Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function

Abstract

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.

Characterization of intrinsically disordered regions in proteins informed by human genetic diversity

“…This showed that 41% and 18% of the motifs overlapping with IDRs in our dataset are ligand sites (LIG), which mediate binding of the ligand protein to its interaction partner, and subcellular targeting sites (TRG), respectively (S6A Fig and S8 Table ). Moreover, by investigating the Gene Ontology terms (GO version 2021-11-20 [57]) for the motifs available in the ELM resource, we found that 53% of these motifs in IDRs are involved in different biological processes such as DNA repair/replication/damage and cell division/death (S6B Fig) , in agreement with previous studies showing the link between IDPs and these biological processes [9,58,59].…”

Human Enzyme PADI4 Binds to the Nuclear Carrier Importin α3

“…As an example, in the case of the crystallographic complex between Impα3 and the NLS sequence of the Ran-binding protein 3 [ 65 ], the anchoring sequence constituting the binding interface was significantly shorter. For this reason, according to a screening protocol we already successfully employed in other cases [ 78 , 79 , 80 , 81 ], the docking was performed by considering seven-residue fragments of the two peptides. Each of these fragments differed by a shift of two consecutive residues with respect to the following one, and the whole set of fragments covered the whole sequence of the two NLS peptides.…”

Deciphering the Binding of the Nuclear Localization Sequence of Myc Protein to the Nuclear Carrier Importin α3

“…The entire sequence A 310 APPSTRKDYPAAKRVKLDSVRVLRQISNN 339 of NLS-Myc was screened by considering consecutive shorter fragments of six residues, following a protocol we have introduced to model in an exhaustive way the binding of relatively long peptide/protein sequences to their molecular targets [ 58 , 59 , 60 , 61 ]. Each of the fragments was shifted with respect to the previous one by two residues, up to include the whole sequence of the NLS-Myc, for a total of 13 fragments (A 310 APPST 315 , P 312 PSTRK 317 , …, R 334 QISNN 339 ).…”