Mechanistic insights into EGFR-Targeted: [¹²⁵I]I-rociletinib as a model small-molecule radioligand.

Rociletinib is a third-generation tyrosine kinase inhibitor that selectively targets mutant epidermal growth factor receptor (EGFR). Here, we use its radioiodinated analogue, [¹²⁵I]I-rociletinib, as a mechanistic model to examine how molecular binding and pharmacokinetic determinants jointly govern EGFR-targeted radiotracer behavior. Molecular docking against EGFR L858R/T790M showed that both rociletinib and [¹²⁵I]I-rociletinib preserve key hydrogen bonds with hinge residues Met793 and Cys797 and exhibit comparable binding energies, indicating that iodine substitution maintains ATP-binding pocket interactions while enabling additional peripheral contacts. In vitro uptake studies demonstrated preferential accumulation of [¹²⁵I]I-rociletinib in H1975 cells (EGFR L858R/T790M; 106.50% dose/mg protein) relative to H3255 (EGFR L858R; 36.41% dose/mg protein) and H441 (wild-type; 14.00% dose/mg protein), and competitive inhibition with excess rociletinib confirmed target-specific binding. In vivo biodistribution revealed tumor uptake accompanied by rapid hepatobiliary clearance, with predominant fecal excretion (∼70%). ADMET analysis provided a mechanistic framework for these findings: very high plasma protein binding (PPB > 98%), and an elevated volume of distribution (VDss) promote rapid tissue partitioning and gastrointestinal-hepatic accumulation, limiting sustained systemic exposure and yielding a tumor-to-blood ratio of approximately unity. Favorable transporter characteristics, including predicted P-glycoprotein inhibition without substrate liability, support intracellular retention but do not override systemic clearance pathways. Minimal brain uptake is consistent with predicted blood-brain barrier restriction. Collectively, these results demonstrate that [¹²⁵I]I-rociletinib retains mutant-EGFR selectivity while revealing limited in vivo tumor contrast due to predominant hepatobiliary clearance and high plasma protein binding. Further structural optimization is needed to improve its pharmacokinetic profile and imaging performance.