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.
Highlights
Disordered proteins (IDPs) do not have stable secondary or tertiary structures in several regions, or throughout their whole sequence[4,5,6], since they exist as an ensemble of rapidly inter-converting structures
NUPR1 expression controls pancreatic cancer cell migration, invasion and adhesion, three processes required for metastasis through CDC42, which is a major regulator of cytoskeleton organization[11,13]; apoptosis by interacting with prothymosin α14; and chemo-resistance[15]
The compounds were assayed in pancreatic ductal adenocarcinoma (PDAC)-derived cell-based experiments to test whether they inhibited the interaction between male-specific-lethal protein 1 (MSL1) and NUPR1 in vivo; this interaction is critical during DNA-repair processes
Summary
Disordered proteins (IDPs) do not have stable secondary or tertiary structures in several regions, or throughout their whole sequence[4,5,6], since they exist as an ensemble of rapidly inter-converting structures Because of their plasticity IDPs act as hubs in interaction networks carrying out several functions in cell-signaling routes and regulation (“moonlighting”)[5,6], they are very often involved in important diseases. We started by screening 1120 Food and Drug Administration (FDA)-approved drugs (Prestwick Chemical Library) searching for compounds capable of binding to NUPR1 using fluorescence thermal-denaturation Those triggering the largest changes in the thermal-denaturation profile (15 compounds) were examined by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) to determine their binding affinity and the interacting region in NUPR1. Compound-15, the most effective one, was tested in vivo and completely arrested PDAC development in mice with tumor induced by xenografting PDAC-derived cells
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