Azoline Compound Library Construction via Cell-Free Translation
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Solution Overview
Problem
Current methods for constructing peptide libraries, such as chemical synthesis, biosynthetic enzyme methods, and translation systems, face challenges in diversity, efficiency, and stability when screening for compounds binding to target substances, especially those with protease activity, due to limitations in enzyme specificity and substrate range.
Innovation Solution
A method is developed to create an azoline compound library by expressing a PatE library in a cell-free translation system using a precursor peptide with a leader sequence and recognition sequences for azoline backbone-introducing enzymes, allowing for the introduction of azoline backbones into Cys, Ser, Thr, and 2,3-diamino acids, and subsequent conversion to azole backbones, enabling a more diverse and stable library for screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chemical synthesis method is used to construct peptide library, then manufacturing precision can be achieved, but diversity of the library cannot be increased and time consumption increases
Solution Approach 1:
The precursor peptide is divided into functional segments: leader sequence for enzyme recognition, cassette domain for diversity generation, and recognition sequences for modification. This segmentation allows each part to serve its specific function while maintaining overall library diversity and precision
Solution Approach 2:
A cell-free translation system serves as an intermediary between genetic information and peptide synthesis. This system enables rapid peptide production with precise backbone construction while maintaining library diversity through in vitro modification enzymes
2Productivity
If biosynthetic enzyme method is used, then rapid construction and precise backbone can be achieved, but kinds of synthesizable compounds are limited due to enzyme substrate specificity
Solution Approach 1:
The azoline backbone-introducing enzyme is engineered to have broad substrate specificity, recognizing multiple recognition sequences and modifying various amino acid residues (Cys, Ser, Thr, 2,3-diamino acid). This multi-functionality allows a single enzyme to generate diverse compound types while maintaining rapid construction speed
3Adaptability or versatility
If translation system is used to construct peptide library, then diversity and construction speed can be improved, but only peptidic backbone compounds can be produced
Solution Approach 1:
The chemical structure of the backbone is changed from peptidic to azoline/azole through enzymatic modification. This parameter change transforms the compound's chemical properties, making it resistant to protease cleavage while preserving the diversity and rapid construction advantages of the translation system
4Manufacturing precision
If post-translational modification is performed in vitro, then desired structure can be obtained, but enzyme activity may be lost and purification is required
Solution Approach 1:
The translation system and post-translational modification steps are merged into a single in vitro reaction process. The precursor peptide is translated and modified in the same system without purification between steps, reducing process complexity while maintaining structural precision through the use of engineered enzymes with broad specificity
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 results in a highly diverse azoline and azole compound library that can effectively bind to target substances with protease activity, facilitating efficient screening and analysis of structure-activity relationships.
Implementation Method 1
a translation synthesis system
Implementation Method 2
reacting the azoline backbone-introducing enzyme and the peptide library in the presence of a peptide comprising a leader sequence of a substrate of the azoline backbone-introducing enzyme and thereby introducing an azoline backbone into at least one of Cys, Ser, Thr, and 2,3-diamino acid
Implementation Method 3
a step of reacting the library having an azoline backbone introduced therein with an azole backbone-introducing enzyme in the presence or absence of a peptide comprising a leader sequence of a substrate of the azole backbone-introducing enzyme and thereby converting at least one of the azoline backbones into an azole backbone
Data Source
AI summary
An object of the present invention is to provide a method of efficiently constructing a library abundant in diversity and also usable for screening of a compound that binds to a target substance having protease activity.The present invention provides a method of constructing an azoline compound library containing two or more azoline compounds having an azoline backbone introduced into at least one of Cys, Ser, Thr, and 2,3-diamino acid, and analogs thereof of Xaa0 of a peptide represented by the following formula (I):A-(Xaa0)n-B  (I)[wherein, m numbers of Xaa0s respectively represent arbitrary amino acids, at least one of which is an amino acid selected from the group consisting of Cys, Ser, Thr, and 2,3-diamino acid, and analogs thereof, m represents an inter selected from 2 to 40, and A and B each independently represent a peptide composed of from 0 to 100 amino acids].