Computational design and biophysical validation of macrocyclic peptides as inhibitors of SLIT2/ROBO1 interaction

The SLIT2/ROBO1 signaling axis regulates cellular migration and angiogenesis but also contributes to tumor progression and immune evasion in glioblastoma. Targeting this pathway with small molecules or antibodies remains challenging due to the shallow and extended nature of the SLIT2/ROBO1 interface. Here, we report the first computational design and experimental validation of macrocyclic peptides that inhibit SLIT2/ROBO1 binding. Twenty peptides were generated through a structure-guided interface mapping approach (Des3PI 2.0) and ranked using a contact-based scoring function. The top candidates were synthesized and evaluated using time-resolved fluorescence resonance energy transfer (TR-FRET) and biolayer interferometry (BLI) assays. Among the SLIT2-targeting peptides, SP4 and SP3 showed the most pronounced inhibition in TR-FRET (62% and 46%, respectively), with dose-dependent IC₅₀ values of 41 ± 5.2 μM and 68 ± 7.5 μM by BLI, confirming direct binding to the SLIT2/ROBO1 interface. The lead peptide SP4 also demonstrated favorable in vitro pharmacokinetic properties, including strong stability in simulated intestinal fluid (t₁/₂ = 7.6 h), high plasma integrity (89% at 1 h), and moderate metabolic stability in rat liver microsomes (t₁/₂ = 4.7 h). Collectively, this work provides a foundation for developing next-generation SLIT2/ROBO1 modulators for cancer and neuroimmune disorders.