Identification and Rational Redesign of Peptide Ligands to CRIP1, A Novel Biomarker for Cancers

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Abstract
Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of cancers. Here we report on the use of phage display in combination with molecular modeling to identify a high-affinity ligand for CRIP1. Panning experiments using a circularized C7C phage library yielded several consensus sequences with modest binding affinities to purified CRIP1. Two sequence motifs, A1 and B5, having the highest affinities for CRIP1, were chosen for further study. With peptide structure information and the NMR structure of CRIP1, the higher-affinity A1 peptide was computationally redesigned, yielding a novel peptide, A1M, whose affinity was predicted to be much improved. Synthesis of the peptide and saturation and competitive binding studies demonstrated approximately a 10–28-fold improvement in the affinity of A1M compared to that of either A1 or B5 peptide. These techniques have broad application to the design of novel ligand peptides. Breast cancer is one of the most frequently diagnosed malignancies in American females and is the second leading cause of cancer deaths in women. Several improvements in diagnostic protocols have enhanced our ability for earlier detection of breast cancer, resulting in improvement of therapeutic outcome and an increased survival rate for breast cancer patients. However, current early screening techniques are neither comprehensive nor infallible. Imaging techniques that improve breast cancer detection, localization, and evaluation of therapy are essential in combating the disease. Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of breast cancers. Here, we report the use of phage display and computational molecular modeling to identify a high-affinity ligand for CRIP1. Phage display panning experiments initially identified consensus peptide sequences with modest binding affinity to purified CRIP1. Using ab initio modeling of binding peptide structures, computational docking, and recently developed free energy estimation protocols, we redesigned the peptides to increase their affinity for CRIP1. Synthesis of the redesigned peptide and binding studies demonstrated approximately a 10–28-fold improvement in the binding affinity. The combination of computational and experimental techniques in this study demonstrates a potentially powerful tool in modulating protein–protein interactions.