Heptares Therapeutics Limited

Smithkline Beecham Corporation

Nxera Pharma UK Limited

Fiona Hamilton Marshall

Seyed Ali Jazayeri-Dezfuly

Malcolm Peter Weir

Christopher Gordon Tate

Steven Michael Foord

Alan Wise

Catherine Jane Hutchings

Neil James Fraser

Ashley Antony Barnes

Guillaume Pierre Lebon

Julia Helen Margaret White

Jayesh Chhotubhai Patel

Nathan Robertson

The invention provides a fusion protein comprising, from N-terminus to C-terminus: a) a first portion of a Family B G-protein coupled receptor (GPCR) that comprises transmembrane helix (TM)-1, TM2 and TM3 of the GPCR; b) a stable protein domain; and c) a second portion of the GPCR comprising TM4, TM5, TM6 and TM7 of the GPCR. The invention also provides a method of crystallising a GPCR comprising providing the fusion protein of the invention and crystallising it to obtain crystals.

Stable proteins

The invention provides an assay for assessing the conformational stability of a membrane protein, comprising: (a) providing a sample comprising a first population and a second population of a membrane protein; wherein the membrane protein in the first population is labelled with a donor label and the membrane protein in the second population is labelled with an acceptor label, or the membrane protein in the first population is labelled with an acceptor label and the membrane protein in the second population is labelled with a donor label, (b) exposing the first and second populations of the membrane protein to a stability modulating agent and/or condition, (c) and assessing aggregation between membrane proteins of the first and second populations by activating the donor label to permit a distance-dependent interaction with the acceptor label, which interaction produces a detectable signal.

Assay for assessing conformational stability of membrane protein

The invention provides a fusion protein comprising, from N-terminus to C-terminus: a) a first portion of a Family B G-protein coupled receptor (GPCR) that comprises transmembrane helix (TM)-1, TM2 and TM3 of the GPCR; b) a stable protein domain; and c) a second portion of the GPCR comprising TM4, TM5, TM6 and TM7 of the GPCR. The invention also provides a method of crystallizing a GPCR comprising providing the fusion protein of the invention and crystallizing it to obtain crystals.

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

There is provided a G protein coupled receptor (GPCR) or a polynucleotide encoding said GPCR for use as a vaccine. There is also provided methods of antagonizing or agonizing A GPCR in vivo comprising the administration of a GPCR or a polynucleotide encoding a GPCR to a subject. The invention further provides a GPCR for use in inhibiting an activity of a GPCR binding partner in a subject.

Vaccines comprising mutant GPCRs with increased conformational stability relative to parent receptors

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 conformation. Methods of producing GPCRs with increased stability relative to a parent GPCR are also disclosed.

Screening

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.

Mutant proteins and methods for producing them

The present invention relates to the novel GABA receptor subtypes GABA -R1 and GABA -R2 as well as to a novel, functional GABA receptor which comprises a heterodimer of GABA -R1 and GABA -R2 receptor subunits. The present invention also relates to variants of the receptors, nucleotide sequences encoding the receptors and variants thereof and novel vectors, stable cell lines, antibodies, screening methods, methods of treatment and methods of receptor production.

Growing community of inventors

Hertfordshire, United Kingdom

Fiona Hamilton Marshall