Optimization and <i>in Vivo</i> Validation of Peptide Vectors Targeting the LDL Receptor

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., <i>J. Med. Chem.</i> <b>2012</b>, <i>55</i> (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL­[CMPRLRGC]_cDPR) and structure–activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]_c) which specifically binds hLDLR with a <i>K</i>_D of 76 nM and has an <i>in vitro</i> blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-“Pen”M“Thz”RLRGC]_c), which showed the highest <i>K</i>_D value of 9 nM. However, this latter analogue displayed the lowest <i>in vitro</i> blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 10⁶ s^–1.M^–1 range, and off-rates varying from the low 10^–2 s^–1 to the 10^–1 s^–1 range. Furthermore, all these new analogues showed increased blood half-lives <i>in vitro</i>, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM“Thz”RLRG“Pen”]_c), showing a <i>K</i>_D of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled ³H-VH4127 in wild-type or <i>ldlr</i> −/– mice confirmed their active LDLR targeting <i>in vivo</i>. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential <i>in vivo</i>