Aug 2026· Current Protocols· Vol 6· 0 citations· 71 references
Medicine
TL;DR
A protocol for discovering protein‐binding peptides using a very large, target‐agnostic yeast surface display library containing approximately 6.1 × 109 unique clones and providing broad coverage of short peptide sequence space is described.
Protein-protein interactions underpin most cellular processes, and engineered binders present powerful tools for probing biology and developing novel therapeutics. However, scalable, quantitative characterization of large numbers of candidates remains a major bottleneck. Here we show that ADAPT-M (Affinity Determination by Adaptation of ProTein binders for Microfluidics) enables rapid, parallel measurement of binding affinities and dissociation behavior directly from enriched display libraries in under one week, without requiring gene synthesis or hands-on protein purification. Applied to a computationally designed library targeting the SARS-CoV-2 Omicron BA.1 receptor binding domain, ADAPT-M recovered most highly enriched variants and revealed that many display-enriched binders lacked measurable binding in vitro, highlighting limitations of screening alone. ADAPT-M enabled quantitative characterization of dozens of binders in parallel and selection of lead candidates for structural analysis. Unexpectedly, structural and mutational studies revealed that designed binding interfaces were preserved despite engaging alternative epitopes. By bridging screening and scalable in vitro validation, ADAPT-M accelerates protein binder discovery and supports data-driven protein engineering. ADAPT-M is a workflow combining design and high-throughput experimentation. It overcomes the testing bottleneck and enables rapid quantitative affinity measurements of thousands of designer proteins enriched from yeast surface display libraries.
Carla P. Perez, N. DelRosso, Cameron L. Noland et al.· Nature Communications· 0 citations
Chemical proteomics has identified covalent ligands targeting cysteine residues across many hundreds of human proteins. The functional effects of these liganding events, however, remain challenging to assign at scale. Here we describe ESCAPE (Endogenous Site-specific Competition Assays using Prime Editors), a platform for the site-resolved functional analysis of covalent ligands in cells. In this method, cysteine-to-serine substitutions are generated by prime editing to abrogate covalent ligand-protein interactions, and the impact of these edits on ligand-induced cellular phenotypes is quantified through allele frequency-based resistance scores. Applied to ligandable cysteines mapped by activity-based protein profiling in 50+ proteins, ESCAPE identified multiple covalent ligand-protein interactions that impair cancer cell growth, including azetidine butynamides that target a non-orthosteric cysteine in the RNA helicase DDX49 to disrupt 18S rRNA processing, 40S ribosome assembly, and protein synthesis. ESCAPE thus provides a scalable framework for the functional characterization of covalent ligands targeting structurally and mechanistically diverse proteins.
Jason Tse, William R. Brothers, Rachel E. Hayward et al.· bioRxiv· 0 citations
Fragment-based drug discovery (FBDD) is a powerful workflow for the development of drug candidates and probe molecules that begins with the discovery of one or more low affinity, low molecular weight ligands for a protein of interest (POI). Recently, we developed a simple fragment discovery platform in which TentaGel beads displaying many copies of the fragment are incubated with a fluorescently labeled multimeric protein. Association of two or more bead-displayed ligands with the multimeric target stabilizes the complex sufficiently to allow its detection even after rigorous washing. However, this simple “pull-down” assay cannot be used for monomeric protein targets since these complexes are too kinetically labile to survive being taken out of equilibrium. Here we explore various strategies to multimerize monomeric targets, thus allowing them to be used as targets in this fragment discovery platform. We find that a particularly effective and convenient strategy is to express a fusion of the POI with FOLDON, a 30-residue, homo-trimeric peptide.
Joel Tong, Isuru M. Jayalath, James V. Parsons et al.· RSC Chemical Biology· 0 citations
Experimental validation and functional optimization remain bottlenecks in AI-based protein design. We present a scalable workflow for developing AI-designed minibinders against cancer-associated surface proteins. Screening thousands of designs using mammalian cell-surface display identifies several high-affinity PD-L1 minibinders but far fewer for CD276 (B7-H3) and VTCN1 (B7-H4), highlighting substantial target dependence. Interface predicted template modeling (ipTM) scores generated by Chai-1 with ESM embeddings correlate with binding success and capture deleterious effects of interface mutations. Fluorophore-labeled AI-minibinders enable flow-cytometric staining comparable to conventional antibodies. However, when incorporated into chimeric antigen receptors (CAR), some show poor cell-surface trafficking and limited functionality. Redesign through a genetic algorithm-based diversification strategy that preserves the binding interface while changing non-binding surfaces experimentally reveals an isoelectric point (pI) window that improves CAR expression and enhances target-selective tumor cell killing. Our findings identify biochemical optimization beyond the binding interface as a critical requirement for translating AI-minibinders into functional applications. In this work the authors present a scalable mammalian cell-display workflow to identify AI-designed minibinders against cancer surface targets. AI-guided optimization beyond the binding interface improves their expression as chimeric antigen receptors and target-selective killing.
B. Broske, B. McEnroe, S. C. Frechen et al.· Nature Communications· 0 citations
This review provides an updated overview of cyclic peptides as modulators of PPIs, outlining current opportunities, methodological advances, and remaining challenges in the development of cyclic peptide-based PPI modulators.
Sara Salvi, P. Linciano, S. Collina et al.· International Journal of Mol...· 0 citations
Abstract Engineering proteins for therapeutic applications is a field that has seen substantial growth in the past two decades, but challenges remain. A major deficiency in current strategies is the lack of a means to efficiently screen for binding to key epitopes to yield a desired function. To meet this challenge, we designed a tethered yeast surface display construct that leverages high local concentrations by tethering a candidate protein binder to a target protein of interest (POI) via a flexible peptide linker. These high local concentrations enable screening of epitope-specific binders based on decreased signal due to inhibitory function when the construct is assayed alongside a competitive POI binder. We demonstrate that epitope-specific screening and enrichment is possible based on fluorescent output and study key optimization parameters. We anticipate this technique will accelerate binder development by reducing the need for downstream low throughput epitope mapping of isolated proteins.
Rojhae A Panton, J. Lind, Amanda Richardson et al.· Protein engineering, design...· 0 citations
