Screening a Molecular Fragment Library to Modulate the PED/PEA15-Phospholipase D1 Interaction in Cellular Lysate Environments

Abstract

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.

Molecular interactions between a diphenyl scaffold and PED/PEA15: Implications for type II diabetes therapeutics targeting PED/PEA15 – Phospholipase D1 interaction

“…The obtained file was employed for docking simulations performed by Grid-based ligand docking with energetics (GLIDE) [34] , [35] , [36] , [37] , [38] . In order to properly take into account putative conformational rearrangements of the protein binding site during molecular recognition, Induced Fit Docking (IFD) [39] , [40] simulations were performed using the SP mode (all the used parameters are available in Table S1 - Supporting information ) and all the default settings, building a cubic grid centered on the center of mass of H52 (predicted to be protonated during the protein preparation step) and H65 (predicted to be neutral and with the δ -N atom bound to an hydrogen atom), namely the residues mostly affected by the interaction with BPH03 as indicated by NMR data [29] . Furthermore, an inner box of 10 Å × 10 Å × 10 Å and an outer box of 30 Å × 30 Å × 30 Å were employed.…”

“…15 N-Labeled PED was expressed in Escherichia coli and purified as previously reported [28] . The In-cell NMR analysis of the protein-small molecules interactions [53] , [52] were conducted on cell lysates prepared as reported in Farina et al [29] .…”

“…These structural insights have facilitated the screening of a library of small molecules, leading to the identification of the 4-[(4-Methylphenyl)thio]-3-nitrobenzoic acid, a compound named BPH03 ( Fig. 1 B ), as a promising candidate for the development of modulators capable of directly interfering with the binding interface between PED and PLD1 [29] . As the interference of protein-protein interactions with a small molecule is expected to be more successful in presence of compactly clustered interaction hot spots [30] we here report the detailed description of BPH03 interaction with PED evidencing, within its D4 binding surface, the presence of a hidden druggable pocket as well as the conformational changes taking place during PED/BPH03 interaction.…”

“…These results were achieved using a combined experimental and in-silico approach. Starting from the recently conducted experimental screening of a molecular fragments library by NMR [29] , molecular modeling techniques (cavity mapping, molecular docking, and molecular dynamics) were employed in continuous interplay with NMR experiments conducted to support the in-silico results. Given the overexpression of PED in various tissues of individuals with T2D [19] , [27] all the obtained results are discussed in the perspective of being used as a starting point for rationally designing and developing the first inhibitors of the PED/PLD1 interaction for the treatment of T2D.…”

Polystyrene nanoplastics affect the human ubiquitin structure and ubiquitination in cells: a high-resolution study

“…16 The signals of Ubq bound to NPs are, as expected, invisible due to line-broadening but information regarding the bound state can be obtained due to the exchange-mediated signal averaging. 16,39,40 Chemical shi perturbation and signal intensities with respect to reference spectra as a function of ligand addition thus provide useful dynamic and conformational information about the binding event. For this reason, the chemical shis and intensity variations of the different nuclei in the presence and absence of NPs were evaluated.…”