The discovery of compounds that induce novel interactions between two proteins is a critical goal in modern drug development. These molecules, known as molecular glues, function by stabilizing a ternary complex formed between an E3 ubiquitin ligase and a target protein. Unlike traditional inhibitors that bind to a single, well-defined pocket, molecular glues must simultaneously cooperatively engage two distinct protein surfaces. This requirement makes their discovery exceptionally challenging. Conventional approaches often rely on serendipitous findings or require extensive prior knowledge of binding sites and known ligands for both proteins.
Fig.1 Trimerizer Helicon-induced cooperative binding between MDM2 and β-catenin.1
A recent study has introduced a systematic, two-step phage display strategy that overcomes these limitations. This method enables the discovery of α-helical peptides, termed "trimerizer Helicons," that can induce cooperative binding between an E3 ligase and a target protein, even when no prior affinity exists between them. The approach eliminates the need for structural data or known binders, providing a robust and generalizable platform for the discovery of molecular glues. At Creative Biolabs, we offer the specialized phage display library construction services and biopanning and screening strategies necessary to implement this advanced methodology for your research on E3 ligase reprogramming and novel substrate identification.
Traditional phage display experiments are designed to identify high-affinity binders for a single target molecule. This binary approach is efficient for discovering inhibitors or antagonists. However, it is statistically improbable to find a peptide that randomly possesses the precise sequence required to bind two different proteins simultaneously with high affinity and cooperativity. The probability of such a dual-binding event occurring by chance within a library of billions of unique sequences is extremely low. To address this fundamental challenge, the researchers developed a sequential, two-stage screening process. The first stage focuses on identifying peptides that bind to the E3 ligase, which acts as the "presenter" protein. The second stage uses the insights from the first screen to construct a highly focused library designed to find peptides that can bind the target protein, but only when the E3 ligase is present. This strategic division of the discovery process transforms an impossible random search into a directed engineering problem.
Fig.2 General phage display workflow for trimerizer Helicon discovery.1
The process begins with a naive phage display screen. Researchers use an extensive and diverse library containing approximately 108 unique, cysteine-stapled α-helical peptides, known as Helicons. This library is screened against the target E3 ubiquitin ligase. The goal of this initial screen is not to find the final therapeutic agent, but to identify a set of peptides that successfully bind to the E3 ligase surface. After several rounds of selection and amplification, Next-Generation Sequencing (NGS) is used to analyze the enriched phage population. The resulting sequences are grouped into clusters based on similarity. From these clusters, a sequence logo is generated, which visually represents the amino acid preferences at each position within the peptide. Analysis of this sequence logo reveals two distinct types of residues.
The core innovation of this strategy lies in the design of the focused phage library. Researchers use the information from the naive screen's sequence logo to engineer a new library. This library retains the ability to bind the E3 ligase while maximizing diversity at the positions that will interact with the target protein. This engineering process follows three specific design principles:
The final stage involves peptide library screening to identify trimerizer helicons. These are peptides that promote the formation of a ternary complex. The screening protocol distinguishes between simple binders and cooperative stabilizers by performing two parallel selection experiments:
Data analysis compares the sequencing counts from both conditions. Peptides that show significant enrichment in the complex selection but low or no recovery in the control selection are identified as trimerizers. This binding profile indicates that the peptide requires the E3 ligase to bind the target, confirming the formation of a cooperative ternary complex. Peptides that bind in both conditions are discarded as nonspecific binders.
This approach is not limited to the specific E3 ligases studied in the original research. It can be applied to any E3 ligase or protein target that can be expressed and purified in a functional form. By leveraging this platform, researchers can systematically explore the proteome and identify novel substrates for targeted protein degradation, moving beyond the current reliance on VHL and CRBN. The successful execution of this workflow requires precise control over library generation and screening conditions. Creative Biolabs offers a comprehensive suite of enabling technologies designed to support your targeted protein degradation projects from concept to lead identification. Our integrated platform provides the specialized resources needed to overcome the technical hurdles associated with finding cooperative binders.
Our experts provide the technical capabilities necessary to engineer novel protein-protein interactions. to discuss how we can apply these advanced phage display methods to your specific therapeutic targets.
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