The crystal structures of these RET-bound TKIs showed that these drugs uniquely wrap around RET.

  • Major Finding: The crystal structures of these RET-bound TKIs showed that these drugs uniquely wrap around RET.

  • Concept: These drugs did not enter the substrate binding pocket and were not affected by gatekeeper mutations.

  • Impact: Along with patient data in this study, these structures show how resistance to these TKIs can occur.


Many RET-inhibiting tyrosine kinase inhibitors (TKI) act either by passing through the substrate binding site, contacting the front and back clefts of the drug binding pocket, or by binding only the front cleft. This makes these inhibitors subject to resistance mediated by mutation of the gatekeeper residue to block access to the substrate binding pocket. However, the mechanisms underlying resistance in the RET-selective TKIs selpercatinib and pralsetinib have not been fully established. Subbiah, Shen, Terzyan, Liu, and colleagues analyzed cell-free DNA (cfDNA) from a patient with RET-mutant medullary thyroid cancer (MTC) and a patient with CCDC6RET-fusion non–small cell lung cancer (NSCLC) who had substantial initial responses to selpercatinib followed by resistance. In line with preclinical observations that Y806C/N and G810C/S mutations in RET confer selpercatinib resistance, cfDNA analyses showed that the patient with MTC developed RETY806C mutation after two months of treatment, and RETG810C and RETG810S mutations arose after 22 and 24 months, respectively. In the patient with NSCLC, CCDC6RETG810C mutation was detected in cfDNA at the time of disease progression after 18 cycles of treatment. The structures of RET–pralsetinib and RET–selpercatinib as determined by X-ray crystallography at resolutions of 2.1 and 1.9 Å, respectively, revealed that both drugs bound RET in a similar way that was unique compared with the binding modes of other TKIs. Selpercatinib and pralsetinib did not occupy the substrate binding pocket of RET, instead binding the front cleft and wrapping around the protein to simultaneously bind the back cleft; as such, they are not subject to the resistance-inducing effects of gatekeeper mutations that confer resistance to other TKIs. Selpercatinib's nine-membered pyrazolo ring bound RET's adenosine pocket, which is bordered by the Y806 residue altered in one of the previously described resistant RET mutants, and the G810C resistance mutation would cause steric clashes with pralsetinib's methylpyrimidine ring. In summary, this work elucidates unique, previously unknown modes of resistance to the RET-selective TKIs selpercatinib and pralsetinib.

Subbiah V, Shen T, Terzyan SS, Liu X, Hu X, Patel KP, et al. Structural basis of acquired resistance to selpercatinib and pralsetinib mediated by non-gatekeeper RET mutations. Ann Oncol 2021;32:261–8.

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