Amphipathic helical peptides hamper protein-protein interactions of the intrinsically disordered chromatin nuclear protein 1 (NUPR1)

Abstract

Background: NUPR1 is a multifunctional intrinsically disordered protein (IDP) involved, among other functions, in chromatin remodelling, and development of pancreatic ductal adenocarcinoma (PDAC). It interacts with several biomolecules through hydrophobic patches around residues Ala33 and Thr68. The drug trifluoperazine (TFP), which hampers PDAC development in xenografted mice, also binds to those regions. Because of the large size of the hot-spot interface of NUPR1, small molecules could not be adequate to modulate its functions. Methods: We explored how amphipathic helical-designed peptides were capable of interacting with wild-type NUPR1 and the Thr68Gln mutant, inhibiting the interaction with NUPR1 protein partners. We used in vitro biophysical techniques (fluorescence, circular dichroism (CD), nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC)), in silico studies (docking and molecular dynamics (MD)), and in cellulo protein ligation assays (PLAs) to study the interaction. Results: Peptide dissociation constants towards wild-type NUPR1 were ~ 3 M, whereas no interaction was observed with the Thr68Gln mutant. Peptides interacted with wild-type NUPR1 residues around Ala33 and residues at the C terminus, as shown by NMR. The computational results clarified the main determinants of the interactions, providing a mechanism for the ligand-capture that explains why peptide binding was not observed for Thr68Gln mutant. Finally, the in cellulo assays indicated that two out of four peptides inhibited the interaction of NUPR1 with the C-terminal region of the Polycomb RING protein 1 (C-RING1B). Conclusions: Designed peptides can be used as lead compounds to inhibit NUPR1 interactions. General significance: Peptides may be exploited as drugs to target IDPs.

Ligand-based design identifies a potent NUPR1 inhibitor exerting anticancer activity via necroptosis

“…The resulting complexes were equilibrated by 1 ns of MD simulation performed using the GAFF force field (46) for the compounds. Simulation conditions including treatment of the electrostatics and van der Waals interactions, and reference values and coupling times for both the thermostat and barostat were as previously reported (26,28). After the MD simulation runs, the binding affinity of the compounds was evaluated using the scoring function of AutoDock Vina (45), by performing a redocking of the ligand in the position occupied within the average structure of the simulated complex.…”

“…Structurally, NUPR1 is an intrinsically disordered protein (IDP) with an entirely disordered conformation (5,(25)(26)(27)(28). Consequently, the target-based high throughput screening for drug selection toward this protein is highly challenging.…”

Targeting intrinsically disordered proteins involved in cancer

“…It has two hot spot regions (identified by in silico procedures and protein engineering studies [98]) involved in binding to its natural partners (DNA, prothymosin α, male-specific lethal protein, and the C-terminal domain of RING1B): (1) the 30′s region, where the two tyrosine residues of the protein are located; and (2) the region around Thr68. Both regions are among the most hydrophobic polypeptide patches in the chain [96,98,99]. However, in all the complexes, NUPR1 remains disordered.…”

“…Structurally, NUPR1 is an 82-residue-long, monomeric, basic IDP [95][96][97][98][99]. It has two hot spot regions (identified by in silico procedures and protein engineering studies [98]) involved in binding to its natural partners (DNA, prothymosin α, male-specific lethal protein, and the C-terminal domain of RING1B): (1) the 30′s region, where the two tyrosine residues of the protein are located; and (2) the region around Thr68.…”

“…We have selected those compounds with larger variations either in the thermal denaturation midpoints or in the denaturation profiles, and next, we have measured their affinity for NUPR1 using isothermal titration calorimetry (ITC). We have chosen those with the most favourable affinities, with values similar to those found for the NUPR1 natural [78,96,98] or non-natural [99] binding partners. We then have combined SAR (structure-activity relationships) by NMR results and MD simulations, carried out independently of each other, to verify that a similar set of residues were affected by the binding of the compound.…”