Patent Application
20250164508
The text of the computer readable sequence listing filed herewith, titled “39893_601_SequenceListing”, created Feb. 16, 2023, having a file size of 44,350 bytes, is hereby incorporated by reference in its entirety.
Provided herein are methods, compositions, kits and systems for in-solution assays of G-Protein Coupled Receptor (GPCR) activity. In particular, provided herein are methods, compositions, kits and systems comprising a fusion protein comprising a GPCR and a first nanoluciferase subunit, a GPCR conformation specific binder bound to a second nanoluciferase subunit wherein the GPCR conformation specific binder binds to an active GPCR, and a bioluminescent substrate to detect GPCR activation when the GPCR is bound to a ligand or drug in solution.
G-protein coupled receptors (GPCRs) are critical participants in diverse cellular signaling pathways. (Irannejad, R.; Tomshine, J. C.; Tomshine, J. R.; Chevalier, M.; Mahoney, J. P.; Steyaert, J.; Rasmussen, S. G. F.; Sunahara, R. K.; El-Samad, H.; Huang, B.; von Zastrow, M. Conformational Biosensors Reveal GPCR Signalling from Endosomes. Nature 2013, 495 (7442), 534-538.) GPCRs function by converting an extracellular signal into intracellular signaling cascades by a ligand-induced transmembrane conformational change that leads to G-protein binding and activation. G-protein coupled receptors (GPCRs) are a class of seven transmembrane proteins that function as essential intracellular signal transducers. (Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3, 639-650 (2002). Approximately 30% of FDA approved therapeutics target GPCRs. Santos, R. et al. A comprehensive map of molecular drug targets. Nat Rev Drug Discov 16, 19-34 (2017). Congreve, M., de Graaf, C., Swain, N. A. & Tate, C. G. Impact of GPCR Structures on Drug Discovery. Cell 181, 81-91 (2020). Due to the complexity of their signaling pathways and high modularity, GPCRs remain crucial targets for new therapeutic development. Wacker, D., Stevens, R. C. & Roth, B. L. How Ligands Illuminate GPCR Molecular Pharmacology. Cell 170, 414-427 (2017). Live cell-based assays have been instrumental for GPCR drug screening, as well as GPCR signaling and mechanistic studies. (Yasi, E. A., Kruyer, N. S. & Peralta-Yahya, P. Advances in G protein-coupled receptor high-throughput screening. Current Opinion in Biotechnology 64, 210-217 (2020), Siehler, S. Cell-based assays in GPCR drug discovery. Biotechnol. J. 3, 471-483 (2008). Martins, S. A. M. et al. Towards the miniaturization of GPCR-based live-cell screening assays, Trends in Biotechnology 30, 566-574 (2012), Haider, R. S., Godbole, A. & Hoffiann, C. To sense or not to sense new insights from GPCR-based and arrestin-based biosensors. Current Opinion in Cell Biology 57, 16-24 (2019), Sakamoto, S., Kiyonaka, S. & Hamachi, I. Construction of ligand assay systems by protein-based semisynthetic biosensors. Current Opinion in Chemical Biology 50, 10-18 (2019). Olsen, R. H. J. et al. TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome. Nat Chem Biol 16, 841-849 (2020). Olsen, R. H. J. & English, J. G. Advancements in G protein-coupled receptor biosensors to study GPCR-G protein coupling. British Journal of Pharmacology (2022), Horing, C. et al. A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes. IJMS21, 8440 (2020), Hauge Pedersen, M. et al. A novel luminescence-based β-arrestin recruitment assay for unmodified receptors. Journal of Biological Chemistry 296, 100503 (2021), Inoue, A. et al. Illuminating G-Protein-Coupling Selectivity of GPCRs. Cell 177, 1933-1947.e1925 (2019).) However, there is still a lack of accessible and generalizable methods for detecting GPCR activation for broad applications. For example, it is infeasible with live cell assays to validate extracted GPCR protein's functionality for biochemical and structural studies.
As noted, over 34% of Food and Drug Administration (FDA) approved drugs target more than 100 members of the GPCR family. (Hauser A. S.; Chavali S.; Masuho I.; Jahn L. J.; Martemyanov K. A.; Gloriam D. E.; Babu M. M. Pharmacogenomics of GPCR Drug Targets. Cell. 2018 172, (1-2): 41-54.e19.) Over 830 different human genes comprising over 4% of the protein-coding genome are predicted to code for GPCRs based on genome sequence analysis. (Bjarnadóttir T. K.; Gloriam D. E.; Hellstrand S. H.; Kristiansson H.; Fredriksson R.; Schiöth H. B. Comprehensive repertoire and phylogenetic analysis of the G-protein-coupled receptors in human and mouse Genomics. 2006 88 (3): 263-73.) To identify therapeutic GPCRs, versatile and robust methods are needed to acquire information about how ligands activate GPCRs, and to screen ligands for agonist and antagonist discovery. (Laschet, C.; Dupuis, N.; Hanson, J. A Dynamic and Screening-Compatible Nanoluciferase-Based Complementation Assay Enables Profiling of Individual GPCR-G Protein Interactions. Journal of Biological Chemistry 2019, 294 (11), 4079-4090. Zeng, W.; Guo, L.; Xu, S.; Chen, J.; Zhou, J. High-Throughput Screening Technology in Industrial Biotechnology. Trends Biotechnol. 2020, 38 (8), 888-906.) A preferred screening platform is adaptable to test hundreds of thousands of candidate GPCR ligands. (Soave, M.; Heukers, R.; Kellam, B.; Woolard, J.; Smit, M. J.; Briddon, S. J.; Hill, S. J. Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies. Cell Chem. Biol. 2020, 27(10), 1250-1261.)
Methods to test GPCR activation that exploit shared properties of GPCRs including GPCR structure, downstream signaling, and binding partners are labor-intensive and require use of toxic radioactive chemicals for drug screening. (Zhang, R.; Xie, X. Tools for GPCR Drug Discovery. Acta Pharmacol Sin 2012, 33 (3), 372-384. Manglik, A.; Kobilka, B. K.; Steyaert, J. Nanobodies to Study G Protein-Coupled Receptor Structure and Function. Annu. Rev. Pharmacol. Toxicol. 2017, 57 (1), 19-37.) Other approaches comprising radio-labeled ligand binding and activation-dependent fluorescent sensors require individualized engineering of each GPCR of interest. (Dixon, A. S.; Schwinn, M. K.; Hall, M. P.; Zimmerman, K.; Otto, P.; Lubben, T. H.; Butler, B. L.; Binkowski, B. F.; Machleidt, T.; Kirkland, T. A.; Wood, M. G.; Eggers, C. T.; Encell, L. P.; Wood, K. V. NANOLUC Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem. Biol. 2016, 11 (2), 400-408.) Additional methods rely on detection of downstream signaling effects including production of cyclic-AMP (cAMP), as well as an increase in concentration of calcium (Ca2+). (Laschet, ibid) However, detection of downstream signaling events may result in non-specific and unreliable indicators of GPCR activation. Alternative assays employ ligand-induced protein interactions between a GPCR and a G-protein, or between a GPCR and arrestin. (Nuber, S.; Zabel, U.; Lorenz, K.; Nuber, A.; Milligan, G.; Tobin, A. B.; Lohse, M. J.; Hoffmann, C. β-Arrestin Biosensors Reveal a Rapid, Receptor-Dependent Activation/Deactivation Cycle. Nature 2016, 531 (7596), 661-664.) Though often used for in vivo screening of GPCR ligands, obstacles arise from adaptation of these method to high-throughput, live-cell screening platforms that require culture and handling of large batches of adherent cells while maintaining consistency between different batches of cells. A first obstacle is that culturing large batches of adherent cells for high-throughput screening (HTS) is time-consuming. A second obstacle is that in vivo GPCR assay results often vary due to changes in protein expression levels, and a lack of uniform membrane trafficking. Such a lack of consistency from batch to batch of cells results in inconsistent activation readouts. Accordingly, there is a need for in-solution assays for GPCR ligand HTS of greater efficiency, convenience in cell harvest and storage, uniformity of reagents, and consistency between screening assays.
Provided herein are methods, compositions, kits and systems for in-solution assays of G-Protein Coupled Receptor (GPCR) activity. In particular, provided herein are methods, compositions, kits and systems comprising a fusion protein comprising a GPCR and a first nanoluciferase subunit, a GPCR conformation specific binder bound to a second nanoluciferase subunit wherein the GPCR conformation specific binder binds to an active GPCR, and a bioluminescent substrate to detect GPCR activation when the GPCR is bound to a ligand or drug in solution.
In some embodiments, the present invention provides a method of measuring ligand activation of a G-protein coupled receptor (GPCR) in solution, comprising: expressing a GPCR fusion protein comprising a GPCR and a NANOLUC LgBit subunit or a NANOLUC SmBit subunit in a cell, extracting the GPCR fusion protein from the cell, generating a reaction mixture in solution comprising the GPCR fusion protein with a conformation specific nanobody wherein the nanobody is bound to a NANOLUC LgBit subunit, or NANOLUC SmBit subunit complementary to the NANOLUC SmBit subunit, or NANOLUC LgBit subunit fused to the GPCR, wherein binding of the NANOLUC LgBit to the NANOLUC SmBit generates a LgBit:SmBit luciferase, a bioluminescent substrate, and a buffer, adding the ligand to the reaction mixture in the solution, and measuring luminescence of the reaction mixture in the solution comprising the ligand wherein luminescence intensity indicates ligand activation of the GPCR. In some embodiments, the GPCR comprises a B2AR GPCR, a mu-opioid receptor (MOR) GPCR, or a dopamine receptor D1 (DRD1) GPCR. In some embodiments, the cell is a prokaryotic cell or a mammalian cell. In some embodiments, the conformation specific nanobody comprises Nb80, Nb39 or Nb40. In some embodiments, the NANOLUC LgBit subunit is 17.5 kDa. In some embodiments, the NANOLUC SmBit comprises 11 amino acids.
In some embodiments, the present invention provides method of measuring ligand activation of a G-protein coupled receptor (GPCR) in solution, comprising: expressing a GPCR fusion protein comprising the GPCR and a NANOLUC LgBit subunit or a NANOLUC SmBit subunit in a cell, extracting the GPCR fusion protein from said cell, generating a reaction mixture in solution comprising the GPCR fusion protein with a conformation specific peptidomimetic wherein the conformation specific peptidomimetic is bound to a NANOLUC LgBit subunit, or NANOLUC SmBit subunit complementary to the NANOLUC SmBit subunit, or NANOLUC LgBit subunit fused to the GPCR, wherein binding of the NANOLUC LgBit to the NANOLUC SmBit generates a LgBit:SmBit luciferase, a bioluminescent substrate, and a buffer, adding the ligand to the reaction mixture in the solution; and measuring luminescence of the reaction mixture in the solution comprising the ligand wherein luminescence intensity indicates ligand activation of the GPCR. In some embodiments, the GPCR comprises a beta-2-adrenergic receptor (B2AR) GPCR, a mu-opioid receptor (MOR) GPCR, or a dopamine receptor D1 (DRD1) GPCR. In some embodiments, the cell is a prokaryotic cell or a mammalian cell. In some embodiments, the conformation specific peptidomimetic is selected from the group consisting of SEQ ID NO.: 1 to SEQ ID NO.: 46. In some embodiments, the NANOLUC LgBit subunit is 17.5 kDa. In some embodiments, the NANOLUC SmBit comprises 11 amino acids.
In some embodiments, the present invention provides a kit, comprising: a GPCR fusion protein comprising the GPCR and a NANOLUC LgBit subunit or a NANOLUC SmBit: a conformation specific nanobody wherein the nanobody is bound to a NANOLUC LgBit subunit or NANOLUC SmBit subunit complementary to the NANOLUC LgBit subunit or NANOLUC SmBit subunit fused to the GPCR. a bioluminescent substrate; and a buffer. In some embodiments, the kit comprises one or more test ligands and/or one or more control ligands.
In some embodiments, the present invention provides a kit, comprising: a GPCR fusion protein comprising the GPCR and a NANOLUC LgBit subunit or a NANOLUC SmBit: a conformation specific peptidomimetic wherein the conformation specific peptidomimetic is bound to a NANOLUC LgBit subunit or NANOLUC SmBit subunit complementary to the NANOLUC LgBit subunit or NANOLUC SmBit subunit fused to the GPCR. a bioluminescent substrate; and a buffer. In some embodiments, the kit comprises one or more test ligands and/or one or more control ligands.
In some embodiments, the present invention provides a composition, comprising a GPCR fusion protein comprising the GPCR and a NANOLUC LgBit subunit or a NANOLUC SmBit, a conformation specific nanobody, wherein the nanobody is bound to a NANOLUC LgBit subunit or NANOLUC SmBit subunit complementary to the NANOLUC LgBit subunit or NANOLUC SmBit subunit fused to the GPCR, a bioluminescent substrate, and a buffer. In some embodiments the composition comprises one or more test ligands and/or one or more control ligands.
In some embodiments, the present invention provides a composition, comprising a GPCR fusion protein comprising the GPCR and a NANOLUC LgBit subunit or a NANOLUC SmBit, a conformation specific peptidomimetic, wherein the conformation specific peptidomimetic is bound to a NANOLUC LgBit subunit or NANOLUC SmBit subunit complementary to the NANOLUC LgBit subunit or NANOLUC SmBit subunit fused to the GPCR, a bioluminescent substrate, and a buffer. In some embodiments the composition comprises one or more test ligands and/or one or more control ligands.
In some embodiments, activated-GPCR-conformation-specific binders and G-protein mimics, including nanobodies, antibodies, mini-Gs proteins and peptidomimetics are bound to the split NANOLUC subunits to assay GPCR activity. In some embodiments, any protein that selectively binds with greater affinity to the activated form of the GPCR find use in the assay. In some embodiments, nanobodies are bound to either the N- or C-terminus of the split NANOLUC subunits. In some embodiments, mini-G proteins and G-protein peptidomimetics are fused to the C-terminus of the split NANOLUC subunits such that their C-terminus is free of fusion to interact with an activated GPCR.
In some embodiments, the present invention provides in vitro assay with a detergent-free solubilization buffer to resuspend the cell-pellet, and sonication to lyse the cells to prepare a homogeneous cell lysate containing the native GPCR membrane component. In some embodiments, when using a peptidomimetic as a conformation-specific binder, a GPCR membrane component and a SmBit-peptidomimetics fusion peptide solution are prepared separately and mixed during assays. In some embodiments, when using nanobodies and mini-G proteins as conformation specific binders, the nanobody-SmBit and SmBit-mini-G protein fusion proteins are co-expressed together as a single reagent or expressed separately as two reagents.
In some embodiments, the assay is performed with a saturated concentration of agonists in parallel with a no-drug control group comprising vehicle only. In some embodiments, a T-test is used to compare the 2 groups of samples. If a >1-fold drug-dependent luminescence increase and statistical significance (p<0.05) using student T-test is achieved, the reagents and conditions meet performance standards.
In some embodiments, the present invention provides a generalizable and accessible In vitro GPCR split NANOLUC ligand Triggered Reporter (IGNiTR), having broad and diverse applications. IGNiTR leverages the interaction between a conformation-specific binder and agonist-activated GPCR to reconstitute a split nanoluciferase. In some embodiments, IGNiTR comprises three Gs-coupled GPCRs and a Gi-coupled GPCR with three classes of conformation-specific binders: nanobodies, miniG proteins, and G-protein peptidomimetics. IGNiTR demonstrates binding efficacy and potency values of diverse Dopamine Receptor D1 (DRD1) ligands. IGNiTR further supports use of a synthetic G protein peptidomimetic, thereby providing easily standardized reagents for characterizing GPCRs and ligands. In some embodiments IGNiTR finds use in: 1) characterizing GPCR functionality during Nanodisc-based reconstitution process; 2) high-throughput screening of ligands against DRD1; and 3) detection of opioids for in the field applications. Because of convenience, accessibility and consistency, IGNiTR supports extensive applications in GPCR ligand detection, screening and GPCR characterization.
In some embodiments, the present invention provides an in vitro assay, based on GPCR protein in cell lysate in a simple and easily adaptable format for broad applications. Existing in vitro assays, including radioligand binding, monitor GPCR-ligand binding, but do not measure ligand efficacy for inducing the active conformation, which recruits downstream G-proteins. (Yasi, E. A., Kruyer, N. S. & Peralta-Yahya, P. Advances in G protein-coupled receptor high-throughput screening. Current Opinion in Biotechnology 64, 210-217 (2020), Wiseman, D. N. et al. Expression and purification of recombinant G protein-coupled receptors: A review. Protein Expression and Purification 167, 105524 (2020), Enrico Rovati, G. Ligand-binding studies: old beliefs and new strategies. Trends in Pharmacological Sciences 19, 365-369 (1998). Masureel, M. et al. Structural insights into binding specificity, efficacy and bias of a β2AR partial agonist. Nat Chem Biol 14, 1059-1066 (2018). Keen, M. in Signal Transduction Protocols Vol. 41 1-16 (Humana Press, 1995), Saumell-Esnaola, M. et al. Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods. Microb Cell Fact 21, 192 (2022), Volz, M. R. & Moosmann, B. Development of a non-radioactive mass spectrometry-based binding assay at the μ-opioid receptor and its application for the determination of the binding affinities of 17 opiates/opioids as well as of the designer opioid isotonitazene and five further 2-benzylbenzimidazoles. Analytica Chimica Acta 1219, 339978 (2022).) An alternative approach, reconstitution of split bioluminescent enzymes, is used to report on protein-protein interactions. We harness the robust luminescent signal that is quantifiable in a complex biological environment to track a ligand-induced binding interaction. (Soave, M. et al. Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies. Cell Chemical Biology 27, 1250-1261.e1255 (2020), Israeli, H. et al. Structure reveals the activation mechanism of the MC4 receptor to initiate satiation signaling. Science 372, 808-814 (2021). Laschet, C., Dupuis, N. & Hanson, J. A dynamic and screening-compatible nanoluciferase-based complementation assay supports profiling of individual GPCR-G protein interactions. Journal of Biological Chemistry 294, 4079-4090 (2019).)
In some embodiments, IGNiTR leverages agonist-dependent GPCR conformational change and subsequent recruitment of G-proteins and other conformation-specific binders (Roth, B. L., Irwin, J. J. & Shoichet, B. K. Discovery of new GPCR ligands to illuminate new biology. Nat Chem Biol 13, 1143-1151 (2017), Weis, W. I. & Kobilka, B. K. The Molecular Basis of G Protein-Coupled Receptor Activation. Annu. Rev. Biochem. 87, 897-919 (2018).) to reconstitute split nanoluciferase (NANOLUC) (Roth, B. L., Irwin, J. J. & Shoichet, B. K. Discovery of new GPCR ligands to illuminate new biology. Nat Chem Biol 13, 1143-1151 (2017), Weis, W. I. & Kobilka, B. K. The Molecular Basis of G Protein-Coupled Receptor Activation. Annu. Rev. Biochem. 87, 897-919 (2018).) (Dixon, A. S. et al. NANOLUC Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem. Biol. 11, 400-408 (2016). Ni, Y., Arts, R. & Merkx, M. Ratiometric Bioluminescent Sensor Proteins Based on Intramolecular Split Luciferase Complementation. ACS Sens. 4, 20-25 (2019).) Unlike live cell assays, IGNiTR components are easily stored with cell pellets expressing GPCR components frozen to preserve the integral, native lipid environment. Additionally, IGNiTR allows the use of peptidomimetics as conformation-specific binders, broadening assay applications.
In some embodiments, IGNiTR supports three applications. First, IGNiTR provides a robust platform for high-throughput screening of ligands against Dopamine Receptor D1. Second, IGNiTR rapidly detects the μ-opioid receptor agonist fentanyl at nanomolar range in an easy and portable setup for potential field applications. Third, IGNiTR is used characterize protein functionality of GPCR protein samples at different stages of the Nanodisc-based GPCR extraction and reconstitution process. IGNiTR's adaptability supports unique applications complementary to live cell assays or existing in vitro assays.
To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. While the invention will be described in conjunction with certain representative embodiments, it will be understood that the invention is not limited to these illustrative examples. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein may be used in the practice of the present invention. Unless defined otherwise, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the invention, certain methods, devices, and materials are described herein. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.
As used in this disclosure, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.”
As used herein, the term “about” represents an insignificant modification or variation of the numerical value such that the basic function of the item to which the numerical value relates is unchanged.
As used herein, “protein” is used synonymously with “peptide,” “polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from the cell, tissue, or cell-free source from which the amino acid sequence is obtained, or substantially free from chemical precursors or other chemicals when chemically synthesized.
As used herein, “modulate” means to alter, either by increasing or decreasing, the activity of a gene or protein. The term “inhibit”, as used herein, means to prevent or reduce the activity of gene or protein.
As used herein, the term “bioactivity” indicates an effect on one or more cellular or extracellular process (e.g., via binding, signaling, etc.) that can impact physiological or pathophysiological processes.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise). Any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated.
As used herein, the term “cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
As used herein, the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell lysate. The term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
Provided herein are methods, compositions, kits and systems for in-solution assays of G-Protein Coupled Receptor (GPCR) activity. In particular, provided herein are methods, compositions, kits and systems comprising a fusion protein comprising a GPCR and a first nanoluciferase subunit, a GPCR conformation specific binder bound to a second nanoluciferase subunit wherein the GPCR conformation specific binder binds to an active GPCR, and a bioluminescent substrate to detect GPCR activation when the GPCR is bound to a ligand or drug in solution.
In some embodiments, the present invention provides versatile methods, compositions, systems and kits to perform an in-solution assay that serves as a robust platform to screen GPCR ligands, and to characterize activation of GPCR proteins isolated from the membrane. Some embodiments comprise a quantifiable, bioluminescent signal coupled to the conformational change of a GPCR induced by ligand binding (
In some embodiments, the present invention comprises conformation-specific binders that bind only to the active state of the GPCR to detect GPCR activity. Some embodiments comprise reconstitution of the protein interaction-dependent split protein using the bioluminescent enzyme nanoluciferase (NANOLUC, Promega, Madison, WI). (Dixon, A. S.; Schwinn, M. K.; Hall, M. P.; Zimmerman, K.; Otto, P.; Lubben, T. H.; Butler, B. L.; Binkowski, B. F.; Machleidt, T.; Kirkland, T. A.; Wood, M. G.; Eggers, C. T.; Encell, L. P.; Wood, K. V. NANOLUC Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem. Biol. 2016, 11 (2), 400-408, Ni, Y.; Arts, R.; Merkx, M. Ratiometric Bioluminescent Sensor Proteins Based on Intramolecular Split Luciferase Complementation. ACS Sens. 2019, 4 (1), 20-25.) On activation of the GPCR by an agonist, a conformation-specific binder binds to the active-state GPCR and reconstitutes the split NANOLUC. (Soave, M.; Heukers, R.; Kellam, B.; Woolard, J.; Smit, M. J.; Briddon, S. J.; Hill, S. J. Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies. Cell Chem. Biol. 2020, 27(10), 1250-1261.) In some embodiments, in the presence of the substrate furimazine the reconstituted enzyme produces a quantifiable bioluminescent signal. (Dixon, ibid, Ni, ibid.) Rapid reconstitution of split NANOLUC and the resulting bioluminescent signal produces a quantifiable indicator of GPCR functionality and agonist activation in solution. In some embodiments, the present invention provides an in-solution assay for the characterization and quantification of GPCR activation, and for screening candidate activating, inactivating, and neutral ligands. Advantages of an in-solution assay over existing technologies include: 1) increased efficiency of GPCR expression in suspended mammalian cells on a large scale compared to adherent cells; 2) expression of GPCRs in different batches of cells; and 3) extraction of the proteins and their combination antecedent to aliquoting for different assays to enhance assay consistency. Mixing of the proteins before aliquoting provides uniformity of reagents, and greater consistency between different experiments than can be achieved using live cell protocols. Because the GPCR protein extract may be harvested and stored in advance, reduced is required for cell culture. In some embodiments, the GPCRs are isolated from cell lysate. Accordingly, the split NANOLUC-based in-solution assays of the present invention provide a cost-effective and convenient platform to characterize the functionality, folding and activation of diverse membrane-extracted GPCRs.
In some embodiments, the present invention provides a generalizable in vitro GPCR assay, IGNiTR, able to characterize a GPCR's structural integrity and activity by detecting the agonist-induced interaction of the GPCR with a conformation-specific binder. IGNiTR comprises features that are both complementary to and advantageous over live cell-based assays. First, IGNiTR components, including the GPCR and the conformation-specific binder components may be prepared in advance and stored frozen until usage. Second, IGNiTR may be performed without the restrictions of working with live mammalian cells following biosafety level 2 regulations. Third, the preparation of IGNiTR in a cell lysate solution supports use of a synthetic fusion G protein peptidomimetic, with concentrations that are well-controlled for assay fine-tuning, including optimization of DDR. Fourth, mixing of the components supports standardization of the reaction conditions in thousands of wells to achieve consistency across HTS plates. IGNiTR provides multiple advantages over existing in vitro assays. IGNiTR's bioluminescent readout is quantifiable in a single step, and therefore is easily scaled up and performed in the field while existing in vitro GPCR assays, including the radioligand assay, require a complex protocol and platform. In some embodiments, the present invention support diverse applications in, or example: 1) HTS of GPCR ligands; 2) characterization and detection of GPCR ligands in the lab and in the field; and 3) verification of GPCR structural integrity antecedent to and during in vitro diverse GPCR characterizations.
In some embodiments, the present invention provides in-solution GPCR ligand screening using conformation-specific binders. In some embodiments, the conformation-specific binder comprises one or more nanobodies. In some embodiments, the GPCR is a Mu-opioid receptor (MOR). MORs regulate pain modulation. Opioid molecules that target MORs comprise the most potent pain medications. (Darcq, E.; Kieffer, B. L. Opioid Receptors: Drivers to Addiction? Nat. Rev. Neurosci. 2018, 19 (8), 499-514.) However, available synthetic opioids have severe side effects, including addiction and ventilatory depression. (Wang, Y.; Fan, Z.; Shao, L.; Kong, X.; Hou, X.; Tian, D.; Sun, Y.; Xiao, Y.; Yu, L. Nanobody-Derived Nanobiotechnology Tool Kits for Diverse Biomedical and Biotechnology Applications. Int. J. Nanomedicine 2016, Volume 11, 3287-3303.) Conventional in vivo assays for screening opioids rely on late-stage events in the MOR signaling cascade. A robust and easily scalable in-solution assay for screening opioid ligands by detecting the early-stage signaling events is needed to screen alternative non-addictive opioids. In some embodiments, the present invention provides in-solution MOR ligand screening assay. As shown in
In some embodiments, the present invention provides in-solution GPCR ligand screening methods, compositions, systems and kits comprising G-protein peptidomimetic conformation-specific binders. In some embodiments, a G-protein peptidomimetic is fused with LgBit of the NANOLUC Assay, and SmBit of the NANOLUC assay is fused with a GPCR. In some embodiments, a G-protein peptidomimetic is fused with SmBit of the NANOLUC Assay, and LgBit of the NANOLUC assay is fused with a GPCR. G-protein peptidomimetic conformation-specific binders support in-solution assays of the present invention that are adaptable with a broad diversity of GPCRs of value in high-throughput ligand screening. Use of G-protein peptidomimetics is highly advantageous given its capacity to use unnatural amino acids to achieve increased stability and higher binding affinity for the GPCR target. (Kapolka, N. J.; Taghon, G. J.; Rowe, J. B.; Morgan, W. M.; Enten, J. F.; Lambert, N. A.; Isom, D. G. DCyFIR: A High-Throughput CRISPR Platform for Multiplexed G Protein-Coupled Receptor Profiling and Ligand Discovery. Proc. Natl. Acad. Sci. 2020, 117(23), 13117.) In turn, peptidomimetic conformation-specific binders provide economic advantages that arise from the low molecular weight of the peptide, and use of peptide synthesis to bypass protein expression and purification. In some embodiments, a 10 mg peptidomimetic synthesis may be used for at least 10,000 assay reactions. Accordingly, peptidomimetic conformation-specific binders for in-solution ligand screening transform the GPCR screening process to identify candidate agonists for GPCR targets including, for example, GPCRs that participate in drug dependency.
The present disclosure provides kits comprising components of the GPCR ligand binding assays described herein. Such kits may comprise, for example, two or more of 1) at least one conformation specific nanobody, mini-G or peptidomimetic binder; 2) at least one nanoluciferase comprising a LgBit and a SmBit; 3) a substrate; and 4) reagents and cells for protein expression. Additional kit components may optionally include, for example: 1) stabilizers and/or a buffer; 2) at least one container, vial or similar apparatus for holding and/or mixing the kit components; 3) a reagent transfer apparatus; and/or 4) instructions for using the components of the kit to perform a GPCR binding assay.
In some embodiments, the present invention provides a kit with a membrane-bound GPCR and a cytosolic nanobody-SmBit and SmBit-mini-G protein fusion protein. In some embodiments, in a kit with a membrane-bound GPCR, the cytosolic nanobody-SmBit and SmBit-mini-G protein fusion proteins are expressed separately for greater versatility to pair the GPCR with either a nanobody, a mini-G protein or a peptidomimetic. In some embodiments, a peptidomimetic is derived from the C-terminal helical domain of a G-proteins, or the chimeric form of multiple G-protein C-terminal helical domains, and/or comprises mutations or other unnatural amino acids.
In some embodiments, a kit of the present invention comprises positive and negative control reagents and ligands to identify possible sources of error and contamination. In some embodiments, a kit of the present invention comprises calibration reagents and ligands including, for example, a luminescence calibration reagent. In some embodiments, a kit of the present invention comprises a known GPCR assay with a same-class GPCR and same class ligand for comparison to a new ligand to be screened. In some embodiments, a kit provides a synthetic HiBit peptide to titrate with the GPCR-expressing cell pellet as a positive control. In the presence of the NANOLUC substrate, the HiBit reconstitutes with the LgBit in a cell pellet expressed and attached to a GPCR, and a luminescent signal confirms the correct expression of the protein.
In some embodiments, a kit comprises customizable options. In some embodiments, a kit of the present invention comprises frozen (e.g., prepared with glycerol for storing at −20 C degrees) mammalian cell pellets containing GPCRs expressed and attached to LgBit. To perform an activation screening in vitro, a client may order a kit with a GPCR(s) of choice. In addition to the cell pellet, the kit may include a corresponding binding partner, for example, a nanobody attached to SmBit or a peptidomimetic.
In some embodiments, a kit comprises:
In some embodiments, a kit comprises a solution of SmBit bound to a peptidomimetic, and isoproterenol solution for activating 1 B2AR GPCR. The positive control may be provided to ensure that the cell pellet is well-stored and the protocol works.
In some embodiments, a kit comprises:
In some embodiments, a kit of the present invention comprises:
In some embodiments, a kit of the present invention provides:
In some embodiments, a positive control provides B2AR-LgBit and SmBit bound to Nb80, mini-Gs, and Gs-peptidomimetic with >2-fold isoproterenol (10 uM)-dependent luminescence increase, achieving statistical significance (p<0.05) using a student's T-test comparing the +isoproterenol condition to the no-drug vehicle group, indicating that the positive control experiment performs to specification.
The present disclosure provides compositions and reaction mixtures comprising components of the GPCR ligand binding assays described herein. Such compositions and reaction mixtures may comprise, for example, two or more of 1) at least one conformation specific nanobody, mini-G or peptidomimetic binder; 2) at least one nanoluciferase comprising a LgBit and SmBit; 3) a substrate; and 4) reagents and cells for protein expression. Additional composition and reaction mixture components may optionally include stabilizers and/or a buffer.
The following examples are provided to demonstrate and further illustrate certain embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.
A NANOLUC assay in HEK293T cell culture was performed to screen diverse split NANOLUC fusion constructs to test whether orientation of the protein components affects the relative signal output (
Reconstitution of split NANOLUC was used to track the binding of Nb39 to the Mu-opioid receptor (MOR). (Stoeber, M.; Jullié, D.; Lobingier, B. T.; Laeremans, T.; Steyaert, J.; Schiller, P. W.; Manglik, A.; von Zastrow, M. A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action. Neuron 2018, 98 (5), 963-976.e5, Vasudevan, L.; Stove, C. P. A Novel Nanobody-Based Bio-Assay Using Functional Complementation of a Split Nanoluciferase to Monitor Mu-Opioid Receptor Activation. Anal. Bioanal. Chem. 2020, 412 (29), 8015-8022.) The MOR was expressed with the SmBit attached to the C-terminus and Nb39 was expressed attached to LgBit on the C-terminus. Protein constructs were expressed in mammalian cell culture using the same method developed for the B2AR/Nb80 protein interacting partners above. Nb44 with binding affinity for the agonist-bound MOR was also tested. (Stoeber, M.; Jullié, D.; Lobingier, B. T.; Laeremans, T.; Steyaert, J.; Schiller, P. W.; Manglik, A.; von Zastrow, M. A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action. Neuron 2018, 98 (5), 963-976.e5.) As shown in
Next, a NANOLUC assay in-solution assay was tested using the B2AR-based GPCR. (
To test that the membrane protein component was folded correctly and able to be activated effectively by its agonist, the lysed solution was mixed with the cytosolic protein component solution. Initial testing used 10 uL of each solution added to a 96-well plate, followed by a brief incubation period with the agonist (˜10 μM per well). Substrate for the reconstituted NANOLUC is added in a buffer solution (1:19 substrate: buffer) as the final step before using the plate reader to detect and quantify the intensity of subsequent bioluminescence. If the well contained properly folded and functional membrane protein, the cytosolic protein (nanobody attached to LgBit) binds in the presence of the agonist, thereby allowing reconstitution of the split NANOLUC. As shown in
MOR-SmBit fusion in HEK293T cells and extraction of the membrane-bound MOR protein using the DDM/CHS detergent-based solution are performed as developed for the B2AR-based in-solution assay. Both Nb39 and Nb44 are fused to the LgBit fragment, and the protein composite is expressed in HEK293T cells and extracted as the soluble cell extract. Western Blots are used to confirm the presence of MOR-SmBit in the membrane component extract and Nb39/Nb44-LgBit in the soluble protein extract. The split NANOLUC assay is tested using different ratios of the MOR-SmBit and Nb39/Nb44-LgBit protein mix to establish optimal conditions for generating a large opioid-dependent signal change.
To optimize the dynamic range (or signal-to-noise ratio SNR) of the opioid-dependent bioluminescent signal increase, the optimal ratio of the membrane protein component to the cytosolic protein component is tested. The ratios are adjusted to measure which component is the limiting factor. Mixing the cell lysates together thoroughly before aliquoting into the 96-well plate is used to ensure assay to assay consistency. The agonist-incubation period is tested to assess effects on the signal fold-change. The amount of time between addition of the NANOLUC substrate addition and the bioluminescence measurement using the plate reader is varied to establish an optimal time delay.
A limiting factor when using adherent mammalian cells to produce membrane proteins such as GPCRs is that the monolayer of cells grown in the flasks limits the quantity of proteins to be extracted, and also the scale of the in-solution experiments. The in-solution assay is optimized by selection of a stable cell line for expressing the GPCR membrane proteins in non-adherent K562 cells to scale expression and extraction of the GPCR proteins. K562 cells in suspension cells provide larger batch expression compared to the single layer of cells grown using, for example, HEK293T. Protein expression is measured using Western Blots, and the correlation between membrane protein concentration and signal fold-change is determined.
Nanobody Expression in E. coli.
To simultaneously screen multiple opioid agonists at a time, production of the Nanobody-LgBit components on a large scale and their storage for easy access is desired. To this end, the Nb39-LgBit is expressed in the periplasm of E. coli following established protocols. (Mannes, M.; Martin, C.; Triest, S.; Pia Dimmito, M.; Mollica, A.; Laeremans, T.; Menet, C. J.; Ballet, S. Development of Generic G Protein Peptidomimetics Able to Stabilize Active State Gs Protein-Coupled Receptors for Application in Drug Discovery. Angew. Chem. Int. Ed. 2021, 60 (18), 10247-10254.) The Nb39-LgBit protein is purified using a Ni-NTA column, followed by Fast Protein Liquid Chromatography using a size exclusion column. The purity of the nanobody component is measured by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, and the concentration of the purified protein is determined by Bicinchoninic Acid (BCA) Protein Assay.
In-Solution Luminescent Assay with Different Concentrations of Protein Mix
Based on determined concentrations of MOR-SmBit, Nb39-LgBit, and Nb44-LgBit and the previously obtained optimal ratio of protein mix, assays with different concentrations of the split NANOLUC protein component mixture are evaluated to determine the assay's opioid-dependent fold change, and their opioid-sensitivity in correlation to the split NANOLUC protein concentrations. Opioid dose-response curves are generated from 0.01 nM to 1 mM opioids with GPCR-MOR concentrations at 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM to identify the minimum protein concentrations needed to obtain robust opioid-dependent luminescent signal for drug screening.
This experimental example provides an in-solution luminescent assay to screen opioid-dependent MOR activation by its induced interaction with Nb39 and resulting reconstitution of split NANOLUC. Two protocols for large scale production of the MOR-SmBit protein from suspension K562 cells are provided, antecedent to the expression and purification of Nb39-LgBit protein from E. coli cells. The working concentration range of the extracted proteins is determined to establish the minimum required protein concentrations necessary to obtain a robust opioid-dependent signal. The resulting in-solution assay provides a consistent and robust method for screening candidate MOR agonists.
In some embodiments, insect cells are used for the production of the MOR-SmBit protein. (Rasmussen, S. G. F.; Choi, H.-J.; Fung, J. J.; Pardon, E.; Casarosa, P.; Chae, P. S.; DeVree, B. T.; Rosenbaum, D. M.; Thian, F. S.; Kobilka, T. S.; Schnapp, A.; Konetzki, I.; Sunahara, R. K.; Gellman, S. H.; Pautsch, A.; Steyaert, J.; Weis, W. I.; Kobilka, B. K. Structure of a Nanobody-Stabilized Active State of the B2 Adrenoceptor. Nature 2011, 469 (7329), 175-180.) In some embodiments, suspension mammalian cells are used to express Nb-LgBit.
In some embodiments, a G-protein peptidomimetic is provided as a conformation-specific binder. In some embodiments, a Gs-protein mimics and that binds with high affinity for GPCRs that couple to Gs-proteins is provided. (Mannes, M.; Martin, C.; Triest, S.; Pia Dimmito, M.; Mollica, A.; Laeremans, T.; Menet, C. J.; Ballet, S. Development of Generic G Protein Peptidomimetics Able to Stabilize Active State Gs Protein-Coupled Receptors for Application in Drug Discovery. Angew. Chem. Int. Ed 2021, 60 (18), 10247-10254.) The peptides are designed based on the conserved features of the α5-helix of both the Gs-protein and the engineered mini-G protein bound to crystalized B2AR. (Mannes, ibid.) The α5-helix interacts with a cavity between transmembrane domains 3 and 5 of B2AR after activation of the GPCR, comprising, for example, a stapled peptide (Ac-KKKFNc[PraCRDAzk]IQRMHLRQYEChaL-OH) (SEQ ID NO.: 4). (Mannes, ibid.) In some embodiments, a peptide of the present invention comprises the unnatural amino acid cyclohexylalanine (Cha).
The Gs-protein mimic peptide is small and its generalizable for diverse Gs-GPCRs while still maintaining high affinity. In some embodiments, a protein component for an in-solution assay is generated using peptide synthesis of a Gs-mimic peptide (22 amino acids) fused with the SmBit peptide (11 amino acids) thereby eliminated the need for separately expressing a second protein component for the in-solution assays of the present invention. To streamline the synthesis of the hybrid peptide, we used the unstapled peptidomimetic that contains an unnatural amino acid (VTGYRLFEEILGSFNDCRDIIQRMHLRQYE-{Cha}-L) (SEQ ID NO.: 2). The SmBit portion of the hybrid peptide was tested for the capacity to reconstitute with extracted B2AR-LgBit. The membrane bound proteins were extracted using the membrane solubilization method described above (see “Testing split NANOLUC in-solution”. Cell lysate containing the fusion B2AR-LgBit protein was mixed with 10 μM of the hybrid SmBit-Gs-mimic peptide, and then assayed in a 96-well plate. The conditions were tested both with and without addition of B2AR agonist, isoproterenol (Iso). Furimazine substrate for NANOLUC was added last immediately before plate reading to quantify the bioluminescence. As shown in
In some embodiments, Gs-peptide mimic-based in-solution luminescent assays are of use in determining the activation state of diverse Gs-coupled GPCRs. The versatility of the Gs-mimic peptide-based is tested assay using additional Gs-coupled GPCRs. For example, the Dopamine Receptor D1 (DRD1) is fused to LgBit to evaluate dopamine-dependent bioluminescence (
Increased versatility of the in-solution split NANOLUC assay of the present invention for diverse GPCRs is provided through modification of the existing Gs-mimic peptide to recognize other G-protein coupled GPCRs, including Gi, Gq, and G12/13-coupled GPCRs, in an agonist dependent manner. Analysis of the binding interaction between the α5-helix of these G-proteins and the GPCR interface from the available crystal structures indicates that the last 5 amino acids of the α5-helix are important to confer specificity for different G-proteins. For example in a method termed Dynamic Cyan Induction by Functional Integrated Receptors (DCyFIR) replacement of the terminal 5 amino acids of the G-protein α5-helix changes its specificity for different types of GPCRs. (Kapolka, N. J.; Taghon, G. J.; Rowe, J. B.; Morgan, W. M.; Enten, J. F.; Lambert, N. A.; Isom, D. G. DCyFIR: A High-Throughput CRISPR Platform for Multiplexed G Protein-Coupled Receptor Profiling and Ligand Discovery. Proc. Natl. Acad. Sci. 2020, 117 (23), 13117.)
To test a Gi-mimic peptide, a peptide was tested with incorporation of the unnatural amino acid, Cha, at the penultimate position of the peptide mimic. A leucine (Leu) residue at the penultimate position of G-proteins is conserved in G-proteins, indicating its importance in binding to GPCRs. (Mannes, ibid.) Changing Leu to Cha improves the binding affinity of Gs-protein mimic for B2AR. We therefore incorporated Cha to the Gi peptidomimetic with sequence: VTGYRLFEEILGSKKKFDCVTDCIIKNNLKDSGLF (SEQ ID NO.: 5). The cysteine mutations were initially introduced to install disulfide bond to stabilize the peptidomimetics. However, we found that the disulfide bond formation made the G protein peptidomimetics less selective for the active conformation of the GPCR. Therefore, when testing this Gi peptidomimetic, we added DTT to reduce the disulfide bond.
Current assays for evaluating the correct folding of extracted GPCRs mainly rely on the radioligand binding assay. (Rasmussen, S. G. F.; Choi, H.-J.; Fung, J. J.; Pardon, E.; Casarosa, P.; Chae, P. S.; DeVree, B. T.; Rosenbaum, D. M.; Thian, F. S.; Kobilka, T. S.; Schnapp, A.; Konetzki, I.; Sunahara, R. K.; Gellman, S. H.; Pautsch, A.; Steyaert, J.; Weis, W. I.; Kobilka, B. K. Structure of a Nanobody-Stabilized Active State of the B2 Adrenoceptor. Nature 2011, 469 (7329), 175-180.) However, the ligand binding assay is tedious and difficult to perform in a high-throughput manner. Additionally, the ligand binding assay does not detect whether the GPCR is able to confer the agonist-induced conformational change that leads to G-protein binding, and is not suitable for high-throughput analysis. To ensure the correct folding and functionality of the extracted GPCR, high-throughput luminescent assays are needed to characterize the reconstituted GPCRs. (Dixon, A. S.; Schwinn, M. K.; Hall, M. P.; Zimmerman, K.; Otto, P.; Lubben, T. H.; Butler, B. L.; Binkowski, B. F.; Machleidt, T.; Kirkland, T. A.; Wood, M. G.; Eggers, C. T.; Encell, L. P.; Wood, K. V. NANOLUC Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem. Biol. 2016, 11 (2), 400-408.) As shown in
In some embodiments, the present invention provides an agonist-dependent fold change of at least 1.5-fold for in-solution assays to achieve a robust readout. In some embodiments, Z′ values are calculated by comparing the positive and negative binding results with multiple replicates to obtain a Z′ value >0.5, thereby indicating robustness of the assay for drug and ligand screening, and confirmation of the structural integrity of the extracted GPCR.
In some embodiments, mini-G proteins are used instead of and/or in addition to peptidomimetics. (Horing, C.; Seibel, U.; Tropmann, K.; Grätz, L.; Mönnich, D.; Pitzl, S.; Bernhardt, G.; Pockes, S.; Strasser, A. A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes. Int. J. Mol. Sci. 2020, 21 (22), 8440, Carpenter, B.; Tate, C. G. Engineering a Minimal G Protein to Facilitate Crystallisation of G Protein-Coupled Receptors in Their Active Conformation. Protein Eng. Des. Sel. 2016, 29 (12), 583-594.) Mini-G proteins are smaller versions of their G-protein counterparts to bind to corresponding GPCRs. (Nehmé, R.; Carpenter, B.; Singhal, A.; Strege, A.; Edwards, P. C.; White, C. F.; Du, H.; Grisshammer, R.; Tate, C. G. Mini-G Proteins: Novel Tools for Studying GPCRs in Their Active Conformation. PLoS ONE 2017, 12 (4), e0175642.) By virtue of their size and stabilizing effects. Mini-G proteins are candidates for expression and co-crystallization. Similar to the nanobody-based assay, the LgBit-mini-G or SmBit-mini-G fusion protein may be expressed, purified, and stored separately.
Bulk growth of HEK293 cell culture was performed in T-150 flask to 80-100% confluence (
Cell pellets were thawed on ice and resuspended in 200 uL membrane resuspension buffer (resuspension buffer comprising 20 mM HEPES, pH 7.5, 2 mM MgCl2, protease inhibitor (50×) and benzoase (2 uL/5 mL of buffer); 4.5 mL resuspension buffer, and 45 uL protease inhibitor, benzonase 1.8 uL. The solution was then sonicated at an amplitude of 20 (3×1 sec pulse) and immediately placed on ice.
Cell pellets were thawed on ice and resuspended in 200 uL mammalian protein extraction reagent (M-PER) buffer. The solution was then sonicated at an amplitude of 20 (3×1 sec pulse). The sample was placed on ice before transferring for centrifugation at 10,000 rpm for 10 minutes, and placed on ice until use.
To prepare the Nano-Glo Live Cell Assay reagents (Promega, Madison, WI), a buffer and substrate was mixed at a 19:1 ratio in an amount sufficient to provide a target set of protein conditions. For example, with one GPCR and Nanobody protein combination to test, an amount sufficient for 7 reactions at 20 uL per reaction=133 uL buffer/7 uL furimazine was prepared to comprise 6 replicates for include 3 wells each for +/− test drug or ligand, and one well to compensate for possible lost volume.
The protein mixture was then added to the 140 uL of buffer+substrate. 70 uL of each protein component was added i.e., 70 uL of membrane protein cell lysate+70 uL of nanobody cytosolic protein in the supernatant. The total volume added was 140 uL sufficient for 10 uL of each protein component to be aliquoted per well i.e., 20 uL for both protein components together with thorough mixing throughout.
The total volume of the mixture was 280 uL partitioned into 7 wells to yield the final volume of 40 uL to aliquot into the 96-well plate. After 6 wells were filled with 40 uL each, the the activator drug/ligand was added to the “+drug” wells/conditions. For example, if the GPCR is β2AR, the activating drug was isoproterenol (Iso). The stock solution of the drug was 20 uM, and 20 uL of the stock solution was added to the “+drug” wells. For the “-drug” wells, 20 uL of the membrane resuspension buffer was added to maintain a standardized volume. The total concentration of Iso in the wells was 6.67 uM.
Optimization revealed that 8 minutes of incubation time is needed for the maximal luminescent signal output. After this interval, the 96-well plate was placed in the BioTek plate-reader (BioTek Instruments, Winooski, Vermont). An auto gain luminescence protocol measured the relative luminescence from each well before providing the final readings. Data output was processed and graphed using Prism 9.
Graphs in the
GPCR membrane protein steps of the assay were as described above i.e., the buffer and substrate of the Promega Nano-Glo Live Cell Assay reagents were mixed in a 19:1 ratio. (
Next, the protein mixture was added to the 140 uL of buffer+substrate. 70 uL of GPCR membrane protein cell lysate was mixed into the solution. The peptidomimetic, was stored as other small molecule or peptide drugs as stock solutions frozen in the −80° freezer. (LifeTein, Somerset, New Jersey) 17.5 uL of 100 uM stock solution was added to the assay mixture with an equal volume (17.5 uL) of membrane resuspension buffer to provide [35 uM] per well. The added peptidomimetic brought the total volume to 245 uL i.e., 7 samples at 35 uL per well.
After aliquoting 35 uL per well into the 96-well plate the activating ligand/drug was added. If the GPCR is β2AR, the activating drug was isoproterenol (Iso). 15 uL of the 20 uM was added to the “+drug” wells. For the “-drug” wells, 15 uL of membrane resuspension buffer was added to maintain a standardized volume. The total Iso concentration per well was 6 uM.
Optimization revealed that 8 minutes of incubation time is needed for maximal luminescent signal output. After this interval, the 96-well plate was placed inside the BioTek plate-reader. (BioTek Instruments, Winooski, Vermont) An auto gain luminescence protocol measured the relative luminescence from each well before providing the final readings. Data output was processed and graphed using Prism 9.
A co-transfected version of the NANOLUC assay in-solution assay was tested using the MOR-based GPCR. (
To test that the membrane protein component was folded correctly and able to be activated effectively by its agonist, 20 uL of the complete lysed solution was added to a 96-well plate, followed by a brief incubation period with the MOR agonist, Loperamide (˜10 μM per well). Substrate for the reconstituted NANOLUC is added in a buffer solution (1:19 substrate: buffer) as a final step before using the plate reader to detect and quantify the intensity of subsequent bioluminescence. If the well contains properly folded and functional membrane protein, the cytosolic protein (i.e., Nb39 linked to LgBit) binds in the presence of the agonist, thereby allowing reconstitution of the split NANOLUC. As shown in
As shown in
All IGNiTR constructs (prepared via sonication,
We tested two different fusion geometries with β2-adrenergic Receptor (β2AR) fused to either the large portion of the split NANOLUC, LgBiT, or the small portion, SmBit, and the G-protein mimic fused to the other half of the split NANOLUC. As shown in
Data in
IGNiTR composed of β2AR fused to LgBiT and SmBiT fused to Nb80 or miniGs each yielded significant DDRs (
To increase the versatility of IGNiTR, we used a G-protein peptidomimetic as the conformation-specific binder in IGNiTR (
We fused the SmBiT (11 amino acids) to the G, peptidomimetic to create a SmBiT-G, peptidomimetic fusion peptide. To test the peptidomimetic version of IGNiTR, β2AR-LgBiT protein in sonicated cell lysate was mixed with the G, fusion peptide and NANOLUC substrate. Then, agonist or vehicle was added to evaluate the DDR (
We then designed a Gi peptidomimetic using a parallel strategy because the α-5-helix of the Gαi-protein also interacts with a highly hydrophobic binding pocket based on the Gαi-protein structure. (Koehl, A. et al. Structure of the μ-opioid receptor-Gi protein complex. Nature 558, 547-552 (2018).) The Gi fusion peptide is composed of the SmBiT fused to the Gi peptidomimetic. We tested IGNiTR with the Gi fusion peptide and a Gi-coupled GPCR, the μ-opioid receptor (μ-OR). A significant DDR was observed for μ-OR IGNiTR (
To establish IGNiTR's ability to characterize the diverse conformational states of a GPCR induced by various ligands, we applied the technique to DRD1 IGNiTR with full agonists, partial agonists and antagonists. The full agonist dopamine produced higher DDR than the partial agonist fenoldopam at saturated concentrations, with both producing a DDR>1. The result validates that both full and partial agonists induce the active conformational state (Teng, X. et al. Ligand recognition and biased agonism of the D1 dopamine receptor. Nat Commun 13, 3186 (2022), Teng, X. et al. Structural insights into G protein activation by D1 dopamine receptor. Sci. Adv. 8, eabo4158 (2022).), and that IGNiTR differentiates ligand efficacies. DRD1 antagonist, SCH 23390, does not increase luminescence compared to the no drug condition. These results further validate the Gs peptidomimetic's selective binding to the active conformation of DRD1. As well, DRD1 titration with dopamine and fenoldopam produced EC50 values of 2.6 μM and 145 nM, respectively (
In some embodiments, IGNiTR provides an alternative for HTS of GPCR ligands, especially because IGNiTR components may be mixed in a batch, increasing consistency across large-scale screens. We optimized DRD1 IGNiTR assay conditions by varying the DRD1 and G, fusion peptide concentrations (
The IGNiTR assay was then characterized with different concentrations of the fusion Gs and Gi fusion peptides. To minimize the cell pellet needed for characterization, we used the 0.5× of the GPCR-LgBiT lysate dilutions. We varied the concentration of fusion peptides added. For both Gs and Gi fusion peptides, the highest concentration (10 μM) resulted in a lower DDR compared to the lower concentrations due to higher background luminescence (
The optimized DRD1-IGNiTR assay was then used to scale up to screen for potential agonists using 1,916 compounds from an FDA-approved & Passed Phase I Drug Library from SelleckChem library. The Z′ value was consistent across the plates with an average of 0.79 (FIG. 27) which is within the range of optimal Z′ value for HTS (1>Z′>0.5). (Zhang, J.-H., Chung, T. D. Y. & Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. SLAS Discovery 4, 67-73 (1999).) Even though no hit molecules were identified from this library of compounds, this proof-of-principle screening demonstrated the feasibility and robustness of IGNiTR in GPCR ligand screening.
In some embodiments, IGNiTR is packaged as an accessible kit for detecting GPCR agonists, such as opioids, outside of a biosafety level 2 laboratory space. The ongoing opioid crisis is being fueled by the emergence of additional synthetic opioids. (Cuitavi, J., Hipólito, L. & Canals, M. The Life Cycle of the Mu-Opioid Receptor. Trends in Biochemical Sciences 46, 315-328 (2021). Mattson, C. L. et al. Trends and Geographic Patterns in Drug and Synthetic Opioid Overdose Deaths—United States, 2013-2019. MMWR Morb Mortal Wkly Rep 70, 202-207 (2021).) Accordingly, there is a pressing need for accessible methods to detect opioid derivatives which often are highly potent and thus have the potential to cause lethal overdoses. To address this need, we tested using μ-OR-based IGNiTR to detect the synthetic opioid, fentanyl.
First, we optimized the μ-OR and Gi fusion peptide concentrations for μ-OR IGNiTR (
Nanodiscs are widely applied for GPCR reconstitution by embedding the GPCR in a lipid bilayer, forming stable GPCR-lipid complexes. (Rouck, J. E., Krapf, J. E., Roy, J., Huff, H. C. & Das, A. Recent advances in nanodisc technology for membrane protein studies (2012-2017). FEBS Letters 591, 2057-2088 (2017).) It remains critical but challenging to track the GPCR structural integrity and function throughout the Nanodisc assembly process. (Serebryany, E., Zhu, G. A. & Yan, E. C. Y. Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors. Biochimica et Biophysica Acta (BBA)—Biomembranes 1818, 225-233 (2012).) Therefore, we tested IGNiTR's ability to characterize β2AR functionality during the three crucial steps of POPC-based Nanodisc formation (Mitra, N. et al. Calcium-Dependent Ligand Binding and G-protein Signaling of Family B GPCR Parathyroid Hormone 1 Receptor Purified in Nanodiscs. ACS Chem. Biol. 8, 617-625 (2013).) as indicated in
Standard cloning procedures, including NEB restriction enzyme digest, Q5 polymerase PCR amplification, T4 ligation and Gibson assembly, were used. Oligonucleotide primers were purchased from Sigma Aldrich. The plasmid DNA encoding MC4R was a gift from the Roger Cone Lab. The plasmid DNA encoding MiniGs and Nanobody 80 was purchased from Twist Biosciences.
Plasmid constructs were transformed into Escherichia coli cells via heat shock. XL1-Blue competent cells were used for all constructs, except for MBP-LgBit, which is described in “Expression and Purification of MBP-LgBit” below. Sequences were confirmed by Sanger Sequencing (Eurofins, GeneWiz)
HEK 293T/17 cell lines (ATCC, cat #: CRL-11268) used in these experiments were cultured at 37° C. and 5% CO2. Cells were grown in complete growth media (1:1 MEM (Eagle's Minimal Essential Medium): DMEM (Dulbecco's Modified Eagle Medium, Gibco): with 50 mM HEPES (Gibco), 10% Fetal Bovine Serum (Sigma), and 1% Penicillin-Streptomycin (Gibco).
Cells at 80-90% confluence were plated into a flask that had been pre-incubated with human fibronectin for 10 minutes at 37° C. An hour after seeding, these cells were transfected using FBS-free MEM and polyethylenimine (PEI, 1 mg/ml, Polysciences) with a ratio of 1:10 between μL of PEI and pg of plasmid DNA. The cells were then incubated at 37° C. for 20-24 hours.
The cell pellet was harvested by aspirating the media and resuspending the cells with a cell scraper in Dulbecco's Phosphate Buffered Saline (DPBS) buffer. 18 mL of DPBS was used to resuspend the cells within a T-75 flask, with this ratio kept constant for other flask sizes. 1.5 mL of resuspended cells were placed into an Eppendorf tube, which was centrifuged at 6,010 g for 3 minutes. The supernatant was aspirated, and the pellet was resuspended in DPBS, then centrifuged again under the same conditions. The supernatant was aspirated again, and the cell pellet was flash frozen in liquid N2 and stored at −80° C. until ready for use.
The cell pellets were prepared immediately before the assay, by first placing the cell pellet onto ice. For cell pellets containing GPCR constructs, the pellet was treated with 210 μL of Membrane Resuspension Buffer (MRB). MRB is comprised of incomplete membrane resuspension buffer (resuspension buffer comprising 20 mM HEPES, pH 7.5 and 2 mM MgCl2) and benzoase (EMD Millipore, 70746) in a 4.5 mL to 1.8 μL ratio. For cell pellets containing cytosolic proteins (Nanobody 80 or MiniGs), the pellet was treated with Mammalian Protein Extraction Reagent buffer. After this, 2 μL of 100× protease inhibitor (Sigma Aldrich, P1860 #) was added to a final concentration of 1× and the pellet was resuspended by pipetting up and down. The solution was then sonicated using Model 50 Sonic Dismembrator (Fischer Brand) at a 20% amplitude (3×1 sec pulse) and returned to ice.
A master mix containing Nano-Glo Buffer and substrate (Promega, N2012), GPCR construct, and G protein mimic was prepared. For one well, the ratio was 9.75 μL of Nano-Glo Buffer, 0.25 μL of Nano-Glo substrate, 5 μL of GPCR pellet and 5 μL of the conformation specific binder. The conformation specific binder may be either peptidomimetic (LifeTein), miniGs pellet or Nanobody 80 pellet. The GPCR construct cell pellet was prepared separately and added to the master mix immediately prior to adding the master mix to the well. The concentrations were varied for the optimization assays (
To scale up the IGNiTR assay for high throughput screening, we used the Echo 655 (Labcyte) to load 150 nL of drug to each well of the 384 well plate. Then, the Multidrop Combi Reagent Dispenser (Thermo Scientific) was used to add 10 μL of MRB to each well. The same machine was used to add 20 μL of master mix, prepared as described in “IGNiTR Assay” using 2 μM fusion peptidomimetic and the 0.5× dilution of DRD1-LgBiT found to be optimal from the characterization in
We validated the drug hits from an initial screen of six 384 well plates by testing four replicates of each compounds using the same method. From this validation, a dose response curve was constructed for promising candidates by loading 150 nL at a range of concentrations, using a mosquito X1 (SPT Labtech) to obtain final concentrations in well from 661 nM to 25.1 μM. 10 μL of MRB was loaded to the compounds, followed by 20 μL of master mix. After a 30 minute incubation, the luminescence values were measured using an EnVision 2104 Multilabel Reader (Perkin Elmer).
The DNA encoding MBP-LgBit was transformed into BL21 cells. A colony of these cells was inoculated in 5 mL Luria-Bertani broth with ampicillin at 37° C. overnight. The culture was then transferred to a 500 mL flask of Luria-Bertani broth with ampicillin and placed in a 37° C. shaker until OD-600 reached 0.4 to 0.8. Protein expression was induced by addition of 1000×0.1 g/mL IPTG, to a final concentration of 1×. The culture was then shaken overnight at room temperature.
The cells were centrifuged at 4,248 g for 5 minutes at 4° C. The cell pellet was lysed by resuspension in cold Bacterial Protein Extraction Reagent (B-PER, Fisher) buffer with 1 mM dithiothreitol (DTT) and 1× protease inhibitor (BioBasic, BS386). 15 mL of B-PER was used for every 500 mL of bacteria culture. 3-4 μL of benzoase was added to the cells, followed by a 5 min incubation on ice to ensure full cell lysis. The cells were then centrifuged at 16,994 g for 10 minutes at 4° C.
50 mL of clear lysate was added to 2 mL of Ni-NTA resin slurry and incubated at 4° C. for 10 minutes. This mixture was then purified via an Ni-NTA column. The purity of MBP-LgBiT was then established using gel electrophoresis and a Coomassie stain analysis.
The molar extinction coefficient of MBP-LgBiT was calculated using Expasy PratParam to be 89,270 M−1 cm−1. This value was then used in conjunction with Bier's Law to establish the concentration of the protein by the absorbance at A280. The absorbance was re-calculated for each time MBP-LgBiT was used.
The standard curve was created using the same 384 well plates. First, a master mix containing a ratio of 5 μL of 30 μM HiBiT, 0.125 μL furimazine and 4.875 μL NanoGlo Buffer was prepared. 10 μL of this master mix was added to each well. 5 μL of MBP-LgBit or GPCR-LgBit was added, in the dilution ratio indicated in the figure legends. Times reported on figure captions were recorded from the addition of the MBP-LgBit or GPCR-LgBit to the first well of the plate.
All statistical analysis was performed using GraphPad Prism 9 software. This software was also used to construct plots. The sample size is indicated in figure legends (where n is the number of independent replicates). The mean and standard error of the mean were calculated for each condition. Two-sided Student's t-tests were used to evaluate the significance between data points. Z′ values were calculated using the following equation:
where SD is the standard deviation.
The frozen pellet expressing μ-OR-LgBiT was thawed, resuspended, and mixed with the Gi fusion peptide and NANOLUC substrate. The reaction mix was aliquoted to separate wells of an opaque white 96-well plate. A range of concentrations of fentanyl were added to the wells and the plate was imaged using an Azure Biosystems c600 for chemiluminescence. The resulting images were analyzed using imageJ.
The pellet (β2AR-LgBiT) was resuspended in 400 uL membrane resuspension buffer (MRB) and sonicated. The lysate was quantified using the Pierce BCA protein assay kit (Thermo Scientific). The protein concentration was calculated using an average molecular weight of 40 kDa for membrane proteins. Nanodiscs were made as described previously. POPC (Avanti Polar Lipids) was dried under nitrogen and stored in a desiccator overnight. POPC was solubilized to 50 mM with 100 mM sodium cholate. Nanodiscs were assembled by adding MSP1E3D1 (Millipore Sigma) and lysate to the solubilized lipids up to a final volume of 350 μL in standard disc buffer (20 mM Tris, 100 mM NaCl, 0.5 mM EDTA, 0.01% NaN3) supplemented with sodium cholate to a final concentration of 20 mM. The final lysate concentration in the mixture was 10 uM, the MSP:lysate was 4:1, and the lipid:MSP was 90:1. The component mixture was incubated on an end over end mixer at 4° C. for 45 minutes. 150 mg of Amberlite XAD-2 beads (Millipore Sigma) were added, and the component mixture was incubated at 4° C. overnight before the beads were removed. The resulting Nanodiscs were then purified with Ni-NTA spin columns (NEB). The purified Nanodiscs were then exchanged into standard disc buffer with Bio-Spin P-6 Gel Columns (Bio-Rad) to remove imidazole.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.
The present application is a § 371 National Entry application of PCT/US2023/062766, filed Feb. 16, 2023, which claims priority to U.S. Provisional Application Ser. No. 63/311,216 filed Feb. 17, 2022, each of which are herein incorporated by reference in its entirety.
This invention was made with government support under MH132939, and DA056192 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/062766 | 2/16/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63311216 | Feb 2022 | US |
