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A facile strategy for the construction of a phage display cyclic peptide library for the selection of functional macrocycles.

ABSTRACT: Cyclic peptides represent invaluable scaffolds in biological affinity, providing diverse collections for discovering functional molecules targeting challenging biological entities and protein-protein interactions. The field increasingly focuses on developing cyclization strategies and chemically modified combinatorial libraries in conjunction with M13 phage display, to identify macrocyclic peptide inhibitors for traditionally challenging targets. Here, we introduce a cyclization strategy utilizing ortho-phthalaldehyde (OPA) for the discovery of active macrocycles characterized by asymmetric scaffolds with side-chain cyclization. Through this approach, aldehyde groups attached to free molecules sequentially attack the ε-amine of lysine and the thiol of cysteine, facilitating the rapid cyclization of genetically encoded linear precursor libraries displayed on phage particles. The construction of a 109-member library and subsequent screening successfully identified cyclic peptide binders targeting three therapeutically relevant proteins: PTP1B, NEK7, and hKeap1. The results confirm the efficacy in rapidly obtaining active ligands with micromolar potency. This work provides a fast and efficient operable high-throughput platform for screening functional peptide macrocycles, which hold promise for broad application in therapeutics, chemically biological probes, and disease diagnosis.

SUBMITTER: Xiang H

PROVIDER: S-EPMC11290325 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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A facile strategy for the construction of a phage display cyclic peptide library for the selection of functional macrocycles.

Xiang Hua H Bai Liwen L Zhang Xindan X Dan Ting T Cheng Peng P Yang Xiaoqin X Ai Honglian H Li Kai K Lei Xinxiang X

Chemical science 20240701 30

Cyclic peptides represent invaluable scaffolds in biological affinity, providing diverse collections for discovering functional molecules targeting challenging biological entities and protein-protein interactions. The field increasingly focuses on developing cyclization strategies and chemically modified combinatorial libraries in conjunction with M13 phage display, to identify macrocyclic peptide inhibitors for traditionally challenging targets. Here, we introduce a cyclization strategy utilizi ...[more]

PMID: 39092106

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Project description:A large human natural single‑chain fragment variable (scFv) phage library was constructed based on Cre‑LoxP recombination, and used to successfully identify antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9). The library was derived from 400 blood samples, 30 bone marrow samples, and 10 cord blood samples from healthy donors. Lymphocytes were isolated from each sample and cDNA was synthesized using reverse transcription‑quantitative PCR. Two‑step overlap PCR was then used for scFv synthesis using a LoxP peptide as the linker. The scFv gene was inserted into the phagemid vector pDF by enzymatic digestion and ligation, and then transformed into Escherichia coli (E. coli) SS320 to establish a primary antibody library in the form of scFvs. A primary antibody library consisting of 5x107 peripheral blood and umbilical cord blood sources, as well as a primary antibody library of 5x107 bone marrow samples were obtained. By optimizing the recombination conditions, the primary phage library was used to infect E. coli BS1365 strain (which expresses the Cre enzyme), and a human scFv recombinant library with a size of 1x1011 was obtained through Cre‑LoxP enzyme‑mediated heavy and light chain replacement and recombination. This constructed recombinant library was employed to screen for antibodies against recombinant PCSK9. After four rounds of selection, a fully human antibody (3D2) was identified with a binding affinity of 1.96±1.56ⅹ10‑10 M towards PCSK9. In vitro, the PCSK9/low‑density lipoprotein receptor (LDLR) pathway of Hep‑G2 cells was inhibited by 3D2 treatment, thereby increasing LDL uptake in these cells. In addition, combination treatment with 3D2 and statin was more effective at increasing LDLR levels than treatment with 3D2 or statin alone. Furthermore, 3D2 resulted in a 3‑fold increase in hepatic LDLR levels, and lowered total serum cholesterol by up to 61.5% in vivo. Taken together, these results suggest that the constructed human Cre‑LoxP scFv phage display library can be used to screen fully human scFv, and that 3D2 may serve as a candidate hypolipidemic therapy.

Dataset Information

A facile strategy for the construction of a phage display cyclic peptide library for the selection of functional macrocycles.

Publications

A facile strategy for the construction of a phage display cyclic peptide library for the selection of functional macrocycles.

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