DNA-Encoded Rapafucin Macrocycles for Proteome-Wide Binding Screens
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Solution Overview
Problem
The challenge in chemical biology is to discover new small molecule probes for uncharacterized proteins to elucidate their functions, as existing chemical libraries are not feasible for screening against human protein chips due to the lack of a universal readout for detecting ligand-protein binding, and adding artificial tags often interferes with ligand activity.
Innovation Solution
Development of a library of Rapafucin compounds, specifically DNA-encoded libraries of hybrid cyclic molecules based on FK506 and rapamycin, which retain high binding affinity for FKBP while allowing for screening against the human proteome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing chemical libraries are used for screening against human protein chips, then the screening process cannot be performed, but the lack of a universal readout for detecting ligand-protein binding prevents screening
Solution Approach 1:
The patent introduces DNA tags as intermediary markers attached to ligands. These DNA tags serve as universal readout signals that can be detected through various methods (sequencing, qPCR, etc.), enabling the detection of ligand-protein binding events across different protein targets on the chip without requiring target-specific detection reagents.
Solution Approach 2:
The patent transforms the detection parameter from protein-specific signals to universal DNA-based signals. By converting the detection readout to DNA sequences that can be universally amplified and sequenced, the system achieves a standardized measurement approach that works across the entire human proteome.
2Difficulty of detecting and measuring
If artificial tags are added to ligands for detection, then a universal readout is provided, but the tags interfere with ligand activity
Solution Approach 1:
The patent divides the ligand system into two separate functional components: the small molecule ligand that binds to the protein target, and the DNA tag that serves as the detection signal. This segmentation allows each component to perform its specific function without interfering with the other, as the DNA tag is attached via a linker that maintains ligand binding capability.
Solution Approach 2:
The DNA tag acts as an intermediary detection element that does not directly participate in ligand-protein binding. It is attached to the ligand through a spacer or linker that maintains the ligand's binding activity while providing a separate, non-interfering detection handle for universal readout.
3Adaptability or versatility
If a comprehensive chemical library is screened against the entire human proteome, then complete protein function elucidation is possible, but the complexity and resource requirements become prohibitive
Solution Approach 1:
The patent creates a universal detection system using DNA tags that can read out binding events for any protein on the chip. This single detection approach replaces the need for multiple protein-specific detection reagents, simplifying the overall system while maintaining the ability to screen against the entire human proteome.
Solution Approach 2:
The patent uses DNA sequence copying and amplification (through sequencing or qPCR) to generate detectable signals from ligand-protein binding events. This copying approach allows for signal amplification and detection without requiring complex direct detection methods for each protein target.
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 efficient screening of chemical libraries against the human proteome by maintaining ligand activity and providing a universal readout for binding detection, facilitating the elucidation of protein functions.
Implementation Method 1
They act by recruiting an abundant and ubiquitously expressed cellular protein, the prolyl cis-trans isomerase FKBP, and the binary complexes subsequently bind to and allosterically inhibit their target proteins
Data Source
AI summary
The present disclosure provides macrocyclic compounds inspired by the immunophilin ligand family of natural products FK506 and rapamycin. The generation of a Rapafucin library of macrocycles that contain FK506 and rapamycin binding domains should have great potential as new leads for developing drugs to be used for treating diseases.