Drugs targeting cyclin-dependent kinases (CDKs) have been a hot topic in the development of cancer treatments. Kinases in the CDK family play a significant role in the cell cycle and transcription, and thus in tumor development. The first drug targeting a CDK, a CDK4 inhibitor called palbociclib (Pfizer), was approved in February of 2015 for treatment of patients with estrogen receptor positive advanced breast cancer under the FDA’s accelerated Priority Review and Breakthrough Therapy programs. Until now, the majority of drugs targeting CDKs have been competitive inhibitors that temporarily block the enzyme’s ATP binding site, disrupting cell division. These inhibitors eventually disengage, and the enzyme’s function is restored. A permanent, covalently bound inhibitor is an attractive approach for achieving a more durable therapeutic response and less severe side effects. Toward this end, Elizabeth Anscombe et al. at the Department of Biochemistry, University of Oxford and her team have recently designed and characterized the first irreversible inhibitor of the protein kinase CDK2.
CDK2 regulates the transition from the G1 phase of the cell cycle to S phase by phosphorylation of retinoblastoma tumor suppressor protein (Rb). Inhibition of CDK2 and loss of the Rb signaling pathway often results in deregulation of the cell cycle. Anscombe et al. identified the sulfone NU6300 as an intermediate in the synthetic manipulation of a previously identified reversible inhibitor of CDK2, NU6102. NU6300 possesses a vinyl group that acts as a Michael acceptor, thus creating a covalent bond with the Lys89 residue that lies just outside of the CDK2 ATP binding site. Its ethyl counterpart, NU6310, was used in the study as a non-covalent ATP-competitive CDK2 inhibitor.
Electrospray ionization mass spectrometry (ESI-MS) and equilibrium binding studies using surface plasmon resonance (SPR) demonstrated that NU6300 bound to CDK2/cyclin A, and the MS data was consistent with a covalent complex. Covalent binding was confirmed by testing the activity of CDK2/cyclin A for phosphorylation of Rb via in vitro kinase assays. After overnight incubation with NU6300 and exhaustive dialysis, CDK2 activity was not recovered. However, CDK2 activity was restored after a similar treatment with the reversible inhibitor, RU6310.
Though Anscombe et al. demonstrated covalent binding of NU6300 and CDK2 in vitro, the potency of this reaction in vivo was modest. In fact, the original reversible inhibitor NU6102 was found to have a much higher inhibition rate in uterine tumor cells. Incubation with NU6300 only decreased phosphorylation of Rb protein by 43%, compared to 85% inhibition when incubated with NU6102 at the same concentration. However, NU6300 retained 75% of its inhibitory activity after a 1 hour drug washout, while over half of NU6102 activity was lost. The more durable residence time for NU6300 is consistent with covalent binding.
The authors acknowledged a recent report of a covalent inhibitor for CDK7 that binds at the ATP site, suggesting the approach taken by the group can be applied to other targets in the CDK family. Irreversible inhibitors have also been developed for tyrosine kinases such as the epidermal growth factor receptor (EDFR) for treatment of non-small cell lung cancer and Bruton’s tyrosine kinase for leukemia. Much is left to be explored in the use of these “second-generation” kinase inhibitors, but if perfected, they could lead to more effective cancer treatments with less severe side effects.
– Hannah Lucas