Altered Glycosylation of Human Alpha-1-Acid Glycoprotein as a Biomarker for Malignant Melanoma

A high-resolution HILIC-MS/MS method was developed to analyze anthranilic acid derivatives of N-glycans released from human serum alpha-1-acid glycoprotein (AGP). The method was applied to samples obtained from 18 patients suffering from high-risk malignant melanoma as well as 19 healthy individuals. It enabled the identification of 102 glycan isomers separating isomers that differ only in sialic acid linkage (α-2,3, α-2,6) or in fucose positions (core, antenna). Comparative assessment of the samples revealed that upregulation of certain fucosylated glycans and downregulation of their nonfucosylated counterparts occurred in cancer patients. An increased ratio of isomers with more α-2,6-linked sialic acids was also observed. Linear discriminant analysis (LDA) combining 10 variables with the highest discriminatory power was employed to categorize the samples based on their glycosylation pattern. The performance of the method was tested by cross-validation, resulting in an overall classification success rate of 96.7%. The approach presented here is significantly superior to serological marker S100B protein in terms of sensitivity and negative predictive power in the population studied. Therefore, it may effectively support the diagnosis of malignant melanoma as a biomarker.

Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling

Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ–Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure–activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50∼50 µM) and Western blotting of β-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (KD=2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.

Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling

Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ/Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure-activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50 ~50 µM), and Western blotting of β-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (Kd = 2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.

Conformational analysis and molecular design of anthranilic acid derivatives as partial agonists of the Farnesoid X Receptor (FXR) with favorable predicted pharmacokinetic profiles

We report here virtual design of new anthranilic acid derivatives (AAD) identified as potent partial Farnesoid X recep-tor (FXR) agonists with favorable predicted pharmacokinetic profiles. By in situ modification of the crystal structure (PDB ID: 3OLF) of FXR complex with a benzimidazole-based partial agonistic ligand, 3D models of 17 FXR:AADx complexes with known observed activity (EC50exp) were prepared to establish a quantitative structure–activity (QSAR) model and linear correla-tion between relative Gibbs free energy (GFE) of receptor-ligand complex formation (Gcom) and EC50exp: pEC50exp = -0,1146 Gcom + 8,175 (#); R2 = 0.98. A 3D QSAR pharmacophore model (PH4) derived from the QSAR directed our effort to design novel AAD analogs. During the design, an initial virtual library of 94501 AAD was focused down to 33134 drug-like compounds and finally, PH4 screened to identify 100 promising compounds. Theoretical EC50 (EC50pre) values of all analogs compounds were predicted by means of equation (#) and their pharmacokinetics (ADME) profiles were computed. More than 12 putative AADs display EC50pre 300 times superior to that of the reported most active training set inhibitor AAD1.