Method for Determining Mutateable Ligand-Gpcr Binding at Single Amino Acid Resolution and Pairs of Mutated Ligand and Gpcr

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This patent discloses a method for precisely determining how mutations in ligand molecules influence their binding to G-protein coupled receptors (GPCRs), such as rhodopsin, at single amino acid resolution. Using a microtiter plate assay, libraries of mutant ligands—especially arrestins—are systematically tested against GPCR targets. The approach enables creation of detailed functional maps of ligand-receptor binding interactions, supporting the development of mutant ligand-GPCR pairs with tailored binding strengths for research, diagnostics, and drug discovery.

Use CasesContent extracted from patent full text and abstract with AI.

• High-throughput screening and mapping of ligand-receptor interactions for GPCRs.
• Optimization of ligand (e.g., arrestin) variants for improved or reduced GPCR binding in drug discovery.
• Development of custom ligand-GPCR pairs for diagnostic assays.
• Structure-guided drug design targeting GPCR-ligand interactions.
• Silencing or enhancing GPCR signaling pathways for functional studies or therapeutic applications.
• Screening for drugs that modulate GPCR-ligand binding, such as in beta-arrestin recruitment assays.

BenefitsContent extracted from patent full text and abstract with AI.

• Allows single amino acid resolution mapping of ligand-GPCR binding interfaces, enabling precise functional insights.
• Enables rapid generation and testing of extensive mutant ligand libraries in a cost-effective, high-throughput format.
• Facilitates rational design of ligands with customized binding strengths for various applications in pharmaceuticals and diagnostics.
• Reduces experimental costs and technical barriers compared to generating many GPCR mutants.
• Provides a platform for identifying ligands or ligand-GPCR pairs with enhanced or weakened binding for modulating biological processes.
• Supports a better understanding of GPCR signaling modulation and mechanism, aiding basic research and drug development.

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Patent Abstract

The present invention provides a method for determining the binding ability of a G-protein coupled receptor, hereinafter referred to as GPCR, and a mutateable ligand, said method comprising the steps of: a) providing a well microtiter plate having the wells disposed in a array having a first number of rows and a second number of columns; b) providing a GPCR, such as rhodopsin, in each of said wells; c) providing a number of mutants of the parent ligand, wherein the parent ligand being one that binds to the GPCR when the GPCR is residing in a particular conformation; d) bringing the mutants of the parent ligand in the wells into contact with the GPCR under conditions where the parent ligand would couple to the GPCR; and e) determining for each mutant whether the mutant ligand has a weaker or stronger binding ability as compared to the standard binding ability of the parent ligand and the GPCR by determining the amount of coupled mutant-GPCR complex in said wells. In an assay, rhodopsin binding of 403 mutants covering the complete arrestin sequence has been investigated. This information provides a functional 4th dimension to the crystal structures of inactive, preactivated and active arrestins. The resulting single amino acid resolution functional maps reveal a series of critical interactions in the polar core and along the C-tail of arrestin that are interrupted during arrestin activation. Our data further reveals several patches of amino acids that strongly reduce binding and act as direct binding interfaces to rhodopsin. This information in combination with computational molecular docking of active arrestin4 and light-activated rhodopsin allows to develop a model of the arrestin-rhodopsin complex. Combination of mutants allows modification of binding affinity and stability of the GPCR-ligand complex for diagnostic purposes or pharmacological intervention.