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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/129998 Al 3 August 2017 (03.08.2017) P O P C T (51) International Patent Classification: (72) Inventors: CARPENTER, Byron; Medical Research C07K 14/47 (2006.01) Council, Polaris House, North Star Avenue, Swindon SN2 1FL (GB). LESLIE, Andrew; Medical Research Council, (21) International Application Number: Polaris House, North Star Avenue, Swindon SN2 1FL PCT/GB20 17/050221 (GB). NEHME, Rony; Medical Research Council, Polaris (22) International Filing Date: House, North Star Avenue, Swindon SN2 1FL (GB). 27 January 2017 (27.01 .2017) TATE, Christopher Gordon; Medical Research Council, Polaris House, North Star Avenue, Swindon SN2 1FL (25) Filing Language: English (GB). WARNE, Antony; Medical Research Council, Po (26) Publication Language: English laris House, North Star Avenue, Swindon SN2 1FL (GB). (30) Priority Data: (74) Agent: PEARS, Michael; Potter Clarkson LLP, The Bel- 1601 690.9 29 January 20 16 (29.01.2016) GB grave Centre, Talbot Street, Nottingham NG1 5GG (GB). (71) Applicant: HEPTARES THERAPEUTICS LIMITED (81) Designated States (unless otherwise indicated, for every [GB/GB]; BioPark, Broadwater Road, Welwyn Garden kind of national protection available): AE, AG, AL, AM, City Hertfordshire AL7 3AX (GB). AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. [Continued on nextpage] (54) Title: G PROTEINS (57) Abstract: The invention provides a mutant of a parent heterotrimeric G protein alpha (Ga) subunit, which mutant (i) lacks at least one helix of the helical domain of the parent Ga subunit; (ii) is capable of binding to a GPCR in the ab sence of a heterotrimeric G protein beta ( β) subunit and a heterotrimeric G protein gamma (Gy) subunit; and (iii) has an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the parent heterotri meric Ga subunit, which mutations are selected from a dele tion, a substitution and an insertion. w o 2017/129998 Ai I il II II 11 I I 11 III III III lllll lllll lllll lllll lllll 111 llll 11llll (84) Designated States (unless otherwise indicated, for every Published: kind of regional protection available): ARIPO (BW, GH, — with international search report (Art. 21(3)) GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, — before the expiration of the time limit for amending the TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, claims and to be republished in the event of receipt of DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΤ , LT, amendments (Rule 48.2(h)) LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). G PROTEINS The present invention relates to mutant G proteins, and particularly to mutant alpha subunits of a heterotrimeric G protein. It also relates to products comprising such mutants, uses of the mutants, and methods involving such mutants. G proteins bind guanine nucleotides and act as molecular switches in a number of signalling pathways by interconverting between a GDP-bound inactive and a GTP-bound active state. They consist of two major classes: monomeric small G proteins and heterotrimeric G proteins. While small G proteins and the alpha subunit (Ga) of heterotrimeric G proteins both contain a GTPase domain (G-domain), Ga contains an additional helical domain (H-domain) and also forms a complex with G beta (G ) and G gamma (Gy) subunits. Although they undergo a similar signalling cycle, their activation differs in one important aspect. The guanine nucleotide exchange factors (GEFs) of small G proteins are largely cytosolic proteins, whereas the GEFs of Ga subunits are usually membrane-bound G protein coupled receptors (GPCRs). While GEFS of small G proteins interact directly with the GDP binding region, GPCRs bind to Ga at a site almost 30 A away from the GDP binding region and allosterically trigger GDP release to activate them. GPCRs constitute a very large family of proteins that control many physiological processes and are the targets of many effective drugs. Thus, they are of considerable pharmacological importance. Reference is made particularly to Overington et al (2006) Nature Rev. Drug Discovery 5, 993-996 which indicates that over a quarter of present drugs have a GPCR as a target. A list of GPCRs is given in Foord et al (2005) Pharmacol Rev. 57, 279-288, which is incorporated herein by reference. Three decades of biochemical and biophysical research have produced a model of G protein activation by GPCRs. Agonist binding to a GPCR induces subtle changes in the receptor structure 1 4, allowing a productive interaction to occur with the G protein. This process is likely to comprise at least two stages: an initial docking interaction, possibly involving the G protein βγ subunits or lipid moieties, induces a conformational change in the extreme C-terminus of the subunit5 6. The C-terminus, which is the major receptor-binding region7 and determinant of receptor specificity8,9 , is then able to fully engage the receptor. This interaction triggers mutually induced conformational changes in both the G protein and receptor 10 . In the G protein these changes are propagated to the nucleotide-binding pocket, resulting in the release of GDP10-1 1 . In the receptor the conformational changes feedback to the ligand-binding pocket, reducing the dissociation rate of the ligand, which results in significantly increased agonist binding affinity 2 13 . The mechanism of this affinity shift is likely to result from either subtle reorganisation of the ligand-binding pocket, or transition of the complex to a lower energy state due to the conformational stabilisation imparted by G protein binding. In this ternary complex the receptor acts as a chaperone, protecting the thermally labile nucleotide-free G protein from denaturation 14,15 . In the absence of guanine nucleotides this complex is stable; however in vivo, GTP is rapidly bound due to its high cellular concentration 12'14 . This triggers a conformational change, which causes dissociation of the G protein from the receptor 1 '17 , and separation of the Ga and βγ subunits 1416. The activated a-GTP and βγ subunits are then able stimulate their respective downstream signalling pathways. Atomic resolution mapping of the ligand-binding pocket is of significant importance for the design of drugs to modulate GPCR activity. Thus, methodology to crystallise receptors in their high-affinity agonist-bound conformation is a key prerequisite for efficient structure-based design of agonist compounds. To date, three approaches have accomplished this: first, the C-terminal peptide of transducin was crystallised in complex with both opsin 36 and metarhodopsin II37; second, a camelid antibody (Nb80), which induces the high-affinity agonist-bound state, has been crystallized in complex with 2A 38; third, heterotrimeric Gs has been crystallised in complex with 2A R . Despite the valuable insight into GPCR activation provided by these structures, they have several major disadvantages for wider structure-based drug design applications. The opsin and metarhodopsin II complexes were solved at 3.2 A and 2.85 A respectively, and in both cases electron density around the chromaphore-binding pocket was strong 36 7. However, the use of G protein C-terminal peptides to stabilise the active conformation of other GPCRs has been unsuccessful 10 . Furthermore, the conformational changes induced by the transducin peptide are much smaller than those observed in the P2AR-GS complex 10 , indicating that these structures represent an intermediate conformation along the activation pathway. The Nb80-fi2AR complex was solved at 3.5 A resolution, and also exhibited good electron density around the ligand-binding pocket 38 . However, the conformational changes induced by Nb80 are smaller than those observed in the 2AR- Gs complex 10 , suggesting that this structure may also represent an intermediate conformation. Furthermore, although Nb80 was derived specifically to bind AR and is therefore likely to efficiently couple to other closely related GPCRs (eg AR), new nanobodies likely need to be raised against more distantly related receptors. The P2AR- Gs complex was solved at 3.2 A resolution, however, in this structure electron density around the ligand-binding pocket was very poor. Furthermore, the complexity of crystallising G protein-GPCR complexes means this strategy is of limited use for wider structure-based drug design applications. All of the aforementioned complexes were solved at medium-high resolution, however, they provided insufficient detail around the ligand-binding pocket to accurately define the structural changes associated with the high-affinity agonist-bound conformation. Therefore, in order to accurately define these changes, and to allow optimal structure-based drug design, there is a strong requirement to solve the structures of G protein-GPCR complexes at greater than 2 A resolution. The separate GTPase and helical domains of the stimulatory G protein (Gas) have been previously transfected into COS-7 cells4 1 .
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