Stable APO active fusion of a g protein coupled receptor (GPCR) and a g protein alpha subunit helix 5

Abstract

Disclosed herein is a stable apo active fusion of a G protein coupled receptor (GPCR) and a G protein alpha subunit (Gα) helix 5 (Gα,H5), wherein the GPCR-Gα,H5 fusion is formed without a co-expression of a beta subunit (Gβ) or a gamma subunit (Gγ). The apo active GPCR-Gα,H5 fusion can be isolated from a cell membrane in a purified form and maintains its stability in the purified form.

Description

STABLE APO ACTIVE FUSION OF A G PROTEIN COUPLED RECEPTOR (GPCR) AND A G PROTEIN ALPHA SUBUNIT HELIX 5TECHNICAL FIELD

[0001] Disclosed herein is a stable apo active fusion of a G protein coupled receptor (GPCR) and a G protein alpha subunit (Ga) helix 5 (H5), wherein the GPCR-Ga H5 fusion is formed without a coexpression of a beta subunit (G ) or a gamma subunit (Gy). The apo active GPCR-Ga H5 fusion can be isolated from a cell membrane in a purified form and maintains its stability in the purified form.BACKGROUND OF THE INVENTION

[0002] G proteins bind guanine nucleotides and act as molecular switches in several signaling pathways by interconverting between a GDP-bound inactive and a GTP-bound active state. Heterotrimeric G proteins contain a helical domain (H-domain) and forms a complex with G beta (G[3) and G gamma (Gy) subunits. The guanine nucleotide exchange factors (GEFs) of Ga subunits are usually membrane-bound G protein coupled receptors (GPCRs). GPCRs bind to Ga at a site almost 30 A away from the GDP binding region and allosterically trigger GDP release to activate them.

[0003] GPCRs constitute a very large family of proteins that control many physiological processes and are the targets of many effective drags. As a result, they are of considerable pharmacological importance. It is estimated that about 30-50% of the targets of currently marketed drugs involve GPCRs. Salon et al. (2011) Pharm. Rev. 63, 901-937; Santos et. al. (2017) Nat. Rev. Drug Discov. 16, 19-34.

[0004] Ligand binding to a GPCR induces subtle changes in the receptor structure, allowing a productive interaction to occur with the G protein. This process is believed to comprise at least two stages: an initial docking interaction, possibly involving the G and Gy subunits, induces a conformational change in the extreme C-terminus of the Ga subunit. The C-terminus, which is the major receptor-binding region and determinant of receptor specificity, is then able to fully engage the receptor. This interaction triggers mutually induced conformational changes in both the G protein and receptor. In the G protein these changes are propagated to the nucleotide-binding pocket, resulting in the release of GDP. In the receptor the conformational changes feedback to the ligand-binding pocket, reducing the dissociation rate of the ligand, which results in increased agonist binding affinity.

[0005] GPCR-Ga fusion construct was first reviewed by Kobilka et al (1999) Trends Pharmacol. Sci., Vol. 20, Issue 9, 383-389. At the time, such constructs were only studied in whole cell functional assays. Biochemical isolation of GPCR-Ga fusion for structural study was not described until recently by Lees et al (2023) where a receptor GPR61 was fused to a GaS subunit and co-purified with Gp / Gy subunits.Less, et al. (2023) Nature Communications, 14:5938. In addition to GPCR-G protein fusion constructs, priming active conformation of GPCRs by sole interactions of the terminal helix of Ga subunit of the G protein complex has been demonstrated by Sivaramakrishnan et al (2019) Nat Communications, 10, 4836. However, translation of these designs to class B GPCRs and more broadly, their utility in stabilizing purified GPCRs from class A or B has been lacking.

[0006] While GPCRs comprise an important class of drug targets, discovering, developing and characterizing new therapeutics against these receptors is often challenged by their low stability and other considerations during protein expression and / or purification. Although high throughput screening (HTS) of large libraries in biochemical assays has led to identification and development of molecules which later became clinical compounds, such discoveries in GPCRs have been primarily limited to family A receptors that are typically modulated by endogenous small molecule ligands, while successful applications of such HTS experiments to highly validated clinical targets including peptide receptors of family B GPCRs has been limited. Additionally, in successful cases, thermostabilized receptors have been used which carry several point mutations and bias the conformation of the ligand to active or inactive state which ultimately biases the pharmacology of the protein and its response to ligands with different pharmacological properties (agonist, inverse agonist, or antagonists) which in turn reduced the sensitivity of the assay to a diverse array of fragments / ligands. Ideally, a close-to-native system would produce most reliable characterization of the ligand / fragments relevant to protein-ligand complex in the cells. Additionally, the conformational flexibility of the protein (not artificially stabilized in one conformation) can allow for identification and / or characterization of ligands with novel mechanisms of binding.

[0007] While NMR, crystallization and other biophysical techniques can be used for such fragment screenings and characterizations, their applications are even more limited due to high sample volume demand and challenges in obtaining sufficient amount of stable and pure functionally folded GPCRs required for these experiments. Therefore, identifying new approaches to obtain pure and stable protein amenable to HTS and / or characterization techniques such as Surface Plasmon Resonance (SPR) and Affinity Selection Mass Spectrometry (ASMS) can greatly facilitate the process of drug discovery and development.SUMMARY OF THE INVENTION

[0008] There is an unmet need for a purified and stable apo active GPCR for ligand screening and characterization studies. Disclosed herein is an approach to obtain such a purified and stable apo active GPCR by fusing the GPCR with a single helix of its effector protein, a G protein alpha subunit (Ga) helix 5 (Ga,H5), wherein the GPCR-Ga,H5 fusion is formed without a co-expression of a beta subunit (G[3) ora gamma subunit (Gy). The fused GPCR-Ga,H5 protein can be isolated from a cell membrane in a purified and stable form and maintains its stability without the presence of a GPCR ligand.

[0009] In one embodiment, a GPCR is fused to an effector protein of the GPCR, wherein the effector protein is an alpha subunit (Ga) of the G protein; wherein the N-terminus of Ga terminal helix 5 (H5) is fused to the C-terminus of the GPCR to form the GPCR-Ga,H5 fusion; and wherein the fusion of the Ga subunit to the GPCR stabilizes the GPCR in an apo active state.

[0010] In one embodiment, the GPCR-Ga,H5 fusion protein further comprises a junction region, wherein the junction comprises one or more linkers. In one embodiment, each linker comprises one or more GSG units.BRIEF DESCRIPITON OP DRAWINGS

[0011] Fig. 1 shows a chromatogram from a size exclusion column Superose 6 10 / 300 GL from Cytiva on a headless AMY3R-Ga,H5 fused protein.

[0012] Fig. 2 shows a chromatogram from a size exclusion column Superose 6 10 / 300 GL from Cytiva on a full length AMY3R-Ga,H5 fused protein.

[0013] Fig. 3 shows a chromatogram from a size exclusion column Superose 6 10 / 300 GL from Cytiva on a headless GCGR-Ga,H5 fused protein.

[0014] Fig. 4 shows a chromatogram from a size exclusion column Superose 6 10 / 300 GL from Cytiva on a full length GCGR-Ga,H5 fused protein.

[0015] Fig. 5 shows an SPR sensorgram of purified GCGR and AMY3R avi-tagged proteins associating to a streptavidin chip.

[0016] Fig. 6 Shows a schematic of compound #4742 binding to two AMY3R-AVI- Ga,H5 constructs in a concentration dependent manner by SPR (A and B). Panels C and D show the affinity fits for the sensorgrams from panels A and B.

[0017] Fig. 7 shows a plot of full-length AMY3R-avi-Ga,H5 activity over 12h, as determined by repeated injections of 10 uM compound #4742.

[0018] Fig. 8 shows an ASMS saturation curve for compound #3175 from 1 nM to 50 uM.

[0019] Fig. 9 Shows a ASMS saturation curve for compound #3175 which substantiates specific binding across two different constructs.DESCRIPTION OF THE INVENTION

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the disclosure pertains.

[0021] Moreover, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one.”

[0022] As used herein, “about” means within a statistically meaningful range of a value or values such as, for example, a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.

[0023] As used herein, the term “apo” means ligand-free when used to describe a GPCR. For example, an apo active GPCR means a GPCR in an active conformation when no ligand is bound to the GPCR.

[0024] Historically, it has been challenging to biochemically prepare a purified apo (ligand free) GPCR due to the inherent instability of the receptor after detergent extraction from a cell membrane. While this inherent instability can be resolved by adding a high affinity ligand to the receptor prior to detergent extraction, the addition of the ligand would greatly limit the utility of the purified GPCR in structural or ligand screening studies since a GPCR needs to be purified in a stable form in the absence of a ligand for such studies.

[0025] It has been surprisingly found that a GPCR can be stabilized in an ‘apo-activc’ state by engineering the GPCR to engage with a terminal helix 5 (or “H5”) of the alpha subunit of a G protein (Ga,H5) to form a fused protein without the presence of a ligand. Specifically, a GPCR can be engineered, stabilized, and purified in the ‘apo-active’ state via a construct of the GPCR with its C- terminus fused to the N-terminus of the Ga,H5. By fusing the C-terminus of the GPCR to the N-terminus of the Ga,H5, the GPCR protein and Ga,H5 are placed in close proximity, and the receptor can be transiently stabilized in the active state in the absence of a ligand. In this design, G0 and Gy are not coexpressed with Ga in the GPCR-Ga,H5 fusion.

[0026] The fusion design disclosed herein utilizes a terminal helix H5 in the alpha subunit of GPCR’ s primary effector protein, G protein, to stabilize the active conformation of the GPCR. Instead of using the entire heterotrimeric G protein, only the Ga subunit is used to prime the GPCR in its apo active conformation and transiently stabilize the active conformation. Due to weak interactions between the GPCR and the Ga,H5 (as opposed to with the entirety of the heterotrimeric G protein), the interactions aretransient and hence do not lock the receptor in one conformation or another. This transient nature of the apo active GPCR confirmation allows for GPCR dynamics which can be important in ligand bindings. In one embodiment, a suitable G protein is selected from a subtype S, subtype Q, subtype I, and subtype O.

[0027] The fusion design here also allows for engineering of an accessible affinity tag on the primary target GPCR which is used to immobilize the protein, such as on a streptavidin sensor chip to characterize ligand binding by a surface plasmon resonance (SPR) chip. The engineering of Ga,H5 allows for the isolation and purification of a stable and functionally active GPCR which can be utilized in downstream characterization screenings while eliminating the need to use elaborate protein complexes which may complicate readouts and, in some cases, limit downstream applications.

[0028] Potential advantages of the designs disclosed herein include, but are not limited to, the following. First, stabilization of an apo active protein is maintained throughout protein purification to generate monomeric and active proteins for SPR experiments without using thermostabilizing mutations which may alter the native-state and behavior of the protein. Second, the smaller size of the GPCR-Ga,H5 fusion can make the system more sensitive to mass changes upon small ligand binding. Third, receptors primed in active conformation while retaining dynamics allow investigations of a scries of ligands with diverse pharmacological profiles, ideal for agonist profiling and potentially amenable to antagonist and inverse agonist profiling as well.

[0029] In one embodiment, the fusion point between the GPCR and the Ga subunit is optimized. In one embodiment, the N-terminus of the Ga subunit is fused at 50-70 amino acids past transmembrane helix 8 of the receptor as defined by the secondary structure prediction shown in gpcrdb.org. In one embodiment, the distance between the regions specified in the body includes the linker and tag region.

[0030] For detergent extraction, several detergents are suitable for the purification of the GPCR-Ga,H5 fusions and the application of downstream experiments such as biophysical screens using Surface Plasmon Resonance, Affinity Selection Mass Spec, and DNA-encoded Library. Non-limiting examples of suitable detergents such as LMNG / CHS.

[0031] In one embodiment, disclosed herein is a fusion of a G protein coupled receptor (GPCR) and a terminal helix from an effector protein of the GPCR; wherein the effector protein is an alpha subunit (Ga) of a G protein complex; wherein an N-terminus of Ga terminal helix 5 is fused to a C-terminus of an amino acid of the GPCR to form the GPCR-Ga fusion; and wherein the fusion of the Ga subunit to the GPCR stabilizes the GPCR in an apo active state without the presence of a GPCR ligand.

[0032] In one embodiment of the fusion described above, the terminal helix 5 of the Ga subunit comprises about 27 amino acids. In another embodiment, the terminal helix 5 of the Ga subunit comprises less than 27 amino acids. In another embodiment, the terminal helix 5 of the Ga subunit comprises about 15 to about 27 amino acids.

[0033] In one embodiment, the GPCR-Ga,H5 fusion does not comprise a beta (G ) subunit or a gamma (Gy) subunit.

[0034] In one embodiment of the GPCR-Ga,H5 fusion, the G protein is of a subtype selected from S, I, O and Q.

[0035] In one embodiment of the GPCR-Ga,H5 fusion, the GPCR is selected from a class A GPCR and a class B GPCR.

[0036] In one embodiment of the GPCR-Ga,H5 fusion, the GPCR is selected from an amylin / calcitonin receptor, a glucagon receptor, a glucose-dependent insulinotropic polypeptide (GIP) receptor, a glucagon- like peptide 1 ( GI.P- 1 ) receptor, and a neuropeptide Y type 2 receptor (NPY2R).

[0037] In one embodiment of the GPCR-Ga,H5 fusion, the fusion comprises a junction between the GPCR and the Ga helix 5; and wherein the junction comprises one or more linkers.

[0038] In one embodiment of the GPCR-Ga,H5 fusion, each linker comprises one or more GSG units.

[0039] In one embodiment of the GPCR-Ga,H5 fusion, the fusion of the Ga,H5 to the GPCR stabilizes the GPCR in an apo active state after the fused protein is extracted from a cell membrane.

[0040] In one embodiment of the GPCR-Ga,H5 fusion, the fusion is suitable for a downstream characterization or screening study.

[0041] In one embodiment of the GPCR-Ga,H5 fusion, the characterization or screening study uses an Affinity Selection Mass Spectrometry (ASMS) as an analytical tool.

[0042] In one embodiment of the GPCR-Ga,H5 fusion, the characterization or screening study uses a Surface Plasmon Resonance (SPR) as an analytical tool.

[0043] In one embodiment of the GPCR-Ga,H5 fusion, wherein: the fusion comprises a junction between the GPCR and the Ga,H5; the junction comprises an affinity tag which allows for a capture of the purified fusion protein (e.g. on an SPR chip); and the junction comprises a linker between the GPCR and the affinity tag and a linker between the affinity tag and the Ga,H5.

[0044] In one embodiment of the GPCR-Ga,H5 fusion, each linker comprises 4 GSG units.

[0045] In one embodiment of the GPCR-Ga,H5 fusion, the fusion is used for screening a ligand that binds to the apo active GPCR.

[0046] In one embodiment of the GPCR-Ga,H5 fusion, the ligand is a small molecule ligand or a biologic ligand.

[0047] In one embodiment of the GPCR-Ga,H5 fusion, wherein the GPCR is an amylin / calcitonin receptor.

[0048] In one embodiment of the GPCR-Ga,H5 fusion, wherein the GPCR is a glucagon receptor.

[0049] In one embodiment of the GPCR-Ga,H5 fusion, wherein the GPCR is a glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0050] In one embodiment of the GPCR-Ga,H5 fusion, wherein the GPCR is a glucagon-like peptide 1 (GLP-1) receptor.

[0051] In one embodiment of the GPCR-Ga,H5 fusion, wherein the GPCR is a neuropeptide Y type 2 receptor (NPY2R).

[0052] SPR is a reliable biophysical method for measuring affinity and binding kinetics of a ligand to its target protein and can be applied to a variety of protein targets, including membrane proteins and among them, G protein-coupled receptors (GPRCs). SPR has proven useful for both screening of fragment libraries of small molecular weight fragments as well as studying binding kinetics of already known ligands with measurements of their pharmacological properties. Properties of ligand and protein-ligand interactions include association rate (ka), dissociation rate (kd), affinity (KD) (e.g., by equilibrium or kinetic analysis), and the stoichiometry of the interaction.

[0053] Drugs with slow dissociation rates often show superior clinical efficacy. Therefore, the ability to identify fragments with slow dissociation rates may help prioritize starting points for lead compound optimization. SPR analysis can also be used to determine thermodynamic parameters of interactions (delta G or DG, delta H or DH and delta S or DS) using van't Hoff equation, by measuring the KD over a temperature range (Myszka, 2000; Navratilova et al., 2007; Papalia et al., 2008). The equilibrium dissociation and thermodynamic constants determined by SPR analysis correlate well with the values determined using isothermal titration calorimetry, with the use of significantly less protein (Navratilova et al., 2007).

[0054] SPR utilizes an optical biosensor detection method that measures changes in the refractive index of the biosensor interface upon direct molecular interaction of a ligand and its target protein at the biosensor’s surface (as opposed to an indirect measurement based on displacing a labeled ligand; either fluorescent or isotope). In order to conduct an SPR experiment, a target protein needs to be immobilized on the biosensor surface and exposed to a flow of a mobile phase which contains a ligand or a fragment of interest. Various immobilization strategies range from covalent linkages like amine coupling to non- covalent coupling including the use of affinity tags (e.g. biotin streptavidin).

[0055] While purified untagged proteins can be used for covalent coupling to the SPR chip, the engineering of affinity tags provides the unique opportunity of enriching the specific protein target of interest at the SPR chip, in some cases eliminating the need to obtain purified proteins with the high standards required for other screening methods to ensure a successful SPR experiment while minimizing false positives from off-target response. However, despite low sample requirements for SPR and multiple coupling approaches available for utility of target protein in this biophysical experiment, applications of SPR to GPCR targets has been limited to a few class A GPCRs, partly owing to their low expression levels and partly due to their instability in detergent solutions that are to this date the most successful extraction method for solubilizing this class of membrane protein from the lipid bilayer of cells for downstream purification and characterization. This is while SPR as a direct measurement tool could be instrumental in identifying and characterizing fragments and ligands for GPCRs, as opposed to commonly used indirect methods for such characterizations including displacement of existing known ligands or readout of downstream signaling response in cell-based assays often used in HTS of large libraries of compounds.

[0056] Affinity selection mass spectrometry (ASMS) is a label-free technique used to measure the binding affinity of ligands to a biomolecular target. Since this biophysical technique offers a direct measurement of binding, ASMS can be used to identify ligands binding to allosteric as well as orthosteric sites. ASMS can be used as a screening platform and also to provide affinity measurements and kinetic information by running saturation curves and association / dissociation experiment respectively.

[0057] As can be seen from the embodiments and examples disclosed herein, just the c-terminal helix of the Ga subunit from the hctcrotrimcric G protein is enough to prime the GPCR in its active conformation. The weak interactions between the GPCR and Ga,H5 are tr ansient and do not lock the receptor in one conformation or another, thus allowing for GPCR dynamics which can be important in ligand binding.

[0058] The engineering of AviTag in the junction of GPCR and Ga,H5 allows for successful capture of purified GPCR-Ga,H5 fusion on an SPR chip. The engineering of 4x(GSG) linkers in between each protein / peptide (GPCR / 4x(GSG) / AviTag / 4x(GSG) / Ga,H5) allows for flexibility and movement of each part and for Ga,H5 to associate / dissociate from the GPCR throughout experiments while captured on the SPR chip. Finally, the Ga,H5 stabilizes the receptor throughout the purification and downstream characterizations. This makes for a suitable system to screen and investigate ligands with agonistic properties but due to dynamic nature of the system should allow for binding and characterization of ligands with other pharmacological behavior as well.EXAMPLES

[0059] The following examples are provided for illustration purposes only. The abbreviations used in the preparations and examples are as defined below unless otherwise defined in the specification.°C degree CelsiusCHS cholesteryl hemisuccinateGLP-1R glucagon like peptide- 1 receptorGCGR glucagon receptorGIPR glucose-dependent insulinotropic polypeptide (GIP) receptorGPCR G protein coupled receptorCALCR calcitonin receptor h hour(s)IPA isopropyl alcoholHEPES 2-[4-(2-hydroxyethyl)piperazin-1 -yl]ethane-l -sulfonic acidLCMS liquid chromatography mass spectrometryLMNG Lauryl Maltose Neopentyl Glycol kDa kilodaltonMALDI matrix-assisted laser desorption / ionizationMALDITOF MALDI coupled to time-of-flight mass spectrometry min minute(s) mg milligram mL milliliter mM millimolar rpm revolutions per minute sec. second(s)SEC size exclusion chromatography pL microliterExperimental Protocols and Parameters

[0060] The Baculovirus Expression protocol described below was used for the preparation of GPCR- Ga,H5 fusion proteins.

[0061] Baculovirus Expression: Spodoptera frugiperda (S 9) were grown in Sf-900II SFM media(Thermo Fisher) at 27 °C and 140 rpm. Standard baculovirus expression using a modified version of theBac-to-Bac system protocol (Thermo Fisher) in combination with the DHIOEMBacY bacmid (Geneva Boiotech) was used to generate high-titer recombinant baculovirus for the receptor-Ga,H5 fusion (GCGR- Ga,H5 or CALCR-Ga,H5 and RAMP3), and BirR. Cells were grown to a density of 2 x 10 6 cells / mL and then infected with 2-3 separate baculoviruses, one containing GPCR-Ga,H5 fusion (as well as one containing RAMP3 in the case of CALCR) and the other containing birA. GPCR-Ga,H5 and BirA were co-expressed by infecting Sf9 cells at a ratio of 1 : 1 (and 0.5 RAMP3 in the case of CALCR), in the presence of 100 pM Biotin to facilitate biotinylation of GPCR-Ga,H5 by BirA. After 48-hour incubation at 27 °C, the cells were collected by centrifugation and stored at -80 °C for purification.

[0062] The experimental protocols described below were used for the isolation and purification of purified GPCR-Ga,H5 fusion proteins.

[0063] Flag Affinity Purification and Size Exclusion Chromatography (SEC): Cells harvested from 5 liters of cell culture were lysed in the lysis buffer containing 20 mM HEPES pH 7.5, 50 mM NaCl, 5 mM CaCL. 2 mM MgCL. 1:100 dilution of Halt Protease inhibitors (Thermo Scientific), 1:1000 dilution of Cocktail VII protease inhibitors (Calbiochcm) and homemade Turbonuclcasc. Protein was then solubilized with a final concentration of 0.5% LMNG / 0.05% CHS (Lauryl Maltose Neopentyl Glycol and Cholesteryl Hemisuccinate from Anatrace) for 2 h at 4 °C. Supernatant was collected by centrifugation at 16,000 x g for 60 min at 4 °C and then stirred with 2 mL of FLAG resin (Genscript) overnight at 4 °C. After packing, the resin was washed with 20 CV (column volumes) of wash buffer containing 10 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCL, 2 mM MgCL and 0.1% LMNG / 0.01% CHS. Protein was eluted from the FLAG resin with 15 mL of elution buffer containing 10 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCL, 0.1 mg / inl Flag peptide and 0.05% LMNG / 0.005% CHS after 1 h incubation with this buffer at 4 °C. Protein was concentrated to 500 pL using a 100 kDa MWCO Amicon Ultra Centrifugal Filter. Size exclusion chromatography was carried out by loading the protein sample onto Superose 6 Increase 10 / 300 GL (Cytiva) column and using SEC buffer containing 10 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCL and 0.01% LMNG / 0.001% CHS, to obtain a pure and monodispersed protein. Fractions of interest from this step were collected and further analyzed by protein SDS-PAGE and MALDI (Figs.1-4).

[0064] Fig. 1 shows a chromatogram from size exclusion column Superose 6 10 / 300 GL from Cytiva on a headless AMY3R-Ga,H5 fused protein. The highlighted region on the chromatogram corresponds to the monomeric protein used for downstream applications. Specifically, The highlighted peak belongs to heterodimer of headless AMY3R-Ga,H5 (CALCR-Ga,H5 + RAMP3) where the molecular weight (MW) of CALCR is ~42 kDa and the MW of RAMP3 is ~5.6 kDa. The protein gel further confirms thepresence of both CALCR and RAMP3 in the collected fraction as highlighted in the figure. Lastly, the presence of CALCR-Ga,H5 is verified by MALDI and the correct mass of the protein of interest was confirmed in the sample using MALDI.

[0065] Fig. 2 shows a chromatogram from size exclusion column Superose 6 10 / 300 GL from Cytiva on a full length AMY3R-Ga,H5 fused protein. The highlighted region on the chromatogram corresponds to the monomeric protein used for downstream applications. The protein gel further confirms the presence of both CALCR and RAMP3 in the collected fraction as highlighted in the figure. Glycosylated RAMP3 appeals as a smeared region on the gel. Lastly, correct mass of the protein of interest was confirmed in the sample using MALDI. Specifically, the highlighted peak belongs to heterodimer of full length Amy3R-Ga,H5 (CALCR- Ga,H5 + RAMP3) where the MW of CALCR is -55.6 kDa and the MW of RAMP3 is -15 kDa. This fraction and the components are shown in the protein gel. RAMP3 band appears as a smear due to glycosylation of this protein. The presence of CALCR-Ga,H5 and RAMP3 is also verified by MALDI.

[0066] Fig. 3 shows a chromatogram from a size exclusion column Superose 6 10 / 300 GL from Cytiva on a headless GCGR-Ga,H5 fused protein. The highlighted region on the chromatogram corresponds to the monomeric protein used for downstream applications. In particular, the highlighted peak belongs to monomeric headless GCGR-Ga,H5 where the MW of GCGR is -43 kDa. Lastly, the protein gel further confirms the presence of GCGR in the collected fraction as highlighted in the figure and the correct mass of the protein of interest was confirmed in the sample using MALDI.

[0067] Fig. 4. Shows a chromatogram from a size exclusion column Superose 6 10 / 300 GL from Cytiva on a full length GCGR-Ga,H5 fused protein. The highlighted region on the chromatogram corresponds to the monomeric protein used for downstream applications. In particular, The highlighted peak belongs to monomeric full length GCGR-Ga,H5 where the MW of GCGR is -54.5 kDa. Presence of GCGR-Ga,H5 in this fraction is confirmed in the protein gel and the correct mass of GCGR-Ga.H5 is also verified by MALDI.

[0068] The Mass Spectrometry protocol and parameters described below were used for analyzing and characterizing the fusion and other proteins.

[0069] MALDI Protocol: 2-8 mg of matrix (Alpha-Cyano-4-hydroxycinnamic Acid) was dissolved to 20 mg / mL with mixing and heating in a mixture of 16.67% LCMS grade water (1 pail), 33.33% LCMS grade IPA (2 parts), and 50% formic acid (3 parts). The solution was centrifuged at 10,000 rpm for 15 seconds to remove insoluble material. Then, 1 pL of protein sample (at 1 mg / mL minimum) was mixed with 9 pL of the matrix solution, and 0.2 pL of that mixture was transferred and spotted onto a MALDIplate. The sample was allowed to dry on the plate, and the dried sample was then washed with 0.2 pL of 1 % formic acid. The excess liquid was removed carefully, and the MALDI plate was inserted into the MALDI instrument for MALDITOF analysis.Mass Spectrometry Parameters Using MALDI:Mass Spec: Bruker Ultraflextreme MALDI- TOF and TOF / TOF MALDI MTP target plate: Bruker Daltonics, Part No:8280784 Chemical s / Materials:LCMS Grade Isopropanol (CAS 67-63-0)LCMS Grade Water (CAS: 7732-18-5)LCMS Grade Formic Acid (CAS: 64-18-6)Alpha-Cyano-4-hydroxycinnamic Acid (CAS:28166-41 -8)SPR Experiments

[0070] Proteins were diluted in non-DMSO running buffer (lOmM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCh, 0.01% LMNG, 0.001% CHS) to 0.1 nig / mL. Compound#4742 was diluted from 10 mM in 100% DMSO to 10 uM in DMSO running buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCh, 0.01% LMNG, 0.001% CHS, 3% DMSO), and 3x titration was performed 5 times for a total of 6 ligand concentrations from 41.2 nM to 10 uM. A Cytiva Series S SA sensorchip was loaded into a Biacore T200 instrument, which was then set to 25 °C and primed with DMSO running buffer. Proteins were captured for 250s on the following flow cells to the response units (RUs) stated: GCGR-avi-Ga,H5 on Fcl (6200RU); HL GCGR-avi-Ga,H5 on Fc2 (6700RU); AMY3R-avi-Ga,H5 on Fc3 (6200RU); and HL AMY3R-avi-Ga,H5 on Fc4 (5200RU) (Fig. 5).

[0071] Fig. 5. A schematic of GPCR capture by AVI tag to a streptavidin SPR chipCapture sensorgrams from left to right: GCGR-AVI-Ga,H5 (purple); headless GCGR-AVI-Ga,H5 (blue); AMY3R-AVI-Ga,H5 (green); and headless AMY3R-AVI-Ga,H5 (orange).

[0072] After protein capture, all four flowcells were conditioned 8 times with DMSO running buffer, and DMSO corrections were tested according to Biacore standards. Compound #4742 was injected across all four flowcells for 60 sec of association and 60 sec of dissociation time between injections. Ligand injections began with two blanks (DMSO running buffer), then concentrations increased starting from 41.2 nM and ending with 10 uM (Fig. 6).

[0073] Fig. 6. A schematic of compound #4742 binding to two AMY3R-AVI-GaH5 constructs in a concentration dependent manner by SPR (A and B). Panels C and D show the affinity fits for the sensorgrams from panels A and B.

[0074] Following collection of the concentration response curve, single point injections of 10 uM continued for 12h, spaced out with 6 blanks between injections to determine the receptor half-life (Fig 7).

[0075] Fig. 7. A schematic of decreasing activity of two AMY3R-AVI-Ga,H5 constructs as determined by receptors binding to compound #4742 over 12 hours by SPR.ASMS Protocol:

[0076] ASMS can be performed in a number of different methods and is typically chosen based on the protein Target. In the case of GPCRs established approaches such as ultrafiltration and magnetic beads have been most often used. For this study, an ultrafiltration method running saturation curves was used to calculate the Kds of compound. Ultrafiltration does not require the protein to be tagged in any way. This saturation curve was conducted by incubation 1 pg of purified GPCR Ga,H5 in 200 pL purification buffer (lOmM HEPES, 100 mM NaCl, 2 mM MgCF. 0.01% LMNG 0.001% CHS) with the ligand from 100 nM to 50 pM in a 10 point does response curve in half-log units. This was always tested against another GPCR purified protein as a negative control, as well as the ligand by itself through the filter plate to consider any interaction with the filter plate itself. After incubation (1 hour at room temperature) the samples were transferred to a 1 pm glass fiber filter that sat on top of a vacuum manifold. The samples were then subjected to rapid wash with cold buffer 5-times the sample volume as the vacuum was open to wash off the non-binding ligands. The plate was dried, and then transferred back to the vacuum manifold. The contents in each well were then resuspended with 50% methanol and 50% ammonium formate to denature the protein and release the bound ligand. This time with a collection plate underneath, the ligand that once was bound is now collected. This plate was now ready for HPLC MS / MS analysis to detect the amount of ligand at the corresponding incubation concertation. HPLC MS / MS was conducted on an Agilent 1290 HPLC system hooked up to a Sciex 6500+ QQQ. Multiquant was used for data analysis and Graphpad Prism for processing to obtain Kd values (Figs. 8-9).

[0077] Fig. 8. ASMS saturation curve for compound #3175 from 1 nM to 50 uM. Fig. 5A shows the full range of experiment. Fig. 8B shows the zoomed in portion where specific binding is observed.

[0078] Fig. 9. ASMS saturation curve for compound #3175 showing specific binding across two different constructs with their calculated Kd values.Example 1: CALCR-Ga,H5 Fusion

[0079] Below are two examples of the instant design as applied to a fusion of a calcitonin receptor (CALCR)HA Tag + Flag Tag + CALCR (25-430) + 4xGSG C-terminal linker + aviTag + 4x GSG linker + 27 terminal AAs from Ga,s. The fused protein has the SEQ ID No. 1 shown helow.MKTIIALSYIFCLVFADYKDDDDKAFSNQTYPTIEPKPFLYVVGRKKMMD AQYKCYDRMQQLPAYQGEGPYCNRTWDGWLCWDDTPAGVLSYQFCPD YFPDFDPSEKVTKYCDEKGVWFKHPENNRTWSNYTMCNAFTPEKLKNAYVLYYLAIVGHSLSIFTLVISLGIFVFFRSLGCQRVTLHKNMFLTYILNSMII IIHLVEVVPNGELVRRDPVSCKILHFFHQYMMACNYFWMLCEGIYLHTLI VVAVFTEKQRLRWYYLLGWGFPLVPTTIHAITRAVYFNDNCWLSVETHLLYIIHGPVMAALVVNFFFLLNIVRVLVTKMRETHEAESHMYLKAVKATM ILVPLLGIQFVVFPWRPSNKMLGKIYDYVMHSLIHFQGFFVATIYCFCNNE VQTTVKRQWAQFKIQWNQRWGRRPSNRSARAAAGSGGSGGSGGSGGLNDIFEAQKIEWHEGSGGSGGSGGSGDTENIRRVFNDCRDIIQRMHLRQYELL*HA Tag + Flag Tag + CALCR (138-430) + 4xGSG C-terminal linker + aviTag + 4x GSG linker + 27 terminal AAs from Ga,s. The fused protein has the SEQ ID No. 2 shown below.MKTIIALSYIFCLVFADYKDDDDKATPEKLKNAYVLYYLAIVGHSLSIFTLVISLGIFVFFRSLGCQRVTLHKNMFLTYILNSMIIIIHLVEVVPNGELV RRDPVSCKILHFFHQYMMACNYFWMLCEGIYLHTLIVVAVFTEKQRL RWYYLLGWGFPLVPTTIHAITRAVYFNDNCWLSVETHLLYIIHGPVMAALVVNFFFLLNIVRVLVTKMRETHEAESHMYLKAVKATMILVPLLGIQ FVVFPWRPSNKMLGKIYDYVMHSLIHFQGFFVATIYCFCNNEVQTTVK RQWAQFKIQWNQRWGRRPSNRSARAAAGSGGSGGSGGSGGLNDIFEAQKIEWHEGSGGSGGSGGSGDTE NIRRVFNDCR DIIQRMHLRQ YELL*

[0080] These proteins were successfully co-expressed with RAMP3 separately following the expression protocol described above and the resulting AMY3R was purified / characterized following the purification and mass spectrometry protocols outlined above. The resulting protein sample was used in follow-upASMS and SPR studies to profile small molecule ligands developed for this receptor. Both ASMS and SPR experiments were conducted in parallel to another sample serving as a negative control.

[0081] Construct design: GPCRs, both full length and with truncated extracellular domain, can be used for this design, depending on the specific downstream applications. The N-terminus of the last helix of the G protein alpha subunit (Ga,H5 ) which is ~27 amino acids is fused to the C-terminus of the GPCR while spaced with flexible linkers and affinity tag, if needed. Here, 4x repeats of (GSG) linker were used on either side of an affinity tag (Avi tag) to increase flexibility of fused Ga,H5 to facilitate its binding to the GPCR. The C-term of GPCR can be truncated down to 8-10 amino acids downstream of helix 8(according to the predicted secondary structures on gpcrdb.org). In cases where the native sequence of the GPCR forms a flexible coil, one may choose to preserve the native sequence instead of replacing it with the sequence of GSG repeats.SEQUENCESSEP ID No. 1MKTIIALSYIFCLVFADYKDDDDKAFSNQTYPTIEPKPFLYVVGRKKMMDAQYKCYDRMQQLPAYQGEGPYCNRTWDGWLCWDDTPAGVLSYQFCPDYFPDFDPSEKVTKYCDEKGVWFKHPENNRTWSNYTMCNAFTPEKLKNAYVLYYLAIVGHSLSIFTLVISLGIFVFFRSLGCQRVTLHKNMFLTYILNSMIIIIHLVEVVPNGELVRRDPVSCKILHFFHQYMMACNYFWMLCEGIYLHTLIVVAVFTEKQRLRWYYLLGWGFPLVPTTIHAITRAVYFNDNCWLSVETHLLYIIHGPVMAALVVNFFFLLNIVRVLVTKMRETHEAESHMYLKAVKATMILVPLLGIQFVVFPWRPSNKMLGKIYDYVMHSLIHFQGFFVATIYCFCNNEVQTTVKRQWAQFKIQWNQRWGRRPSNRSARAAAGSGGSGGSGGSGGLNDIFEAQKIEWHEGSGGSGGSGGSGDTENIRRVFNDCRDIIQRMHLRQYELL*SEO ID No. 2MKTIIALSYIFCLVFADYKDDDDKATPEKLKNAYVLYYLAIVGHSLSIFTLVISLGIFVFFRSLGCQRVTLHKNMFLTYILNSMIIIIHLVEVVPNGELVRRDPVSCKILHFFHQYMMACNYFWMLCEGIYLHTLIVVAVFTEKQRLRWYYLLGWGFPLVPTTIHAITRAVYFNDNCWLSVETHLLYIIHGPVMAALVVNFFFLLNIVRVLVTKMRETHEAESHMYLKAVKATMILVPLLGIQFVVFPWRPSNKMLGKIYDYVMHSLIHFQGFFVATIYCFCNNEVQTTVKRQWAQFKIQWNQRWGRRPSNRSARAAAGSGGSGGSGGSGGLNDIFEAQKIEWHEGSGGSGGSGGSGDTE NIRRVFNDCR DIIQRMHLRQ YELL*

Claims

CLAIMS1. A fusion of a G protein coupled receptor (GPCR) and an effector protein of the GPCR; wherein the effector protein is an alpha subunit of a G protein (Ga); wherein the N-terminus of Ga terminal helix 5 is fused to the C-terminus of an amino acid of the GPCR to form the GPCR-Ga,H5 fusion; and wherein the fusion of the Ga subunit to the GPCR stabilizes the GPCR in an apo active state without the presence of a GPCR ligand.

2. The GPCR-Ga,H5 fusion of claim 1 , wherein the fusion does not comprise a beta (G0) subunit or a gamma (Gy) subunit.

3. The GPCR-Ga,H5 fusion of any of claims 1-2, wherein the G protein is of a subtype selected from S, I, O and Q.

4. The GPCR-Ga,H5 fusion of any of claims 1-3, wherein the GPCR is selected from a class A GPCR and a class B GPCR.

5. The GPCR-Ga,H5 fusion of any of claims 1-4, wherein the GPCR is selected from an amylin / calcitonin receptor, a glucagon receptor, a glucose-dependent insulinotropic polypeptide (GIP) receptor, a glucagon-like peptide 1 (GLP-1 ) receptor, and a neuropeptide Y type 2 receptor (NPY2R).

6. The GPCR-Ga,H5 fusion of any of claims 1-5, wherein the fusion comprises a junction between the GPCR and the Ga terminal helix 5; and wherein the junction comprises one or more linkers.

7. The GPCR-Ga,H5 fusion of claim 6, wherein each linker comprises one or more GSG units.

8. The GPCR-Ga,H5 fusion of any one of claims 1-7, wherein the fusion of the Ga,H5 to the GPCR stabilizes the GPCR in an apo active state after the fused protein is extracted from a cell membrane.

9. The purified and stable GPCR-Ga,H5 fusion of claim 8, wherein the fusion is suitable for a downstream characterization or screening study.

10. The purified and stable GPCR-Ga.H5 fusion of claim 9, wherein the characterization or screening study uses an Affinity Selection Mass Spectrometry (ASMS) as an analytical tool.

11. The pur ified and stable GPCR-Ga,H5 fusion of claim 9, wherein the char acterization or screening study uses a Surface Plasmon Resonance (SPR) as an analytical tool.

12. The purified and stable GPCR-Ga,H5 fusion of claim 1 1 , wherein: the fusion comprises a junction between the GPCR and the Ga,H5; the junction comprises an affinity tag which allows for a capture of the purified fusion protein on an SPR chip; and the junction comprises a linker between the GPCR and the affinity tag and a linker between the affinity tag and the Ga,H5.

13. The purified and stable GPCR-Ga,H5 fusion of claim 12, wherein each linker comprises 4 GSG units.

14. The purified and stable GPCR-Ga,H5 fusion of claim 13, wherein the fusion is used for screening a ligand that binds to the apo active GPCR.

15. The purified and stable GPCR-Ga,H5 fusion of claim 14, wherein the ligand is a small molecule ligand or a biologic ligand.

16. The GPCR-Ga,H5 fusion of claim 8, wherein the GPCR is an amylin / calcitonin receptor.

17. The GPCR-Ga,H5 fusion of claim 8, wherein the GPCR is a glucagon receptor.

18. The GPCR-Ga,H5 fusion of claim 8, wherein the GPCR is a glucose-dependent insulinotropic polypeptide (GIP) receptor.

19. The GPCR-Ga.H5 fusion of claim 8, wherein the GPCR is a glucagon-like peptide 1 (GLP-1 ) receptor.

20. The GPCR-Ga,H5 fusion of claim 8. wherein the GPCR is a neuropeptide Y type 2 receptor (NPY2R).

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