GPCR Conformational Selectivity via Segmented Cell Sorting
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Solution Overview
Problem
Current methods fail to selectively bind to transmembrane receptors in specific conformations, limiting therapeutic applications and structural studies of these receptors.
Innovation Solution
The development of methods to produce and selectively sort cells that bind to transmembrane receptors in either active or inactive conformations using cell sorting and extracellular capture agents, along with the use of G-protein or mimetic labeling to identify conformation-specific binders.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional binding methods are used, then agents can bind to transmembrane receptors, but they cannot selectively bind to specific conformations (active or inactive states)
Solution Approach 1:
The patent segments the binding problem by separately selecting for agents that bind to active-state receptors versus inactive-state receptors. This is achieved by producing two separate libraries of capture agents - one selected against active-state receptors and another against inactive-state receptors - allowing conformation-specific binding to be achieved through segmented selection processes rather than a single unspecific binding approach
Solution Approach 2:
The patent changes the binding parameter from non-specific affinity binding to conformation-specific binding by altering the selection criteria. Active-state receptors are selected with agents that bind preferentially to the active conformation, while inactive-state receptors are selected with agents that bind preferentially to the inactive conformation. This parameter change in selection criteria enables conformational selectivity to be achieved
2Measurement precision
If cell sorting is used to select conformation-specific binders, then selective binding can be achieved, but the process complexity increases
Solution Approach 1:
The patent uses cells as intermediary carriers to display libraries of capture agents on their surfaces. This intermediary approach allows complex conformation-specific binding selections to be performed at the cell level using flow cytometry, where cells expressing specific capture agents can be sorted based on their binding characteristics. The cell serves as a mediator that simplifies the selection process by providing a platform for high-throughput screening
Solution Approach 2:
The patent replaces complex mechanical separation methods with flow cytometry-based cell sorting. Instead of using physical or chemical separation techniques that would be cumbersome for selecting conformation-specific binders, the system uses fluorescent labeling and flow cytometry to identify and sort cells based on their binding properties, significantly simplifying the selection process
3Measurement precision
If a library of capture agents is screened, then conformation-specific binders can be identified, but the time required for screening increases
Solution Approach 1:
The patent performs preliminary actions by pre-producing and pre-characterizing the active and inactive state receptors before beginning the selection process. The receptors are produced in controlled conditions, stabilized in their respective conformations, and ready for immediate use in selection. This preliminary preparation eliminates time-consuming steps during the actual screening process
Solution Approach 2:
The patent maintains continuous useful action by establishing a streamlined workflow where cells displaying capture agent libraries are continuously exposed to receptors, bound, sorted, and re-stimulated in an uninterrupted sequence. The process flows continuously from binding through sorting to functional testing without idle periods, maximizing the efficiency of the screening process
Data Source
AI summary
Provided herein are several methods for selecting agents that bind to transmembrane receptors in a conformationally-selective way. In some embodiments, the method may comprise producing: a transmembrane receptor in an active conformation; and said transmembrane receptor in an inactive conformation and using cell sorting to select, from a population of cells comprising a library of cell surface-tethered extracellular capture agents, cells that are specifically bound to either the transmembrane receptor in its active conformation or the transmembrane receptor in its inactive conformation, but not both. In other embodiments, the method may comprise: contacting a GPCR with a population of cells that comprise a library of surface-tethered extracellular proteins; labeling the cell population with a conformationally-specific binding agent, e.g., a G-protein or mimetic thereof; and using cell sorting to select from the cell population cells that bind to the agent.