br Results and discussion br Conclusion In summary a series
Results and discussion
Conclusion In summary, a series of indazole-based derivatives were synthesized and SAR studies conducted, with view to the development of a novel Aurora kinases inhibitor. The carboxylic nitric oxide synthase inhibitors group extending from the C-3 position of the aniline and substituted groups at the C-5 position of the indazole core are two newly identified pharmoacophore to interact with binding region for potency improvement, respectively. More importantly, we combined knowledge-based substructure search and in silico fragment-based approach to rapidly identify a novel series of Aurora kinases inhibitors such as the dual Aurora A and B inhibitor (compound 17), selective Aurora B inhibitor (compound 21), and selective Aurora A inhibitor (compound 30). Computational modeling was used to understand how isoform selectivity could be affected by targeting specific residue in the Aurora kinases binding pocket in particular targeting residues Arg220, Thr217 or Glu177. Overall, the excellent enzymatic activity of compound 17 together with the high isoform selectivity of compound 30 validated our SAR findings, and should greatly inform future design of Aurora kinases inhibitors.
Acknowledgement Financial support from the National Health Research Institutes and Ministry of Science and Technology, Taiwan (MOST-101-2113-M-400-002-MY4 and MOST-103-2325-B-400 -021) are gratefully acknowledged.
Aurora kinase family Aurora family of ser/threonine kinases has been recognized as important regulators of mitosis with essential roles in the progressive stages ranging from mitotic entry to cytokinesis . These kinases have been highly conserved through evolution playing essential roles in ensuring accurate coordination of chromosomal and cytoskeletal events including centrosome maturation and separation as well as proper spindle assembly leading to faithful partitioning of the chromosomes into daughter cells. The number of Aurora kinase family members varies in different animal phyla. Yeast has one prototypic member Ipl1/Ark1 while in majority of higher eukaryotes the family has two related members Aurora-A and -B represented by conserved orthologs in different species. Aurora-A and -B kinases display different sub-cellular localizations and functions. A third member of the kinase family, Aurora-C, is present only in mammals and is predominantly expressed in testes but has also been reported to rescue in vitro grown human cells depleted of Aurora-B indicating a possible functional overlap between the two kinases in somatic cells. The three members of the mammalian Aurora kinase family share similar carboxyl terminus catalytic domains but divergent amino terminal ends of variable lengths displaying little or no similarity. Although all three Aurora kinases have been found to be over expressed in human cancer cells, possible involvement of Aurora-C in the development of tumorigenic phenotypes, if any, remains unknown in view of its minimal expression and function detected in somatic cells. This review, therefore, discusses only Aurora-A and -B as potential anticancer drug targets along with the description of the inhibitors being developed as anticancer molecules targeting the two kinases. A number of comprehensive reviews have been written on the structure and function of Aurora kinases and for the purpose of this article we will be mainly focusing on the cancer relevant functional interactions of Aurora-A and -B kinases with a brief description of structural characteristics and functional involvement in specific cellular pathways. Aurora-A and -B share about 70% identity in the carboxyl terminus catalytic domain and three conserved Aurora box motifs (A-box I, A-box II and A-box III) in their varying amino terminal domain. The functional significance of A-box motifs is not yet well defined although dephosphorylation of a serine residue in the A-box II is required for degradation of Aurora-A and there is suggestive evidence that the A-box motifs are involved in substrate recognition and sub-cellular localization of the two kinases. Despite conserved structural characteristics, Aurora-A and -B manifest predominantly different localization and function during mitosis interacting with a distinct set of proteins. Aurora-A is localized primarily on spindle poles and transiently along the spindle microtubules as cells progress through mitosis. The kinase functions in mitotic entry, centrosome maturation–separation, bipolar spindle organization and recovery from spindle damage . Aurora-B is associated with the Chromosomal Passenger Complex (CPC) comprising of the scaffolding protein INCENP and the targeting proteins Survivin and Borealin/DasraB. The CPC localizes to the inner centromere during prophase through metaphase and then transfers to the spindle midzone and the midbody during late mitosis and cytokinesis . Aurora-B functions in regulating attachment of kinetochore to spindle microtubules, sister chromatid cohesion and cytokinesis , . The diverse localization and functions of the two related kinases are determined by their binding partners some of which also regulate their kinase activities. Activation of Aurora-A has been shown to be regulated by multiple protein binding cofactors among which the role of TPX2 is well characterized. While the N-terminus of TPX2 induces conformational change in Aurora-A facilitating activation through auto-phosphorylation of Thr288 in the T-loop, the TPX2 bound kinase is also protected from dephosphorylation by PP1 on entry into mitosis , . Aurora-B activation involves auto-phosphorylation of Thr232 in the T-loop and requires interaction with the CPC consisting of the INCENP, Survivin and the Borealin/DasraB proteins. The three CPC proteins in a stable core complex target to the centromere  interacting with Aurora-B through the C-terminus IN-box of the INCENP protein . Intriguingly, most of the interacting proteins with Aurora-A and -B associate with conserved residues in their similar catalytic domains rather than in the variable amino terminus domains and a single amino acid difference in the catalytic domain of the two kinases (G198 in human Aurora-A and N142 in human Aurora-B) was shown to be critical in controlling the intrinsic activity and selective activation of Aurora-A by its binding partner and activator TPX2 , . A site directed mutant of this Aurora-A residue (G198N) revealed classical Aurora-B localization and association with the CPC components INCENP and Survivin partially rescuing Aurora-B loss of function , . Such subtle structural specificity regulating interaction of Aurora-A and -B with binding proteins explains limited functional interchangeability, including some shared substrates such as MCAK, INCENP, Kif2 and RASSF1A, observed for the two kinases. Although functional interactions with the common substrates, most likely, occur at different times during mitosis yet such overlapping activities between the two kinases are expected to have significant implications in terms of affecting proliferation and chromosomal ploidy when the two proteins are aberrantly expressed, as is the case in many human malignant cell types.