Constrained Peptide Library Construction Service

Positional Stabilization Positional Cyclization Phage Display Peptide Library Published Data FAQ Resources

Creative Biolabs developed a constrained peptide library construction technology, which offers constrained peptides with target-adapted cross-links. Our technology involves generating natural or semi-synthetic peptides with constrained structures to retain and improve the properties (e.g. stability, resistance to protease activity, protein binding affinity, intrinsic cell permeability, etc.) of the peptides. Our platform has been optimized over the years to include Positional Stabilization Method, Positional Cyclization Method and Phage Display Peptide Library Method.

Positional Stabilization Method

Constrained peptide positional stabilization. (Creative Biolabs Original) Fig. 1 Schematic diagram of constrained peptide position stabilization.

Creative Biolabs has developed a broadly applicable technology for “freezing” the 3D-structure of short peptides (20-30 amino acids) using a small rigid entity that carries 2~4 anchor points as a scaffold. The peptide slowly adopts a well-defined three-dimensional structure around the scaffold and loses flexibility with the scaffold entity in the center, forming not limited to single loop peptide without fully aqueous conditions at neutral pH. This platform technology not only rigidifies the structure of the peptide but also improves its binding activity and proteolytic stability.

Positional Cyclization Method

Positional Cyclization Method is another new peptide conformation stabilizing technology. This technique involves synthesis of peptides with constrained structures in the form of lactam bridge or disulfide bond and stapling at various positions to fix the overall structure conformation of the protein. This technology not only offers peptides with desirable pharmacokinetic properties but also retains the sequence flexibility that permits the introduction of new and diverse functionalities.

Phage Display Peptide Library

Phage display constrained peptide library. (Creative Biolabs Original) Fig. 2 Schematic diagram of phage display restricted peptide library.

We have nature monomeric proteins as scaffolds displayed on the surface of filamentous bacteriophage M13 with one or more loops of the scaffold framework randomized using PCR mutagenesis as a constrained random peptide library. We have also constructed random nonapeptide libraries in the N-terminal region of the coat protein of bacteriophage with lactam bridge or disulfide bond flanking the insert. All these constrained peptide libraries represent structural diversity and biochemical stability intended for therapeutic use.

We are professional in providing constrained peptides of extremely high diversity and specificity. Our drug-like peptides are highly stable and of chemical structures that offer extremely high intrinsic cell permeability.

Published Data

IL-7Rα-targeted Peptides Identification Using A Disulfide-constrained Combinatorial Phage Display Library

Fig. 3 Apparent dissociation constant (K d) values of the synthesized selected peptides and of IL-7 were determined for their binding to IL-7 receptor (IL-7R) and fibronectin (FN).¹

The research investigates the use of a disulfide-constrained combinatorial phage display library to identify peptides targeting IL-7Rα, a biomarker relevant for rheumatoid arthritis (RA) diagnosis and treatment. The study successfully identified two IL-7Rα-specific heptapeptides, P258 and P725. P258, optimized for molecular imaging, was conjugated with ultra-small superparamagnetic particles of iron oxide (USPIO) and demonstrated effective targeting of IL-7Rα in vivo, enabling early detection of RA through MRI. P725, showing potential as a blocking agent, inhibited IL-7Rα interactions, suggesting its therapeutic relevance. The constrained peptide library was crucial in this study, offering a diverse yet focused repertoire of peptides that facilitated the rapid identification of highly specific and functional peptides for both diagnostic imaging and potential therapeutic interventions, thus underscoring its importance in precision medicine for autoimmune diseases.

Reference

Burtea, Carmen, et al. "Screening for peptides targeted to IL-7Rα for molecular imaging of rheumatoid arthritis synovium." Arthritis research & therapy 18 (2016): 1-19. Distributed under Open Access license CC BY 4.0, without modification.

FAQ

What is a constrained peptide library, and how does it differ from other peptide libraries?
A constrained peptide library is a collection of peptides that are engineered to maintain a specific structure, typically through the introduction of disulfide bonds or other chemical modifications. This constraint limits the flexibility of the peptides, allowing them to adopt and maintain a defined conformation. This structural rigidity can enhance the binding affinity and specificity of peptides for their target molecules, making constrained peptide libraries particularly useful in drug discovery and biomolecular research. Unlike linear peptide libraries, constrained libraries often result in higher stability and reduced off-target interactions.
What are the advantages of using a constrained peptide library in drug discovery?
Constrained peptide libraries offer several advantages in drug discovery, including enhanced stability, increased binding affinity, and improved specificity for target proteins. The structural rigidity of constrained peptides often mimics the natural conformation of protein-protein interactions, making them ideal candidates for developing inhibitors or modulators of these interactions. Additionally, constrained peptides are often more resistant to proteolytic degradation, which is crucial for in vivo applications. These properties make constrained peptide libraries a powerful tool for identifying lead compounds in therapeutic development.
How is a constrained peptide library constructed?
Construction of a constrained peptide library involves synthesizing peptides with specific structural constraints, such as disulfide bonds between cysteine residues, cyclization, or incorporation of non-natural amino acids. These constraints are designed to stabilize the peptide's conformation, enhancing its ability to interact with specific targets. The process typically includes the design of a diverse set of peptide sequences, followed by chemical synthesis and verification of the peptide structures. The resulting library is then screened against a target of interest to identify peptides with desirable binding properties.
What are the key applications of constrained peptide libraries in biomedical research?
Constrained peptide libraries are used in various biomedical research applications, including the identification of peptide ligands for receptors, enzymes, and other proteins; the development of peptide-based drugs; and the study of protein-protein interactions. These libraries are particularly valuable in cancer research, where they can be used to discover peptides that inhibit oncogenic pathways, and in infectious disease research, where they can help identify peptides that block viral entry into host cells. Additionally, constrained peptides are useful in diagnostic imaging and targeted drug delivery.
What are the challenges associated with constrained peptide library construction?
One of the primary challenges in constructing constrained peptide libraries is ensuring that the constraints do not overly restrict the peptide's flexibility, which could limit its ability to bind to the target. Additionally, synthesizing peptides with specific constraints, such as disulfide bonds or cyclization, can be technically challenging and time-consuming. Ensuring the structural integrity and diversity of the library while maintaining the desired constraint is another challenge, as this balance is crucial for successful screening and identification of functional peptides.
How is the diversity of a constrained peptide library ensured?
The diversity of a constrained peptide library is ensured by incorporating a wide range of amino acid sequences within the constrained framework. This diversity is achieved by varying the sequence of amino acids while maintaining the structural constraints, such as disulfide bonds or cyclization. Additionally, the use of randomization techniques during synthesis allows for the generation of a large number of unique peptide sequences, increasing the likelihood of identifying peptides with high affinity and specificity for the target molecule.
What role do disulfide bonds play in constrained peptide libraries?
Disulfide bonds help stabilize the peptide's three-dimensional structure. These covalent bonds form between cysteine residues within the peptide, creating loops or cycles that restrict the peptide's conformational flexibility. This structural stability enhances the peptide's ability to maintain a consistent shape, which is often critical for high-affinity binding to target proteins. The introduction of disulfide bonds is a common strategy in constrained peptide library construction to achieve desired structural and functional properties.
How are constrained peptide libraries screened for target binding?
Screening constrained peptide libraries for target binding typically involves techniques such as phage display, yeast display, or in vitro selection methods like SELEX (Systematic Evolution of Ligands by Exponential Enrichment). In these techniques, the library is exposed to the target protein, and peptides that exhibit strong binding are isolated and enriched through iterative rounds of selection. The binding affinity and specificity of the selected peptides are then further characterized using biochemical assays, such as surface plasmon resonance (SPR) or enzyme-linked immunosorbent assays (ELISA).
Can constrained peptide libraries be used for developing therapeutic antibodies?
Constrained peptide libraries can be used to develop therapeutic antibodies by serving as templates for generating peptide mimics that bind to specific epitopes on target proteins. These peptide mimics can then be used to generate monoclonal antibodies with high specificity and affinity for the target. The constrained nature of the peptides helps ensure that the mimicked epitopes closely resemble the natural structure of the target protein, increasing the likelihood of generating effective antibodies. This approach is particularly valuable in cases where traditional antibody generation methods may be challenging.
What innovations are being made in the field of constrained peptide library construction?
Innovations in constrained peptide library construction include the incorporation of non-natural amino acids, the development of new chemical linkers for cyclization, and the use of advanced screening techniques like high-throughput sequencing. These innovations aim to increase the diversity, stability, and functional potential of constrained peptide libraries. Additionally, computational approaches are being employed to design peptides with optimized constraints and predicted binding properties, further enhancing the efficiency of library construction and screening processes. These advancements are driving the development of more effective peptide-based therapeutics and diagnostic tools.