Heptares Therapeutics Limited

Medical Research Council

United Kingdom Research and Innovation

Nxera Pharma UK Limited

Christopher Gordon Tate

Malcolm Peter Weir

Richard Henderson

Antony Johannes Warne

Yoko Shibata

Maria Josefa Serrano-Vega

Francesca Magnani

Byron Carpenter

Andrew Leslie

Rony Nehmé

Fiona Hamilton Marshall

Seyed Ali Jazayeri-Dezfuly

Jonathan Richard Heal

Edward Christopher Hulme

Guillaume Pierre Lebon

Nathan Robertson

Anthony Johannes Warne

Antony Warne

The invention relates to mutant G-protein coupled receptors with increased conformational stability, and methods of use thereof. In some aspects, polynucleotides encoding the mutant G-protein coupled receptors are provided. In some aspects, host cells comprising the polynucleotides are provided. In some aspects, the invention relates to crystallized forms of the mutant G-protein coupled receptors, and methods of preparing the same.

Mutant G-protein coupled receptors and methods for selecting them

The invention provides a mutant of a parent heterotrimeric G protein alpha (G&#x3b1;) subunit, which mutant (i) lacks at least one helix of the helical domain of the parent G&#x3b1; subunit; (ii) is capable of binding to a GPCR in the absence of a heterotrimeric G protein beta (G&#x3b2;) subunit and a heterotrimeric G protein gamma (G&#x3b3;) subunit; and (iii) has an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the parent heterotrimeric G&#x3b1; subunit, which mutations are selected from a deletion, a substitution and an insertion.

G proteins

The present invention relates to mutant transmembrane proteins which have increased conformational stability when compared to their parent protein, methods of selection and production. In particular the invention relates to mutant transmembrane proteins which are mutated in or in the proximity of the transmembrane alpha helices or in a kinked region or in an alpha-helix adjacent to a kink. The mutant transmembrane proteins have use in crystallisation studies and also in screening to identify compounds for use in drug discovery and therapy.

Mutant proteins and methods for their production

A method of producing a conformational specific binding partner of a GPCR, the method comprising: a) providing a mutant GPCR of a parent GPCR, wherein the mutant GPCR has increased stability in a particular conformation relative to the parent GPCR; b) providing a test compound; c) determining whether the test compound binds to the mutant GPCR when residing in a particular conformation; and d) isolating a test compound that binds to the mutant GPCR when residing in the particular formation. Methods of producing GPCRs with increased stability relative to a parent GPCR are also disclosed.

Methods for screening for binding partners of G-protein coupled receptors

The present invention relates to mutant transmembrane proteins which have increased conformational stability when compared to their parent protein, methods of selection and production. In particular the invention relates to mutant transmembrane proteins which are mutated in or in the proximity of the transmembrane alpha helices or in a kinked region or in an alpha-helix adjacent to a kink. The mutant transmembrane proteins have use in crystallization studies and also in screening to identify compounds for use in drug discovery and therapy.

A method for selecting a membrane protein with increased stability, the method comprising: a) providing one or more mutants of a parent membrane protein in a membrane-containing composition, wherein the one or more mutants are exposed to an amount of a membrane destabilizing agent which is effective to destabilize the parent membrane protein in-situ, b) determining whether the or each mutant membrane protein has increased stability with respect to its structure and/or a biological activity compared to the stability of the parent membrane protein with respect to its structure and/or the same biological activity, and c) selecting the one or more mutants which have increased stability compared to the stability of the parent membrane protein.

Mutant proteins and methods for selecting them

The present invention relates in some aspects to mutant G protein coupled receptors (GPCRs) and methods for selecting those with increased stability. In certain aspects, the invention relates to the selection and preparation of mutant GPCRs which have increased stability under a particular condition compared to their respective parent proteins.

Aspects of the invention relate to methods of producing a mutant GPCR with increased stability relative to its parent GPCR.

Mutant proteins and methods for producing them

A method for producing a mutant G-protein coupled receptor (GPCR) with increased stability relative to a parent GPCR, the method comprising making one or more mutations in the amino acid sequence that defines a parent GPCR, wherein (i) the one or more mutations are located within a window of/plus or minus 5 residues, where/is the position of amino acid residue 2.46 in the parent GPCR when the parent GPCR is a Class 1 GPCR, or where/is the position of an equivalent amino acid residue in the parent GPCR when the parent GPCR is a Class 2 or 3 GPCR, and/or (ii) the one or more mutations are located within an amino acid sequence of transmembrane helix 7 in the parent GPCR which amino acid sequence interacts with the window of/plus or minus 5 residues, to provide one or more mutants of the parent GPCR with increased stability.

Growing community of inventors

Cambridge, United Kingdom

Christopher Gordon Tate