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The Sam (sterile alpha motif) domain from the lipid phosphatase Ship2 binds the Sam domain from the EphA2 receptor to negatively regulate receptor endocytosis and degradation. This interaction is primarily linked to pro-oncogenic effects. We report on the design and evaluation of EphA2-Sam/Ship2-Sam peptide inhibitors provided with positive charges and different aromatic characters. Starting from the sequence of previously identified Ship2-Sam targeting peptides, an in silico approach was set up to predict higher affinity peptide ligands. A few peptides were experimentally tested through an interdisciplinary approach. Interaction studies were performed by nuclear magnetic resonance spectroscopy and biolayer interferometry. 3D models of Ship2-Sam/peptide complexes were predicted by AlphaFold2. Cell-based assays were carried out to investigate whether such peptide sequences might have an influence on EphA2 signaling. The approach led to the identification of novel Ship2-Sam ligands and shed further light on original approaches to design inhibitors of the Ship2-Sam/EphA2-Sam interaction.

The overexpression of PED/PEA15, the phosphoprotein enriched in diabetes/phosphoprotein enriched in the astrocytes 15 protein (here referred simply to as PED), observed in some forms of type II diabetes, reduces the transport of insulin-stimulated glucose by binding to the phospholipase D1 (PLD1). The inhibition of the PED/PLD1 interaction was shown to restore basal glucose transport, indicating PED as a pharmacological target for the development of drugs capable of improving insulin sensitivity and glucose tolerance. We here report the identification and selection of PED ligands by means of NMR screening of a library of small organic molecules, NMR characterization of the PED/PLD1 interaction in lysates of cells expressing PLD1, and modulation of such interactions using BPH03, the best selected ligand. Overall, we complement the available literature data by providing detailed information on the structural determinants of the PED/PLD1 interaction in a cellular lysate environment and indicate BPH03 as a precious scaffold for the development of novel compounds that are able to modulate such interactions with possible therapeutic applications in type II diabetes.

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of antidiabetic drugs acting through this nuclear receptor. We have employed a multidisciplinary approach, including protein–protein docking, X-ray crystallography, NMR, HDX, MD simulations, and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternative inhibition mechanism adopted by a ligand bound far from the phosphorylation site.

A RGD motif was identified in the N‐terminal region of cE5, a potent salivary thrombin inhibitor from the African malaria vector Anopheles gambiae. A peptide (APQ30) encompassing the first 30 amino acids residues of the protein and including the RGD tripeptide was tested in cell adhesion assays and found to inhibit αvβ3and αvβ5mediated adhesion. A shorter peptide (APQ16), strongly conserved among members of the A. gambiaespecies complex and including only the first 16 residues, retained adhesion inhibitory properties, however with enhanced specificity toward αvβ5. In addition, migration and invasion assays showed its capacity to inhibit the invasiveness of the malignant cell lines HepG2 and MDA‐MB231. Altogether our data point to APQ16 as a new promising candidate as theranostic agent.