How yeast surface display technology is revolutionizing the discovery of macrocyclic peptide drugs
In the relentless pursuit of new medicines, scientists are constantly exploring molecular bridges—therapies that combine the best attributes of different biological compounds. Macrocyclic peptides represent one such exciting bridge, offering the high specificity of large protein-based drugs like antibodies with the stability and synthetic ease of smaller molecules 1 . The challenge, however, lies in efficiently finding the rare, effective peptide needles in a vast molecular haystack.
Enter an unlikely hero: the common baker's yeast, Saccharomyces cerevisiae. Through a sophisticated technique known as yeast surface display, researchers are turning this simple organism into a powerful engine for drug discovery 4 .
Create vast pool of peptide variants
Screen against therapeutic targets
Recover and reproduce successful candidates
Sequence and characterize hits
A landmark study published in Nature Communications in 2025 perfectly illustrates the power of yeast display 1 .
Engineered yeast with cysteine-free GPI anchor for peptide display 1 .
Constructed "one-ring" (CX~m~C) and "two-ring" (CX~m~CX~n~C) libraries with NNK codons 1 .
Used fluorescently labeled target proteins and two-color assay for sorting 1 .
Sequenced genetic material from selected cells and validated binding affinity 1 .
Isolated macrocyclic peptide ligands with good binding properties against all five protein targets tested 1 .
Discovered potent hACE2 inhibitor with:
Structural Insight: X-ray crystallography revealed optimal shape complementarity and multiple inter-molecular interactions 1 .
| Library Topology | Peptide Format | Description | Theoretical Diversity |
|---|---|---|---|
| One-Ring | CX~7~C | Two fixed cysteines form a single ring, with 7 random amino acids in between | Up to 2 × 10⁹ |
| One-Ring | CX~9~C | Two fixed cysteines form a single ring, with 9 random amino acids in between | Up to 2 × 10⁹ |
| Two-Ring | CX~3~CX~9~C, CX~6~CX~6~C, CX~9~CX~3~C | Three fixed cysteines form two disulfide-bonded rings, with variable loop sizes | ~3 × 10⁸ |
| Protein Target | Binding Affinity (Kd) | Key Finding |
|---|---|---|
| Human ACE2 (hACE2) | 16.1 nM | Potent inhibition (IC₅₀ = 7.5 nM) |
| Multiple Other Targets | "Good binding properties" | Isolation of ligands for all 5 tested targets |
| Reagent / Material | Function in the Experiment | Specific Examples / Notes |
|---|---|---|
| Yeast Strain & Display System | Provides the cellular platform for peptide expression and surface anchoring | S. cerevisiae with Aga1-Aga2 system 4 or cysteine-free GPI anchor system 1 |
| Epitope Tags | Allows detection and quantification of peptide expression on the yeast surface | Hemagglutinin (HA) tag, c-myc tag. Used with fluorescently-labeled antibodies 1 4 |
| Fluorophore-Conjugated Reagents | Enable detection and sorting via Flow Cytometry (FACS) | Labeled target protein; antibodies against epitope tags for expression detection 1 4 |
| Fluorescence-Activated Cell Sorter (FACS) | The core instrument for high-throughput screening and analysis of the yeast library | Used to sort cells based on binding and expression signals 1 4 |
| PCR and Cloning Reagents | For library construction, diversification, and recovery of peptide sequences from sorted cells | Enzymes for DNA amplification and manipulation; plasmid vectors 7 |
| NNK Degenerate Codons | The genetic code used to create random amino acid sequences at defined positions | NNK (N = A/T/G/C; K = G/T) allows for all 20 amino acids and one stop codon 1 |
Creating diverse peptide libraries with NNK codons for maximum sequence variation
High-throughput sorting based on binding affinity and expression levels
Yeast surface display has firmly established itself as a formidable tool in the molecular engineer's arsenal. By transforming simple yeast cells into living libraries, scientists can now navigate the vast sequence space of peptides with unprecedented control and quantitative precision.
The successful discovery of low-nanomolar macrocyclic peptide inhibitors against challenging targets like hACE2 underscores the technology's potential to accelerate the development of new therapeutics .
As methods continue to evolve—opening doors to even more complex peptide architectures and streamlined screening processes—this synergy of biology and engineering promises to unlock a new era of peptide-based drugs, bridging the gap between small molecules and biologics to tackle diseases that have long eluded effective treatment.
References will be populated here.