Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, driven by the BCR-ABL1 fusion oncoprotein. The discovery of orthosteric BCR-ABL1 tyrosine kinase inhibitors (TKIs) targeting its active ATP-binding pocket, such as first-generation Imatinib and second-generation Nilotinib (NIL), has profoundly revolutionized the therapeutic landscape of CML. However, currently targeted therapeutics still face considerable challenges with the inevitable emergence of drug-resistant mutations within BCR-ABL1. One of the most common resistant mutations in BCR-ABL1 is the T315I gatekeeper mutation, which confers resistance to most current TKIs in use. To resolve such conundrum, co-administration of orthosteric TKIs and allosteric drugs offers a novel paradigm to tackle drug resistance. Remarkably, previous studies have confirmed that the dual targeting BCR-ABL1 utilizing orthosteric TKI NIL and allosteric inhibitor ABL001 resulted in eradication of the CML xenograft tumors, exhibiting promising therapeutic potential. Previous studies have demonstrated the cooperated mechanism of two drugs. However, the conformational landscapes of synergistic effects remain unclear, hampering future efforts in optimizations and improvements. Hence, extensive large-scale molecular dynamics (MD) simulations of wide type (WT), WT-NIL, T315I, T315I-NIL, T315I-ABL001 and T315I-ABL001-NIL systems were carried out in an attempt to address such question. Simulation data revealed that the dynamic landscape of NIL-bound BCR-ABL1 was significantly reshaped upon ABL001 binding, as it shifted from an active conformation towards an inactive conformation. The community network of allosteric signaling was analyzed to elucidate the atomistic overview of allosteric regulation within BCR-ABL1. Moreover, binding free energy analysis unveiled that the affinity of NIL to BCR-ABL1 increased by the induction of ABL001, which led to its favorable binding and the release of drug resistance. The findings uncovered the in-depth structural mechanisms underpinning dual-targeting towards T315I BCR-ABL1 to overcome its drug resistance and will offer guidance for the rational design of next generations of BCR-ABL1 modulators and future combinatory therapeutic regimens.

The RET protein is a transmembrane receptor tyrosine kinase (RTK) whose oncogenic mutations or fusions are closely related to human cancers such as thyroid and non-small cell lung cancer. Vandetanib as a clinical-approved protein-tyrosine kinase inhibitor (TKI) exhibits anti-cancer efficacy by blocking the RET ATP-binding site, but drug resistance was observed for the RET^G810A mutant. Recent studies have identified another TKI nintedanib as an effective molecule to inhibit vandetanib-resistant RET^G810A. However, there is no clear evidence of why nintedanib and vandetanib displayed different inhibitory activities towards RET^G810A. Here, we exploited molecular dynamic (MD) simulations to compare the interactions of the RET^G810A mutant with nintedanib and vandetanib. A higher structural flexibility of the activation loop was observed in the nintedanib-bound RET^G810A, which may result in discrepant autophosphorylation activity in the nintedanib- and vandetanib-bound RET kinase, causing differentiated pharmacological effects of the two compounds. Molecular mechanics/Poisson-Bolzmann surface area method suggested that nintedanib had a higher affinity towards RET^G810A over vandetanib, accounting for its better inhibitory effect as an ATP-competitive compound. These results depicted the underlying mechanism for the different inhibitory efficacy of nintedanib and vandetanib on RET^G810A from both conformational and energetic aspects. Furthermore, we also found that both compounds maintained the ‘DFG-in, αC-helix-in, and activation loop-open’ conformation of RET^G810A, which is the characteristic of the active state. Together, our results provide comprehensive mechanistic insights into nintedanib’s capability in inhibiting vandetanib-resistant RET mutant and enlighten future structural-based optimisation of RET TKIs to overcome drug resistance.