Constrained Polypeptide Binders via Multi-Point Covalent Linking
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Solution Overview
Problem
Existing methods for generating small molecule binders with high affinity and specificity are limited, particularly for biological targets, as they often rely on large biopolymer structures and struggle with conformational flexibility and proteolytic degradation, and current strategies for attaching connector compounds to polypeptides are labor-intensive and not suitable for genetically encoded phage libraries.
Innovation Solution
The development of a method that links a connector compound, such as tris-(bromomethyl)benzene, to a polypeptide via at least three covalent bonds, creating conformationally constrained polypeptide loops that can interact with targets, thereby enhancing binding affinity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If biological in vitro selection techniques are used to isolate binding ligands, then large biopolymeric structures such as antibodies can be isolated, but the method is not practicable for isolating small molecule drugs and suffers from conformational flexibility and proteolytic degradation
Solution Approach 1:
The invention divides the polypeptide into multiple segments by introducing cysteine residues at specific positions, allowing each segment to be independently constrained by connector compounds. This segmentation enables the creation of structurally defined small molecule-like binders while maintaining the genetic encodability and selection capabilities of biological systems.
Solution Approach 2:
The invention creates composite structures by combining genetically encoded polypeptide segments with chemically synthesized connector compounds (such as tris-(bromomethyl)benzene). This composite approach integrates the advantages of both biological molecules (specificity, encodability) and chemical molecules (structural constraint, stability).
2Reliability
If connector compounds are attached to polypeptides using existing methods, then binding affinity can be improved, but the process is labor-intensive and not suitable for genetically encoded phage libraries
Solution Approach 1:
The invention enables self-service by incorporating reactive cysteine residues directly into the genetically encoded polypeptide sequence. The polypeptide itself provides the necessary functional groups (thiol groups of cysteine) that react with connector compounds, eliminating the need for separate modification steps and enabling automated library generation.
Solution Approach 2:
The invention changes the chemical parameters of the polypeptide by introducing cysteine residues at specific positions, which provides reactive thiol groups. This parameter change enables efficient conjugation with connector compounds under mild conditions, making the process suitable for high-throughput library generation from genetically encoded phage libraries.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for the creation of polypeptides with multiple binding loops, increasing binding affinity and specificity, and enables the generation of large libraries of constrained peptides that can bind to targets through multiple interactions, overcoming limitations of previous methods.
Implementation Method 1
a connector compound attached to said polypeptide, wherein said connector compound is attached to said polypeptide by at least three covalent bonds
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3C
AI summary
The invention relates to a complex comprising a phage particle, said phage particle comprising (i) a polypeptide; (ii) a nucleic acid encoding the polypeptide of (i); (iii) a connector compound attached to said polypeptide wherein said connector compound is attached to the polypeptide by at least three discrete covalent bonds. The invention also relates to libraries, and to methods for making complexes and to methods of screening using same.