Atropisomeric pyrrolopyrimidine inhibitor as a targeted approach for RET tyrosine kinase in neuroblastoma.

Abstract

3118 Background: Increased RET expression is associated with poor prognosis in children with solid tumors such as neuroblastoma (NB), prompting an interest in RET inhibition. A number of kinase inhibitors currently in use for cancer patients have RET inhibitory activity, but these inhibitors also display activity against other kinases, resulting in unwanted side effects and limiting their safety and efficacy. However, developing more specific RET inhibitors remains a drug design challenge due to high levels of conservation between kinase binding pockets. Using novel chiral chemistry leveraging atropisomerism to convert a promiscuous, rapidly interconverting pyrrolopyrimidine compound into an atropisomerically stable analog, we have developed a new atropisomerically stable, highly selective and specific RET inhibitor, getretinib, with similar potency and improved selectivity to that of other next generation RET inhibitors but with half the molecular weight and significantly improved ligand efficiencies towards RET. Methods: Associations of gene expression with patient survival and prognostic features were performed on available neuroblastoma tumor databases using the R2 Genomics Analysis and Visualization Platform. The efficacy of RET inhibition was assessed against a panel of NB cell lines using live cell imaging and cell viability assays, comparing results with the active and selective RET kinase inhibitor, (R)-getretinib to results with the inactive atropisomer, (S)-getretinib. Mechanisms of cell death and impacts on RET signaling in cells treated with (R)- and (S)-getretinib were evaluated by Western blots. Results: (R)-getretinib reduced NB cell confluence in a dose-dependent manner, while (S)-getretinib had no significant effect on cell confluence over time. R-getretinib treatment of NB cells resulted in reduced phosphorylation of RET in a dose-dependent manner, while treatment with (S)-getretinib resulted in paradoxical increase in RET phosphorylation. Conclusions: We present (R)-getretinib as an atropisomerically stable and potent inhibitor of RET and have shown its efficacy in in vitro models of NB. The high selectivity of (R)-getretinib towards RET has the potential to minimize unwanted side effects caused by off-target kinase binding, thereby increasing its potential for clinical utility.