and ß-cyclodextrin than stPho1. The crystal structure of stPho1ΔL78 has been resolved at 2.2 Å resolution and revealed similarities and differences with the mammalian enzymes. The structural fold is conserved as is the active site, while other binding sites such as the inhibitor, the glycogen storage, the quercetin, and the allosteric are not. The binding of α-d-glucose, caffeine, and ß-cyclodextrin to stPho1 has been studied by X-ray crystallography and revealed significant differences from those of the mammalian phosphorylases. As stPho1 is capable of catalyzing both starch synthesis and degradation, our studies suggest that the direction of stPho1 activity is regulated by the proteolytic degradation of the L78 peptide.

Strong Binding of C-Glycosylic1,2-Thiodisaccharides to Galectin-3─Enthalpy-Driven Affinity Enhancement by Water-Mediated Hydrogen Bonds.

Galectin-3 is involved in multiple pathways of many diseases, including cancer, fibrosis, and diabetes, and it is a validated pharmaceutical target for the development of novel therapeutic agents to address unmet medical needs. Novel 1,2-thiodisaccharides with a C-glycosylic functionality were synthesized by the photoinitiated thiol-ene click reaction of O-peracylated 1-C-substituted glycals and 1-thio-glycopyranoses. Subsequent global deprotection yielded test compounds, which were studied for their binding to human galectin-3 by fluorescence polarization and isothermal titration calorimetry to show low micromolar Kd values. The best inhibitor displayed a Kd value of 8.0 µM. An analysis of the thermodynamic binding parameters revealed that the binding Gibbs free energy (ΔG) of the new inhibitors was dominated by enthalpy (ΔH). The binding mode of the four most efficient 1,2-thiodisaccharides was also studied by X-ray crystallography that uncovered the unique role of water-mediated hydrogen bonds in conferring enthalpy-driven affinity enhancement for the new inhibitors. This 1,2-thiodisaccharide-type scaffold represents a new lead for galectin-3 inhibitor discovery and offers several possibilities for further development.

In Vitro Chondrogenesis Induction by Short Peptides of the Carboxy-Terminal Domain of Transforming Growth Factor ß1.

Τransforming growth factor ß1 (TGF-ß1) comprises a key regulator protein in many cellular processes, including in vivo chondrogenesis. The treatment of human dental pulp stem cells, separately, with Leu83-Ser112 (C-terminal domain of TGF-ß1), as well as two very short peptides, namely, 90-YYVGRKPK-97 (peptide 8) and 91-YVGRKP-96 (peptide 6) remarkably enhanced the chondrogenic differentiation capacity in comparison to their full-length mature TGF-ß1 counterpart either in monolayer cultures or 3D scaffolds. In 3D scaffolds, the reduction of the elastic modulus and viscous modulus verified the production of different amounts and types of ECM components. Molecular dynamics simulations suggested a mode of the peptides' binding to the receptor complex TßRII-ALK5 and provided a possible structural explanation for their role in inducing chondrogenesis, along with endogenous TGF-ß1. Further experiments clearly verified the aforementioned hypothesis, indicating the signal transduction pathway and the involvement of TßRII-ALK5 receptor complex. Real-time PCR experiments and Western blot analysis showed that peptides favor the ERK1/2 and Smad2 pathways, leading to an articular, extracellular matrix formation, while TGF-ß1 also favors the Smad1/5/8 pathway which leads to the expression of the metalloproteinases ADAMTS-5 and MMP13 and, therefore, to a hypertrophic chondrocyte phenotype. Taken together, the two short peptides, and, mainly, peptide 8, could be delivered with a scaffold to induce in vivo chondrogenesis in damaged articular cartilage, constituting, thus, an alternative therapeutic approach for osteoarthritis.

Affinity Crystallography Reveals Binding of Pomegranate Juice Anthocyanins at the Inhibitor Site of Glycogen Phosphorylase: The Contribution of a Sugar Moiety to Potency and Its Implications to the Binding Mode.

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.