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Targeting the Atypical Chemokine Receptor ACKR3/CXCR7: Phase 1 - Phage Display Peptide Identification and
R D Vestal1, D R LaJeunesse, E W Taylor
1Department of Nanoscience, University of North Carolina at Greensboro (UNCG), Greensboro, North Carolina 27412, USA..
Abstract:
One of the greatest challenges in fighting cancer is cell targeting and biomarker selection. The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed on many cancer cell types, including breast cancer and glioblastoma, and binds the endogenous ligands SDF1/CXCL12 and ITAC/CXCL11. A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and targeted with the NEB PhD-7 Phage Display Peptide Library. Twenty-nine phages were isolated and heptapeptide inserts sequenced; of these, 23 sequences were unique. A 3D molecular model was created for the ACKR3/CXCR7 N-terminus by mutating the corresponding region of the crystal structure of CXCR4 with bound SDF1/CXCL12. A ClustalW alignment was performed on each peptide sequence using the entire SDF1/CXCL12 sequence as the template. The 23-peptide sequences showed similarity to three distinct regions of the SDF1/CXCL12 molecule. A 3D molecular model was made for each of the phage peptide inserts to visually identify potential areas of steric interference of peptides that simulated CXCL12 regions not in contact with the receptor's Nterminus. An ELISA analysis of the relative binding affinity between the peptides identified 9 peptides with statistically significant results. The candidate pool of 9 peptides was further reduced to 3 peptides based on their affinity for the targeted N-terminus region peptide versus no target peptide present or a scrambled negative control peptide. The results clearly show the Phage Display protocol can be used to target a synthesized region of the ACKR3/CXCR7 N-terminus. The 3 peptides chosen, P20, P3, and P9, will be the basis for further targeting studies.
Insights
Researchers developed a phage display method to identify peptides targeting the ACKR3/CXCR7 receptor, crucial for cancer cell targeting. Three promising peptides (P20, P3, P9) were identified for future cancer therapy development.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Targeting cancer cells and identifying biomarkers remain significant challenges in oncology.
- The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed in various cancer types, including breast cancer and glioblastoma.
- ACKR3/CXCR7 binds endogenous ligands such as SDF1/CXCL12 and ITAC/CXCL11.
Purpose of the Study:
- To synthesize a region of the ACKR3/CXCR7 N-terminus and target it using a phage display peptide library.
- To identify specific peptides that bind to the ACKR3/CXCR7 N-terminus for potential cancer targeting applications.
Main Methods:
- A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and used to screen the NEB PhD-7 Phage Display Peptide Library.
- Isolated phage heptapeptide inserts were sequenced, and 3D molecular models were generated for the ACKR3/CXCR7 N-terminus and peptide inserts.
- ELISA analysis was performed to determine the binding affinity of the identified peptides to the target N-terminus region.
Main Results:
- Twenty-nine phages were isolated, yielding 23 unique heptapeptide sequences.
- Sequence alignment revealed similarities between peptides and three distinct regions of the SDF1/CXCL12 molecule.
- ELISA identified 9 peptides with significant binding affinity, further narrowed down to 3 peptides (P20, P3, P9) with high specificity.
Conclusions:
- The Phage Display protocol is effective for targeting synthesized regions of the ACKR3/CXCR7 N-terminus.
- The identified peptides P20, P3, and P9 demonstrate potential as novel agents for ACKR3/CXCR7-mediated cancer targeting.
- These peptides provide a foundation for further development in cancer therapy strategies.
