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Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor
Guillaume Jacquot1, Pascaline Lécorché1, Jean-Daniel Malcor1
1VECT-HORUS SAS, Faculté de Médecine secteur Nord , 51 Boulevard Pierre Dramard, CS80011, 13344 Marseille Cedex 15, France.
Abstract:
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., J. Med. Chem. 2012, 55 (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 KD of 76 nM and has an in vitro 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 KD value of 9 nM. However, this latter analogue displayed the lowest in vitro 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 in vitro, 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 KD 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 ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.
Insights
Researchers developed new peptide ligands targeting the LDL receptor (LDLR) for enhanced drug delivery. These peptides show improved binding and longer blood half-lives, demonstrating effective LDLR targeting in vivo for potential therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Active targeting of pathophysiological organs is crucial for drug delivery and reducing side effects.
- The human LDL receptor (LDLR) is a cell-surface receptor with high uptake activity and tissue enrichment, making it an attractive target.
- Previous work identified peptide ligands for LDLR, with ongoing optimization for improved properties.
Purpose of the Study:
- To design and characterize novel peptide analogues with enhanced biological properties for LDLR targeting.
- To evaluate the binding kinetics, blood half-life, and cell uptake of new peptide analogues.
- To confirm the in vivo targeting capability of optimized peptide analogues in a mouse model.
Main Methods:
- Design and synthesis of new peptide analogues (VH4127 to VH4131) based on previous LDLR ligands.
- Analysis of binding kinetics (on-rates, off-rates, KD) and in vitro blood half-life.
- Cell-based assays to assess cell-surface binding and endocytosis.
- In vivo targeting studies using tritium-radiolabeled peptides in wild-type and LDLR-deficient mice.
Main Results:
- New peptide analogues exhibited very high on-rates (106 s-1M-1) and varied off-rates.
- Several analogues demonstrated significantly increased in vitro blood half-lives (up to ~10 hours for VH4131).
- The optimized analogue VH4127 showed a KD of 18 nM and a half-life of ~4.3 hours, with increased cell binding and endocytosis.
- Intravenous injection of 3H-VH4127 confirmed active LDLR targeting in vivo in wild-type mice.
Conclusions:
- The study successfully developed a diversified portfolio of peptide vectors targeting the LDLR.
- New analogues demonstrate improved binding kinetics and extended blood half-lives compared to previous versions.
- The optimized peptide VH4127 shows promising potential for active LDLR targeting and delivery in vivo.
