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    • To test the compounds metabolic

    2024-01-05

    To test the compounds metabolic stability, we measured the half-life of compounds and in mouse hepatic microsomes (1mg/mL) and found them to have modest stability, with half-lives of 7.5 and 2.9min respectively, indicating these compounds may be unsuitable for studies. In order to gain structural insights into the mode of binding and to rationalize the selectivity of the molecules, we solved the co-crystal structure of CTX0294885 in complex with Aurora A at a resolution of 2.6Å (, PDB: ). The structure of the complex of compound with Aurora A revealed the expected ATP competitive binding mode of pyrimido-benzodiazepinone inhibitors, comprising two hydrogen bonds with the main chain amide and carbonyl group of the hinge residue A213. A similar binding mode was observed in docking studies for compound (). The 4-propanoylaminobenzamid moiety is rests over the αD helix, with the propyl group oriented towards the solvent exposed area. The kink created by the benzodiazepinone ring inserts the aromatic toluene portion of this core ring system into a deep hydrophobic pocket created by L263 and an unusual inactive conformation of the activation segment which assumed a DFG “in” conformation with residues C-terminal to the DFG motif packing against the aromatic ring of the benzodiazepinone moiety. The methyl group addition to the scaffold cannot be accommodated in this region by many kinases, including ERK5, which contributes to the selectivity of compounds and . Comparison of the binding mode of compound with that of the Aurora A specific inhibitor Alisertib (PDB: ) revealed that both inhibitors share the same hinge binding interactions. However, the different coupling of the aromatic ring systems to the azepine ring results in orientation of the aliserib benzazepine towards the P-loop while the fluoro-methoxyphenyl ring is oriented towards the DFG motif (). The pyrimido-benzodiazepinone scaffold has also been utilized for the development of a selective ERK5 inhibitor by our laboratory., Superimposition of the structure of with Aurora A with the complex of compound with ERK5 (PDB: ) showed that insertion of a proline residue (P214) in the hinge region results in a considerable re-orientation of in Aurora A. In addition, the introduction of a cyclopentyl moiety to the nitrogen of the benzodiazepinone ring in compound most likely results in steric exclusion of the ERK5 selective inhibitor from the Aurora A ATP binding site (). We have previously described multi-targeted Aurora inhibitors based upon a pyrimido-benzodiazepinone scaffold, in this work we characterize advanced analogs from this scaffold series, with vastly improved selectivity. We envision these molecules will be invaluable for studying Aurora kinase signaling and the therapeutic potential of pan-Aurora inhibition in cellular studies. We also use structural studies to identify key selectivity hot-spots of the Aurora A binding pocket, providing a blueprint for improving selectivity of Aurora kinase targeting inhibitors. Author contributions
    Acknowledgments We would like to acknowledge funding from NIHR01 U54 HL127365, the Linde center for chemical biology and the KU-KIST Graduate School of Converging Science and Technology Program.
    Introduction Aurora kinases are serine/threonine kinases which regulate mitotic progression, centrosome maturation, and spindle assembly [1,2]. Overexpression of Aurora kinases has been associated with increased tumor progression, and thus they are appealing targets for the development of anti-cancer therapies [[3], [4], [5], [6], [7]]. Despite similarities in protein sequence, the sub-cellular localization, expression patterns, timing, and activity of Aurora-A are markedly different to Auroras -B and -C [1]. Aurora-A primarily localizes to centrosomes and mitotic spindles where it regulates centrosome duplication, centrosome maturation, and mitotic spindle formation. Disruption of Aurora-A kinase activity arrests cells in the G2/M stage of the cell cycle, thereby activating the spindle assembly checkpoint (SAC) and thus growth inhibition [8,9]. On the other hand, Aurora-B is part of the chromosomal passenger complex (CPC), a hetero-tetrameric complex composed of Aurora-B, inner centromere protein (INCENP), Borealin and Survivin [10,11]. In prometaphase, the CPC localizes at the centromeric region of the chromosome, monitoring proper attachment of microtubules emanating from opposite poles of the cell, activating SAC, as well as promoting chromosome congression to the metaphase plate [[12], [13], [14]]. Inhibiting the kinase activity of Aurora-B or depleting INCENP results in dephosphorylation of histone H3 on serine 10 and override of SAC [15,16], leading to premature decondensation of the chromatin, an increased degree of polyploidy, cell senescence, and apoptosis [17,18].