High-Throughput Yeast Display Platform for Macrocyclic Peptide Discovery

Macrocyclic peptides, characterized by high affinity, high selectivity, favorable protease stability, and appreciable membrane permeability, have emerged as an ideal therapeutic modality positioned between small molecules and antibodies. Currently, approximately 80 peptide-based drugs have been approved globally, with over 40 being macrocyclic peptides, and the number of annual approvals continues to rise. However, conventional macrocyclic peptide screening technologies, such as phage display and mRNA display, are hampered by difficulties in process monitoring, lengthy selection cycles, low success rates, and high downstream costs associated with chemical synthesis and characterization.

Recently, a study published in Nature Communications reported a high-throughput screening platform based on yeast display technology for the rapid discovery and characterization of disulfide-constrained macrocyclic peptides with high affinity and selectivity. This platform not only overcomes the limitations of traditional display methods but also enables real-time monitoring and quantitative analysis of the selection process, providing a powerful tool for peptide-based drug development.

I. Technological Breakthrough: Yeast Display Coupled with Quantitative Flow Cytometry

Sara Linciano and colleagues fused a cysteine-rich (highlighted in blue) macrocyclic peptide, along with a linker and HA tag, to the N-terminus of a cysteine-free GPI-anchored protein (depicted as a black line). This strategic design completely prevents unwanted intermolecular disulfide bond formation between the macrocyclic peptide's cysteines and the conventional Aga1-Aga2 system, thereby ensuring correct folding and display of the macrocyclic peptide. By varying the number or position of cysteines, they constructed single- or double-loop libraries with different ring sizes and spacings, achieving library sizes ranging from 3 × 10? to 2 × 10?, which ensures the likelihood of identifying high-affinity binders. By integrating flow cytometry and utilizing fluorescently labeled antibodies to detect the HA tag, they enabled rapid quantification of macrocyclic peptide ligand expression levels and normalization of binding signals to expression levels.

图1大环肽库设计与多样性分析.png

Figure 1: Macrocyclic peptide library design and diversity analysis.

II. Validation of Technical Versatility Across Multiple Targets

The researchers selected five highly diverse proteins with unrelated sequences and structures. Five yeast libraries (CX?C, CX?C, CX?CX?C, CX?CX?C, and CX?CX?C) were pooled and individually incubated with each protein target (PT). Initial screening involved two rounds of magnetic bead selection using streptavidin beads to capture binding peptides, followed by four rounds of fluorescence-activated cell sorting (FACS) to further enrich for high-affinity binders. For example, enrichment for PT5 increased from 11% to 82% after four FACS rounds (Figure 3C). Ultimately, specific binding macrocyclic peptides were obtained for all five targets, demonstrating the broad applicability of this technology.

图2五种不同蛋白靶点（PT）的三维结构.png

Figure 2: Three-dimensional structures of the five distinct protein targets (PTs).

Figure 3: General screening workflow and FACS enrichment efficiency.

III. Affinity and Specificity Validation

To determine the binding affinities of the isolated macrocyclic peptides, the researchers treated yeast cells displaying specific macrocyclic peptides with a series of known concentrations of biotinylated PT solutions. Subsequent fluorescence detection allowed for quantitative binding curve generation and calculation of dissociation constants (KD values). This technique enables rapid characterization of selected macrocyclic peptide ligands directly on the yeast cell surface, eliminating the need for chemical synthesis and purification.

图4酵母表面滴定法测亲和力.png

Figure 4: Yeast surface titration assay for affinity measurement.

Furthermore, surface plasmon resonance (SPR) was employed to validate the binding affinities between the selected macrocyclic peptides and their PTs. Results showed that most peptides exhibited KD values in the nanomolar range, with some reaching the picomolar level. The yeast surface titration assay (macrocyclic peptide immobilized, PT in solution) and SPR (PT immobilized, peptide in solution) represent orthogonal measurement directions, effectively ruling out the possibility of avidity effects that might overestimate binding affinity.

图5 SPR确定结合亲和力.png

Figure 5: SPR determination of binding affinities.

IV. Technological Comparison: Yeast Display vs. Conventional Methods

The researchers then compared the binding affinities of macrocyclic peptides identified via yeast display with those of previously reported linear or cyclic peptides targeting the same molecules. The results indicated that, compared to phage display or mRNA display, the yeast display platform consistently yielded higher-affinity binders even with comparable or smaller library sizes.

图6与传统技术的性能对比.png

Figure 6: Performance comparison with conventional technologies.

Summary

This study successfully established a macrocyclic peptide screening platform based on yeast display. The constructed display libraries (~10?) surpass previous limits for yeast display libraries; while still smaller than mRNA display libraries in magnitude, they are comparable to or slightly larger than the largest phage display libraries. This platform allows for rapid and efficient identification and characterization of macrocyclic peptides directly on the cell surface, obviating costly and time-consuming chemical synthesis and purification. All identified macrocyclic peptides exhibited binding affinities below 1 μM. While this study demonstrates screening capabilities for disulfide-constrained macrocyclic peptides, yeast display can be integrated with other in vitro directed evolution tools to leverage their respective advantages, potentially enabling applications not explored previously.

TekBiotech (Tianjin) Co., Ltd. , with its core services in phage display and yeast display-based antibody development, also provides complementary upstream and downstream services including antibody sequencing, targeted peptide discovery, affinity maturation and measurement. Leveraging our proprietary core technology platforms, we are committed to being the most trusted partner for global pharmaceutical companies, biotechnology firms, and research institutions in drug target identification and precision drug development.

Reference
[1] Linciano, S., Mazzocato, Y., Romanyuk, Z. et al. Screening macrocyclic peptide libraries by yeast display allows control of selection process and affinity ranking. Nat Commun 16, 5367 (2025).

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