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Updated: May 16, 2026
Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions
Published on: January 7, 2019
High-throughput identification of putative receptors for cancer-binding peptides using biopanning and microarray
Daniel J Ferraro1, Sandeep R Bhave, Rama P Kotipatruni
1Department of Radiation Oncology, Mallinckrodt Institute of Radiology, and Siteman Cancer Center, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63110, USA.
Abstract:
Phage-display peptide biopanning has been successfully used to identify cancer-targeting peptides in multiple models. For cancer-binding peptides, identification of the peptide receptor is necessary to demonstrate the mechanism of action and to further optimize specificity and target binding. The process of receptor identification can be slow and some peptides may turn out to bind ubiquitous proteins not suitable for further drug development. In this report, we describe a high-throughput method for screening a large number of peptides in parallel to identify peptide receptors, which we have termed "reverse biopanning." Peptides can then be selected for further development based on their receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically to cancers after irradiation. The reverse biopanning process identified 2 peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for the protein tax interacting protein 1 (TIP-1), a protein previously identified in our laboratory to be expressed in tumors and upregulated after exposure to ionizing radiation. We used computational modeling as the initial method for rapid validation of peptide-TIP-1 binding. Pseudo-binding energies were calculated to be -360.645 kcal mol(-1), -487.239 kcal mol(-1), and -595.328 kcal mol(-1) for HVGGSSV, TTRYSRV, and NVFFCSV respectively, suggesting that the peptides would have at least similar, if not stronger, binding to TIP-1 compared to the known TIP-1 binding peptide HVGGSSV. We validated peptide binding in vitro using electrophoretic mobility shift assay, which showed strong binding of RKFLMTTRYSRV and the truncated form TTRYSRV. This method allows for the identification of many peptide receptors and subsequent selection of peptides for further drug development based on the peptide receptor.
Insights
Reverse biopanning is a new high-throughput method to identify cancer-targeting peptide receptors. This approach aids in selecting peptides for drug development by revealing their specific protein interactions, like TIP-1.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- Phage-display peptide biopanning identifies cancer-targeting peptides but requires receptor identification for mechanism validation and optimization.
- Current receptor identification methods can be slow, and some peptides bind non-specific proteins, hindering drug development.
- Identifying specific peptide receptors is crucial for understanding cancer-targeting peptide mechanisms and improving drug design.
Purpose of the Study:
- To introduce a high-throughput method, termed "reverse biopanning," for parallel screening of numerous peptides to identify their receptors.
- To demonstrate the utility of reverse biopanning by screening previously identified cancer-binding peptides.
- To enable selection of peptides for further development based on identified receptor interactions.
Main Methods:
- Developed and applied a high-throughput "reverse biopanning" technique for parallel peptide receptor screening.
- Screened a library of 39 cancer-specific peptides using the reverse biopanning method.
- Employed computational modeling and electrophoretic mobility shift assay (EMSA) for validation of peptide-receptor binding.
Main Results:
- Reverse biopanning identified two peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for tumor-expressed protein 1 (TIP-1).
- Computational modeling indicated strong binding affinities for selected peptides to TIP-1, comparable to a known TIP-1 binder.
- In vitro EMSA confirmed robust binding of RKFLMTTRYSRV and its truncated form TTRYSRV to TIP-1.
Conclusions:
- Reverse biopanning is an effective high-throughput method for identifying peptide receptors.
- This method facilitates the selection of peptides for drug development based on specific receptor interactions.
- The identified peptide-TIP-1 interactions offer potential for targeted cancer therapies.
