Membrane-Permeable Construct for In Vivo Enzyme Capture
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for studying enzyme-substrate interactions are limited by their ability to only identify these interactions in vitro, lacking the capability to detect them in vivo, which is crucial for understanding and manipulating cellular processes and developing therapeutic tools.
Innovation Solution
A membrane-permeable construct is developed, comprising a substrate moiety with a photoreactive moiety and a cell-penetrating peptide, allowing for the identification of enzymes that interact with a target substrate in vivo by cross-linking with intracellular enzymes and facilitating their isolation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard biochemical methods (coimmunoprecipitation, pull-down, chemical crosslinking) are used to study enzyme-substrate interactions, then binding interactions can be identified in vitro, but the ability to detect interactions in vivo is lost
Solution Approach 1:
The patent introduces a membrane-permeable construct as an intermediary that bridges the gap between in vitro detection methods and in vivo detection needs. This construct comprises a substrate moiety with a photoreactive moiety attached to a cell-penetrating peptide, allowing it to enter cells and detect enzyme-substrate interactions in vivo while maintaining the detection capabilities of traditional methods
Solution Approach 2:
The invention creates a composite construct by combining three distinct components: a substrate moiety (mimicking natural enzyme substrates), a photoreactive moiety (enabling cross-linking and detection), and a cell-penetrating peptide (facilitating cellular entry). This composite structure integrates the functional advantages of each component to achieve in vivo detection capability
2Adaptability or versatility
If a membrane-permeable construct with photoreactive moiety is used for in vivo enzyme capture, then in vivo identification of enzymes is enabled, but the construct complexity increases
Solution Approach 1:
The construct is divided into distinct functional segments: a substrate moiety that interacts with the target enzyme, a photoreactive moiety that enables cross-linking upon light activation, and a cell-penetrating peptide that facilitates cellular entry. This segmentation allows each component to perform its specific function while keeping the overall design modular and manageable
Solution Approach 2:
Different regions of the construct possess specialized properties tailored to their functions: the substrate moiety has biochemical compatibility with target enzymes, the photoreactive moiety has light-induced reactivity, and the cell-penetrating peptide has membrane-translocation capability. This local optimization of properties enables the construct to achieve multiple functions simultaneously
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
Enables the in vivo identification of enzymes interacting with specific substrates, providing a detailed understanding of enzyme-substrate interactions and enabling the development of therapeutic tools for manipulating cellular processes.
Implementation Method 1
the substrate moiety includes at least one photoreactive moiety
Implementation Method 2
a peptide moiety, comprising R1-CPP-R2, wherein CPP is a cell-penetrating peptide
Data Source
AI summary
The invention includes compositions, methods and kits for the in vivo identification of an enzyme that binds to a substrate. The invention comprises, in part, a photoreactive moiety to aid in identification of such an enzyme.