Abstract
The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.
Highlights
Kinase mutations drive many cancers through deregulated activity, and inhibitors of these kinases have revolutionized cancer treatment
In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the aC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop
A synthetic library of vNAR domains based on a scaffold isolated from Wobbegong shark was screened for binding to the kinase domain (KD) of human wild-type (WT) Aurora-A
Summary
Kinase mutations drive many cancers through deregulated activity, and inhibitors of these kinases have revolutionized cancer treatment. Most current kinase inhibitors target the ATP-binding site, which is relatively straightforward to block with small molecules [1]. This site is highly conserved among all protein kinases, and many inhibitors have off-target effects. Combinations of kinase inhibitors might address these issues, but combining these drugs safely and effectively is a challenge. This is thought to be, at least to some extent, due to the unfocused selectivity profiles of ATP-competitive inhibitors. Kinase inhibitors that bind to allosteric sites are more selective than ATP-competitive inhibitors [5]. Allosteric inhibitors are more challenging to develop because kinase targets do not always have a clearly suitable allosteric site, and approaches to targeting such sites through screening and synthetic chemistry
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
