Evaluation of phage display discovered peptides as ligands for prostate-specific membrane antigen (PSMA).
The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD~1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy.
Radiology, Washington University, St Louis, Missouri, United States of America.
Duanwen
D
Shen
Fei
F
Xie
W Barry
WB
Edwards
D000595:Amino Acid Sequence; D001665:Binding Sites; D014408:Biomarkers, Tumor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005452:Fluoresceins; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007610:Kallikreins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008297:Male; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D019151:Peptide Library; D011467:Prostate / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D017430:Prostate-Specific Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D017422:Sequence Analysis, DNA
D014408:Biomarkers, Tumor; D005452:Fluoresceins; D055767:Immobilized Proteins; D008024:Ligands; D010446:Peptide Fragments; D019151:Peptide Library; C045132:4-carboxyfluorescein; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen