Radiology, Washington University, St Louis, Missouri, United States of America.
PLoS One. 2013 Jul 25;8(7):e68339. doi: 10.1371/journal.pone.0068339. Print 2013.
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.
本研究旨在鉴定适用于进一步开发的 PSMA 潜在配体，这些配体可作为新型 PSMA 靶向肽，使用噬菌体展示技术。将人 PSMA 蛋白固定为靶标，然后与 15 肽噬菌体展示随机肽库孵育。经过一轮预筛选和两轮筛选，在第三轮淘选中进行高严格性筛选，以鉴定最高亲和力结合物。分离出与人前列腺癌细胞中 PSMA 具有特异性结合活性的噬菌体，并对相应的 15 肽 DNA 进行测序，提供三个共有序列：GDHSPFT、SHFSVGS 和 EVPRLSLLAVFL 以及其他未显示共有序列的序列。从结合噬菌体的 DNA 测序推导的两个肽序列，SHFSVGSGDHSPFT 和 GRFLTGGTGRLLRIS 用 5-羧基荧光素标记，并通过荧光显微镜显示与人前列腺癌细胞上的 PSMA 结合和共内化。高严格性要求产生了亲和力 KD~1 µM 或更高的肽，这些肽是亲和力成熟的合适起点。虽然这些值低于预期，但高严格性确实产生了显然与 PSMA 不同表面结合的肽序列。这些肽序列可能是进一步开发用于前列腺癌肿瘤成像和治疗的肽的基础。
