Disulfide-constrained Peptide Scaffolds Enable a Robust Peptide-therapeutic Discovery Platform
Lijuan Zhou, Fei Cai, Yanjie Li, Xinxin Gao, Yuehua Wei, Anna Fedorova, Daniel Kirchhofer, Rami N. Hannoush, Yingnan Zhang
Abstract
Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 1011. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and in vitro folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease.
Introduction
Many natural peptides have been identified as important regulators of biological functions, acting as hormones, ion channels and GPCR modulators, growth factors, and neurotransmitters. They are usually selective and high-affinity ligands that bind to cell surface receptors and induce intracellular signaling. Therefore, peptides are an attractive modality for developing therapeutics to either agonize or antagonize cellular pathways associated with disease [1]. However, peptides often exhibit poor chemical and physical stability and a short plasma circulating half-life, limiting their therapeutic potential [2]. To overcome some of these peptide-intrinsic shortcomings, we considered using naturally occurring and highly stable disulfide constrained peptides (DCPs) as frameworks coupled with display technology aimed at generating de novo binders of therapeutically relevant target proteins [3]. Notebly, DCP library strategies has been effectively employed using mRNA display with MCoTI-II as a scaffold [4]. In this study, we employed phage display across seven distinct scaffolds to extent the applicability of this approach.
Methods
Protein reagents
Proteins that were purchased commercially are listed in the following in format of protein name (vendor name, catalog number): Her3-Fc (R&D, 348-RB); Fz7CRD-Fc (R&D, 6178-FZ); Insulin (Sigma-Aldrich, I2643) and CD28-Fc (R&D, 342-CD-200). The following human and bacterial (BamA) proteins were produced in house (Genentech) are listed as following in format of protein name (construct, gene ID, expression system): HGF activator (HGFA) (V373-S655, 3083, Hi5 Baculovirus) [41]; HtrA1 (Q23-P480, 5654, Tni Baculovirus) [35]; ApoL1 (D61-L398, 8542, SF9 Baculovirus) [42]; human R-spondin (A32-Q243, 340419, CHO); Lgr5ECD-Fc (M1-W558, 8549, CHO); PCSK9 (M1-Q692, 255738, CHO) [43]; PCSK9ΔCRD (Q31-G452 = PCSK9 lacking the C-terminal Cys-rich domain CRD, 255738, Tni Baculovirus) [44]; BamA-amphipol (BamA protein encapsulated with biotinylated amphipol) (V418-W810, 944870, E. Coli.) [45]; CD16A (G17-Q208, 2214, CHO); hEpCAM (Q24-K265, 4072, CHO); Notch2Long (Notch2 EGF6-EGF12) (D219-E418, 4853, CHO); Notch2NRR (Notch2 NRR domain) (A1423-Q1677, 4853, CHO). Proteins expressed in Baculovirus and E. Coli. systems were reported previously as referenced.
Results
Display of DCP scaffolds on M13 surface
Phage display with filament M13 phage utilizes the secretion pathway of E. Coli to translocate the DCPs fused to phage coat protein p8 or p3 to the periplasmic space, where the DCPs are folded. Initially, we selected one CKP scaffold, namely EETI-II [16], and two cyclotide scaffolds, MCoTI-II [17], and kalata B1 [18], to assess the feasibility of displaying DCPs on the surface of M13 phage. The DCPs, incorporating an N-terminal gD-tag for quantification of display levels, were fused at the C-terminal to the major M13 coat protein p8 or the C-terminal domain of minor coat protein p3 (Fig 1A). Moreover, as both EETI-II and MCoTI-II are potent trypsin inhibitors, we measured phage binding to trypsin-coated plates to verify the correct folding of these phage surface-displayed DCPs. For phage display of the cyclotides MCoTI-II and kalata B1, ring opening was necessary to enable fusion with the phage coat proteins, as detailed in the methods section.
Discussion
In this study, we first successfully displayed four DCP scaffolds on the M13 phage surface by utilizing both the major coat protein p8 and the minor coat protein p3. We then demonstrated the binding capabilities of two specific scaffolds, EETI-II and McoTI-II, to their native binding partner trypsin while being displayed on the p8 protein on the phage surface. This finding indicates that these scaffolds are able to retain their correct folding when presented on the phage surface. Kalata B1 is a CKP containing cyclotide. We displayed this cyclotide by breaking one ring and generated peptide with new N- and C-termini, so that it can be fused to phage coat proteins for surface display. The successful display of this cyclotide in the format of DCP opened up the possibility of including cyclotides as DCP scaffolds for library construction. These findings provide evidence for the feasibility of utilizing phage display as a valuable platform for constructing a diverse library of DCP variants for purpose of engineering new functionalities.
Citation: Zhou L, Cai F, Li Y, Gao X, Wei Y, Fedorova A, et al. (2024) Disulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platform. PLoS ONE 19(3): e0300135. https://doi.org/10.1371/journal.pone.0300135
Editor: Maria Gasset, Higher Council for Scientific Research, SPAIN
Received: October 3, 2023; Accepted: February 21, 2024; Published: March 28, 2024
Copyright: © 2024 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0300135#abstract0
