Targeting Transcription in Neuroblastoma: Development of Inhibitors of Non-POU Domain-Containing Octamer-Binding Protein NONO

The non-POU domain-containing octamer-binding protein (NONO emerges as a master regulator of gene expression in physiological states and an anticancer target. Phenotypic screening identified (R)-4-(2-chloroacetyl)-N-(4-methoxybenzyl)-1-((4-methoxyphenyl)sulfonyl)piperazine-2-carboxamide ((R)-SKBG-1)) as a pioneer pharmacological NONO inhibitor. Herein we, for the first time, report a thrifty synthetic route to (R)-SKBG-1 from the available (R)-(tert-butoxycarbonyl)piperazine-2-carboxylic acid. In search of structure-activity relationship within the (R)-SKBG-1 chemotype, we synthesized a series of original derivatives. For docking into the model of the NONO homodimer, the geometrical parameters of the complex (R)-SKBG-1–NONO were minimized using OPLS4 force field. Covalent docking was performed using Glide algorithm. Docking poses were ranged according to IFD score. These procedures revealed that the covalent bond between the chloroacetyl moiety at the position 4 of the piperazine ring (‘warhead’) and Cys145 residue proved to be mandatory for NONO inhibition. The cytotoxicity was assessed by MTT tests, colony formation assays and flow cytometry assisted analysis of cell death (YO-PRO-1/propidium iodide staining). NONO inhibition was translated into preferential potency for human neuroblastoma cell lines Kelly, SH-SY5Y, SK-N-AS and SK-N-SH (at low micromolar concentrations) compared to non-malignant, bone marrow derived HS-5 fibroblasts. Molecular dynamics simulations of NONO-RNA complexes were analyzed based on RMSD and RMSF fluctuations and time of contact between ligand atoms and neighboring amino acid residues; ΔG_bind values for representative clusters were estimated by MM-GBSA method (solvent: water). We found that binding to Cys145 ensured the close proximity of (R)-SKBG-1 to critical Phe residues involved in protein-RNA interaction. The derivative with the acryloyl moiety at the position 4 of the piperazine ring formed the covalent bond with Cys145 via Michael addition and was indiscriminately potent for tumor and non-malignant cell lines. In contrast, modifications of the chloroacetyl fragment with propionyl, trifluoroacetyl or cyclopropionyl moieties led to a significant decrease of cytotoxicity since these compounds were unable to bind Cys145. Thus, binding to Cys145 and stable ligand positioning in the binding cavity are necessary and sufficient for NONO inhibition and neuroblastoma cell death.