Display cloning: functional identification of natural product receptors using cDNA-phage display

Sche, Paul P; McKenzie, Kathleen M.; White, Jennifer Duffield; Austin, David

doi:10.1016/s1074-5521(00)80018-6

Cited by 140 publications

(63 citation statements)

References 85 publications

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“…In both affinity chromatography and matrix-free methods, proteins are incubated with the modified small molecule, and the binding proteins are revealed by mass spectrometry after gel electrophoresis. Genetic and other versions of matrix-based affinity chromatography, e.g., yeast three-hybrid (37) and phage display cloning (38), require tagged small molecules as well. Thus, all current affinity methods are limited to small molecules that contain derivatizable functionalities and whose bioactivity/binding is unaffected by the modification (SI Text).…”

Section: Discussionmentioning

confidence: 99%

Target identification using drug affinity responsive target stability (DARTS)

Lomenick¹,

Hao²,

Jonai³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

760

707

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Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.aging ͉ label-free ͉ proteomics ͉ small molecules

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Section: Discussionmentioning

confidence: 99%

Target identification using drug affinity responsive target stability (DARTS)

Lomenick¹,

Hao²,

Jonai³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

760

707

View full text Add to dashboard Cite

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“…Despite the limitations of the use of phage display systems for the expression of eukaryotic proteins related to the lack of eukaryotic posttranslational modification machinery in bacteria, this system has been successfully used for the identification of several eukaryotic membrane proteins in which these modifications are critical. Receptors for natural products (23), for phosphatidylserine-specific clearance of apoptotic cells (24), and for endothelial surface protein aminopeptidase P (25) have been identified using T7-based cDNA expression libraries. Therefore, we relied on this approach and have exploited it for the identification of molecules expressed on the surface of PC cells, which were binders for the peptides previously selected from random phage display library.…”

Section: Discussionmentioning

confidence: 99%

Prostate Cancer Cell Adhesion to Bone Marrow Endothelium

Romanov¹,

Whyard²,

Adler³

et al. 2004

Cancer Research

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Bone metastasis is the most frequent complication of prostate cancer (PC). Elucidation of the biological basis of this specificity is required for the development of approaches for metastatic inhibition. We investigated the possibility that the preferential attachment of PC cells to bone marrow endothelium (as opposed to endothelium from other organs) affects this specificity. We selected, from peptide phage-displayed libraries, peptide ligands to surfaces of PC cells (C4-2B) attenuated (30 -40%) binding of C4-2B cells to bone marrow endothelial cells (BMECs). We then determined the molecules on the surface of C4-2B cells interacted with the selected peptides using column affinity chromatography and a cDNA expression phage-displayed library generated from C4-2B cells in T7 phage. We identified a phage from the cDNA library that specifically bound to one of the selected peptides-L11. This phage displayed the amino acid sequence homologous for the COOH-terminal portion of prostatespecific antigen (PSA). To examine the possible direct involvement of PSA in the interactions between PC and BMECs, we performed a cell-cell adhesion assay. Antibodies to PSA attenuated PC cells adhesion to BMECs. In addition, exogenous proteolytically active PSA modulated this adhesion. Finally, inactivation of mRNA coding PSA by a small interfering RNA (siRNA) diminished C4-2B cell adhesion to BMECs. These results indicate that PSA expressed as secreted and surface-associated molecules in C4-2B cells is involved in cell-cell interactions and/or digests components of bone marrow endothelium for preferential adhesion and penetration of PC cells. The suggested experimental approach is a promising strategy for identification of cell surface molecules involved in intercellular interactions.

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“…A cDNA library displayed on T7 phage (23)(24)(25) was used to clone proteins binding to the CPGPEGAGC peptide. The peptide was synthesized in a Symphony synthesizer (Rainin Instruments) at our peptide facility, and cyclized and purified by HPLC.…”

Section: Methodsmentioning

confidence: 99%

Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

Essler

Ruoslahti

2002

Proc. Natl. Acad. Sci. U.S.A.

174

103

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In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPE-GAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.endothelial cells ͉ vascular targeting ͉ breast cancer T he vascular beds of individual tissues differ in structure and metabolic function, and in expression of organ-specific adhesion molecules (1). Such specialization is particularly striking in lymphoid tissues where the high endothelia are composed of cells that express unique adhesion molecules for lymphocyte homing (2). Furthermore, metastasis into preferred organs by certain tumors may depend on interactions between tumor cells and organ-specific molecules in the vasculature of the target organ (3).Our laboratory has shown that peptide libraries displayed on phage can be screened in vivo for phage that home to a specific target, and we and others have shown that peptides capable of homing to the vasculature of various individual tissues can be isolated in this manner (4 -6). In vivo selections have also successfully identified peptides that home to tumor vasculature and the vasculature of other pathological lesions (7,8). Some of the tumor-homing peptides recognize known markers of sprouting endothelial cells and angiogenic tumor vasculature, such as the ␣ v ␤ 3 , ␣ v ␤ 5 , and ␣ 5 ␤ 1 integrins (7, 9), matrix metalloproteases (10 -12), and NG2 proteoglycan (13,14). A group of tumor-homing peptides containing the sequence motif NGR were used to identify aminopeptidase N as a new marker of angiogenic vasculature (15). Only one homing peptide receptor in the vasculature of normal tissues has been identified so far; membrane dipeptidase is selectively expressed in lung vasculature and serves as a receptor for a set of lung-homing peptides (16).Homing peptides show promise in the delivery of drugs and other therapies into designated target sites. Thus, peptides identified by phage display have been used to target doxorubicin, proap...

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Display cloning: functional identification of natural product receptors using cDNA-phage display

Cited by 140 publications

References 85 publications

Target identification using drug affinity responsive target stability (DARTS)

Target identification using drug affinity responsive target stability (DARTS)

Prostate Cancer Cell Adhesion to Bone Marrow Endothelium

Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

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