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EP-4737478-A1 - MODIFIED G PROTEIN COUPLED RECEPTOR AND USE THEREOF

EP4737478A1EP 4737478 A1EP4737478 A1EP 4737478A1EP-4737478-A1

Abstract

The present application belongs to the technical field of protein engineering, and particularly relates to a modified G protein coupled receptor, a complex comprising the modified G protein coupled receptor, and use thereof. The present application provides a modified G protein coupled receptor which successively comprises, from an N-terminus to a C-terminus, the N terminus, a first transmembrane domain, a first intracellular loop, a second transmembrane domain, a first extracellular loop, a third transmembrane domain, a second intracellular loop, a fourth transmembrane domain, a second extracellular loop, a fifth transmembrane domain, a third intracellular loop, a sixth transmembrane domain, a third extracellular loop, a seventh transmembrane domain and the C terminus, wherein at least a part of the first intracellular loop, the second intracellular loop and/or the third intracellular loop is replaced with an optional first linker, a first part of a fluorescent protein and an optional second linker, wherein the first linker and the second linker each independently comprise one or more amino acids.

Inventors

  • LI, MINGHUI

Assignees

  • Alphelix Biotech Co., Ltd.

Dates

Publication Date
20260506
Application Date
20240626

Claims (15)

  1. A modified G protein coupled receptor, the modified G protein coupled receptor successively comprising, from the N terminus to the C terminus, the N terminus, a first transmembrane domain, a first intracellular loop, a second transmembrane domain, a first extracellular loop, a third transmembrane domain, a second intracellular loop, a fourth transmembrane domain, a second extracellular loop, a fifth transmembrane domain, a third intracellular loop, a sixth transmembrane domain, a third extracellular loop, a seventh transmembrane domain and the C terminus, wherein at least a part of the first intracellular loop, the second intracellular loop and/or the third intracellular loop is replaced with an optional first linker, a first part of a fluorescent protein and an optional second linker, wherein the first linker and the second linker each independently comprise one or more amino acids.
  2. The modified G protein coupled receptor according to claim 1, wherein the fluorescent protein comprises one or more of a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein and an enhanced green fluorescent protein.
  3. The modified G protein coupled receptor according to claim 1 or 2, wherein the first part of the fluorescent protein comprises β-strands 10-11, β-strands 1-2, β-strands 8-11 or β-strands 1-4 of the fluorescent protein.
  4. The modified G protein coupled receptor according to any one of claims 1-3, wherein the first part of the fluorescent protein comprises an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and at least 99.5% sequence identity to the amino acid sequence as shown in SEQ ID NO.1.
  5. The modified G protein coupled receptor according to any one of claims 1-4, wherein an amino acid at the position 34.51 of a Ballesteros-Weinstein numbering system in the second intracellular loop is cysteine.
  6. The modified G protein coupled receptor according to any one of claims 1-5, wherein the modified G protein coupled receptor comprises an amino acid sequence as shown in any one of SEQ ID NOs. 3-71 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and at least 99.5% sequence identity to the amino acid sequence as shown in any one of SEQ ID NOs. 3-71.
  7. A complex, the complex comprising the modified G protein coupled receptor according to any one of claims 1-6 and a second part of a fluorescent protein, wherein the first part of the fluorescent protein binds to the second part of the fluorescent protein.
  8. The complex according to claim 7, wherein the second part of the fluorescent protein comprises β-strands 1-9, β-strands 3-11, β-strands 1-7 or β-strands 5-11 of the fluorescent protein.
  9. The complex according to claim 7 or 8, wherein the second part of the fluorescent protein comprises an amino acid sequence as shown in SEQ ID NO. 2 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and at least 99.5% sequence identity to the amino acid sequence as shown in SEQ ID NO. 2.
  10. The complex according to any one of claims 7-9, wherein the complex further comprises a clamp protein which binds to the first part of the fluorescent protein and the second part of the fluorescent protein.
  11. The complex according to claim 10, wherein the clamp protein comprises an amino acid sequence as shown in any one of SEQ ID NOs. 72-74 and SEQ ID NO. 82 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and at least 99.5% sequence identity to the amino acid sequence as shown in any one of SEQ ID NOs. 72-74 and SEQ ID NO. 82.
  12. The complex according to claim 11, wherein cysteine is located at the position 186 of the clamp protein, and the position refers to SEQ ID NO. 72.
  13. The complex according to claim 11 or 12, wherein a disulfide bond is formed between the amino acid at the position 186 of the clamp protein and the amino acid at the position 34.51 of the Ballesteros-Weinstein numbering system in the second intracellular loop.
  14. Use of the modified G protein coupled receptor according to any one of claims 1-6 or the complex according to any one of claims 7-13 in ligand affinity determination and drug discovery or screening.
  15. Use of the complex according to any one of claims 7-13 in analysis of a 3D structure via cryogenic electron microscopy.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims the priority of application Chinese patent CN2023107655060 filed on June 27, 2023 and entitled "modification method of G protein receptor and use", the disclosure of which is incorporated by reference herein in its entirety. TECHNICAL FIELD The present application belongs to the technical field of protein engineering, and particularly relates to a modified G protein coupled receptor, a complex comprising the modified G protein coupled receptor, and use thereof. BACKGROUND A majority of drug molecules exert effects by binding to corresponding target proteins in vivo. They activate or inhibit the function of a target protein, and change the physiological process of a human body, thereby achieving the purpose of disease treatment. A G protein coupled receptor (GPCR) is a type of target proteins having an important drug development and research value in the human body. About 40% of the drugs currently existing in the market are based on the G protein coupled receptor as a target. Many drugs using these proteins as targets are still being developed. A large number of GPCRs exist in the human body, more than 800 of which have been found. These GPCRs share certain commonalities in their spatial structures: they all comprise 7 transmembrane helices; the N-terminus and 3 interhelical loop structures are located at the outer side of the cell, and the C-terminus and the other 3 interhelical loop structures are located at the inner side of the cell. These proteins are located on a cell membrane, and as receptors of signal molecules, transmit signals to the interior of the cell. A foreign signal molecule binds to a part of GPCR located at the outer side of the cell, causing the conformational change of the transmembrane helices of the receptor so that the intracellular part of the receptor binds to the G protein and triggers the physiological effect in the cell. GPCR is involved in a wide variety of physiological processes and plays a crucial role in maintaining normal life activities in the human body. The drug molecules can exert agonistic or inhibitory effects on GPCR by binding to GPCR, thereby regulating the physiological processes inside the cell. Therefore, in theory, GPCR can be widely used in the discovery and screening of the drugs. However, many GPCRs cannot stably exist in the ligand-free state, thereby restricting their uses in the discovery, screening and other aspects of the drugs. DNA encoding compound library screening rapidly developed in recent years and identifying compounds binding to specific target proteins from compound mixtures utilizing a mass spectrometric technique propose extremely high requirements on screened target proteins, including: good stability and similar qualities of binding to natural wild type protein ligands, especially unoccupied ligand binding sites. For the discovery of therapeutic antibody drugs, regardless of a hybridoma technology, a single B cell technology or various display technologies, compared with cells that overexpress target proteins, the purified target proteins with the aforementioned characteristics have significant advantages, greatly accelerate the research and development process, and avoid the interference of other background proteins. In another aspect, accurately determining the affinity between target GPCR and a compound or an antibody by utilizing these stable purified proteins in combination with biological and physical technologies such as surface plasmon resonance (SPR) is extremely conducive to optimization and improvement of the drugs. In another aspect, analyzing the structures of complexes formed by drug molecules or potential drug molecules and their target GPCR proteins is of great guiding significance for studying their mechanism of action and further research and development of drugs. At present, the cryogenic electron microscopy has been widely applied in structural analysis of GPCR, thereby yielding a large number of GPCR structures. However, the most of these structures are composite structures formed by GPCRs binding to agonists and coupled with G proteins, and such complexes can meet the requirements of the cryogenic electron microscopy on the sizes of target protein molecules. When GPCRs are in a non-agonistic state due to binding to antagonists and inverse agonists or negative modulators, they together with G proteins do not form stable complexes. The molecular weight of a single GPCR protein is in a range of 30-60 kD in most cases, and therefore its structure is difficultly analyzed via cryogenic electron microscopy. Hence, there is an urgent need in the art to improve the stability of the G protein coupled receptor in the ligand-free state and address the issue of analyzing the structure of GPCR in the inactive state via cryogenic electron microscopy. SUMMARY In order to solve the above problems, the present application provides a modified G protein coupled receptor, a c