Unbiased Selection of Peptide–Peptoid Hybrids Specific for Lung Cancer Compared to Normal Lung Epithelial Cells

Abstract

To develop widely-applicable diagnostic and potentially therapeutic approaches overcoming protein heterogeneity in human cancer, we have developed a technology to unbiasedly select high specificity compound(s) that bind any bio-molecule (e.g., proteins, lipids, carbohydrates) presented on the cancer cell surface but not on normal cells. We utilized a peptidomimetic based on-bead two-color (OBTC) combinatorial cell screen that can detect differences between two cell surfaces at high accuracy by looking for beads (where each bead in the library had one peptide-peptoid hybrid on the surface) that only bound cancer but not normal cells. We screened a library of 393,216 compounds targeting HCC4017 lung adenocarcinoma cells (labeled in red) in the presence of HBEC30KT normal bronchial epithelial cells (labeled in green) derived from the same tissue of the same patient. This screen identified a peptide-peptoid hybrid called PPS1 which displayed high specific binding for HCC4017 cancer cells over HBEC30KT cells. Specificity was validated through: on-bead, ELISA-like and magnetic bead pulldown studies; while a scrambled version of PPS1 did not show any binding. Of interest, the simple dimeric version (PPS1D1) displayed cytotoxic activity on HCC4017 cells, but not on normal HBEC30KT cells. PPS1D1 also strongly accumulated in HCC4017 lung cancer xenografts in mice over control constructs. We conclude that such combinatorial screens using tumor and normal cells from the same patient have significant potential to develop new reagents for cancer biology, diagnosis, and potentially therapy.

Identification of the minimum pharmacophore of lipid–phosphatidylserine (PS) binding peptide–peptoid hybrid PPS1D1

“…All the dimers were synthesized using the complete on-bead synthesis protocol described in our previous reports. 10,27 Briefly, peptide portions were synthesized using the standard Fmoc synthesis approach, and peptoid residues were coupled using the standard microwave-assisted peptoid synthesis protocol 28, 29 All compounds were purified using semi-preparative HPLC. Purity was confirmed by analytical HPLC, and synthesis was confirmed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Supplementary Figure S2–S9).…”

“…9 PPS1 was identified via an unbiased selection approach using our unique on-bead two-color cell screen (OBTC), which is capable of recognizing the difference between two cell surfaces. 10 In our previous study, we targeted red stained HCC4017 lung cancer cells in the presence of green stained HBEC30KT normal bronchial epithelial cells derived from the same patient. We mixed these two cell types in a 1:1 ratio and exposed to one-bead one-compound peptide-peptoid hybrid library synthesized on tentagel beads.…”

A mini-library system to investigate non-essential residues of lipid-phosphatidylserine (PS) binding peptide–peptoid hybrid PPS1

“…It is found in the inner layer of normal cell membranes, but in cancerous and tumor endothelial cells it is reported to be transferred (i.e., flipped) to the outer layer of the lipid membrane, thereby providing a promising cancer biomarker target with increased specificity over that of normal cells 4, 7–12 . More importantly, this PS flipping in cancer and tumor endothelial cells in the tumor microenvironment is universal, indicating that PS targeted drugs may have efficacy as common anticancer drugs 7, 8, 13–17 .…”

“…PPS1 displayed specific binding towards lipids that had an overall negative charge that were present on cancer cells, but not normal cells 14, 17 . The simple dimeric version of PPS1, PPS1D1 displayed cancer cell killing activity, but had no effect on normal cells 13 . Our recently reported alanine/sarcosine scan studies identified two positively charged residues at the second and third position and four C-terminal hydrophobic residues that composed the minimum pharmacophore 15 (Fig.…”

“…We recently identified a peptide–peptoid hybrid, PPS1, which recognized PS on series of lung cancer cells 13, 14 . PPS1 displayed specific binding towards lipids that had an overall negative charge that were present on cancer cells, but not normal cells 14, 17 .…”