Orally Bioavailable 4-Phenoxy-quinoline Compound as a Potent Aurora Kinase B Relocation Blocker for Cancer Treatment.

We investigated a novel 4-phenoxy-quinoline-based scaffold that mislocalizes the essential mitotic kinase, Aurora kinase B (AURKB). Here, we evaluated the impact of halogen substitutions (F, Cl, Br, and I) on this scaffold with respect to various drug parameters. Br-substituted LXY18 was found to be a potent and orally bioavailable disruptor of cell division, at sub-nanomolar concentrations. LXY18 prevents cytokinesis by blocking AURKB relocalization in mitosis and exhibits broad-spectrum antimitotic activity in vitro. With a favorable pharmacokinetic profile, it shows widespread tissue distribution including the blood-brain barrier penetrance and effective accumulation in tumor tissues. More importantly, it markedly suppresses tumor growth. The novel mode of action of LXY18 may eliminate some drawbacks of direct catalytic inhibition of Aurora kinases. Successful development of LXY18 as a clinical candidate for cancer treatment could enable a new, less toxic means of antimitotic attack that avoids drug resistance mechanisms.

In-vitro metabolism of LXY18, an orally available, potent blocker of AURKB relocation in mitosis.

This study investigated the metabolism of LXY18, a quinolone-based compound that suppresses tumorigenesis by blocking AURKB localization. Metabolite profiling of LXY18 in liver microsomes from six species and human S9 fractions revealed that LXY18 undergoes various conserved metabolic reactions, such as N-hydroxylation, N-oxygenation, O-dealkylation, and hydrolysis, resulting in ten metabolites. These metabolites were produced through a combination of CYP450 enzymes, and non-CYP450 enzymes including CES1, and AO. Two metabolites, M1 and M2 were authenticated by chemically synthesized standards. M1 was the hydrolyzed product catalyzed by CES1 whereas M2 was a mono-N-oxidative derivative catalyzed by a CYP450 enzyme. AO was identified as the enzyme responsible for the formation of M3 with the help of AO-specific inhibitors and LXY18 analogs, 5b and 5c. M1 was the intermediate of LXY18 to produce M7, M8, M9, and M10. LXY18 potently inhibited 2C19 with an IC50 of 290 nM but had a negligible impact on the other CYP450s, indicating a low risk of drug-drug interaction. Altogether, the study provides valuable insights into the metabolic process of LXY18 and its suitability as a drug candidate. The data generated serves as a significant reference point for conducting further safety assessments and optimizing drug development.

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.

Discovery and Optimization of Seven-Membered Lactam-Based Compounds to Phenocopy the Inhibition of the Aurora Kinase B.

We used mechanism-informed phenotypic screening to identify and optimize compounds that phenocopy the genetic depletion of the mitotic aurora kinase B (AURKB) kinase. After assaying nine aryl fused seven-membered lactam compounds, we identified a hit compound 6a that was subsequently optimized to five lead compounds with low nanomolar activity, represented by the lead compound 6v (19 nM). With excellent drug-like properties, these compounds reproduced the loss of function in phenotypes of AURKB and exhibited potent cytotoxic activities in various cancer cell lines. Collectively, these data support that seven-membered lactam-based analogs might be valuable for further development as a new type of antimitotic agents for the treatment of cancer.

The Phytochemical Scoulerine Inhibits Aurora Kinase Activity to Induce Mitotic and Cytokinetic Defects.

To identify novel bioactive compounds, an image-based, cell culture screening of natural product extracts was conducted. Specifically, our screen was designed to identify phytochemicals that might phenocopy inhibition of the chromosomal passenger protein complex in eliciting mitotic and cytokinetic defects. A known alkaloid, scoulerine, was identified from the rhizomes of the plant Corydalis decumbens as being able to elicit a transient mitotic arrest followed by either apoptosis induction or polyploidy. In examining the mitotic abnormality further, we observed that scoulerine could elicit supernumerary centrosomes during mitosis, but not earlier in the cell cycle. The localization of NUMA1 at spindle poles was also inhibited, suggesting diminished potential for microtubule recruitment and spindle-pole focusing. Polyploid cells emerged subsequent to cytokinetic failure. The concentration required for scoulerine to elicit all its cell division phenotypes was similar, and an examination of related compounds highlighted the requirement for proper positioning of a hydroxyl and a methoxy group about an aromatic ring for activity. Mechanistically, scoulerine inhibited AURKB activity at concentrations that elicited supernumerary centrosomes and polyploidy. AURKA was only inhibited at higher concentrations, so AURKB inhibition is the likely mechanism by which scoulerine elicited division defects. AURKB inhibition was never complete, so scoulerine may be a suboptimal AURK inhibitor or work upstream of the chromosomal passenger protein complex to reduce AURKB activity. Scoulerine inhibited the viability of a variety of human cancer cell lines. Collectively, these findings uncover a previously unknown activity of scoulerine that could facilitate targeting human cancers. Scoulerine, or a next-generation analogue, may be useful as a nontoxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.