6656 In this study we designed and synthesized

In this study, we designed and synthesized two ALK PROTACs (degraders), 5 (MS4077) and 6 (MS4078), by linking ceritinib and pomalidomide [45] through two different linkers. Using human ALCL and NSCLC cells, we characterized both compounds in a battery of assays to demonstrate their effects on reducing fused ALK proteins, inhibiting ALK downstream signaling, and inhibiting cancer cell proliferation. In addition, we studied the mechanism of ALK degradation induced by both compounds through a series of rescue assays. We also evaluated compound 6 in a mouse pharmacokinetic (PK) study. Moreover, we developed 7 (MS4748) and 8 (MS4740), close analogs of 5 and 6, respectively, which are incapable of degrading ALK and can serve as negative controls in cellular studies.
Results and discussion
Conclusions We discovered compounds 5 and 6 as novel, potent and cell-active ALK PROTACs. Compounds 5 and 6 displayed very similar potencies in cellular assays even though they have two different types of linkers. In a competitive binding assay, compounds 5 and 6 exhibited high affinity for ALK with K values of 37 ± 4 nM and 19 ± 3 nM, respectively. In cellular assays, compounds 5 and 6 were able to degrade two different ALK fusion proteins and inhibit phosphorylation of ALK and STAT3 in a concentration- and time-dependent manner in both SU-DHL-1 cells (5: DC50 = 3 ± 1 nM; 6: DC50 = 11 ± 2 nM) and NCI-H2228 cells (5: DC50 = 34 ± 9 nM; 6: DC50 = 59 ± 16 nM). To study the ALK fusion protein degradation mechanisms, we designed and synthesized compounds 7 and 8 as negative controls and performed a series of rescue assays. These experiments have demonstrated that the ALK fusion protein degradation induced by compounds 5 and 6 is CRBN and proteasome dependent. Our washout experiments revealed that the PROTAC-mediated ALK fusion protein degradation is reversible. Finally, compounds 5 and 6 potently inhibited proliferation of SU-DHL-1 cells (5: IC50 = 46 ± 4 nM; 6: IC50 = 33 ± 1 nM). Taken together, these results suggest that compounds 5 and 6 along with control compounds 7 and 8 are valuable tools for the research 6656 to study cellular effects of the PROTAC-induced ALK fusion protein degradation. Furthermore, compound 6 displayed good plasma exposure in mice. Thus, this compound can be a useful chemical tool for in vivo efficacy studies. Our study laid the foundation for developing the next generation of ALK PROTACs.
Experimental section
Acknowledgement X-R.H. and Y.X. acknowledge the support by the grant R01GM067113 from the U. S. National Institutes of Health.
The incidence of thromboembolic events, ranging from deep vein thrombosis to disseminated intravascular coagulation (DIC), is assumed to be 7-fold higher in patients with cancer as compared to incidence in the overall population and is greater within the first months after diagnosis . Importantly, cancer patients with venous thromboembolism (VTE) have an increased risk of early mortality during chemotherapy, elevated risk of disease progression and shortened survival . In patients with non-small cell lung cancer (NSCLC), the incidence of VTE has been estimated to be up to 14% throughout the course of the disease, which is consistent with a 20-fold increase of VTE risk as compared to general population . Patients with adenocarcinoma histology seem to experience the highest rate of VTE . In last decade we witnessed impressive advancements in the understanding of the molecular mechanisms underlying the development and progression of NSCLC, which has led to the discovery of targetable genetic alterations (e.g. ) that promote cancer cell growth and survival . rearrangement is just one of the latest identified driver mutations in NSCLC, but it has already had a striking impact on the way we treat patients with advanced NSCLC . Crizotinib, an orally available ALK, ROS1 and MET tyrosine kinases inhibitor has shown unprecedented (~60%) overall response rates (ORRs) in heavily pretreated patients with -positive NSCLC in single-arm phase I and phase II trials. Subsequently, crizotinib confirmed significantly higher response rates and longer progression-free survival (PFS) compared with chemotherapy as second-line treatment and up-front therapy compared to platinum/pemetrexed chemotherapy in untreated advanced -rearranged NSCLC, becoming the standard of care for these patients . More recently, several next generation ALK TKIs have been developed and have already entered in clinical practice in first- or subsequent lines of treatment .