Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

Abstract

Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

“…Finally, we could argue that the flexibility of the formed complex, where NUPR1 is not folded, was responsible for the absence of thermogram in ITC (37). The persistently disordered conformation of NUPR1 seems to be a general feature (33,34,38): Whatever molecule (synthetic, DNA, or protein) is bound to it, it is not capable of altering its disordered nature. However, binding to C-RING1B had an intermediate-to-slow exchange rate within the NMR time scale (either for C-RING1B or NUPR1).…”

“…3A) Gly16, Glu18, Asp19, Ser22 (Ser10), Ser23 (Asn53), Ser27, Leu29, Tyr30, Ser31, and Arg82 (the residues within parentheses are those where signal overlapping occurs). Most of these residues were close to the hydrophobic 30s region (38) or, alternatively, they were partly sequestered from the solvent by local structure or hydrogen bonding (40,41). Upon addition of C-RING1B, the cross-peaks began to disappear (Fig.…”

“…We also measured the binding of both mutants, Thr68Gln and Ala33Gln/Thr68Gln, to C-RING1B. Both mutants were designed to hamper the interaction based on our previous in silico results of the whole intact NUPR1 (38). The titration curves were always noisier than those of wild type (Fig.…”

“…acid Thr68, which together with Ala33 constitutes the most hydrophobic regions of its sequence (38) and is another anchoring site to NUPR1 molecular partners, showed docking scores of about -6.0 kcal/mol. However, Thr68 has a slightly higher preference for binding to cluster Cl-1 of C-RING1B (Fig.…”

“…We have previously shown (38) that the interaction of wildtype NUPR1 with MSL1 can be hampered by the addition of a drug (trifluoperazine, TFP) competing for the same binding site of the protein. Therefore, we wondered whether the interaction between RING1B and wild-type NUPR1 could be also inhibited by TFP.…”