High-throughput identification of putative receptors for cancer-binding peptides using biopanning and microarray analysis

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 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,” which can then be selected for further development based on their peptide receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically 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 the cell surface 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, −487.239 kcal/mol, and −595.328 kcal/mol 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 in vitro via 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.

Application of Phage-Displayed Peptides in Tumor Imaging Diagnosis and Targeting Therapy

“…Hence, if the screening targets are known, further researches can be conducted to validate peptide properties such as effects on cell cycle, cell proliferation, cell migration and apoptosis (Kim et al 2014 ). In addition, investigators have already developed an efficient method for screening a large number of peptides in parallel to identify peptide receptors or targeting sites, which was termed as “reverse biopanning” (Ferraro et al 2013 ) .…”

“…1 Biopanning steps (modified from Kügler et al). (1) mimicked binding phages to targets, (2) washing away unbound phages, (3) eluting off specific bound phages, (4) enriching the specifically bound phages for next round of biopanning In vitro biopanning is usually chosen for a variety of targets on cell surface or molecules, which includes biopanning on the immobilized molecules (Rahbarnia et al 2017 ; Zhao et al 2018 ), solution biopanning (Fouladi et al 2019 ; Thanongsaksrikul et al 2018 ), selection on whole cells (Carneiro et al 2014 ; Li et al 2017 ; Yeh et al 2016 ), reverse biopanning (Ferraro et al 2013 ), display of established internalizing peptides (Rangel et al 2012 ). Molecules screening by this approach are more likely to keep native conformation, and recognize specific post-translational modifications (Kehoe et al 2006 ; Velappan et al 2019 ).…”

Anti-tax interacting protein-1 (TIP-1) monoclonal antibody targets human cancers

“…In this study, we developed and evaluated 2C6F3, a monoclonal antibody that specifically binds to TIP-1 for radioimmunotherapy. TIP-1 is overexpressed in various cancers including lung cancer [14], breast cancer [9] and glioblastoma [10]. TIP-1 consists of a single PDZ domain and has been showed to have various biological functions.…”

Neoantigen activation, Protein Translocation and Targeted Drug Delivery in Combination With Radiotherapy

“…This observation provided support for the specific accumulation of targeted NPs in irradiated tumors compared with nonradiated tumors and normal tissues. A landmark finding in chemo-radiotherapy was the discovery of radiation-inducible translocation of the predominantly intracellular Tax interacting protein 1 (TIP-1, also known as Tax1 binding protein 3, Tax1BP3) on the plasma membrane of a variety of tumor types (e.g., lung, colon, liver metastases, glioblastoma, breast and pancreatic cancers) [22][23][24][25][26], providing a novel, targetable tumor marker. TIP-1 translocates to the plasma membrane of tumor endothelial cells as early as 3 h and persists for up to 72 h following irradiation in a dose-dependent way [23].…”