Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies.

Cadherins are homophilic cell-cell adhesion molecules whose aberrant expression has often been shown to correlate with different stages of tumor progression. In this work, we investigate the interaction of two peptidomimetic ligands with the extracellular portion of human E-cadherin using a combination of NMR and computational techniques. Both ligands have been previously developed as mimics of the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 of the cadherin adhesion arm, and have been shown to inhibit E-cadherin-mediated adhesion in epithelial ovarian cancer cells with millimolar potency. To sample a set of possible interactions of these ligands with the E-cadherin extracellular portion, STD-NMR experiments in the presence of two slightly different constructs, the wild type E-cadherin-EC1-EC2 fragment and the truncated E-cadherin-(Val3)-EC1-EC2 fragment, were carried out at three temperatures. Depending on the protein construct, a different binding epitope of the ligand and also a different temperature effect on STD signals were observed, both suggesting an involvement of the Asp1-Trp2 protein sequence among all the possible binding events. To interpret the experimental results at the atomic level and to probe the role of the cadherin adhesion arm in the dynamic interaction with the peptidomimetic ligand, a computational protocol based on docking calculations and molecular dynamics simulations was applied. In agreement with NMR data, the simulations at different temperatures unveil high variability/dynamism in ligand-cadherin binding, thus explaining the differences in ligand binding epitopes. In particular, the modulation of the signals seems to be dependent on the protein flexibility, especially at the level of the adhesive arm, which appears to participate in the interaction with the ligand. Overall, these results will help the design of novel cadherin inhibitors that might prevent the swap dimer formation by targeting both the Trp2 binding pocket and the adhesive arm residues.

Structure-Based Virtual Screening Allows the Identification of Efficient Modulators of E-Cadherin-Mediated Cell-Cell Adhesion.

Cadherins are a large family of transmembrane calcium-dependent cell adhesion proteins that orchestrate adherens junction formation and are crucially involved in tissue morphogenesis. Due to their important role in cancer development and metastasis, cadherins can be considered attractive targets for drug discovery. A recent crystal structure of the complex of a cadherin extracellular portion and a small molecule inhibitor allowed the identification of a druggable interface, thus providing a viable strategy for the design of cadherin dimerization modulators. Here, we report on a structure-based virtual screening approach that led to the identification of efficient and selective modulators of E-cadherin-mediated cell-cell adhesion. Of all the putative inhibitors that were identified and experimentally tested by cell adhesion assays using human pancreatic tumor BxPC-3 cells expressing both E-cadherin and P-cadherin, two compounds turned out to be effective in inhibiting stable cell-cell adhesion at micromolar concentrations. Moreover, at the same concentrations, one of them also showed anti-invasive properties in cell invasion assays. These results will allow further development of novel and selective cadherin-mediated cell-cell adhesion modulators for the treatment of a variety of cadherin-expressing solid tumors and for improving the efficiency of drug delivery across biological barriers.

A combined fragment-based virtual screening and STD-NMR approach for the identification of E-cadherin ligands.

Cadherins promote cell-cell adhesion by forming homophilic interactions via their N-terminal extracellular domains. Hence, they have broad-ranging physiological effects on tissue organization and homeostasis. When dysregulated, cadherins contribute to different aspects of cancer progression and metastasis; therefore, targeting the cadherin adhesive interface with small-molecule antagonists is expected to have potential therapeutic and diagnostic value. Here, we used molecular docking simulations to evaluate the propensity of three different libraries of commercially available drug-like fragments (nearly 18,000 compounds) to accommodate into the Trp2 binding pocket of E-cadherin, a crucial site for the orchestration of the protein's dimerization mechanism. Top-ranked fragments featuring five different aromatic chemotypes were expanded by means of a similarity search on the PubChem database (Tanimoto index >90%). Of this set, seven fragments containing an aromatic scaffold linked to an aliphatic chain bearing at least one amine group were finally selected for further analysis. Ligand-based NMR data (Saturation Transfer Difference, STD) and molecular dynamics simulations suggest that these fragments can bind E-cadherin mostly through their aromatic moiety, while their aliphatic portions may also diversely engage with the mobile regions of the binding site. A tetrahydro-ß-carboline scaffold functionalized with an ethylamine emerged as the most promising fragment.

Structural characterization of human O-phosphoethanolamine phospho-lyase.

Human O-phosphoethanolamine phospho-lyase (hEtnppl; EC 4.2.3.2) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the degradation of O-phosphoethanolamine (PEA) into acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism, as PEA is the precursor of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. Here, the crystal structure of hEtnppl in complex with pyridoxamine 5'-phosphate was determined at 2.05 Šresolution by molecular replacement using the structure of A1RDF1 from Arthrobacter aurescens TC1 (PDB entry 5g4i) as the search model. Structural analysis reveals that the two proteins share the same general fold and a similar arrangement of active-site residues. These results provide novel and useful information for the complete characterization of the human enzyme.