Isopeptidase Activity Probe for High Throughput Screening
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Solution Overview
Problem
Current methods for monitoring deubiquitylating enzyme activity in biological samples are low throughput and labor-intensive, limiting their ability to effectively measure isopeptidase activity in cells or tissues and hindering the development of drugs targeting these enzymes.
Innovation Solution
A high throughput assay using an activity probe with a warhead and Ubiquitin or Ubiquitin-like molecule, combined with reagents that generate a detectable signal when bound to isopeptidases, allows for the determination of isopeptidase activity in biological samples, enabling the assessment of target engagement, pharmacodynamic activities, and diagnostic/prognostic applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current methods for monitoring deubiquitylating enzyme activity are used, then measurement capability is achieved, but throughput is low and labor intensity is high
Solution Approach 1:
The patent employs fluorescent probes that undergo detectable signal changes upon binding to deubiquitylating enzymes. The fluorescent signal intensity or pattern changes provide a direct readout of enzyme activity, enabling high-throughput automated measurement without manual intervention, thus resolving the contradiction between throughput and labor intensity
Solution Approach 2:
The patent replaces manual, mechanical assay procedures with an automated fluorescent detection system. The fluorescent probes and detection apparatus enable automated high-throughput screening, eliminating the need for labor-intensive manual operations while maintaining measurement capability
2Measurement precision
If current methods are used, then enzyme activity measurement is possible, but the process is labor-intensive
Solution Approach 1:
The patent replaces manual measurement procedures with an automated fluorescent detection system. The fluorescent probes provide objective, quantifiable signals that can be automatically read and analyzed, maintaining measurement precision while eliminating manual labor requirements
Solution Approach 2:
The patent introduces fluorescent probes as intermediary molecules that mediate between the deubiquitylating enzymes and the detection system. These probes bind specifically to the enzymes and convert enzymatic activity into fluorescent signals, enabling automated, precise measurement without direct manual intervention
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 enables rapid, efficient measurement of isopeptidase activity in biological samples, providing valuable insights into enzyme function and potential inhibitor effects, and can be used for diagnostic and prognostic purposes, facilitating drug development targeting deubiquitylating enzymes.
Implementation Method 1
contacting the extract with an activity probe wherein the activity probe comprises a warhead and a Ubiquitin or a Ubiquitin-like molecule
Implementation Method 2
said reagents comprise a fluorescence donor and a fluorescence acceptor; and wherein the detectable signal is generated by the fluorescence acceptor/donor pair
Data Source
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Figure 5A~5B
AI summary
The present invention relates to materials and methods for high throughput monitoring of target engagement of isopeptidases, such as deubiquitylating enzymes by, inter alia, small molecule inhibitors. In particular the invention relates to development of high throughput assays to measure isopeptidase activity in biological samples, such as cells, animal tissues, animal tumours, human tissue or patient-derived biopsies.