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From in Silico Discovery to intra-Cellular Activity: Targeting JNK-Protein Interactions with Small Molecules.

ABSTRACT: The JNK-JIP1 interaction represents an attractive target for the selective inhibition of JNK-mediated signaling. We report a virtual screening (VS) workflow, based on a combination of three-dimensional shape and electrostatic similarity to discover novel scaffolds for the development of non-ATP competitive inhibitors of JNK targeting the JNK-JIP interaction. Of 352 (0.13%) compounds selected from the NCI diversity set more than 22% registered as hits in a biochemical kinase assay. Several compounds discovered to inhibit JNK activity under standard kinase assay conditions also impeded JNK activity in HEK293 cells. These studies led to the discovery that the lignan (-)-zuonin A inhibits JNK-protein interactions with a selectivity of 100-fold over ERK2 and p38 MAPKα. These results demonstrate the utility of a virtual screening protocol to identify novel scaffolds for highly selective, cell-permeable inhibitors of JNK-protein interactions.

SUBMITTER: Kaoud TS

PROVIDER: S-EPMC3445420 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here, we evaluate the binding and biological activity of a new class of pyrrolidinone and piperidinone compounds, along with derivatives of previously-identified pyrazole and propylamine compounds. Competition assays revealed that the compounds displace a fluorescently labeled peptide (AE147-FAM) with inhibition constant (Ki ) values ranging from 6 to 63??M. Structure-based computational pharmacophore analysis followed by extensive explicit-solvent molecular dynamics (MD) simulations and free energy calculations suggested the pyrazole-based and piperidinone-based compounds adopt different binding modes, despite their similar two-dimensional structures. In cells, pyrazole-based compounds showed significant inhibition of breast adenocarcinoma (MDA-MB-231) and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but piperidinone-containing compounds exhibited no cytotoxicity even at concentrations of 100??M. One pyrazole-based compound impaired MDA-MB-231 invasion, adhesion, and migration in a concentration-dependent manner, while the piperidinone inhibited only invasion. The pyrazole derivative inhibited matrix metalloprotease-9 (gelatinase) activity in a concentration-dependent manner, while the piperidinone showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1? and NF-?B signaling, while pyrrolidinones and piperidinones had no effect. Annexin?V staining suggested that the effect of the pyrazole-based compound on proliferation was due to cell killing through an apoptotic mechanism. The compounds identified represent valuable leads in the design of further derivatives with higher affinities and potential probes to unravel the protein-protein interactions of uPAR.

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From in Silico Discovery to intra-Cellular Activity: Targeting JNK-Protein Interactions with Small Molecules.

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