ORFeome Library Screening for Proximity Effector Identification
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Solution Overview
Problem
Current methods for targeted protein degradation using PROTACs and molecular glues are limited by the reliance on a small number of known E3 ligases, leading to unpredictable efficacy and a restricted range of targetable proteins.
Innovation Solution
A method is developed to identify proximity effector polypeptides by transducing an ORFeome library into cells, expressing the encoded ORFs fused to a targeting moiety, and measuring the abundance or subcellular localization of a target polypeptide to determine if the ORF acts as a proximity effector.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If only a limited number of known E3 ligases (Cereblon and VHL) are used for targeted protein degradation, then the current PROTAC and molecular glue approaches can be implemented with existing knowledge, but the applicability is restricted and predictability of efficacy is poor
Solution Approach 1:
The patent changes the parameter of E3 ligase diversity from a limited set (Cereblon, VHL) to a comprehensive screen of hundreds of potential E3 ligases. By systematically varying which E3 ligase is paired with the target protein in the screening assay, the method identifies novel E3 ligase-target combinations that exhibit reliable and predictable degradation efficacy, thus resolving the contradiction between reliability and versatility.
Solution Approach 2:
The patent creates a comprehensive library of potential E3 ligase-target protein pairs by copying and testing multiple combinations in a standardized screening assay. This systematic copying of different E3 ligase-target interactions allows identification of the most effective pairs, expanding the toolkit while maintaining predictability through standardized assessment criteria.
2Adaptability or versatility
If an ORFeome library is transduced into cells and expressed to identify proximity effectors, then novel proximity effectors can be identified, but the complexity of the screening process increases
Solution Approach 1:
The patent segments the complex task of identifying proximity effectors into manageable components: (1) transduction of ORFeome library into cells, (2) expression of ORFs fused to targeting moieties, (3) measurement of target polypeptide abundance or localization, and (4) determination of effector activity. This segmentation allows systematic identification of novel effectors while managing complexity through structured approach.
Solution Approach 2:
The patent uses fluorescent polypeptides as intermediaries to facilitate detection of target polypeptide abundance and subcellular localization. The fluorescent tags serve as mediators between the proximity effector proteins and the detection system, simplifying the measurement process and enabling high-throughput screening without significantly increasing overall complexity.
3Measurement precision
If target polypeptide abundance is measured to determine proximity effector activity, then selective degradation or stabilization can be identified, but the measurement and analysis requirements increase
Solution Approach 1:
The patent employs fluorescent polypeptides that produce detectable color/fluorescence changes to quantify target polypeptide abundance and subcellular localization. By using fluorescent tags that emit distinct signals, the method achieves precise measurement of protein abundance and positioning, enabling reliable identification of proximity effectors while managing detection complexity through standardized fluorescent readouts.
Data Source
AI summary
Disclosed herein is a method of identifying a proximity effector polypeptide, the method comprising: transducing an ORFeome library into a plurality of cells, the ORFeome library encoding a plurality of ORFs, wherein each of the ORFs is fused to a targeting moiety that binds or can be induced to bind to the target polypeptide directly or indirectly; expressing the plurality of ORFs of the ORFeome library in the transduced plurality of cells, under conditions for the targeting moiety to interact with the target polypeptide; and determining whether any of the plurality of ORFs is a proximity effector polypeptide by measuring abundance, wherein an ORF encodes a proximity effector polypeptide when the ORF increases or decreases the target polypeptide abundance compared to control or is depleted or enhanced in the transduced plurality of cells compared to the control.