A groundbreaking international collaboration between Pompeu Fabra University’s Department of Medicine and Life Sciences and Stanford University has yielded a pioneering method to identify highly selective therapeutic peptides with unparalleled precision. This novel approach leverages biologically and chemically modified bacteriophages, enabling researchers to screen up to one billion peptides simultaneously. Such an expansive and precise screening capacity is instrumental in distinguishing minute differences among closely related proteins that are central to diseases like cancer and type 2 diabetes—conditions often complicated by treatments that lack selectivity and cause unintended side effects.
Central to this breakthrough is the refinement of phage display, a well-established biochemical technique that uses bacteriophages—viruses that infect bacteria—as vehicles to present large peptide libraries on their surfaces. The investigators introduced a critical innovation by incorporating macrocyclic peptides into the library, a strategy that significantly enhances the rigidity of substrate peptides through a ring-shaped conformation. This structural constraint reduces their flexibility, which is pivotal in minimizing nonspecific binding events with off-target proteases, thereby honing in on highly specific molecular interactions.
Furthermore, the addition of a fluorescent tag as an integral part of the peptide library allows for real-time monitoring of peptide-protein interactions during the screening process. This fluorescent element facilitates direct visualization of substrate recognition while assays are performed in live systems or complex biological environments, thereby providing dynamic insight into protein interactions that was previously unachievable with traditional methods. This double modification marks a substantial advancement for selective screening modalities.
The team focused their study on discriminating between two remarkably similar proteases: fibroblast activation protein α (FAPα) and dipeptidyl peptidase 4 (DPP4). These proteins share about 70% structural homology, a similarity that has historically posed a significant challenge for the design of selective drugs. FAPα is notably implicated in oncological contexts, being overexpressed in up to 90% of carcinomas and linked with adverse prognoses. In contrast, DPP4 has critical physiological roles in glucose metabolism, making it a key therapeutic target for type 2 diabetes drugs. However, existing inhibitors often suffer cross-reactivity, affecting both proteins and resulting in undesired pharmacological outcomes.
This phage display-based method proved capable of selectively isolating peptides that recognize FAPα without interacting with DPP4, overcoming a barrier that conventional drug design has struggled to address. The capacity to selectively interact with one protease over a highly homologous counterpart not only paves the way for designing superior diagnostic tools but could also herald therapeutics with dramatically improved efficacy and reduced side effects. These are peptides designed to differentiate with exceptional fidelity, guiding new avenues in precision medicine.
A significant concern with currently approved diabetes medications targeting DPP4 is their unintended interaction with bacterial homologs of the same enzyme, which may disrupt the human microbiome. By contrast, the peptides generated in this study demonstrated such exquisite specificity that the implication for microbiome-safe therapeutic interventions becomes a compelling prospect. This precision could transform how clinicians approach the use of protease inhibitors in metabolic and oncological diseases alike.
The inherent stability and resistance to degradation of the macrocyclic peptides further enhance their feasibility as in vivo diagnostic markers and potential therapeutic agents. Their circular backbone significantly extends their half-life within biological systems compared to their linear counterparts, which are susceptible to rapid enzymatic cleavage. This resilience increases the functional utility of peptide agents in both experimental research and clinical applications.
Because these macrocyclic peptides enable sensitive detection by substantially reducing nonspecific interactions, they permit identification of proteases at lower concentrations. Lower protease quantities are necessary to reveal relevant substrate interactions, enhancing diagnostic sensitivity and reducing assay complexity. This property could revolutionize early detection methods for cancer and other diseases characterized by protease dysregulation.
In addition to medical diagnostics, the fluorescence capabilities embedded within the peptides could be harnessed for advanced surgical techniques, including fluorescence-guided surgery. Such applications would allow real-time visualization of tumor margins or diseased tissues during procedures, ensuring more precise excision and potentially improving patient outcomes through better surgical accuracy.
Proteases themselves hold immense biological significance; they regulate myriad cellular functions by precisely cleaving substrate proteins, thereby controlling processes such as cell proliferation, differentiation, and apoptosis. These enzymes have been extensively implicated in cardiovascular pathology, infectious diseases like HIV, autoimmune conditions, and oncogenesis. Due to their broad influence, selective modulation of protease activity is a promising therapeutic frontier.
However, the high degree of structural and functional redundancy within protease families has complicated the development of selective inhibitors. Many proteases share homologous substrates or active sites, leading to off-target effects and compromised drug efficacy. The selective identification and targeting of protease-substrate pairs, as demonstrated in this study, represent an innovative strategy to circumvent these hurdles, promising more targeted therapeutic interventions.
This novel technology thus stands as a milestone in medicinal chemistry and molecular biology. By exploiting macrocyclic phage display enhanced with real-time fluorescence detection, researchers have unlocked a powerful tool to discriminate between closely related proteases at an unprecedented scale. The method’s capacity to map precise protease-substrate interactions could open up new vistas in drug discovery, diagnostics, and personalized medicine, fostering treatments tailored to individual proteolytic landscapes.
As this methodology is further refined and adapted, its impact could ripple across multiple domains in health sciences, from better biomarker development for cancer prognosis to safer and more effective metabolic disease treatments. It heralds a future where the specificity of peptide therapeutics and diagnostic agents is no longer a limitation but a breakthrough advantage in combating complex diseases.
Subject of Research: Cells
Article Title: Macrocyclic Phage Display for Identification of Selective Protease Substrates
News Publication Date: 18-Jul-2025
Web References: http://dx.doi.org/10.1021/jacs.5c04424
References: Journal of the American Chemical Society, DOI: 10.1021/jacs.5c04424
Image Credits: Pompeu Fabra University
Keywords: Chemistry, Clinical medicine, Cancer, Diabetes, Bacteriophages, Protease
