The blood-brain barrier (BBB) tightly regulates the entry of substances into the CNS (central nervous system). Significant advances in BBB peptide shuttle (BBBpS) research have been achieved in the past; however, the proteolytic instability of these peptides poses a major challenge. Utilizing naturally stabilized peptides as structural frameworks is a promising strategy to enhance the stability and transport kinetics of bioactive peptides. In this study, a prototypic scaffold, sunflower trypsin inhibitor 1 (SFTI-1), was designed to incorporate reported BBB transport-mediating sequences. We utilized structural model predictions to assess the compatibility between the bioactive probes and scaffold. The best probes were assessed using a human brain microvascular endothelial cell transport assay. The apparent permeability P _app = 5.6 × 10 ⁻⁶ was highest for L2-peptide 22, which surpassed 3-fold that of peptide 22 (P _app = 1.9 × 10 ⁻⁶ ) and 2-fold that of native SFTI-1 (P _app = 2.7 × 10 ⁻⁶ ). Subsequently, we evaluated the systemic and neuropharmacokinetic parameters of L2-peptide 22 using the Combinatory Mapping Approach, which determined an unbound brain-to-plasma ratio (K _p,uu,brain ) of ∼0.01 (∼10-fold less than that of SFTI-1). We present workflows for structure- and interaction prediction model-informed medicinal chemistry on natural circular peptides for the development of molecular shuttle systems. Our results suggest that active receptor-mediated transport (RMT) was achieved in vitro ; however, in vivo transport experiments indicated dominant efflux at the BBB counteracting active uptake, highlighting the challenges in translational work arising from the use of imperfect in vitro screening systems and subsequent in vivo proof-of-concept models.