Probing the functions of Nek family kinases using chemical inhibition: towards the development of potent and selective inhibitors of Nek7

Understudied protein kinases such as NIMA-related kinases are crucial for cell cycle progression, being involved in the regulation of microtubule-dependent processes. In particular, Nek7 functions in centrosome separation, nuclear envelope breakdown, mitotic spindle assembly and cytokinesis to ensure timely and accurate progression of mitosis. Therefore, many Nek family kinases including Nek7 have been implicated in numerous cancer types. Despite Nek7 being a potential therapeutic target in cancer, no potent and selective inhibitors are available which has limited functional and target validation studies. Therefore, the development of a chemical tool to probe the function of Nek7 and validate this kinase as a therapeutic target in oncology is of great importance.
Previously identified fragments targeting Nek7 provided starting points for elaboration in activity-directed synthesis, four of which were exploited in 272 reactions over two rounds to identify several reactions which gave bioactive product mixtures. Analysis of these few reactions showed them to be non-productive and the reason for bioactivity was not determined.

In an effort to identify a suitable chemical probe against Nek7, the structure-activity relationship of a previously developed 7‐(1H‐Pyrazol‐4‐yl)‐1,2,3,4‐tetrahydroisoquinolin‐1‐one Nek7 inhibitor was explored further. Firstly, substituents at the 3,5-positions of the pyrazolyl ring were varied with 3-methylation proving beneficial. However any substituent larger than a methyl group was detrimental for potency. Next the 3,4-positions of the tetrahydroisoquinolinone core were optimised, identifying an amino-group at the 3-position to be beneficial with a diverse range of groups tolerated. Exploration of this azetidine moiety identified methylation which gave a further improvement. Combination of 3,5-pyrazole and 3,4-dihydroisoquinolinone substituents proved beneficial and this work has led to the development of a series of low micromolar affinity inhibitors of Nek7, with up to a 12.5-fold improvement over the starting compound.