Integrating a phenotypic screening with a structural simplification strategy to identify 4-phenoxy-quinoline derivatives to potently disrupt the mitotic localization of Aurora kinase B.

We combined a mechanism-informed phenotypic screening (MIPS) assay with a structural simplification strategy to guide the discovery of compounds that disrupt the localization of the mitotic regulator, Aurora kinase B (AURKB), rather than inhibiting its catalytic activity. An initial hit 4-(4-methylthiophen-2-yl)-N-(4-(quinolin-4-yloxy)phenyl)phthalazin-1-amine was identified after screening an in-house library of small molecules and phenocopied the loss of function mutations in AURKB without inhibiting its catalytic activity. We isolated this hit compound activity to its 4-phenoxy-quinoline moiety. The fragment was further optimized into a class of new chemical entities that potently disrupt the mitotic localization of AURKB at low nanomolar concentrations and consequently elicit severe growth inhibition in diverse human cancer cell lines. A lead compound, N-(3-methoxy-5-(6-methoxyquinolin-4-yl)oxy)phenyl)acetamide possessed desirable pharmacokinetic properties such as AUC0-∞: 227.15 [ng∙h/mL/(mg/kg)]; Cmax: 3378.52 ng/mL T1/2: 3.52 h; and F%: 42 % and produced the AURKB-inhibitory phenotypes in a mouse xenograft model. A lead compound is a powerful tool for interrogating the regulation of AURKB and has the potential to be further developed as a first-in-class oncology therapeutic.

2-Phenoxy-3, 4'-bipyridine derivatives inhibit AURKB-dependent mitotic processes by disrupting its localization.

Activity-based drug screens have successfully led to the development of various inhibitors of the catalytic activity of aurora kinases (AURKs), major regulatory kinases of cell division. Disrupting the localization of AURKB, rather than its catalytic activity, represents a largely unexplored alternative approach to disabling AURKB-dependent processes. Localization disruptors could be just as specific as direct inhibitors of AURKB activity, may bypass their off-target and select on-target toxicities, and are likely less susceptible to drug resistance resulting from mutations of the AURKB catalytic site. In this study, we demonstrate that the pan-AURK inhibitor AMG900 works at a low concentration not by inhibiting the phosphorylation of H3 at Ser10, an AURKB substrate, but by disrupting the mitotic localization of AURKB. Structural deletion studies pinpoint this undescribed activity to the 2-phenoxy-3,4'-bipyridine moiety of AMG900. Guided by a mechanism-informed phenotypic screening (MIPS) assay, the drug fragment is optimized into a novel class of inhibitors that, at low nanomolar concentrations, can disable AURKB through disruption of its mitotic localization and have desirable oral PK properties. Hierarchical clustering of cell fitness profiles reveals that these compounds cluster with each other, rather than with known AURK inhibitors such as AMG900 and VX-680. Validation studies in mice demonstrate that compound 15a elicits mitotic arrest and apoptosis in NCI-H23 human lung adenocarcinoma xenografts, resulting in a pronounced suppression of tumor growth. The discovery and optimization of compounds that disrupt AURKB localization are successfully facilitated by MIPS. Our findings suggest that 2-phenoxy-3, 4'-bipyridine derivatives have the potential to be further developed as effective therapeutics for the treatment of malignancy by delocalizing AURKB.