Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine

Abstract

Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7–inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.

Physiological and Pathological Roles of Mammalian NEK7

“…Although some reported inhibitors have shown inhibition of NEK7-mediated inflammasome activation, no published works have shown screening of highly selective and potent inhibitors of NEK7. The latest research showed that use of conventional methods led to failed designs of NEK7 inhibitors owing to its unique conformation and high similarity to its paralog NEK6 ( Byrne et al, 2020 ). Then, this group developed a novel strategy using SRS mutations of NEK7 , which is a promising starting point for developing potent inhibitors targeting NEK7 ( Byrne et al, 2020 ).…”

“…The latest research showed that use of conventional methods led to failed designs of NEK7 inhibitors owing to its unique conformation and high similarity to its paralog NEK6 ( Byrne et al, 2020 ). Then, this group developed a novel strategy using SRS mutations of NEK7 , which is a promising starting point for developing potent inhibitors targeting NEK7 ( Byrne et al, 2020 ). However, the kinase activity inhibition of NEK7 might have no effect on NLRP3 inflammasome activation ( Shi et al, 2016 ).…”

Bioinformatics analysis of the prognostic value of NEK8 and its effects on immune cell infiltration in glioma

“…As cell cycle kinases, NEKs are related to the mitotic regulator ‘never in mitosis, gene A’ (NIMA) 6,7 and regulate the cell cycle progression from the G2 to M phase 8,9 . NEKs are associated with multiple cancers 10,11 . Furthermore, their involvement in specific aspects of microtubule function and the DNA damage checkpoint, a key target pathway for cancer drugs, has led to considerable interest in mitotic enzymes as candidate cancer drug targets.…”

“… 8 , 9 NEKs are associated with multiple cancers. 10 , 11 Furthermore, their involvement in specific aspects of microtubule function and the DNA damage checkpoint, a key target pathway for cancer drugs, has led to considerable interest in mitotic enzymes as candidate cancer drug targets.…”

Micro‐ribonucleic acid‐23a‐3p prevents the onset of type 2 diabetes mellitus by suppressing the activation of nucleotide‐binding oligomerization‐like receptor family pyrin domain containing 3 inflammatory bodies‐caused pyroptosis through negatively regulating NIMA‐related kinase 7

“…Meanwhile, a prior study revealed the decrease of serum level of miR‐23a in type 2 diabetes mellitus 11 . NIMA‐related kinase 7 (NEK7), a serine/threonine protein kinase, regulates proper spindle formation and cytokinesis production 12 . Furthermore, being a mitotic kinase, NEK7 is required for the activation of NLRP3 inflammasomes 13 .…”