Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation

Abstract

Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

Development of a Potent Protein Degrader against Oncogenic BCR-ABL Protein

“…Consistent with the degradation of BCR-ABL protein, SNIPER(ABL)-39 inhibited the signaling pathways initiated by BCR-ABL, and suppressed the growth of BCR-ABL-positive CML cells. 30) These results suggest that SNIPER(ABL)-39 could be a lead as a degradation-based novel anti-cancer drug against BCR-ABL-positive CML.…”

“…To improve the protein knockdown activity, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL). 30) The resulting SNIPER(ABL)-39 (Table 1), in which dasatinib is conjugated to LCL161 derivative by polyethylene glycol ×3 linker, shows potent activity. At 10 nM, SNIPER(ABL)-39 induced dramatic reduction of BCR-ABL protein in CML cell lines such as K562 and KU812.…”

“…Indeed, SNIPER(ABL)-39 showed effective protein knockdown activity at a concentration ≥10 nM and the maximum activity was observed at 100 nM, whereas the activity diminished at 1 µM. 30) This is known as a high-dose hook effect, where a certain pharmaceutical activity is interfered with higher concentration of drugs. This is explained by the formation of ternary complex consisting of target protein-SNIPERs or PROTACs-E3 ubiquitin ligase required for the protein knockdown activity, which would be suppressed by excess amount of the hybrid molecules, and therefore, the protein knockdown activity is attenuated.…”

“…Currently, we and Dr. Crews' group successfully developed degraders against the BCR-ABL protein. 30,40) When dasatinib was incorporated as a ligand for BCR-ABL protein, degraders recruiting IAP and CRBN showed protein knockdown activity against the BCR-ABL protein, whereas VHL-recruiting degrader showed negligible activity (Table 1). However, when HG-7-85-01 65) (another inhibitor of BCR-ABL kinase) was incorporated as a target ligand, VHL-recruiting degrader showed effective protein knockdown activity, whereas IAP-and CRBN-recruiting degraders showed weak and no activity, respectively 64) (Table 1).…”

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL

“…Pioneering studies by the Crews lab have shown that BCR-ABL PROTACs, consisting of CRBN or VHL recruiters linked to BCR-ABL ATP binding pocket-targeting kinase inhibitors such as dasatinib and bosutinib or allosteric inhibitors such as GNF-2, showed preferential degradation of c-ABL over modest degradation of BCR-ABL (Burslem et al, 2019;Lai et al, 2016). Notably, this work has spurred development of various degradation strategies targeting BCR-ABL (Demizu et al, 2016;Shibata et al, 2017Shibata et al, , 2019Shimokawa et al, 2017;Zhao et al, 2019). In this study, we aimed to determine whether our recently discovered covalent RNF114 recruiter nimbolide could be used to kinase-targeting PROTACs, and if so, whether differential selectivity in degrading BCR-ABL, compared to similar BCR-ABL PROTACs employing VHL or CRBN could be attained.…”