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WO2015102541A1 - Optical biosensors for diagnosis and high-throughput drug screening using unique conformational changes of recombinant tagged g protein-coupled receptors for activation - Google Patents

Optical biosensors for diagnosis and high-throughput drug screening using unique conformational changes of recombinant tagged g protein-coupled receptors for activation Download PDF

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Publication number
WO2015102541A1
WO2015102541A1 PCT/SG2014/000632 SG2014000632W WO2015102541A1 WO 2015102541 A1 WO2015102541 A1 WO 2015102541A1 SG 2014000632 W SG2014000632 W SG 2014000632W WO 2015102541 A1 WO2015102541 A1 WO 2015102541A1
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recombinant
gpcr
variant
serotonin
gpcr variant
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Charlotte A.E. Hauser
Somenath Roy
Jun Hui SOH
Jackie Y. Ying
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Agency for Science Technology and Research Singapore
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/942—Serotonin, i.e. 5-hydroxy-tryptamine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention is in the area of biodevices and diagnostics and generally relates to G protein-coupled receptors (GPCRs), GPCR ligands, 5-HT receptors, serotonin biosensor, diagnostic devices, drag screening, ionic lock, molecular beacon, recombinant expression, point-of-care (POC) devices.
  • GPCRs G protein-coupled receptors
  • the present invention relates to recombinant GPCR variants mutated to allow for binding of a fluorophore and an acceptor or quencher, to nucleic acids encoding the GPCR variants, mammalian cells and cell lines and to labeled recombinant GPCR variants.
  • the present invention further relates to a method of obtaining a cmde membrane preparation comprising the recombinant GPCR variants and biosensors.
  • the present invention further relates to the recombinant GPCR variants, the crade membrane preparations, the labeled recombinant GPCR variants, or the biosensor for use in the diagnosis of diseases related to the GPCR.
  • the present invention further relates to a method for detecting the presence and/or measuring the concentration of GPCR ligand(s) in a sample and to a method of drug discovery or screening.
  • GPCRs G-protein-coupled receptors
  • GPCRs are an important class of membrane proteins. They are responsible for all kinds of signal transduction processes in the body. GPCRs mediate signals from native ligands such as hormones, peptides and neurotransmitters, are involved in olfaction and taste, and are coupled to bodily functions such as cardiac and endocrine functions. Furthermore, GPCRs are prime drug targets, recognized by -60% of therapeutic drugs such as /3-blockers, antipsychotics and analgesics.
  • the GPCR superfamily contains well-conserved motifs, indicating a common activation mechanism among all GPCRs.
  • 5-hydroxytryptamine 2A 5-HT2A serotonin receptor
  • 5-HT2A 5-hydroxytryptamine 2A
  • 5-HT2A 5-hydroxytryptamine 2A serotonin receptor
  • this object is solved by a recombinant G-protein coupled receptor (GPCR) variant mutated to allow for binding of a fluorophore and an acceptor or quencher,
  • GPCR G-protein coupled receptor
  • said recombinant GPCR variant comprises
  • one binding site is introduced in the proximity of the Arg residue of the (D/E)RY motif in transmembrane helix 3 (H3) of the GPCR and the other binding site is introduced in the proximity of the acidic amino acid residue in transmembrane helix 6 (H6) of the GPCR, wherein said Arg and said acidic amino acid residue form the ionic lock motif and have an ionic interaction in the inactive state of the GPCR.
  • this object is solved by a nucleic acid encoding a recombinant GPCR variant of the present invention.
  • this object is solved by a mammalian cell or cell line comprising a nucleic acid of the present invention and expressing a recombinant GPCR variant of the present invention on the cell membrane, preferably stably expressing the GPCR variant,
  • this object is solved by a method of obtaining a crude membrane preparation comprising a recombinant GPCR variant of the present invention, comprising the steps of
  • this object is solved by a biosensor comprising a recombinant GPCR variant of the present invention, a crude membrane preparation obtained in the methods of to the present invention or a labeled recombinant GPCR variant of the present invention.
  • this object is solved by using a recombinant GPCR variant of the present invention, a crude membrane preparation obtained in the methods of the present invention, a labeled recombinant GPCR variant of the present invention, or a biosensor of the present invention in ligand binding assays, drug discovery and drug screening,
  • this object is solved by a recombinant GPCR variant of the present invention, a crude membrane preparation obtained in the methods of the present invention, a labeled recombinant GPCR variant of the present invention, or a biosensor of the present invention for use in the diagnosis of diseases related to the GPCR, wherein a disease related to the GPCR is selected from
  • diseases associated or caused by high serotonin levels such as brain injury due to high serotonin levels, violence and aggression, autism, colon cancer,
  • diseases associated or caused by low serotonin levels such as schizophrenia, depression.
  • this object is solved by a method for detecting the presence and/or measuring the concentration of GPCR ligand(s) in a sample,
  • a recombinant GPCR variant of the present invention comprising the use of a recombinant GPCR variant of the present invention, a crude membrane preparation obtained according to the present invention, a labeled recombinant GPCR variant of the present invention, or a biosensor of the present invention.
  • this object is solved by a method of drug discovery or screening, comprising the steps of
  • a decrease of the fluorescence signal is indicative that the compound or drug to be tested is an antagonist of the ligand, i.e. competes for the binding site;
  • an increase of the fluorescence signal is indicative that the compound or drug to be tested is an agonist of the ligand, i.e. increases ligand binding; or that the compound or drug to be tested is an agonist that is capable of activating the GPCR, by triggering the release of the ionic lock.
  • the present invention provides a recombinant G-protein coupled receptor (GPCR) variant imitated to allow for binding of a fluorophore and an acceptor or quencher, wherein said recombinant GPCR variant comprises (1) a binding site or sequence for the fluorophore,
  • one binding site is introduced in the proximity of the Arg residue of the (D/E) Y motif in transmembrane helix 3 (H3) of the GPCR and the other binding site is introduced in the proximity of the acidic amino acid residue in transmembrane helix 6 (H6) of the GPCR, wherein said Arg and said acidic amino acid residue form the ionic lock motif and have an ionic interaction in the inactive state of the GPCR.
  • a binding site or sequence for the fluorophore and/or the acceptor or quencher comprises 3 to 10 amino acids, preferably 5 to 9 amino acids, such as 5, 6, 7 or 9 amino acids.
  • the binding site or sequence differs in length (number of amino acid residues) depending on the fluorophore - acceptor/quencher used.
  • the binding site or sequence for the fluorophore comprises a tetra- cysteine tag (TC tag), preferably an amino acid sequence comprising or consisting of the amino acid sequence
  • CC(X) CC(X) repeatCC (SEQ ID NO. 1) wherein C is cysteine and X is any amino acid and n is 1 to 5.
  • the binding site or sequence for the fluorophore comprises a tetra- cysteine tag (TC tag), more preferably an amino acid sequence comprising or consisting of an amino acid sequence selected from the group of
  • CCPCC SEQ ID NO. 6
  • CCAECC SEQ ID NO. 7
  • the amino acid sequence CCPGCC (SEQ ID NO. 2) is preferred.
  • the fluorophore AsCy3 used is the amino acid sequence CCKAEAACC (SEQ ID NO. 12) is preferred.
  • AsCy3 requires a longer amino acid tag as its interatomic distance of ⁇ 14.5 A is longer than FlAsH-EDT 2 of- 6 A.
  • the binding site or sequence for the iluorophore comprises a tag or fusion selected from the group of
  • Rhodococciis dehalogenase (DhaA) (HaloTag System)
  • LAP Lipoic acid ligase acceptor peptide
  • the GPCRs are tagged with a protein tag, in particular by the insertion of the Rhodococciis dehalogenase (DhaA), which contains an aspartate nucleophile that forms stable covalent bonds with aliphatic hydrocarbons containing a halide, e.g. 1 ,2- dibromoethane.
  • DhaA Rhodococciis dehalogenase
  • Fluorescent chloroalkane ligands include carboxytetramethylrhodamine (TMR).
  • the SNAP-tag a 20 kDa mutant of the DNA repair protein, O 6 - alkylguanine-D A alkyltransferase, is used, which reacts specifically, irreversibly and covalently with benzylguanine (BG) derivatives, The BG derivatives can subsequently be labelled with fluorophore of interests for visualization.
  • the CLIP-tag is used, which is reacts specifically with 0 2 -benzylcytosine (BC) derivatives.
  • BC benzylcytosine
  • fusion proteins of the GPCRs are engineered to contain the human DNA repair protein, 0 6 -alkylguanine-DNA alkyltransferase (wildtype), which can then be labelled with a fluorophore using 0 6 -benzyl guanine (BG) and its derivatives, such as BGBT, BGAF and BGFL.
  • wildtype 0 6 -alkylguanine-DNA alkyltransferase
  • BG 0 6 -benzyl guanine
  • its derivatives such as BGBT, BGAF and BGFL.
  • the tag is lipoic acid ligase acceptor peptide (LAP), 22-amino acid sequence peptide, which can be modified by lipoic acid ligase (LplA) and azide, to present an azide functional group, for further downstream conjugation with fluorophores such as cyclo- octyne- conjugated AlexaFluor 568 or Cy3.
  • LAP lipoic acid ligase acceptor peptide
  • LplA lipoic acid ligase
  • azide azide
  • the binding site or sequence for the acceptor or quencher comprises an amino acid sequence of multiple His (His 6 tag), preferably amino acid sequence HHHHHH (SEQ ID NO. 13).
  • the fluorophore and acceptor/quencher are a fluorescence resonance energy transfer (FRET) pair.
  • FRET fluorescence resonance energy transfer
  • the fluorophore is selected from
  • FlAsH-EDT 2 (4',5'-bis(l ,3,2-dithioarsoIan-2-yl)fluorescein-(l ,2-ethanedithiol) 2 ).
  • bi arsenical fluorophores are:
  • Red fluorescence is attractive for fluorescence microscopy as cellular absorbance, scattering and autofluorescence decrease greater at such longer wavelengths.
  • TMR carboxytetramethylrhodamine
  • the acceptor or quencher is a metal-ion-chelating nitrilotriacetate moiety, preferably nitrilotriacetic acid chromophore (NTA-I),
  • acceptor/quencher are:
  • Au NPs gold - Gold nanoparticles
  • NTA nitrilotriacetic acid
  • Au NPs having its absorption peak at 525 run (green region), will be able to act as fluorescence acceptor of green fluorescence in a FRET pair.
  • fluorophores that are conjugated to a NTA motif can also act as fluorescence acceptor.
  • Red fluorophores such as Cy3 quenches green fluorophores such as FlAsH-EDT 2 , fluorescein, FAM and AlexaFluor 488. Green fluorophores on the other hand, will quench blue fluorescence.
  • the fluorophore and acceptor/quencher used according to the invention are a FRET pair, provided that they are not both green fluorescent protein (GFP) or any of its derivatives, such as CFP/YFP, such as disclosed in Lohse et al. 2012 (Lohse MJ, Nuber S, Hoffmann C. Fluoiescence/bioluminescence resonance energy transfer techniques to study G-protein- coupled receptor activation and signaling. Pharmacol Rev, 2012 Apr;64(2):299-336.)
  • GFP green fluorescent protein
  • the GPCR is a 5HT2A serotonin receptor, a Wnt receptor or an odorant receptor.
  • the GPCR is (human) 5HT2A serotonin receptor.
  • GenBank: X57830 Homo sapiens serotonin 5-HT2 receptor mRNA
  • HTR2A G protein-coupled (HTR2A), transcript variant 1, mRNA
  • the human 5HT2A is preferably encoded by nucleic acid sequence SEQ ID NO. 14: atggatatt ctfctgtgaag aaaatacttc tttgagctca actacgaact ccctaatgca attaaatgat gacaccaggc tctacagtaa tgactttaac tccggagaag
  • the human 5HT2A preferably has the amino acid sequence of SEQ ID NO. 15 or
  • Isoform 1 of 5 -hydroxytiyp famine receptor 2A Homo sapiens
  • MDILCEENTS LSSTTNSLMQ LNDDTRLYSN DFNSGEA TS DAFHWTVDSE NRTNLSCEGC LSPSCLSLLH LQEKNWSALL TAWIILTIA GNILVIMAVS LEKKLQNATN YFLMSLAIAD MLLGFLVMPV SMLTILYGYR WPLPSKLCAV WIYLDVLFST ASIMHLCAIS LDRYVAIQNP IHHSRFNSRT AFLKIIAVW TISVGISMPI PVFGLQDDSK VFKEGSCLLA DDNFVLIGSF VSFFIPLTIM VITYFLTIKS LQKEATLCVS DLGTRAKLAS FSFLPQSSLS SE LFQRSIH RBPGSYTGRR TMQSISNEQK ACKVLGIVFF LFWMWCPFF ITNIMAVICK ESCNEDVIGA LLNVFVWIGY LSSAVNPLVY TLFN TYRSA FSRYIQCQYK ENKKPLQLIL V TIPALAY
  • Isoform 2 of 5-hydroxytryptamine receptor 2A Homo sapiens MQFLKSAKQ PNYYHIMLVE DQEEGTLHQF NYCERCSESQ N KCISCVDP
  • Said (human) 5HT2A serotonin receptor preferably comprises (1) the binding site or sequence for the fluorophore introduced in the proximity of residue Arg 173 at transmembrane helix 3 (H3),
  • GPCR is (human) 5HT2A serotonin receptor as defined above
  • the binding site or sequence for the fluorophore (1) is a tetra-cysteine tag (TC tag), preferably comprising or consisting of amino acid sequence CCPGCC (SEQ ID NO. 2) and wherein the fluorophore is FlAsH-EDT 2
  • the binding site or sequence for the acceptor or quencher (2) is an amino acid sequence of multiple His (His 6 tag), preferably comprising or consisting of amino acid sequence HHHHHH (SEQ ID NO, 13) and wherein the acceptor or quencher is nitrilotriacetic acid chromophore (NTA-I),
  • GPCR is (human) 5HT2A serotonin receptor as defined above
  • the binding site or sequence for the fluorophore (1) is introduced in the proximity of residue Arg 173 in the following way: by insertion C-tem inal of Arg 173 (preferably adjacent to Arg 173) or by replacing the wildtype amino acids C-tenninal of Arg 173 (preferably adjacent to Arg 173),
  • the binding site or sequence for the acceptor or quencher (2) is introduced in the proximity of Glu 318 in the following way: by insertion N-tenninal of Glu 318 (preferably adjacent to Glu 318 or 3 amino acids N-terminal of Glu 318) or by replacing the wildtype amino acids N-terminal of Glu 318 (preferably adjacent to Glu 318 or 10 amino acids N- terminal of Glu 318).
  • the recombinant GPCR variant is selected from the variants comprising an amino acid sequence selected from at least one of SEQ ID NOs. 20 to 29, preferably one of SEQ ID NOs. 19, 21, 23, 25 and 27 and one of SEQ ID NOs. 20, 22, 24, 26 and 28,
  • SEQ ID Nos. 21 and 22 such as in Construct 1; or
  • SEQ ID Nos. 23 and 24 such as in Construct 2; or
  • SEQ ID Nos. 25 and 26 such as in Construct 7; or
  • amino acid sequences of the binding site or sequence for the acceptor or quencher (2) which is an amino acid sequence of multiple His (His 6 tag), comprising or consisting of amino acid sequence HHHHHH (SEQ ID NO. 13) and introduced in the proximity of Glu 318 at transmembrane helix 6 (H6).
  • Table 1 Amino acid sequence of wild-type 5HT2A and the various recombinant 5HT2A constructs after mutagenesis.
  • Arg-173 is highlighted bold, GIu-318 is highlighted bold and underlined;
  • TC tag is highlighted italic and Hisdag is highlighted italic and underlined.
  • the GPCR is serotonin receptor 5HT4.
  • Biosensor cell -based system only
  • the GPCR is Wnt receptor FZD.
  • Ligand Wnt (Wnt 5A for FZD4 and FZD5), Norrin (FZD4), etc.
  • the GPCR is W t receptors FZDs.
  • breast cancer breast cancer, lung cancer, intestine and colorectal cancer, rheumatoid artliritis, etc.
  • Familial exudative Benign ovarian Chromosomal vitreoretinopathy Nome tumor, chronoic duplication, cervical disease, progressive myeloid leukemia, cancer, gastric cancer, cerebellar degeneration, etc. renal tumor, gastric etc,
  • the present invention provides a nucleic acid encoding a recombinant
  • the present invention provides a mammalian cell or cell line comprising a nucleic acid of the present invention and expressing a recombinant GPCR variant of the present invention on the cell membrane, preferably stably expressing the GPCR variant, such as HEK293T.
  • the present invention provides a method of obtaining a crude membrane preparation comprising a recombinant GPCR variant of the present invention, comprising the steps of
  • the present invention provides a recombinant GPCR variant of the present invention labeled with fluorophore and acceptor or quencher,
  • the method further comprises the steps of
  • the present invention provides a biosensor comprising
  • a solid substrate such as nitrocellulose membrane
  • a solid support such as a glass plate, microarray or chip
  • the present invention provides the use of
  • the present invention provides
  • a recombinant GPCR variant of the present invention a crude membrane preparation obtained in the methods of the present invention, a labeled recombinant GPCR variant of the present invention, or
  • a disease related to the GPCR is selected from
  • diseases associated or caused by high serotonin levels such as brain injury due to high serotonin levels, violence and aggression, autism, colon cancer,
  • diseases associated or caused by low serotonin levels such as schizophrenia, depression.
  • the present invention provides the recombinant GPCR variant of the present invention or the biosensor of the present invention
  • GPCR is (human) 5HT2A serotonin receptor
  • diseases related to serotonin activity such as vascular smooth muscle contraction, platelet aggregation, perception, emotion, mental and behavioral disorders, autism, violent behavior, schizophrenia, depression.
  • the present invention provides the recombinant GPCR variant of the present invention or the biosensor of the present invention
  • GPCR is a Wnt receptor
  • the present invention provides the recombinant GPCR variant of the present invention or the biosensor of the present invention
  • GPCR is an odorant receptor
  • the present invention provides a method for detecting the presence and/or measuring the concentration of GPCR ligand(s) in a sample
  • the GPCR ligands are neurotransmitters, like serotonin, an odorant or ligands binding to Wnt receptors, like FZD, amino acids, amines, firagrants.
  • the method comprises the steps of
  • the method comprises
  • the method comprises
  • step (ii) adding the labeled recombinant GPCR variant or crade membrane preparation comprising a labeled recombinant GPCR variant of step (ii) to a 96-well plate, a solid substrate (such as nitrocellulose membrane) or a solid support (such as a glass plate, microan ay or chip).
  • a solid substrate such as nitrocellulose membrane
  • a solid support such as a glass plate, microan ay or chip
  • the method comprises
  • the method comprises the conversion of the fluorescence signal into an electrical signal, such as by the use of photodiode.
  • the present invention provides a method of drug discovery or screeni g, comprising the steps of
  • a decrease of the fluorescence signal is indicative that the compound or drug to be tested is an antagonist of the ligand, i.e. competes for the binding site;
  • an increase of the fluorescence signal is indicative that the compound or drag to be tested is an agonist of the ligand, i.e. increases ligand binding; or that the compound or drug to be tested is an agonist that is capable of activating the GPCR, by triggering the release of the ionic lock.
  • the GPCR ligands are neurotransmitters, like serotonin, an odorant or ligands binding to Wnt receptors, like FZD, amino acids, amines, fragrants.
  • the method comprises adding the labeled recombinant GPCR variant or crude membrane preparation comprising a labeled recombinant GPCR variant of step (ii) to a 96-well plate, a solid substrate (such as nitrocellulose membrane) or a solid support (such as a glass plate, microarray or chip).
  • a solid substrate such as nitrocellulose membrane
  • a solid support such as a glass plate, microarray or chip
  • the method comprises
  • the invention utilizes the sensing platform for facile and rapid detection of GPCR-based ligands, such as neurotransmitters (e.g. serotonin, epinephrine and dopamine), for diagnostic purposes, as well as drug/ligand screening discovery applications.
  • GPCR-based ligands such as neurotransmitters (e.g. serotonin, epinephrine and dopamine)
  • neurotransmitters e.g. serotonin, epinephrine and dopamine
  • We are specifically interested in neurotransmitters that typically exert their cellular effects through activation of cell surface membrane receptor/protein that belongs to the GPCR superfamily.
  • This teclinology can be further integrated with a diagnostic device and a microfluidic platform for the rapid, automated and multiplexed detection of ligands in a device and for on-chip drag screening.
  • G protein-coupled receptors are a large class of membrane receptors characterized by seven, transmembrane a-helices, which are separated by alternating intra- and extracellular loop regions [1]. GPCRs are vital for cellular signaling and function [2]. Furthermore, they serve as important pharmacological targets [3].
  • the 5-hydroxytryptamine 2A (5HT2A) serotonin receptor is a GPCR with essential roles for the actions of serotonin (5HT) in diverse physiological processes, such as vascular smooth muscle contraction, platelet aggregation, perception and emotion [4].
  • the 5HT2A receptor has a high expression tlrroughout all layers of the cortex, particularly in the fifth layer [5].
  • the present study used small fluorophore and quencher molecules, which do not perturb the pharmacological and signaling properties of the 5HT2A receptor, in the detection of serotonin.
  • Our recombinant human 5HT2A receptor has been specifically mutated to incorporate a tetracysteine (TC) tag, with the amino acid sequence of CCPGCC, for specific binding of the fluorophore FlAsH-EDT 2 [19], and a Hiss tag, for the specific binding of a metal-ion-chelating nitrilotriacetate moiety (NTA-I) [20] for quenching of the FlAsH-EDT 2 .
  • TC tetracysteine
  • NTA-I metal-ion-chelating nitrilotriacetate moiety
  • FlAsH-EDT 2 does not fluoresce in free solution, however, it is highly fluorescent when it binds to a specific sequence of 56 amino acids [19, 21], especially CCPGCC [22]. Hence, the use of FlAsH-EDT 2 as a small fluorophore does not produce high background noise, and will not disturb the structure of the 5HT2A receptor.
  • the NTA-I exhibit site-specific labeling properties to His6 tag in proteins of interest, binding to His 6 tags within seconds [20]. Additionally, it quenches fluorescence in the green region (520-560 nm), which corresponds to the emission of FlAsH-EDT 2 (534 nm). Similarly, NTA-I is a non-fluorescent acceptor dye, which does not contribute background noise during fluorescence detection.
  • This separation restores fluorescence, and the presence of serotonin is detected as an increase in fluorescence signal.
  • a method of exploiting the ligand binding lock-unlock motif in GPCRs for the development of highly sensitive optical biosensors for diagnosis and drug disco very/screening.
  • the technology disclosed herein describes for the first time the use of the conformational change in GPCR receptors, induced by unleashing the ionic lock, as exemplified for 5-HT2A receptors, upon ligand activation for the biosensing of GPCR ligands, as for example serotonin in the case of the 5-HT2A receptor.
  • GPCR ligands i.e. serotonin
  • screening of drugs requires only a single-step addition of ligand or drug candidates, without the need of any washing steps.
  • the major advantage of our technology is the use of tiny amounts (microliter volumes) of crude cellular membranes that contain the highly sensitive GPCR receptor of choice. No whole cell assays (short half-life and very labile) or spectroscopy set-ups (expensive equipment that cannot be trimmed down to a POC device) are needed. Furthermore, molecular biological expertise for the design of the appropriate recombinant receptor is needed. It is also practically impossible to get the identical permanent cell clone reproduced.
  • the signal readout is fast, requiring only 5- 15 minutes of incubation time.
  • Construct 0 is obtained by insertion of the TC (Cys-Cys-Pro-Gly-Cys-Cys) tag, for the specific binding of FLAsH-EDT 2 ⁇ after Arg-173 on the second intracellular loop, and by insertion of the His6 tag, for the specific binding of NTA-I, before Glu-318 on the third intracellular loop ( Figure 1 ).
  • Overexpi ession of human recombinant 5HT2A receptors HEK293T cells were transiently transfected with the recombinant 5HT2A receptor constructs for overexpression of recombinant 5HT2A receptors on their cell membranes. Overexpression of the recombinant receptors has been verified and analyzed by Western blotting ( Figure 2a) and immunofluorescence ( Figure 2b). Immunofluorescence images show that the recombinant 5HT2A receptors were specifically expressed on the cell membranes of cells transfected with the recombinant 5HT2A receptor construct.
  • the recombinant .5HT2A receptors were overexpressed at a density of -9.1 ( ⁇ 5.7) million receptors per cell (see Methods). This value is consistent with other reported values [2, 23, 24] of transiently overexpressed GPC s in mammalian cell systems.
  • NTA-I (20 ⁇ ) resulted in significant quenching of fluorescence for the samples ( Figure 2f).
  • the binding of NTA-I to the Hise tag can be reversed with the addition of excess EDTA [20].
  • the restoration of fluorescence also indicates the quenching of FlAsH-EDT 2 fluorescence at the ionic lock motif, where the TC and Hise tags have been introduced.
  • the recombinant 5HT2A receptors that are expressed in HEK293T cells retained the ligand (serotonin) binding ability of 5HT2A receptors as shown in the ligand binding assay ( Figure 2f) conducted using the Quartz Crystal Microbalance with dissipation (QCM-D) system.
  • the QCM-D is a piezoelectric flow cell, whereby binding of biomolecules to the surface of the sensor illicit a decrease in resonant frequency of the quartz crystal sensor.
  • the intensity of fluorescence generated during the detection of serotonin is dependent on the amount of separation between the FlAsH-EDT 2 and NTA-I to produce maximum decrease in fluorescence quenching.
  • we attempted to optimize the fluorescence intensity through physical manipulation of the length of the 3rd intracellular loop (ICL3) as well as the position of the Hise tag, relative to the site of the ionic interaction between Arg-173 and Glu-318, in order to maximize the separation between FlAsH-EDT 2 and NTA-I and to study how the manipulation affects fluorescence intensity.
  • Constracts 0, 2 and 12 where the position of the Hise tag has been varied with respect to Glu-318, which is involved in the ionic interaction with Arg-173.
  • the His 6 tag for Construct 2 is located 3 amino acids away from Glu-318
  • the Hiss tag is located 10 amino acids away from Glu-318. Comparing their perfonnances, Constracts 0 and 2 achieved similar fluorescence signal. Therefore, a slight shift (3 AA) of the H1 ⁇ 4 tag does not affect fluorescence signal.
  • the fluorescence signal for Construct 12 is significantly lower and remains relatively flat from 100 nM-100 ⁇ . This is most likely because the huge shift (10 AA) has significantly affected the quenching efficiency between FlAsH-EDT 2 and NTA-I, and the subsequent increase in fluorescence when the receptor is activated upon serotonin binding.
  • aldehyde-functionalized surface was chosen as the aldehyde group reacts spontaneously with amino groups and retains the protein activity [26]. Fluorescence detection of serotonin on glass slide is largely similar to that in solution (see Methods), with an additional washing step to remove unbound Recomb. 5HT2A sample on the surface of the glass slide.
  • Figure 1 Schematic representation of the detection scheme for the serotonin detection using a human recombinant 5HT2A receptor.
  • construct 0 This construct contains 2 short peptide tags, which are inserted into the wild- type 5HT2A gene through site-directed mutagenesis, for the specific binding of a fluorophore and a quencher.
  • the fluorescence of FIAsH-EDT 2 is quenched due to the close proximity between the fiuorophore and quencher. (Right)
  • the receptor undergoes a conformation change (outward movement illustrated by the arrows), causing the separation of the fiuorophore and quencher, and results in an increase in fluorescence signal as quenching efficiency decreases.
  • 5HT2A sample compared to the HEK293T and Reagents samples. Subsequent addition of the NTA-1 quencher results in a decrease in fluorescence for all samples, especially the Recomb. 5HT2A sample. An increase in fluorescence intensity is observed for the Recomb, 5HT2A sample when 100 pM-1 nM serotonin is added, and fluorescence intensity for the Recomb. 5HT2A experience a steady increase with increasing concentration of serotonin, while the fluorescence intensities for the other 2 samples remain relatively unchanged. (b) Line graph depicting the percentage increase in fluorescence intensity for the Recomb. 5HT2A sample from (a).
  • Fluorescence value at each serotonin concentration is compared to the fluorescence value after addition of 10 ⁇ NTA-I to obtained the percentage increases.
  • the detection limit is 100 pM, with a linear response over the range of 100 pM- 00 ⁇ .
  • (Inset) Fluorescence images illustrating the fluorescence intensity of the Recomb. 5HT2A sample at increasing serotonin concentrations. Notably, an observable increase in fluorescence intensity is achieved at 100 pM serotonin.
  • Constructs 0 and 2 achieved increasing fluorescence signal with increasing serotonin concentration, but not for Construct 12 whereby increase in fluorescence signal becomes relatively fiat from 100 nM-100 ⁇ serotonin concentration. Also, Constructs 0 and 2 achieved similar fluorescence signal, but the fluorescence signal for Construct 12 is significantly lower than the other 2 constructs.
  • Figure 5 Fluorescence detection of serotonin on a solid substrate.
  • top panel Assay procedure, (lower panel) Line graph depicting the increase in fluorescence intensity for the Recomb.
  • FIG. 6 Serotonin detection on an optical and electrical set up.
  • Schematic diagram illustrating (a) a potential portable fluorescent serotonin detection and POC diagnostic device that can be developed through miniaturization of the solution-based serotonin detection assay, and (b) the various electrical and optical components required for the transduction of fluorescence signal, which is generated during serotonin detection, to electrical signal.
  • the fluorescence generated is then passed through a 530 nm bandpass filter so as to reduce the background fluorescence produced by the blue LED and to provide specific detection of serotonin. Fluorescence generated produces a potential drop across a photodiode, and the potential drop is amplified by an operational amplifier (op- amp). Finally, the amplified potential drop can be detected and visualized by an oscilloscope.
  • op- amp operational amplifier
  • Figure 7 Fluorescence study illustrating the effect of antagonist (altanserin) on the increase in fluorescence intensity obtained during serotonin detection using crude membranes prepared fro HEK293T cells transfected with the recombinant 5 ⁇ 2 ⁇ receptor gene (Recomb. 5HT2A).
  • the percentage increase in fluorescence intensity when only 100 ⁇ serotonin is present is 38.1 %, compared to the fluorescence intensity after addition of 20 ⁇ NTA-I. In the presence of 50, 250 and 500 ⁇ altanserin, the increase in fluorescence intensity becomes 30.0%, 23.4% and 7.1% respectively.
  • Figure 8 Fluorescence detection of serotonin within the physiologically relevant range.
  • FIG. 11 Immobilization schemes for GPCR on solid supports.
  • the lipophilic hydrocarbon chain of octylamine penetrates into the phospholipid bilayer and act as cell membrane anchor.
  • Cell membrane is stabilized on hydrophilic PEG surface.
  • Hydrocarbon chains act as spacers between the GPCR and the solid support, and thereby retain the conformation and functionality of the GPCR as in the whole cell,
  • HEK-293T cells Human embryonic kidney (HEK)-293T cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (Gibco), supplemented with 10% fetal bovine serum (FBS) (Gibco).
  • DMEM/F-12 Nutrient Mixture F-12
  • FBS fetal bovine serum
  • Human serotonin 5HT2A receptor gene (NCBI Reference Sequence NM 000621.2) was commercially synthesized (OriGene). The human 5HT2A receptor gene was cut out from its original expression vector, and inserted into the pcDNA3.1 (Invitrogen) expression vector. Site-directed mutagenesis was performed for the inclusion of the tetra- cysteine (TC) and His6 tag, and for creation of the various Constructs ⁇ Table 1). Mutagenesis was carried out using custom-designed primers and the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent), following recommended standard protocol. Nucleotide sequences of the Constructs were verified by restriction enzyme analysis and DNA sequencing.
  • the HEK293T cells were transiently transfected with the recombinant 5HT2A receptor gene using LipofectamineTM 2000 (Invitrogen), following standard procedures. Cells were harvested 72 h after transfection through trypsinization, for crude membrane preparation. Estabiisbing stable cell line. HE 293 cells stably expressing Construct 0 were generated by transfecting the cells with Pvul linearized expression vector (Methods) using Lipofetf amineTM 2000 (Invitrogen), and selection using G418 disulfate salt (Sigma). After selection, independent clones were isolated using limiting dilution. Clones displaying the highest receptor expression levels were used for production of Recomb. 5HT2A samples.
  • the gold- coated sensors are first cleaned with Nanostrip (Cyantek) for 15 min at room temperature, and then with 02 plasma (200 W) for 10 min before use, Immobilization of crude membranes containing the recombinant 5HT2A receptors (Recomb, 5HT2A) onto the sensors occurs by the gold-thiol binding via the tetra-cysteine (TC) tag, After mounting the sensors into the QCM-D flow cell, DPBS is introduced at 7 ⁇ /min until an initial stable baseline is established. Next, the Recomb, 5HT2A sample was introduced at 7 ⁇ /min until a stable baseline is established. Then, the sensor is washed with DPBS at 50 ⁇ /min until a stable baseline is achieved.
  • Nanostrip Cyantek
  • 02 plasma 200 W
  • Immobilization of crude membranes containing the recombinant 5HT2A receptors (Recomb, 5HT2A) onto the sensors occurs by the gold-thiol binding via the tetra-cysteine (
  • the sensors After cleaning with ethanol and then with ultrapure water, the sensors are immersed in PLL solution (0.1 mg/ml), prepared in sodium bicarbonate buffer (pH 9), for 1 h at room temperature. Finally, the sensor is cleaned with ult apure water before being used for experiments.
  • PLL solution 0.1 mg/ml
  • sodium bicarbonate buffer pH 9
  • a fluorophore-labelled antagonist
  • the samples were centrifuged in the membrane filter columns with a 30,000 MWCO at 4,000 g for 20 min to remove excess FlAsH-EDT2 and NTA-I reagents. Subsequently, the samples were transferred to a 96-well plate, with 100 ⁇ of samples per well. Serotonin and/or altanserin were added to the samples, and fluorescence was taken using a fluorescence plate reader (Tecan Infinite® M200). The excitation wavelength is 508 nm, and emission was measured at 534 nm. All experiments were done in triplicates.
  • the Recomb. 5HT2A sample was first incubated with 1 ⁇ FlAsH-EDT2 for 3 h at room temperature and then with 10 ⁇ NTA-I for 1 h. 100 ⁇ of the sample was used for detection of each serotonin concentrations.
  • An optical and electrical set up comprising 3 simple circuits (LED, photodiode and op-amp) was developed to transduce fluorescence signal to an electrical readout on an oscilloscope ( Figure 6b).
  • the Recomb. 5HT2A was excited by a 470 nm high-power blue LED (Power Light Systems).
  • the fluorescence generated from the sample is filtered tlvrough a 532 run laser-line bandpass filter (Edmund optics), so that only green fluorescence is detected. Emission of green fluorescence is detected by a silicon photodiode, which is attached to the filter.
  • the voltage across the photodiode when green fluorescence is detected is further amplified by the op-amp circuit (AD620AN). Three 909 0 resistors were connected in parallel to obtain an amplification gain of -164.
  • the voltage signal is read from a 4-channel digital phosphor oscilloscope (Tektronix TDS 3014B). Power supply to the blue LED and the op-amp is provided by a DC power supply (Topward 6303D).
  • HE 293 cells stably expressing Construct 0 were generated by transfecting the cells with Pvul linearized expression vector (Example 1 - Methods) using Li ofectamineTM 2000 (Invitrogen), and selection using G418 disulfate salt (Sigma), After selection, independent clones were isolated using limiting dilution. Clones displaying the highest receptor expression levels were used for production of Recomb. 5HT2A samples.
  • Small signal ( ⁇ 150 mV) generated by the photodiode is amplified by a high-gain OP AMP, and converted to concentration value of the analyte by a ⁇ -controller.
  • the amount of fluorescence signal generated can be converted to an electrical signal, which is necessary in the development of a diagnostic device using various optical/electrical components and circuits ( Figure 6d, top panel and Figure 6b).
  • a ligand to the human recombinant 5HT2A receptor, which can be resuspended in a vial/tube
  • a highpower blue LED with focusing lens, or a blue laser diode is used to excite the FlAsHEDT2.
  • the fluorescence generated due to ligand activation of the receptor is focused by a collection lens, filtered by a 530-nm band-pass filter, and detected by a photodiode for generation of an electrical signal.
  • the electrical signal can then be amplified by an operational amplifier (op-amp), and transduced by an oscilloscope for direct signal readout.
  • op-amp operational amplifier
  • an oscilloscope for direct signal readout.
  • this technology can be integrated with, a diagnostic device ( Figure 6a), and a microfluidic platform ( Figure 12) for automated and multiplexed detection of ligands in a device and for on-chip drug screening.
  • the diagnostic device comes with vials that are pre-loaded with different human recombinant GPCRs, and is applicable for the quantification of various GPCRrelated ligands using the detection scheme ( Figure 1) for diagnostic purposes. Furthermore, various calibration curves correlating the amount of ligands to the amount of electrical signal generated are stored in microcontrollers found in the device, Users only need to purchase one detection device, and vials loaded with different GPCRs depending on their specific needs. Under standard operating conditions, the device is first switched on. The 7-segment display should read 0 ng/ml, if not, users can press 'Reset' to eliminate any stray values.
  • users can select the ligand of their choice by pressing 'Select' repeatedly until the ligand of their choice is selected.
  • 50 ⁇ of sample e.g. a drop of blood
  • the vial can be placed into the holder of the device.
  • users can press 'Laser' for excitation. Fluorescence signal generated is converted to electrical signal, and the microcontroller processes this electrical signal based on the stored calibration curves to display the amount of ligands present in the sample on the 7-segment display.
  • Ligand detection can also be conducted on a microfluidic platform ( Figure 12) for mghthi ughput drug screening applications.
  • Various human recombinant GPCRs can be prespotted onto a functionalized glass slide using a robotic spotter, which has micrometer spotting resolution.
  • a microfluidic platform can be adapted to the glass slide, and mounted onto a platform that is analogous to a 96-well plate, The platform can then be fed into a high-throughput imaging analysis system for high-throughput analysis of agonist- induced receptor activation or antagonist-induced receptor deactivation, based on the amount of fluorescence generated.

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Abstract

The present invention is in the area of biodevices and diagnostics and generally relates to G protein-coupled receptors (GPCRs), GPCR ligands, 5-HT receptors, serotonin biosensor, diagnostic devices, drug screening, ionic lock, molecular beacon, recombinant expression, point-of-care (POC) devices. The present invention relates to recombinant GPCR variants mutated to allow for binding of a fluorophore and an acceptor or quencher, to nucleic acids encoding the GPCR variants, mammalian cells and cell lines and to labeled recombinant GPCR variants. The present invention further relates to a method of obtaining a crude membrane preparation comprising the recombinant GPCR variants and biosensors. The present invention further relates to the recombinant GPCR variants, the crude membrane preparations, the labeled recombinant GPCR variants, or the biosensor for use in the diagnosis of diseases related to the GPCR. The present invention further relates to a method for detecting the presence and/or measuring the concentration of GPCR ligand(s) in a sample and to a method of drug discovery or screening.

Description

Optical Biosensors for Diagnosis and High-Throughput Drug Screening Using Unique Conformational Changes of Recombinant Tagged
G Protein-Coupled Receptors for Activation
The present invention is in the area of biodevices and diagnostics and generally relates to G protein-coupled receptors (GPCRs), GPCR ligands, 5-HT receptors, serotonin biosensor, diagnostic devices, drag screening, ionic lock, molecular beacon, recombinant expression, point-of-care (POC) devices.
The present invention relates to recombinant GPCR variants mutated to allow for binding of a fluorophore and an acceptor or quencher, to nucleic acids encoding the GPCR variants, mammalian cells and cell lines and to labeled recombinant GPCR variants. The present invention further relates to a method of obtaining a cmde membrane preparation comprising the recombinant GPCR variants and biosensors. The present invention further relates to the recombinant GPCR variants, the crade membrane preparations, the labeled recombinant GPCR variants, or the biosensor for use in the diagnosis of diseases related to the GPCR. The present invention further relates to a method for detecting the presence and/or measuring the concentration of GPCR ligand(s) in a sample and to a method of drug discovery or screening.
BACKGROUND OF THE INVENTION
G-protein-coupled receptors (GPCRs) are an important class of membrane proteins. They are responsible for all kinds of signal transduction processes in the body. GPCRs mediate signals from native ligands such as hormones, peptides and neurotransmitters, are involved in olfaction and taste, and are coupled to bodily functions such as cardiac and endocrine functions. Furthermore, GPCRs are prime drug targets, recognized by -60% of therapeutic drugs such as /3-blockers, antipsychotics and analgesics. The GPCR superfamily contains well-conserved motifs, indicating a common activation mechanism among all GPCRs.
We are particularly interested in the 5-hydroxytryptamine 2A (5-HT2A) serotonin receptor, mainly found in the central nervous system and gastro-intestinal tract. It is the best characterized serotonin receptor. Abnormal blood serotonin levels are associated with diseases such as autism, cancer, head trauma, schizophrenia and depression, making serotonin an important biomarker. It is therefore desirable to provide improved means and methods for serotonin detection, which can be utilized as diagnostic devices and/or drug screening devices.
SUMMARY OF THE INVENTION
According to the present invention this object is solved by a recombinant G-protein coupled receptor (GPCR) variant mutated to allow for binding of a fluorophore and an acceptor or quencher,
wherein said recombinant GPCR variant comprises
(1) a binding site or sequence for the fluorophore,
(2) a binding site or sequence for the acceptor or quencher,
wherein one binding site is introduced in the proximity of the Arg residue of the (D/E)RY motif in transmembrane helix 3 (H3) of the GPCR and the other binding site is introduced in the proximity of the acidic amino acid residue in transmembrane helix 6 (H6) of the GPCR, wherein said Arg and said acidic amino acid residue form the ionic lock motif and have an ionic interaction in the inactive state of the GPCR.
According to the present invention this object is solved by a nucleic acid encoding a recombinant GPCR variant of the present invention.
According to the present invention this object is solved by a mammalian cell or cell line comprising a nucleic acid of the present invention and expressing a recombinant GPCR variant of the present invention on the cell membrane, preferably stably expressing the GPCR variant,
According to the present invention this object is solved by a method of obtaining a crude membrane preparation comprising a recombinant GPCR variant of the present invention, comprising the steps of
(a) providing a nucleic acid of the present invention, transfecting mammalian cells with said nucleic acid or providing a cell or cell line of the present invention,
(b) culturing the cells or cell line and thereby expressing the recombinant GPCR variant in said cell or cell line,
(c) harvesting and homogenizing the cells and centrifuging,
(d) resuspending the pellet to obtain the crude membrane preparation. According to the present invention this object is solved by a recombinant GPCR variant of the present invention labeled with fluorophore and acceptor or quencher.
According to the present invention this object is solved by a biosensor comprising a recombinant GPCR variant of the present invention, a crude membrane preparation obtained in the methods of to the present invention or a labeled recombinant GPCR variant of the present invention.
According to the present invention this object is solved by using a recombinant GPCR variant of the present invention, a crude membrane preparation obtained in the methods of the present invention, a labeled recombinant GPCR variant of the present invention, or a biosensor of the present invention in ligand binding assays, drug discovery and drug screening,
According to the present invention this object is solved by a recombinant GPCR variant of the present invention, a crude membrane preparation obtained in the methods of the present invention, a labeled recombinant GPCR variant of the present invention, or a biosensor of the present invention for use in the diagnosis of diseases related to the GPCR, wherein a disease related to the GPCR is selected from
diseases associated or caused by high serotonin levels , such as brain injury due to high serotonin levels, violence and aggression, autism, colon cancer,
diseases associated or caused by low serotonin levels, such as schizophrenia, depression.
According to the present invention this object is solved by a method for detecting the presence and/or measuring the concentration of GPCR ligand(s) in a sample,
comprising the use of a recombinant GPCR variant of the present invention, a crude membrane preparation obtained according to the present invention, a labeled recombinant GPCR variant of the present invention, or a biosensor of the present invention.
According to the present invention this object is solved by a method of drug discovery or screening, comprising the steps of
(i) providing a compound or drug to be tested;
(ii) providing a recombinant GPCR variant of the present invention or a crude membrane preparation according to the present invention or a biosensor of the present invention, and adding a fluorophore and acceptor or quencher, either at the same time or sequentially, to the recombinant GPCR variant or the crude membrane preparation and incubating;
or
providing a labeled recombinant GPCR variant of the present invention or a crude membrane preparation obtained according to the present invention;
(iii) providing a ligand (or agonist) of the GPCR;
(iv) adding the ligand and compound or drug to be tested to the labeled recombinant GPCR variant or crude membrane preparation comprising a labeled recombinant GPCR variant,
(v) determining whether the drug has an effect on ligand/agonist binding by determining the fluorescence signal,
wherein a decrease of the fluorescence signal (compared to the fluorescence signal of the ligand/agonist) is indicative that the compound or drug to be tested is an antagonist of the ligand, i.e. competes for the binding site;
and wherein an increase of the fluorescence signal (compared to the fluorescence signal of the ligand/agonist) is indicative that the compound or drug to be tested is an agonist of the ligand, i.e. increases ligand binding; or that the compound or drug to be tested is an agonist that is capable of activating the GPCR, by triggering the release of the ionic lock.
DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.
Recombinant GPCR variants
As discussed above, the present invention provides a recombinant G-protein coupled receptor (GPCR) variant imitated to allow for binding of a fluorophore and an acceptor or quencher, wherein said recombinant GPCR variant comprises (1) a binding site or sequence for the fluorophore,
(2) a binding site or sequence for the acceptor or quencher,
wherein one binding site is introduced in the proximity of the Arg residue of the (D/E) Y motif in transmembrane helix 3 (H3) of the GPCR and the other binding site is introduced in the proximity of the acidic amino acid residue in transmembrane helix 6 (H6) of the GPCR, wherein said Arg and said acidic amino acid residue form the ionic lock motif and have an ionic interaction in the inactive state of the GPCR.
In one embodiment, a binding site or sequence for the fluorophore and/or the acceptor or quencher comprises 3 to 10 amino acids, preferably 5 to 9 amino acids, such as 5, 6, 7 or 9 amino acids.
Thereby, the binding site or sequence differs in length (number of amino acid residues) depending on the fluorophore - acceptor/quencher used.
In one embodiment, the binding site or sequence for the fluorophore comprises a tetra- cysteine tag (TC tag), preferably an amino acid sequence comprising or consisting of the amino acid sequence
CC(X)„CC (SEQ ID NO. 1) wherein C is cysteine and X is any amino acid and n is 1 to 5.
For example, the tetra- cysteine tag (TC tag) comprises or consists of the amino acid sequence with SEQ ID NO. 1, wherein n = 2 (i.e. amino acid sequence CCXXCC).
In one embodiment, the binding site or sequence for the fluorophore comprises a tetra- cysteine tag (TC tag), more preferably an amino acid sequence comprising or consisting of an amino acid sequence selected from the group of
CCPGCC (SEQ ID NO. 2),
CCRECC (SEQ ID NO. 3),
CCACC (SEQ ID NO. 4),
CCGCC (SEQ ID NO. 5),
CCPCC (SEQ ID NO. 6), CCAECC (SEQ ID NO. 7),
CCSECC (SEQ ID NO. 8),
CCDECC (SEQ ID NO. 9),
CCGPCC (SEQ ID NO. 10),
CCDEACC (SEQ ID NO. 1 1), or
CCKAEAACC (SEQ ID NO. 12).
In one embodiment, when the iluorophore used is FlAsH-EDT2, the amino acid sequence CCPGCC (SEQ ID NO. 2) is preferred.
In one embodiment, when the fluorophore AsCy3 used is the amino acid sequence CCKAEAACC (SEQ ID NO. 12) is preferred.
Since AsCy3 requires a longer amino acid tag as its interatomic distance of ~ 14.5 A is longer than FlAsH-EDT2 of- 6 A.
In one embodiment, the binding site or sequence for the iluorophore comprises a tag or fusion selected from the group of
Rhodococciis dehalogenase (DhaA) (HaloTag System),
SNAP tag,
CLIP tag,
fusion with 06-alkylguanine-DNA alkyl transferase (wildtype),
Lipoic acid ligase acceptor peptide (LAP).
In one embodiment, the GPCRs are tagged with a protein tag, in particular by the insertion of the Rhodococciis dehalogenase (DhaA), which contains an aspartate nucleophile that forms stable covalent bonds with aliphatic hydrocarbons containing a halide, e.g. 1 ,2- dibromoethane. This is known as the HaloTag System (by Promega, see; http://www.qub. ac.uk/mlpage/courses/level3/meb/halotagcb800025k.pdf). Fluorescent chloroalkane ligands include carboxytetramethylrhodamine (TMR).
In one embodiment, the SNAP-tag, a 20 kDa mutant of the DNA repair protein, O6- alkylguanine-D A alkyltransferase, is used, which reacts specifically, irreversibly and covalently with benzylguanine (BG) derivatives, The BG derivatives can subsequently be labelled with fluorophore of interests for visualization. In one embodiment, the CLIP-tag, is used, which is reacts specifically with 02-benzylcytosine (BC) derivatives. The CLIP-tag was created by engineering the substrate specificity of the SNAP-tag, permitting it to react specifically with 02-benzylcytosine (BC) derivatives
Both SNAP- and CLIP-tags are products of NEB (see: https://w w.neb.com/tools-and- resources/feature-artic3es/snap-tag-teclmologies-novel-tools-to-study-protein-fiinction).
In one embodiment, fusion proteins of the GPCRs are engineered to contain the human DNA repair protein, 06-alkylguanine-DNA alkyltransferase (wildtype), which can then be labelled with a fluorophore using 06-benzyl guanine (BG) and its derivatives, such as BGBT, BGAF and BGFL.
In one embodiment, the tag is lipoic acid ligase acceptor peptide (LAP), 22-amino acid sequence peptide, which can be modified by lipoic acid ligase (LplA) and azide, to present an azide functional group, for further downstream conjugation with fluorophores such as cyclo- octyne- conjugated AlexaFluor 568 or Cy3.
In one embodiment, the binding site or sequence for the acceptor or quencher comprises an amino acid sequence of multiple His (His6 tag), preferably amino acid sequence HHHHHH (SEQ ID NO. 13).
The fluorophore and acceptor/quencher are a fluorescence resonance energy transfer (FRET) pair.
Preferably, the fluorophore is selected from
a fluorescein or derivative,
preferably FlAsH-EDT2 (4',5'-bis(l ,3,2-dithioarsoIan-2-yl)fluorescein-(l ,2-ethanedithiol)2).
Further examples of bi arsenical fluorophores are:
- ReAsH-EDT2 (a Resorufin fluorophore derivative, which has a red fluorescence. Red fluorescence is attractive for fluorescence microscopy as cellular absorbance, scattering and autofluorescence decrease greater at such longer wavelengths.);
- sFlAsH-EDT2 (membrane-impermeable); - F2-FlAsH and F4-FlAsH (photostable fluorophore);
- Carboxy-FlAsH, or CrAsH-EDT2 (more polar/less hydrophobic version of FlAsH-EDT2);
- CHoX-AsH-EDT2 (blue-fluorescing biarsenical);
- Br2REAsH-EDT2;
- ThAsH-EDT2;
- AsCy3;
Further fluorophores are
fluorescein,
FA (Carboxyfluorescein),
AlexaFluor 488,
(cyclo-octyne-conjugated) AlexaFluor 568, or
Cy3,
a rhodamine or derivative
such as carboxytetramethylrhodamine (TMR).
Preferably, the acceptor or quencher is a metal-ion-chelating nitrilotriacetate moiety, preferably nitrilotriacetic acid chromophore (NTA-I),
Further examples of acceptor/quencher are:
- Gold nanoparticles (Au NPs) that are conjugated to a nitrilotriacetic acid (NTA) motif. Au NPs, having its absorption peak at 525 run (green region), will be able to act as fluorescence acceptor of green fluorescence in a FRET pair.
- Other fluorophores that are conjugated to a NTA motif can also act as fluorescence acceptor. Red fluorophores such as Cy3 quenches green fluorophores such as FlAsH-EDT2, fluorescein, FAM and AlexaFluor 488. Green fluorophores on the other hand, will quench blue fluorescence.
Disclaimer:
The fluorophore and acceptor/quencher used according to the invention are a FRET pair, provided that they are not both green fluorescent protein (GFP) or any of its derivatives, such as CFP/YFP, such as disclosed in Lohse et al. 2012 (Lohse MJ, Nuber S, Hoffmann C. Fluoiescence/bioluminescence resonance energy transfer techniques to study G-protein- coupled receptor activation and signaling. Pharmacol Rev, 2012 Apr;64(2):299-336.)
In one embodiment, the GPCR is a 5HT2A serotonin receptor, a Wnt receptor or an odorant receptor.
In one embodiment, the GPCR is (human) 5HT2A serotonin receptor.
Human 5HT2A serotonin receptor:
Gene:
Entrez Gene: 3356
Ensembl: ENSG00000102468
Nucleotide sequences:
GenBank: X57830 (Homo sapiens serotonin 5-HT2 receptor mRNA)
RefSeq: NM_000621 (Homo sapiens 5-hydroxytryptamine (serotonin) receptor
2A, G protein-coupled (HTR2A), transcript variant 1, mRNA)
Protein sequences:
UniProtKB: P28223
The human 5HT2A is preferably encoded by nucleic acid sequence SEQ ID NO. 14: atggatatt ctfctgtgaag aaaatacttc tttgagctca actacgaact ccctaatgca attaaatgat gacaccaggc tctacagtaa tgactttaac tccggagaag
ctaacacttc tgatgcattt aactggacag tcgactctga aaatcgaacc aacctttcct
gtgaagggtg cctctcaccg tcgtgtctct ccttacttca tctccaggaa aaaaactggt
ctgctttact gacagccgta gtgattattc taactattgc tggaaacata ctcgtcatca
tggcagtgtc cctagagaaa aagctgcaga atgccaccaa ctatttcctg atgtcacttg
ccatagctga tatgctgctg ggtttccttg tcatgcccgt gtccatgtta accatcctgt
atgggtaccg gtggcctctg ccgagcaagc tttgtgcagt ctggatttac ctggacgtgc
tcttctccac ggcctccatc atgcacctct gcgccatctc gctggaccgc tacgtcgcca
tccagaatcc catccaccac agccgcttca actccagaac taaggcattt ctgaaaatca
ttgctgtttg gaccatatca gtaggtatat ccatgccaat accagtcttt gggctacagg
acgattcgaa ggtctttaag gaggggagtt gcttactcgc cgatgataac tttgtcctga
tcggctcttt tgtgtcattt ttcattccct taaccatcat ggtgatcacc tactttctaa
ctatcaagtc actccagaaa gaagctactt tgtgtgtaag tgatcttggc acacgggcca
aattagcttc tttcagcttc ctccctcaga gttctttgtc fctcagaaaag cfccttccagc
ggtcgatcca tagggagcca gggtcctaca caggcaggag gactatgcag tccatcagca
atgagcaaaa ggcatgcaag gtgctgggca tcgtcttctt cctgtttgtg gtgatgtggt
gccctttctt catcacaaac atcatggccg tcatctgcaa agagtcctgc aatgaggatg
tcattggggc cctgctcaat gtgtttgttt ggatcggtta tctctcttca gcagtcaacc
cactagtcta cacactgttc aacaagacct ataggtcagc cttttcacgg tatattcagt gtcagtacaa ggaaaacaaa aaaccattgc agttaatttt agtgaacaca ataccggctt tggcctacaa gtctagccaa cttcaaatgg gacaaaaaaa gaattcaaag caagatgcca agacaacaga taatgactgc tcaatggttg ctctaggaaa gcagcattct gaagaggctt ctaaagacaa tagcgacgga gtgaatgaaa aggtgagctg tgtgtga
The human 5HT2A preferably has the amino acid sequence of SEQ ID NO. 15 or
SEQ ID NO. 15:
UniProtKB: P28223- l ,
Isoform 1 of 5 -hydroxytiyp famine receptor 2A, Homo sapiens
MDILCEENTS LSSTTNSLMQ LNDDTRLYSN DFNSGEA TS DAFHWTVDSE NRTNLSCEGC LSPSCLSLLH LQEKNWSALL TAWIILTIA GNILVIMAVS LEKKLQNATN YFLMSLAIAD MLLGFLVMPV SMLTILYGYR WPLPSKLCAV WIYLDVLFST ASIMHLCAIS LDRYVAIQNP IHHSRFNSRT AFLKIIAVW TISVGISMPI PVFGLQDDSK VFKEGSCLLA DDNFVLIGSF VSFFIPLTIM VITYFLTIKS LQKEATLCVS DLGTRAKLAS FSFLPQSSLS SE LFQRSIH RBPGSYTGRR TMQSISNEQK ACKVLGIVFF LFWMWCPFF ITNIMAVICK ESCNEDVIGA LLNVFVWIGY LSSAVNPLVY TLFN TYRSA FSRYIQCQYK ENKKPLQLIL V TIPALAY SSQLQMGQKK NSKQDAKTTD DCSMVALGK QHSEEASKDN SDGVWEKVSC V
SEQ ID NO. 16:
UniProtKJB: P28223-2,
Isoform 2 of 5-hydroxytryptamine receptor 2A, Homo sapiens MQFLKSAKQ PNYYHIMLVE DQEEGTLHQF NYCERCSESQ N KCISCVDP
EDKWYRWPLP SKLCAVWIYL DVLFSTASIM HLCAISLDRY VAIQNPIHHS RFNSRTKAFL KIIAVWTISV GISMPIPVFG LQDDSKVFKE GSCLLADDNF VLIGSFVSFF IPLTIMVITY FLTIKSLQKE ATLCVSDLGT RAKLASFSFL PQSSLSSEKL FQRSIHREPG SYTGRRTMQS ISNEQ AC V LGIVFFLFW MWCPFFITNI MAVIC ESC EDVIGALLNV FVtllGYLSSA VNPLVYTLFN
KTYRSAFSRY IQCQYKENKK PLQLILV TI PALAYKSSQL QMGQKKNSKQ DAKTTDNDCS MVALGKQHSE EASKDNSDGV NEKVSCV
Said (human) 5HT2A serotonin receptor preferably comprises (1) the binding site or sequence for the fluorophore introduced in the proximity of residue Arg 173 at transmembrane helix 3 (H3),
(2) the binding site or sequence for the acceptor or quencher introduced in the proximity of Glu 318 at transmembrane helix 6 (H6).
In one embodiment, wherein the GPCR is (human) 5HT2A serotonin receptor as defined above,
the binding site or sequence for the fluorophore (1) is a tetra-cysteine tag (TC tag), preferably comprising or consisting of amino acid sequence CCPGCC (SEQ ID NO. 2) and wherein the fluorophore is FlAsH-EDT2
and/or
the binding site or sequence for the acceptor or quencher (2) is an amino acid sequence of multiple His (His6 tag), preferably comprising or consisting of amino acid sequence HHHHHH (SEQ ID NO, 13) and wherein the acceptor or quencher is nitrilotriacetic acid chromophore (NTA-I),
In one embodiment, wherein the GPCR is (human) 5HT2A serotonin receptor as defined above,
the binding site or sequence for the fluorophore (1) is introduced in the proximity of residue Arg 173 in the following way: by insertion C-tem inal of Arg 173 (preferably adjacent to Arg 173) or by replacing the wildtype amino acids C-tenninal of Arg 173 (preferably adjacent to Arg 173),
and/or
the binding site or sequence for the acceptor or quencher (2) is introduced in the proximity of Glu 318 in the following way: by insertion N-tenninal of Glu 318 (preferably adjacent to Glu 318 or 3 amino acids N-terminal of Glu 318) or by replacing the wildtype amino acids N-terminal of Glu 318 (preferably adjacent to Glu 318 or 10 amino acids N- terminal of Glu 318).
In one embodiment, wherein the GPCR is (human) 5HT2A serotonin receptor as defined above, the recombinant GPCR variant is selected from the variants comprising an amino acid sequence selected from at least one of SEQ ID NOs. 20 to 29, preferably one of SEQ ID NOs. 19, 21, 23, 25 and 27 and one of SEQ ID NOs. 20, 22, 24, 26 and 28,
preferably comprising SEQ ID NOs. 19 and 20 such as in Construct 0; or
SEQ ID NOs. 21 and 22 such as in Construct 1; or
SEQ ID NOs. 23 and 24 such as in Construct 2; or
SEQ ID NOs. 25 and 26 such as in Construct 7; or
SEQ ID NOs. 27 and 28 such as in Construct 12;
as shown in Table 1.
The amino acid sequences shown in SEQ ID NOs. 1 , 21, 23, 25 and 27
refer to amino acid sequences of the binding site or sequence for the fluorophore (1 ) which is a tetra- cysteine tag (TC tag), comprising or consisting of amino acid sequence CCPGCC
(SEQ ID NO. 2) and introduced in the proximity of residue Arg 173 at transmembrane helix 3
(H3).
The amino acid sequences shown in SEQ ID NOs. 20, 22, 24, 26 and 28
refer to amino acid sequences of the binding site or sequence for the acceptor or quencher (2) which is an amino acid sequence of multiple His (His6 tag), comprising or consisting of amino acid sequence HHHHHH (SEQ ID NO. 13) and introduced in the proximity of Glu 318 at transmembrane helix 6 (H6).
Table 1 Amino acid sequence of wild-type 5HT2A and the various recombinant 5HT2A constructs after mutagenesis.
Arg-173 is highlighted bold, GIu-318 is highlighted bold and underlined;
TC tag is highlighted italic and Hisdag is highlighted italic and underlined.
Figure imgf000014_0001
Construct 2 SLDRCCFGCCYVA IHREPGSYTGRRTMQSISNEQK TC - inserted after Arg- AHHHHHHCKV 173
His6 - inserted 3 AA
[SEQ ID NO. 23] [SEQ ID NO. 24]
before Glu-318
Construct 7 SLDRCCPGCCPIH IHREPGSYTGRRTMQSISNHHH TC - replaced after Arg- HHHEQKAC V 173
His6 - inserted before Glu-
[SEQ ID NO. 25] [SEQ ID NO. 26]
318
Construct SLDRCCPGCCPIH IHRHHHHffflGRRTMQSISNEQK TC - replaced after Arg- 12 ACKVLGIVFF 173
Hiss - replaced 10 AA
[SEQ ID NO. 27] [SEQ ID NO. 28]
before Glu-318
In one embodiment, the GPCR is serotonin receptor 5HT4.
Ligand: Serotonin
BIMU-8, Cisapride, CJ-033, 466, etc.
GR-1 13, 808, GR-125, 487 etc.
(Potential) medicinal application:
anxiety, appetite, GI motility, learning, memory, mood, respiration, ect. Structure information: unknown
Biosensor: cell -based system only
(receptor activity measured through co-expressed channel activity=
In one embodiment, the GPCR is Wnt receptor FZD.
Ligand: Wnt (Wnt 5A for FZD4 and FZD5), Norrin (FZD4), etc.
Involvement in development and diseases:
Stem cell self-renewal, differentiation, stem cell development
No rie disease (FZD4), breast cancer (FZD5), intestine and colorectal
(FZD5/10), lung cancer (FZD5), brain cancer etc.
Structural information: Limited
(N-terminal cysteine-rich domain of mFzd8 known)
Ligand screening: Limited
Sensing device: not available In one embodiment, the GPCR is W t receptors FZDs.
Ligand: WNT, DVL etc.
(Potential) medicinal application:
breast cancer, lung cancer, intestine and colorectal cancer, rheumatoid artliritis, etc.
FZD4 FZD5 FZD10
Familial exudative Benign ovarian Chromosomal vitreoretinopathy, Nome tumor, chronoic duplication, cervical disease, progressive myeloid leukemia, cancer, gastric cancer, cerebellar degeneration, etc. renal tumor, gastric etc,
cancer, etc.
Cell differentiation, stem cell development
Structure information: unknown (except for the N-terminal CRD of mFzd8)
Nucleic acids encoding the recombinant GPCR variants and cell or cell lines
As discussed above, the present invention provides a nucleic acid encoding a recombinant
GPCR variant according to the present invention,
preferably an expression vector or construct.
As discussed above, the present invention provides a mammalian cell or cell line comprising a nucleic acid of the present invention and expressing a recombinant GPCR variant of the present invention on the cell membrane, preferably stably expressing the GPCR variant, such as HEK293T.
Methods for obtaining crude membrane preparation, labeled GPCR variants, biosensors ami their uses
As discussed above, the present invention provides a method of obtaining a crude membrane preparation comprising a recombinant GPCR variant of the present invention, comprising the steps of
(a) providing a nucleic acid of the present invention, transfecting mammalian cells with said nucleic acid or providing a cell or cell line of the present invention, (b) culturing the cells or cell line and thereby expressing the recombinant GPCR variant in said cell or cell line,
(c) harvesting and homogenizing the cells and centrifuging,
(d) resuspending the pellet to obtain the crude membrane preparation.
As discussed above, the present invention provides a recombinant GPCR variant of the present invention labeled with fluorophore and acceptor or quencher,
such as labeled with FlAsH-EDT2 and NTA-I,
such as human 5HT2A serotonin receptor labeled with FlAsH-EDT2 and NTA-I.
In one embodiment, the method further comprises the steps of
(e) adding a fluorophore and acceptor or quencher, either at the same time or subsequently, to the crude membrane preparation and incubating,
(f) obtaining a crude membrane preparation comprising a labeled recombinant GPCR variant,
such as the labeled recombinant GPCR variant of the present invention.
As discussed above, the present invention provides a biosensor comprising
a recombinant GPCR variant of the present invention,
a crude membrane preparation obtained in the methods of the present invention, or a labeled recombinant GPCR variant of the present invention,
preferably comprising a 96-well plate, a solid substrate (such as nitrocellulose membrane) or a solid support (such as a glass plate, microarray or chip).
As discussed above, the present invention provides the use of
a recombinant GPCR variant of the present invention,
a crude membrane preparation obtained in the methods of the present invention, a labeled recombinant GPCR variant of the present invention, or
a biosensor of the present invention,
in ligand binding assays, dmg discovery and drug screening.
Diagnosis of diseases related to the GPCR
As discussed above, the present invention provides
a recombinant GPCR variant of the present invention, a crude membrane preparation obtained in the methods of the present invention, a labeled recombinant GPCR variant of the present invention, or
a biosensor of the present invention,
or use in the diagnosis of diseases related to the GPCR,
wherein a disease related to the GPCR is selected from
diseases associated or caused by high serotonin levels , such as brain injury due to high serotonin levels, violence and aggression, autism, colon cancer,
diseases associated or caused by low serotonin levels, such as schizophrenia, depression.
In one embodiment, the present invention provides the recombinant GPCR variant of the present invention or the biosensor of the present invention,
wherein the GPCR is (human) 5HT2A serotonin receptor,
for use in the diagnosis of diseases related to serotonin activity, such as vascular smooth muscle contraction, platelet aggregation, perception, emotion, mental and behavioral disorders, autism, violent behavior, schizophrenia, depression.
In one embodiment, the present invention provides the recombinant GPCR variant of the present invention or the biosensor of the present invention,
wherein the GPCR is a Wnt receptor,
for use in the diagnosis of cancer, developmental dysregulations.
In one embodiment, the present invention provides the recombinant GPCR variant of the present invention or the biosensor of the present invention,
wherein the GPCR is an odorant receptor,
for use in the diagnosis of dysfunctions in smelling, detection of specific odorants. Detection of GPCR ligandfs) in a sample
As discussed above, the present invention provides a method for detecting the presence and/or measuring the concentration of GPCR ligand(s) in a sample,
comprising the use of
a recombinant GPCR variant of the present invention,
a crude membrane preparation obtained in the methods of the present invention, a labeled recombinant GPCR variant of the present invention, or
a biosensor of the present invention. In one embodiment, the GPCR ligands are neurotransmitters, like serotonin, an odorant or ligands binding to Wnt receptors, like FZD, amino acids, amines, firagrants.
In one embodiment, the method comprises the steps of
(i) providing a sample,
(ii) providing a recombinant GPCR variant of the present invention or a crude membrane preparation obtained according to the present invention or a biosensor of the present invention, and adding a fluorophore and acceptor or quencher, either at the same time or subsequently, to the recombinant GPCR variant or the crade membrane preparation and incubating;
or
providing a labeled recombinant GPCR variant of the present invention or a crade membrane preparation obtained according to the present invention;
(iii) adding the sample to the labeled recombinant GPCR variant or crade membrane preparation comprising a labeled recombinant GPCR variant,
(iv) determining whether ligand is present in the sample by determining a fluorescence signal, wherein no fluorescence signal is indicative that no ligand is present and a fluorescence signal is indicative that ligand is present.
In one embodiment, the method comprises
using or establishing a calibration curve with a ligand, in order to allow quantification or measuring the concentration of said ligand in a sample.
In one embodiment, the method comprises
adding the labeled recombinant GPCR variant or crade membrane preparation comprising a labeled recombinant GPCR variant of step (ii) to a 96-well plate, a solid substrate (such as nitrocellulose membrane) or a solid support (such as a glass plate, microan ay or chip).
In one embodiment, the method comprises
the use of antagonists or agonists, such as in competitive ligand binding assays. In one embodiment, the method comprises the conversion of the fluorescence signal into an electrical signal, such as by the use of photodiode.
Drug discovejy and screening
As discussed above, the present invention provides a method of drug discovery or screeni g, comprising the steps of
(i) providing a compound or drug to be tested;
(ii) providing a recombinant GPCR variant of the present invention or a crude membrane preparation obtained according to the present invention or a biosensor of the present invention, and adding a fluorophore and acceptor or quencher, either at the same time or sequentially, to the recombinant GPCR variant or the crude membrane preparation and incubating;
or
providing a labeled recombinant GPCR variant of the present invention or a crude membrane preparation obtained according to the present invention;
(iii) providing a ligand (or agonist) of the GPCR;
(iv) adding the ligand and compound or drag to be tested to the labeled recombinant GPCR variant or crude membrane preparation comprising a labeled recombinant GPCR variant,
(v) determining whether the drug has an effect on ligand/agonist binding by determining the fluorescence signal,
wherein a decrease of the fluorescence signal (compared to the fluorescence signal of the ligand/agonist) is indicative that the compound or drug to be tested is an antagonist of the ligand, i.e. competes for the binding site;
and wherein an increase of the fluorescence signal (compared to the fluorescence signal of the ligand/agonist) is indicative that the compound or drag to be tested is an agonist of the ligand, i.e. increases ligand binding; or that the compound or drug to be tested is an agonist that is capable of activating the GPCR, by triggering the release of the ionic lock.
In one embodiment, the GPCR ligands are neurotransmitters, like serotonin, an odorant or ligands binding to Wnt receptors, like FZD, amino acids, amines, fragrants.
In one embodiment, the method comprises adding the labeled recombinant GPCR variant or crude membrane preparation comprising a labeled recombinant GPCR variant of step (ii) to a 96-well plate, a solid substrate (such as nitrocellulose membrane) or a solid support (such as a glass plate, microarray or chip).
In one embodiment, the method comprises
the conversion of the fluorescence signal into an electrical signal, such as by the use of photodiode.
Farther description of preferred embodiments
- Abstract:
Here, we present a novel serotonin detection method, by exploiting the ionic interaction between Arg-173 and Glu-318, particularly the activation and subsequent conformational change upon serotonin binding of the highly conserved "(D/E)RY motif of the 5-HT2A receptor. We constructed a recombinant human 5-HT2A receptor, comprising an engineered tetra-cysteine tag for specific binding of the FlAsH-EDT2 fluorophore, and a Hisg tag adjacent to Glu-318 for specific binding of a nitrilotriacetate quencher (NTA-I). We observed piconiolar level of serotonin detection via increase in fluorescence, when conformational changes due to serotonin binding resulted in separation between FlAsH-EDT2 and NTA-I. To our knowledge, this is the first model of a ligand detection system that utilizes the ligand- dependant conformational changes of the ionic lock motif commonly observed in GPCRs. Moreover, we demonstrate the ability of the present method to be miniaturized for the development of a rapid and sensitive diagnostic device, as well as being performed on a functionalized solid substrate for the production of multiplexed drug screening device.
We have developed an optical sensing platform employing specifically designed recombinant tagged GPCRs that are activated by a unique molecular switch mechanism. The invention utilizes the sensing platform for facile and rapid detection of GPCR-based ligands, such as neurotransmitters (e.g. serotonin, epinephrine and dopamine), for diagnostic purposes, as well as drug/ligand screening discovery applications. We are specifically interested in neurotransmitters that typically exert their cellular effects through activation of cell surface membrane receptor/protein that belongs to the GPCR superfamily. This teclinology can be further integrated with a diagnostic device and a microfluidic platform for the rapid, automated and multiplexed detection of ligands in a device and for on-chip drag screening. G protein-coupled receptors (GPCRs) are a large class of membrane receptors characterized by seven, transmembrane a-helices, which are separated by alternating intra- and extracellular loop regions [1]. GPCRs are vital for cellular signaling and function [2]. Furthermore, they serve as important pharmacological targets [3]. The 5-hydroxytryptamine 2A (5HT2A) serotonin receptor is a GPCR with essential roles for the actions of serotonin (5HT) in diverse physiological processes, such as vascular smooth muscle contraction, platelet aggregation, perception and emotion [4]. The 5HT2A receptor has a high expression tlrroughout all layers of the cortex, particularly in the fifth layer [5]. Abnormal serotonin levels in whole blood has been implicated in various mental and behavioural disorders, where high serotonin levels are associated with autism [6] and violent behaviour [7], and low serotonin levels are linked to schizophrenia and depression [8]. Additionally, most typical and nearly all atypical antipsychotic drugs, such as clozapine [9], target the 5HT2A receptor [5]. The 5HT2A receptor is also a major site of actions for hallucinogens, such as lysergic acid diethylamide, which is an agonist [4]. Hence, the 5HT2A receptor represents an important drug target for the treatment of various mental disorders/diseases,
There is a strong ionic interaction between Arg-173 in transmembrane helix 3 (H3), and Glu- 318 in H6 of 5HT2A, which make up an ionic lock motif, and this interaction stabilizes the receptor in its inactive state [4]. Modeling studies show that agonist binding results in the rotation of H6 and subsequent disruption of the ionic interaction between H3 and H64. Additionally, the (D/E)RY motif near the cytoplasmic end of H3 is highly conserved among GPCRs [10], and various studies have suggested that Arg-173 commonly plays an important function in the activation of GPCRs [1 1-15], although without definite mechanisms. As such, we used the conformational changes associated with agonist activation of the 5HT2A receptor, to develop a sensitive serotonin biosensor, and this proof-of-concept can be further extended to develop other GPCR-based biosensors. Studies on conformational change of protein of interest have commonly relied on the Forster (Fluorescence) resonance energy transfer (FRET) technique [16]. However, most studies have adopted the use of green fluorescent protein (GFP), which are relatively large compared to the proteins, and risk affecting the conformation, and hence, activity of labeled proteins [17]. For instance, CFP and YFP were shown to affect certain properties of fusion GPCRs [18]. The 5-HT2A receptor was chosen as a GPC model system to prove the viability of the technology. Thus, the majority of experimental results are obtained with this receptor type. Nonetheless, we have characterized several other GPCRs, 5-HT4 receptor, different Wnt receptors (frizzled), as well as a fish odorant receptor. In general, we are using HEK293T cells to overexpress a recombinant version of the GPCR, We have explored other mammalian expression systems, such as COS cells or BH cells, the insect baculovirus expression system, as well as expression in E. coli.
The present study used small fluorophore and quencher molecules, which do not perturb the pharmacological and signaling properties of the 5HT2A receptor, in the detection of serotonin. Our recombinant human 5HT2A receptor has been specifically mutated to incorporate a tetracysteine (TC) tag, with the amino acid sequence of CCPGCC, for specific binding of the fluorophore FlAsH-EDT2 [19], and a Hiss tag, for the specific binding of a metal-ion-chelating nitrilotriacetate moiety (NTA-I) [20] for quenching of the FlAsH-EDT2. FlAsH-EDT2 does not fluoresce in free solution, however, it is highly fluorescent when it binds to a specific sequence of 56 amino acids [19, 21], especially CCPGCC [22]. Hence, the use of FlAsH-EDT2 as a small fluorophore does not produce high background noise, and will not disturb the structure of the 5HT2A receptor. The NTA-I exhibit site-specific labeling properties to His6 tag in proteins of interest, binding to His6 tags within seconds [20]. Additionally, it quenches fluorescence in the green region (520-560 nm), which corresponds to the emission of FlAsH-EDT2 (534 nm). Similarly, NTA-I is a non-fluorescent acceptor dye, which does not contribute background noise during fluorescence detection.
This study reports the first usage of the confonnational change experienced in the ionic lock motif of the human 5HT2A receptor, upon agonist activation, for the biosensing of serotonin with picomolar limit of detection. Such sensitivity level will definitely be beneficial for detection of serotonin in the body for diagnostic purposes, and opens up opportunities for drug screening purposes as well. In our detection scheme (Figure 1), NTA-I quenches the fluorescence of F]AsHEDT2 due to their close proximity when the 5HT2A receptor is in its inactive state. The binding of agonist, such as serotonin, results in the disruption of the ionic interaction, and subsequent conformation change, which separates FIAsH-EDT2 and NTA-I. This separation restores fluorescence, and the presence of serotonin is detected as an increase in fluorescence signal. Advantageously, there is disclosed a method of exploiting the ligand binding lock-unlock motif in GPCRs for the development of highly sensitive optical biosensors for diagnosis and drug disco very/screening.
The technology disclosed herein describes for the first time the use of the conformational change in GPCR receptors, induced by unleashing the ionic lock, as exemplified for 5-HT2A receptors, upon ligand activation for the biosensing of GPCR ligands, as for example serotonin in the case of the 5-HT2A receptor.
The detection of GPCR ligands, i.e. serotonin, as well as the screening of drugs requires only a single-step addition of ligand or drug candidates, without the need of any washing steps.
The major advantage of our technology is the use of tiny amounts (microliter volumes) of crude cellular membranes that contain the highly sensitive GPCR receptor of choice. No whole cell assays (short half-life and very labile) or spectroscopy set-ups (expensive equipment that cannot be trimmed down to a POC device) are needed. Furthermore, molecular biological expertise for the design of the appropriate recombinant receptor is needed. It is also practically impossible to get the identical permanent cell clone reproduced.
The signal readout is fast, requiring only 5- 15 minutes of incubation time.
- Results
Generation of human recombinant 5HT2A receptor constructs
Through PCR mutagenesis, we generated various human recombinant 5HT2A receptor constructs, consisting of the TC and Hisg tags, based on the human serotonin 5HT2A receptor. The constructs are then cloned into the pcDNA3.1 vector for transient transfection. Experimental data generated for the proof-of-concept were obtained based on our first construct, Construct 0 (see later for optimization results). Construct 0 is obtained by insertion of the TC (Cys-Cys-Pro-Gly-Cys-Cys) tag, for the specific binding of FLAsH-EDT2} after Arg-173 on the second intracellular loop, and by insertion of the His6 tag, for the specific binding of NTA-I, before Glu-318 on the third intracellular loop (Figure 1 ).
Overexpi ession of human recombinant 5HT2A receptors HEK293T cells were transiently transfected with the recombinant 5HT2A receptor constructs for overexpression of recombinant 5HT2A receptors on their cell membranes. Overexpression of the recombinant receptors has been verified and analyzed by Western blotting (Figure 2a) and immunofluorescence (Figure 2b). Immunofluorescence images show that the recombinant 5HT2A receptors were specifically expressed on the cell membranes of cells transfected with the recombinant 5HT2A receptor construct. The recombinant .5HT2A receptors were overexpressed at a density of -9.1 (± 5.7) million receptors per cell (see Methods). This value is consistent with other reported values [2, 23, 24] of transiently overexpressed GPC s in mammalian cell systems.
Specific binding of FLAsH-EDT2 and NTA-I to recombinant receptors
The specific labeling of FlAsH-EDT2 [19, 22] and NTA-I20 to the TC and His6 tag respectively has been previously demonstrated with high affinity, Crude membrane preparation containing the recombinant 5HT2A receptors (Recomb. 5HT2A) showed expression of the His6 tag (Figure 2d). In order to show that FlAsH-EDT2 and NTA-I binds specifically to our recombinant 5HT2A receptors, Recomb. 5HT2A, as well as crude membrane obtained from non-transfected cells (HE 293T) and reagents without any crude membranes (Reagents), were incubated with FlAsH-EDT2 and NTA-I. The HEK293T samples act as negative controls, while the Reagents samples provide background readings.
Initially, the cmde membrane samples (Recomb. 5HT2A and HEK293T) and Reagents exhibit low fluorescence intensity (Figures 2c and 2e) without the addition of FlAsH-EDT2. Therefore, any increase in fluorescence after the addition of FlAsH-EDT2 can be attributed to FlAsH-EDT2. Addition of FlAsH-EDT2 (1 μΜ) results in a significant increase in fluorescence intensity for the Recomb, 5HT2A sample compared to the other samples. Samples were excited at 508 nm [19], and emission is measured at 534 nm. There is a slight increase in fluorescence for the HEK293T. This may be due to the binding of the FlAsH- EDT2 molecules to hydrophobic sites25 of the cmde membrane. However, this increase in fluorescence is insignificant compared to that achieved by the Recomb. 5HT2A,
The binding of FlAsH-EDT2 to the TC tag can be reversed in the presence of excess (miUimolar concentrations) 1-2-ethanedithiol (EDT) [19], as EDT competes with the TC tag for the binding of FlAsH. Addition of excess EDT (5 inM) causes a significant drop in fluorescence intensity for the Recomb. 5HT2A compared to the other two samples (Figure 2d). This ability to reverse the increase in fluorescence reflects the specificity of FlAsH-EDT2 binding to the TC tag.
Subsequently, the addition of NTA-I (20 μΜ) resulted in significant quenching of fluorescence for the samples (Figure 2f). Similarly, the binding of NTA-I to the Hise tag can be reversed with the addition of excess EDTA [20]. The addition of an increasing concentration of EDTA (10-250 mM) caused a steady increase in fluorescence intensity for the Recomb. 5HT2A, while the fluorescence intensity of the controls remained relatively unchanged. This indicates specific binding of the NTA-I to the His tag on the Recomb. 5HT2A sample. Additionally, the restoration of fluorescence also indicates the quenching of FlAsH-EDT2 fluorescence at the ionic lock motif, where the TC and Hise tags have been introduced.
Ligand binding assay
The recombinant 5HT2A receptors that are expressed in HEK293T cells retained the ligand (serotonin) binding ability of 5HT2A receptors as shown in the ligand binding assay (Figure 2f) conducted using the Quartz Crystal Microbalance with dissipation (QCM-D) system. The QCM-D is a piezoelectric flow cell, whereby binding of biomolecules to the surface of the sensor illicit a decrease in resonant frequency of the quartz crystal sensor. Addition of 100 μΜ serotonin to Recomb. 5HT2A sample, which have been immobilized on the QCM-D sample, resulted in an acute drop (-31.9 Hz) in frequency. The first washing with DPBS did not affect the frequency. This indicates that all of the serotonin introduced, was bound to the Recomb. 5HT2A sample. Subsequent additions of increasing concentration of serotonin (500 μΜ and 2 mM) resulted only in minimal decrease of frequency, indicating that the introduction of the initial 100 μΜ serotonin has already saturated all available Recomb. 5HT2A receptors. The overall shift in frequency after the addition of 2 mM serotonin and wash, compared to the baseline at 0 h, is -33.8 Hz (Figure 2f). This difference in frequency indicates the binding of serotonin to the immobilized Recomb. 5HT2A sample.
The specificity of serotonin binding to the Recomb. 5HT2A sample was investigated. Addition of serotonin (2 mM) to crude membrane that do not contain the recombinant 5HT2A receptors (HEK293T), and subsequent washing produced a frequency shift of -0.4 Hz (Figure 2g), which is insignificant compared to that achieved by adding the same amount of serotonin to Recomb. 5HT2A sample. Therefore, the non-specific binding of serotonin to crude membranes is only minimal.
Specificity of serotonin binding is further investigated by the addition of an antagonist (altanserin) to the Recomb. 5HT2A sample (Figure 2h). Due to their higher affinity to the 5HT2A receptor, addition of the antagonist will block the binding site for serotonin, and we expect a lower amount of serotonin binding after blocking. After binding of altanserin (2 niM) to the Recomb. 5HT2A sample, subsequent addition of serotonin (2 mM) and washing produced a frequency shift of only -0.3 Hz, which corresponds to only non-specific binding of serotonin, Therefore, we can conclude that the binding of serotonin to the Recomb. 5HT2A sample is largely specific. Furthermore, the insertion of the short TC and Hisg tags (6 amino acids each), did not seem to affect the ligand binding ability of the 5HT2A receptor, This cannot be said for the insertion of fluorescent proteins such as GFP, which has 238 amino acids, and may potentially perturb [19] and destabilize the conformation of the receptors [18], affecting their ligand binding properties and ability.
Fluorescence detection of serotonin in solution
After characterizing and verifying the expression of the recombinant 5HT2A receptor, TC and His6 tags, the specific binding of FlAsH-EDT2 and NTA-I to their respective tags, as well as specificligand (serotonin) binding to the Recomb. 5HT2A sample, we then investigated whether serotonin can be detected according to our detection scheme (Figure 1), by generating fluorescence signal.
After incubation of the three samples (HEK293T, Recomb. 5HT2A and Reagents) with FlAsHEDT2 (1 μΜ) and NTA-I (10 μΜ), and subsequent removal of excess reagents by filter columns (30,000 MWCO), different concentrations of serotonin were added to the samples, and fluorescence was measured and compared (Figure 3a). The fluorescence intensity for the Recomb. 5HT2A sample started to increase at 100 pM serotonin and reaches a maximum at 100 μΜ serotonin concentration (Figure 3b). The detection limit achieved is 100 pM and the increase in fluorescence shows a linear response over the range of 100 pM-100 Μ serotonin. Additionally, the fluorescence detection has high resolution, with different serotonin concentration levels clearly distinguished from each other (Figure 3b). On the other hand, the fluorescence intensities of the negative control and reagents remained unchanged after the addition of serotonin (Figure 3a), demonstrating the specificity of fluorescence increase for the Recomb. 5HT2A sample. Notably, the increase in fluorescence achieved was visible within the range of serotonin concentration tested (Figure 3b inset).
To our knowledge, this is the first demonstration of exploiting the movement of the ionic lock motif, upon binding of serotonin, as the primary precursor for the detection of serotonin in the picomolar range. Furthermore, the detection limit (100 pM) achieved is much lower than pathological values of blood serotonin levels (700 nM-1 μΜ [6-8]) associated with mental disorders. Therefore, with the low detect limit and fast detection time (5-15 min) of serotonin achieved, we are very optimistic that our detection scheme is very much applicable for the point-of-care diagnostic of neurological diseases and mental disorders.
Optimization of fluorescence signal in solution
The intensity of fluorescence generated during the detection of serotonin is dependent on the amount of separation between the FlAsH-EDT2 and NTA-I to produce maximum decrease in fluorescence quenching. As such, we attempted to optimize the fluorescence intensity through physical manipulation of the length of the 3rd intracellular loop (ICL3) as well as the position of the Hise tag, relative to the site of the ionic interaction between Arg-173 and Glu-318, in order to maximize the separation between FlAsH-EDT2 and NTA-I and to study how the manipulation affects fluorescence intensity.
We engineered various addition constructs (see Methods), which are shown in Table 1, and compared the increase in fluorescence intensities (Figures 4a & 4b) for all the constructs during serotonin detection. We first compared Constructs 0, 1 and 7. Results obtained thus far are based on Construct 0, where the TC tag is inserted after Arg-173, and the His6 tag inserted before Glu-318, hence, the length for both ICL2 and 3 are increased by 6 amino acids. Compared to Construct 0, the Hise tag for Construct 1 is replaced after Glu-318, hence the length of 1CL3 is maintained. For Construct 7, the TC tag is replaced after Arg-173 and hence the length of ICL2 is maintained. Comparing their performance (Figure 4a), Constructs 0 and 7 achieved similar increase in fluorescence, whereas Construct 1 achieved better fluorescence signal compared to the other 2 constructs. This suggests that maintaining the length of ICL3 is more critical for achieving better fluorescence signal, compared to maintaining the length of ICL2. This could be due to the fact that ICL3 is inherently longer, and hence its movement has more fluctuations, compared to ICL2. By increasing the length of ICL3 (in the case of Constructs 0 and 7), we add to that fluctuations in movement when the recombinant receptor is activated upon serotonin binding, and this may have affected the fluorescence performance during serotonin detection.
Next, we compared Constracts 0, 2 and 12, where the position of the Hise tag has been varied with respect to Glu-318, which is involved in the ionic interaction with Arg-173. Compared to Construct 0, the His6 tag for Construct 2 is located 3 amino acids away from Glu-318, and for Construct 12, the Hiss tag is located 10 amino acids away from Glu-318. Comparing their perfonnances, Constracts 0 and 2 achieved similar fluorescence signal. Therefore, a slight shift (3 AA) of the H¼ tag does not affect fluorescence signal. However, the fluorescence signal for Construct 12 is significantly lower and remains relatively flat from 100 nM-100 μΜ. This is most likely because the huge shift (10 AA) has significantly affected the quenching efficiency between FlAsH-EDT2 and NTA-I, and the subsequent increase in fluorescence when the receptor is activated upon serotonin binding.
Fluorescence detection of serotonin on solid substrates
We envisioned that it is possible to translate the solution-based assay of serotonin detection discussed thus far, onto a solid substrate/platform for multiplexed detection of ligands (agonist and antagonist) as well as drug screening application in a microarray format. In order to realize this, we immobilized the recombinant 5HT2A receptors onto an aldehyde- functionalized glass slide to demonstrate the feasibility of detecting serotonin on a solid substrate.
An aldehyde-functionalized surface was chosen as the aldehyde group reacts spontaneously with amino groups and retains the protein activity [26]. Fluorescence detection of serotonin on glass slide is largely similar to that in solution (see Methods), with an additional washing step to remove unbound Recomb. 5HT2A sample on the surface of the glass slide.
We achieved a detection limit of 1 nM (Figure 5a) for the detection of serotonin on aldehyde- functionalized glass slide, Although this detection limit is higher than that achieved by solution-based serotonin detection, it is still much lower than the pathologically relevant values of 700 nM to 1 μΜ, Additionally, there is a linear response over the range of 1 nM to 100 μΜ. The increase in fluorescence can be observed directly (Figure 5b). A slight increase in fluorescence intensity was observed at 1 nM serotonin. Conversion of fluorescence signal to electrical signal
Current detection and quantification of blood serotonin levels require extraction of blood, processing to obtain blood plasma, as well as long purification and quantification steps such as HPLC [7, 8]. This makes rapid point-of-care (POC) diagnosis based on blood serotonin levels impractical and unrealistic. Based on the solution-based serotonin detection described herein, our detection assay possesses the advantages of being specific, fast (5-15 min incubation time), with no washing steps required and fluorescence detection can be directly carried out after incubation. It would hence be greatly beneficial if we were able to transfer and miniaturize the solution-based serotonin detection assay into a portable serotonin detection and diagnostic device (Figure 6a) for POC diagnostic puiposes. To our knowledge, no such device that possesses all our advantages exists. Therefore, we developed an optical and electrical circuit (Figure 6b) in order to convert the fluorescence signal, generated during serotonin detection, to an electrical signal for device development. Using the optical and electrical set up, we were able to obtain a general increase in voltage output across the photodiode with increasing serotonin concentration. The limit of detection achieved is 1 nM (Figure 6c). Although this number is 10-fold higher than that achieved by sophisticated plate- reader, the simple set up shows great potential for the development of a portable device for serotonin detection.
- Discussion
We have developed a novel and sensitive method (Figure 1) for detection of serotonin, which will find much application in clinical diagnostic and drug screening, In this study, we have achieved a relatively high expression of recombinant human 5HT2A receptors, which contains specifically engineered TC and His6 tags for specific binding of small FlAsH-EDT2 (Figure 2c) and NTA-I (Figure 2e) molecules that are applied in serotonin detection. The application of such small molecules preserves the pharmacological and specific ligand binding abilities that are present in native 5HT2A receptors. Ligand binding has been assayed using the QCM-D (Figure 2f), and specificity of serotonin binding is confirmed as non- transfected HEK293T samples show negligible serotonin binding (Figure 2g). Also, ecomb. 5HT2A samples demonstrate low serotonin binding after their binding sites have been blocked by an antagonist (altanserin). Moreover, we show that the presence of altanserin significantly lowers the increase in fluorescence intensity during serotonin detection (Figure 7), further indicating agonist-induced receptor activation and specificity of serotonin detection. In the detection of serotonin (Figure 3a, b), we achieved a wide linear range (100 pM-100 μΜ), and a detection limit of 100 pM, which is much lower than the physiological range (700 nM-1 μΜ6-8) required for diagnostic purposes. Additionally, we demonstrated the feasibility of applying our detection method within the physiological range (Figure 8), where we achieved 200 nM resolution. This level of resolution should allow us to distinguish between low ( <735 nM), normal (800-850 nM) and high (940-1000 nM) serotonin levels, in a semi- quantitative manner. Furthennoie, to improve the resolution, we have established a couple of HEK293 cell lines that stably express Construct 0, and receptors from this cell line show higher expression level with less fluctuation in receptor numbers (Figure 9).
In order to optimize fluorescence signal, we prepared and compared various constructs, and determined that maintaining the length of ICL3 is crucial for good fluorescence signal (Figure 4a). Also, we are only able to shift the position of the Hise tag by about 3 amino acids from the ionic lock motif, without affecting fluorescence signal (Figure 4b).
Finally, we demonstrate the ability to detection serotonin on functionalized glass slide (Figure 5) and to miniaturize our detection system into a simple working optical-electrical circuit that is applicable for serotonin detection as well (Figure 6).
The following examples and drawings illustrate the present invention without, however, limiting the same thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Schematic representation of the detection scheme for the serotonin detection using a human recombinant 5HT2A receptor.
(Construct 0). This construct contains 2 short peptide tags, which are inserted into the wild- type 5HT2A gene through site-directed mutagenesis, for the specific binding of a fluorophore and a quencher.
(a) (top panel) Shown is the production of human recombinant 5HT2A receptors and crude membrane preparations, which are ready for use for serotonin detection,
(middle and bottom panel) Assay procedure of solution-based and solid substrate-based detection of serotonin. (b) (Left) A tetra- cysteine (TC) tag (Cys-Cys-Pro-Gly-Cys-Cys) (red line) is inserted after Arg-173 on the second intracellular loop, while a Hisg tag (cyan line) is inserted before Glu- 318 on the third intracellular loop. The TC tag binds specifically with a fiuorophore, fluorescein arsenical hairpin binder (FlAsH-EDT2), while the Hise tag binds specifically with a quencher (NTA-I). Initially, the fluorescence of FIAsH-EDT2 is quenched due to the close proximity between the fiuorophore and quencher. (Right) Upon serotonin/ligand binding to the receptor, the receptor undergoes a conformation change (outward movement illustrated by the arrows), causing the separation of the fiuorophore and quencher, and results in an increase in fluorescence signal as quenching efficiency decreases.
Figure 2 Characterizing the expression, and specific FIAsR-EDT2, NTA-I and serotonin binding, to recombinant 5HT2A receptor,
(a) Expression of the human recombinant 5HT2A receptor is verified by western blotting. Distinct bands (blue box) at 98 kDa size, which corresponds to dimerized 5HT2A receptor, are observed for the crude membrane preparation of cells transfected with the wild-type and recombinant 5HT2A gene (Recomb. 5HT2A), whereas no bands are seen for the non- transfected HEK293T membrane preparation.
(b) In vitro confocal immuno-fluorescence imaging comparing transfected (top) and non- transfected (bottom) HE 293T cells. Cells transfected with the recombinant 5HT2A gene appear brightly fluorescent, with selective staining of the receptors on the cell membrane (top), whereas insignificant fluorescence is detected for non-transfected cells corresponding to only non-specific staining (bottom).
(c) Fluorescence study to verify TC tag expression and specific binding of FIASH-EDT2 to the TC tag. Three types of samples were compared, namely crude membrane preparation of non- transfected HE 293T cells (HE 293T; blue) and crude membranes of HEK293T cells transfected with the recombinant 5HT2A gene (Recomb. 5HT2A; maroon), and reagents (i.e. PBS, FlAsH-EDT2 etc.) without any crude membranes (Reagents; beige). Initially, the crude membranes (HEK293T and Recomb. 5HT2A) suspended in PBS, and pure PBS (Reagents) exhibit low fluorescence. Addition of Fl AsH-EDT2 to the samples, causes significant increase in fluorescence intensity for the recombinant 5HT2A sample compared to the other 2 samples. Addition of excess (5 niM) EDT causes a significant decrease in fluorescence intensity for the Recomb. 5HT2A sample, compared to the other 2 samples.
(d) Expression of the His6 tag is verified by western blotting. Bands are observed at the 98 kDa band-size only for the Recomb. 5HT2A sample, where a His6 tag is incorporated, whereas no bands are seen for the non-transfected HEK293T and wild-type 5HT2A receptor samples. A GST-His tag protein standard (~26 kDa) serves as positive control.
(e) Fluorescence study to verify specific binding of NTA-I to the His¾ tag. Adding 10 μΜ NTA-I causes quenching, especially for the Recomb. 5HT2A sample (maroon). Addition of increasing amounts of excess (10-250 niM) EDTA results in the steady increase in fluorescence for the Recomb. 5HT2A sample, while the fluorescence intensities for the other two samples remain relatively unchanged.
(f) Recomb. 5HT2A was immobilized on a QCM-D sensor. Addition of 100 μΜ serotonin causes a sharp frequency shift (-31.9 Hz). The overall frequency shift, comparing (i) and (ii), after adding 2 mM serotonin, was -33.8 Hz.
(g) Specificity of serotonin binding was tested with crude membranes of non-transfected HEK293T cells (HEK293T). Adding 2 mM serotonin and washing produced an overall frequency shift of -0.4 Hz (comparing i and ii).
(h) Specificity of serotonin binding was further verified with antagonist blocking of the Recomb. 5HT2A sample. Addition of 2 mM antagonist (altanserin) and subsequent first DPBS wash resulted in a frequency shift of -24.7 Hz (comparing i and ii). The subsequent addition of 2 mM serotonin and second DPBS wash resulted in a frequency shift of only -0.3 Hz (comparing ii and iii).
Figure 3 Fluorescence detection of serotonin.
(a) (top panel) Assay procedure, (lower panel) Fluorescence study comparing the fluorescence intensities of three types of sample, namely crude membranes prepared from non-transfected HEK293T cells (HEK293T; blue) and from HEK293T cells transfected with the recombinant 5HT2A receptor gene (Recomb. 5HT2A; maroon) as
Figure imgf000033_0001
as pure reagents without any crude membranes (Regents; beige). Crude membranes (HEK293T and Recomb. 5HT2A) suspended in PBS and Reagents exhibit low fluorescence. Addition of 1 μΜ FlAsH- EDT2 results in a large increase in fluorescence for the Recomb. 5HT2A sample compared to the HEK293T and Reagents samples. Subsequent addition of the NTA-1 quencher results in a decrease in fluorescence for all samples, especially the Recomb. 5HT2A sample. An increase in fluorescence intensity is observed for the Recomb, 5HT2A sample when 100 pM-1 nM serotonin is added, and fluorescence intensity for the Recomb. 5HT2A experience a steady increase with increasing concentration of serotonin, while the fluorescence intensities for the other 2 samples remain relatively unchanged. (b) Line graph depicting the percentage increase in fluorescence intensity for the Recomb. 5HT2A sample from (a). Fluorescence value at each serotonin concentration is compared to the fluorescence value after addition of 10 μΜ NTA-I to obtained the percentage increases. The detection limit is 100 pM, with a linear response over the range of 100 pM- 00 μΜ. (Inset) Fluorescence images illustrating the fluorescence intensity of the Recomb. 5HT2A sample at increasing serotonin concentrations. Notably, an observable increase in fluorescence intensity is achieved at 100 pM serotonin.
Figure 4 Optimization and comparison of fluorescence signal for various recombinant 5HT2A constructs.
(a) All constructs achieved increasing fluorescence signal with increasing serotonin concentration, Constructs 0 and 7 have similar performance, whereas Construct 1 has the best fluorescence signal.
(b) Constructs 0 and 2 achieved increasing fluorescence signal with increasing serotonin concentration, but not for Construct 12 whereby increase in fluorescence signal becomes relatively fiat from 100 nM-100 μΜ serotonin concentration. Also, Constructs 0 and 2 achieved similar fluorescence signal, but the fluorescence signal for Construct 12 is significantly lower than the other 2 constructs.
(c) Schematic illustration of Constructs 0, 1 and 2.
Figure 5 Fluorescence detection of serotonin on a solid substrate.
(top panel) Assay procedure, (lower panel) Line graph depicting the increase in fluorescence intensity for the Recomb. 5HT2A sample when detection of serotonin is canied out on a aldehyde-functionalized glass slide, instead of in solution. Fluorescence intensity at various serotonin concentrations are compared with the fluorescence intensity observed after the addition of 10 μΜ NTA-I to obtain increase in fluorescence. The limit of detection achieved is 1 nM, with a linear response over the range of 1 nM - 100 μΜ.
(Inset) Fluorescence images depicted the fluorescence response of Recomb. 5HT2A samples, which are immobilized onto the glass slide surface. An increase in fluorescence can be observed after the addition of 1 nM serotonin, and the increase in fluorescence becomes more pronounced with higher concentrations of serotonin.
Figure 6 Serotonin detection on an optical and electrical set up. Schematic diagram illustrating (a) a potential portable fluorescent serotonin detection and POC diagnostic device that can be developed through miniaturization of the solution-based serotonin detection assay, and (b) the various electrical and optical components required for the transduction of fluorescence signal, which is generated during serotonin detection, to electrical signal. After addition of serotonin to the Recomb. 5HT2A sample (with FlAsH- EDT2 and NTA-I attached), a blue LED (λεηι = 470 nm) is used to provide the excitation required for fluorescence detection. The fluorescence generated is then passed through a 530 nm bandpass filter so as to reduce the background fluorescence produced by the blue LED and to provide specific detection of serotonin. Fluorescence generated produces a potential drop across a photodiode, and the potential drop is amplified by an operational amplifier (op- amp). Finally, the amplified potential drop can be detected and visualized by an oscilloscope.
(c) Bar graph showing the increase in voltage across the photodiode achieved with increasing serotonin concentrations.
(d) Schematic diagram illustrating the arrangement of optical components.
Figure 7 Fluorescence study illustrating the effect of antagonist (altanserin) on the increase in fluorescence intensity obtained during serotonin detection using crude membranes prepared fro HEK293T cells transfected with the recombinant 5ΉΤ2Α receptor gene (Recomb. 5HT2A).
The percentage increase in fluorescence intensity when only 100 μΜ serotonin is present is 38.1 %, compared to the fluorescence intensity after addition of 20 μΜ NTA-I. In the presence of 50, 250 and 500 μΜ altanserin, the increase in fluorescence intensity becomes 30.0%, 23.4% and 7.1% respectively.
Figure 8 Fluorescence detection of serotonin within the physiologically relevant range.
(a) Increase in fluorescence intensity during serotonin detection, over a serotonin range of 600-1 100 nM in steps of 100 nM.
(b) Increase in fluorescence intensity during serotonin detection in steps of 200 nM.
Figure 9 Receptor numbers for HEK293 cell lines that stably express Construct 0.
Figure 10 Serotonin detection using stably expressed Construct.
Figure 11 Immobilization schemes for GPCR on solid supports. (a) Membrane stabilization on chip. The lipophilic hydrocarbon chain of octylamine penetrates into the phospholipid bilayer and act as cell membrane anchor. Cell membrane is stabilized on hydrophilic PEG surface. Hydrocarbon chains act as spacers between the GPCR and the solid support, and thereby retain the conformation and functionality of the GPCR as in the whole cell,
(b) Surface bounds 5HT2A receptors labelled with FlAsH-EDT . Figure 12 Detection schemes for GPCR Microarray.
(a) Pie-spotting of various human recombinant GPCRs onto a functionalized glass slide in a microarray format, (b) A microfluidic platform is adapted onto the glass slide, and mounted onto a (c) platform analogous to a 96-well plate. The platform is fed into (d) a high- throughput imaging system for high- throughput analysis based on fluorescence intensity.
EXAMPLES
Example 1 Methods
Cell culture. Human embryonic kidney (HEK)-293T cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (Gibco), supplemented with 10% fetal bovine serum (FBS) (Gibco).
Cloning. Human serotonin 5HT2A receptor gene (NCBI Reference Sequence NM 000621.2) was commercially synthesized (OriGene). The human 5HT2A receptor gene was cut out from its original expression vector, and inserted into the pcDNA3.1 (Invitrogen) expression vector. Site-directed mutagenesis was performed for the inclusion of the tetra- cysteine (TC) and His6 tag, and for creation of the various Constructs {Table 1). Mutagenesis was carried out using custom-designed primers and the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent), following recommended standard protocol. Nucleotide sequences of the Constructs were verified by restriction enzyme analysis and DNA sequencing. The HEK293T cells were transiently transfected with the recombinant 5HT2A receptor gene using LipofectamineTM 2000 (Invitrogen), following standard procedures. Cells were harvested 72 h after transfection through trypsinization, for crude membrane preparation. Estabiisbing stable cell line. HE 293 cells stably expressing Construct 0 were generated by transfecting the cells with Pvul linearized expression vector (Methods) using Lipofetf amine™ 2000 (Invitrogen), and selection using G418 disulfate salt (Sigma). After selection, independent clones were isolated using limiting dilution. Clones displaying the highest receptor expression levels were used for production of Recomb. 5HT2A samples.
Crude membrane preparation. Harvested cells (-200 million cells per transfection batch) were resuspended in 3 ml of DPBS, and homogenised in a Hand Homogeniser (Sartorius) with 30 strokes. The homogenized solution is first centrifuged at 10,000 g and 4°C for 20 min, to remove cell debris, and then at 70,000 g and 4°C for 20 min to pellet the crude membranes containing the recombinant 5HT2A receptors. The pellet is then resuspended in 3 ml DPBS, containing a cocktail protease inhibitor mix (Thermo Scientific), and used immediately for experiments. The expressed recombinant 5HT2A receptors possessed ligand binding and agonist-induced receptor activation like the wild-type receptor.
Ligand binding assay. Quartz crystal microbalance with dissipation (QC -D) was used for the ligand binding assay experiments. All ligand binding assays involving the QCM-D, are carried out at 23°C, and frequency responses from the 5th overtone was recorded. The gold- coated sensors are first cleaned with Nanostrip (Cyantek) for 15 min at room temperature, and then with 02 plasma (200 W) for 10 min before use, Immobilization of crude membranes containing the recombinant 5HT2A receptors (Recomb, 5HT2A) onto the sensors occurs by the gold-thiol binding via the tetra-cysteine (TC) tag, After mounting the sensors into the QCM-D flow cell, DPBS is introduced at 7 μΐ/min until an initial stable baseline is established. Next, the Recomb, 5HT2A sample was introduced at 7 μΐ/min until a stable baseline is established. Then, the sensor is washed with DPBS at 50 μΐ/min until a stable baseline is achieved. For the subsequent introduction of ligands, serotonin (agonist) and/or altanserin (antagonist) is introduced at 7 μΐ/min until a stable baseline is achieved before washing with DPBS at 7 μΐ/min. Immobilization of crude membranes obtained from non- transfected HEK293T cells onto the gold sensors is earned out by coating the sensors with poly-L-lysine (PLL). Cleaned sensors (described above) are immersed in 1 inM ethanolic solution of 3-Mercaptopropionic acid (3-MPA; Sigma-Aldrich) overnight at room temperature with shaking. After cleaning with ethanol and then with ultrapure water, the sensors are immersed in PLL solution (0.1 mg/ml), prepared in sodium bicarbonate buffer (pH 9), for 1 h at room temperature. Finally, the sensor is cleaned with ult apure water before being used for experiments.
Receptor Quantification. 200 μ\ of the crude membrane preparation of Recomb. 5HT2A was incubated with 10 μΜ of a fluorophore-labelled antagonist, BODIPY-4F4PP oxlate (CellAura), which binds selectively to 5HT2A receptors with high affinity (¾ = 5.3 nM), for 2 h at room temperature to ensure complete binding of the antagonist to the Recomb. 5HT2A. Then, the Recomb. 5HT2A was transferred to membrane filter columns (Vivaspin 2; Satorius) with a 10,000 molecular weight cut off (MWCO), and washed with DPBS for removal of unbound fluorophore-labelled antagonist. For washing, the columns were spun down at 4,000 g for 20 mm. The washing step was done 3 times. After washing, the absorbance of the resulting Recomb. 5HT2A solution at 641 mn was recorded, and compared to a calibration curve, for quantification of receptor numbers.
Fluorescence detection of serotonin. All steps were conducted at room temperature unless otherwise stated. Crude membranes containing the recombinant 5HT2A receptor (Recomb. 5HT2A) and those prepared from non-transfected HEK293T cells (HEK293T), and pure reagents (Reagents) were suspended in a PCR or 2 ml microcentrifuge tube. 1 μΜ FlAsH- EDT2 was added to the samples, and incubated for 3 h in the dark. Then, 10 μΜ NTA-I was added to the samples, and also incubated for 1 h in the dark. Next, the samples were centrifuged in the membrane filter columns with a 30,000 MWCO at 4,000 g for 20 min to remove excess FlAsH-EDT2 and NTA-I reagents. Subsequently, the samples were transferred to a 96-well plate, with 100 μϊ of samples per well. Serotonin and/or altanserin were added to the samples, and fluorescence was taken using a fluorescence plate reader (Tecan Infinite® M200). The excitation wavelength is 508 nm, and emission was measured at 534 nm. All experiments were done in triplicates. For the study of the effect of antagonist on fluorescence intensity, altanserin (antagonist) was added with serotonin, and the same excitation and emission wavelengths were used. For the detection of serotonin on aldehyde-functionalized glass slide, the Recomb. 5HT2A samples were first treated by 1 μΜ FlAsH-EDT2 and 10 μΜ NTA-I for 3 h, and then centrifuged in membrane filter columns (30,000 MWCO) at 4,000 g for 20 min, Next, the samples were spotted, by manual pipetting, onto the surface of the glass slide for immobilization of the receptors through aldehyde-amine coupling. The Recomb. 5HT2A samples were left to incubate overnight at 4°C. Then the glass slide was washed in TBST for 4 times, 10 min each with shaking, to remove any unbound sample. Finally, serotonin was spotted, by pipetting, and fluorescence is measured and imaged using a UV transilluminator (Bio-Rad; VersaDoc™ MP 4000), whereby excitation of the FlAsH-EDT2 is produced by a blue LED (470 nm), and the emission was measured and detected using a 530 nm bandpass filter.
Electrical detection of serotonin. Similar to solution-based detection, the Recomb. 5HT2A sample was first incubated with 1 μΜ FlAsH-EDT2 for 3 h at room temperature and then with 10 μΜ NTA-I for 1 h. 100 μ\ of the sample was used for detection of each serotonin concentrations. An optical and electrical set up comprising 3 simple circuits (LED, photodiode and op-amp) was developed to transduce fluorescence signal to an electrical readout on an oscilloscope (Figure 6b). After the addition of serotonin, the Recomb. 5HT2A was excited by a 470 nm high-power blue LED (Power Light Systems). The fluorescence generated from the sample is filtered tlvrough a 532 run laser-line bandpass filter (Edmund optics), so that only green fluorescence is detected. Emission of green fluorescence is detected by a silicon photodiode, which is attached to the filter.
We measured the voltage across the photodiode when green fluorescence is detected. The voltage is further amplified by the op-amp circuit (AD620AN). Three 909 0 resistors were connected in parallel to obtain an amplification gain of -164. The voltage signal is read from a 4-channel digital phosphor oscilloscope (Tektronix TDS 3014B). Power supply to the blue LED and the op-amp is provided by a DC power supply (Topward 6303D).
Example 2
In order to investigate and verify if fluorescence detected was due to the addition of serotonin and subsequent activation of the 5HT2A receptor, we studied the effect of adding antagonist (altanserin) on the fluorescence generated. Due to competitive antagonism, the addition of altanserin blocks the serotonin binding site on the Recomb. 5HT2A sample. Addition of altanserin reduced the amount of fluorescence generated (Figure 7). In the presence of 500 μΜ altanserin, the increase in fluorescence intensity for 100 μΜ serotonin is only 7.1%, which is very low compared to the 38.1 % increase in fluorescence when only 100 μΜ serotonin was added. This reduction in fluorescence generated suggests agonist-induced receptor activation and reflects the specificity of serotonin (agonist) detection. The receptor number for Stable Construct 0-1 is 14,7 (± 1.7) million receptors/cell and that for Stable Construct 0-2 is 15.3 (± 0,8) million receptors/cell (Figure 9). Compared to the receptor number of transiently transfected Construct 0, which is 9.1 (± 5.7) million receptors/cell, stably expressed Constructs 0 show higher receptor expression, as well as lower standard deviation. This lower standard deviation could lead to reduced batch to batch difference in expressed Recomb. 5HT2A samples, and could improve the resolution of serotonin detection within the physiologically relevant range, for practical diagnostic purposes.
HE 293 cells stably expressing Construct 0 were generated by transfecting the cells with Pvul linearized expression vector (Example 1 - Methods) using Li ofectamine™ 2000 (Invitrogen), and selection using G418 disulfate salt (Sigma), After selection, independent clones were isolated using limiting dilution. Clones displaying the highest receptor expression levels were used for production of Recomb. 5HT2A samples.
Example 3
Portable Fluorescence Detection System for GPCR-based Sensors
See Figure 6d,
Excitation of FlAsH-EDT2 is done by a high-power (1.5-W) blue LED with em = 470 nm. Fluorescence signal generated upon ligand-receptor binding is converted to electrical signal by a photodiode.
Small signal (~ 150 mV) generated by the photodiode is amplified by a high-gain OP AMP, and converted to concentration value of the analyte by a μ-controller.
The amount of fluorescence signal generated can be converted to an electrical signal, which is necessary in the development of a diagnostic device using various optical/electrical components and circuits (Figure 6d, top panel and Figure 6b). After the addition of a ligand to the human recombinant 5HT2A receptor, which can be resuspended in a vial/tube, a highpower blue LED with focusing lens, or a blue laser diode, is used to excite the FlAsHEDT2. The fluorescence generated due to ligand activation of the receptor is focused by a collection lens, filtered by a 530-nm band-pass filter, and detected by a photodiode for generation of an electrical signal. The electrical signal can then be amplified by an operational amplifier (op-amp), and transduced by an oscilloscope for direct signal readout. With the ability to provide electrical signal readout for the detection of ligands, this technology can be integrated with, a diagnostic device (Figure 6a), and a microfluidic platform (Figure 12) for automated and multiplexed detection of ligands in a device and for on-chip drug screening.
The diagnostic device (Figure 6a) comes with vials that are pre-loaded with different human recombinant GPCRs, and is applicable for the quantification of various GPCRrelated ligands using the detection scheme (Figure 1) for diagnostic purposes. Furthermore, various calibration curves correlating the amount of ligands to the amount of electrical signal generated are stored in microcontrollers found in the device, Users only need to purchase one detection device, and vials loaded with different GPCRs depending on their specific needs. Under standard operating conditions, the device is first switched on. The 7-segment display should read 0 ng/ml, if not, users can press 'Reset' to eliminate any stray values. Next, users can select the ligand of their choice by pressing 'Select' repeatedly until the ligand of their choice is selected. 50 μί of sample (e.g. a drop of blood) is then added to the vial, and the vial can be placed into the holder of the device. After 5-15 min of incubation, users can press 'Laser' for excitation. Fluorescence signal generated is converted to electrical signal, and the microcontroller processes this electrical signal based on the stored calibration curves to display the amount of ligands present in the sample on the 7-segment display.
Example 4
Ligand detection can also be conducted on a microfluidic platform (Figure 12) for mghthi ughput drug screening applications. Various human recombinant GPCRs can be prespotted onto a functionalized glass slide using a robotic spotter, which has micrometer spotting resolution. Subsequently, a microfluidic platform can be adapted to the glass slide, and mounted onto a platform that is analogous to a 96-well plate, The platform can then be fed into a high-throughput imaging analysis system for high-throughput analysis of agonist- induced receptor activation or antagonist-induced receptor deactivation, based on the amount of fluorescence generated.
Example 5
Purification and Biochemical Characterization ofFZD4 (see Figure 13 A).
Localization of Overexpressed Proteins (see Figure 13 B). Cell-Based Functional Assay (see Figure 13 C).
Expression and Affinity Purification (see Figure 13 D).
■ Best harvest times for 5HT4, FZD4, 5 and 10 are 72, 96, 72 and 72h post infection;
• Estimated yield of 5HT4 and FZD4, 5 and 10: -10 mg protein/2-3 L culture,
Ion-Exchange Chromatography Purification of FZD4 (see Figure 13 E).
Purified FZD4 Binds to Norrin (see Figure 13 F).
Purified FZD4 Does Not Bind to WntSa (see Figure 13 G).
Protein Production and Stabilization (see Figure 13 H).
• Establishing a reliable, up-scaled insect cell suspension culture system
- Culture volumn > 1 L
- Controlled dissolved oxygen (DO)
- Monitored pH
• Evaluating protein stability in detergent micelle (see Figure 13 I).
• Increasing protein concentration for crystallization
1.5 mg mL - 5-10 mg/mL.
The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.
REFERENCES
1. Rosenbaum, D. M., Rasmussen, S. G. & Kobilka, B. K. The structure and function of Gprotein-coupled receptors. Nature 459, 356-363 (2009).
2. Martinez, K. L., Meyer, B. H., Hovius, R., Lundstrom, K. & Vogel, H. Ligand Binding to G Protein-Coupled Receptors in Tethered Cell Membranes. Lang uir 19, 10925-10929 (2003).
3. Bieri, C, Ernst, O. P., Heyse, S., Hofmann, K. P. & Vogel, H. Micropattemed immobilization of a G protein-coupled receptor and direct detection of G protein activation. Nat. Biotechnol 17, 1 105-1 108 (1999). 4. Shapiro, D. A., Kristiansen, .5 Weiner, D. M., Kroeze, W. . & Roth, B. L. Evidence for a model of agonist-induced activation of 5-liydroxyrryptaraine 2A serotonin receptors that involves the disruption of a strong ionic interaction between helices 3 and 6. J. Biol. Chem. 277, 1 1441 - 1 1449 (2002).
5. Raote, I., Bhattacharya, A. & Panicker, M. M. in Serotonin Receptors in Neurobiology (ed Chattopadhyay A) {Boca Raton (FL): CRC Press, 2007).
6. Janusonis, S. Statistical distribution of blood serotonin as a predictor of early autistic brain abnormalities. Theor. Biol. Med. Model 2, 27-42 (2005).
7. Moffitt, T. E. et at. Whole Blood Serotonin Relates to Violence in an Epidemiological Study. Biol. Psychiatry 43, 446-457 (1998).
8. Mann, J. J., McBride, P. A.} Anderson, G. M. & Mieczkowski, T. A. Platelet and Whole Blood Serotonin Content in Depressed Inpatients: Correlations with Acute and Life-Time Psychopathoiogy. Biol. Psychiatry 32, 243-257 (1 92).
9. Roth, B. L., Lopez, E., Patel, S. & Kroeze, W. K. The Multiplicity of Serotonin Receptors: Uselessly Diverse Molecules or an Embarrassment of Riches? Neurosdentisi 6, 252 (2000).
10. Roth, B. L. & Willins, D. L. What's All the RAVE about Receptor Internalization? Neuron 23, 629 (1999).
1 1. Ballesteros, J. et al. Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor. J. Biol, Chem. 273, 10445-10453 (1998).
12. Ballesteros, J. A. et al. Activation of the beta 2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6. J. Biol. Chem. 276, 29171-29177 (2001).
13. Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G. & Cotecchia, S. Constitutively active mutants of the alpha lB-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. BMBO J 15, 3566-3578 (1996). 14. Min, K. C, Zvyaga, T. A., Cypess, A. M. & Sakmar, T. P, Characterization of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Mutations on the cytoplasmic surface affect transducin activation. J. Biol Chem. 268, 9400-9404 (1 93).
15. Ohyama, ., Yamano, Y., Chaki, S., Kondo, T. & Inagami, T. Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis. Biochem. Biophys. Res. Commun. 189, 677-683, doi;0006-291X(92)92254-U [pii] (1992).
16. Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295-305 (2003).
17. Hoffmann, C. et al. A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat. Methods 2, 171-176 (2005).
18. Vilardaga, J.-P., Biinemann, M., Krasel, C, Castro, M. & Lohse, M. J. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat. Biotechnol. 21, 807-812 (2003).
19. Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells. Science 281, 269-272 (1998).
20. Guignet, E. G., Hovius, R. & Vogel, H. Reversible site-selective labeling of membrane proteins in live cells. Nat. Biotechnol. 22, 440-444 (2004).
21. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503-507 (2002).
22. Adams, S. R. et al. New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications. J. Am. Chem. Soc. 124, 6063-6076 (2002).
. Lundstrom, K. Structural genomics and drug discovery. Cell. Ado I. Med. 11, 224-238 )07). 24. Lundstrom, . et al. High-level expression of the human neurokinin- 1 receptor in mammalian cell lines using the Smliki Forest virus expression system. Eur. J. Biochem. 224, 917-921 (1994).
25. Griffin, B. A,, Adams, S. R., Jones, J. & Tsien, R, Y. Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol. 327, 565-578 (2000).
26. Hahn, C. D. et al. Self-assembled monolayers with latent aldehydes for protein immobilization. Bioconjug. Chem. 18, 247-253 (2007).

Claims

Claims
1. A recombinant G-protein coupled receptor (GPCR) variant mutated to allow for binding of a fluorophore and an acceptor or quencher,
wherein said recombinant GPCR variant comprises
(1 ) a binding site or sequence for the fluorophore,
(2) a binding site or sequence for the acceptor or quencher,
wherein one binding site is introduced in the proximity of the Arg residue of the (D/E)RY motif in transmembrane helix 3 (H3) of the GPCR and the other binding site is introduced in the proximity of the acidic amino acid residue in transmembrane helix 6 (H6) of the GPCR, wherein said Arg and said acidic amino acid residue fonn the ionic lock motif and have an ionic interaction in the inactive state of the GPCR.
2. The recombinant GPCR variant of claim 1 , wherein a binding site or sequence for the fluorophore and/or the acceptor or quencher comprises 3 to 10 amino acids, preferably 5 to 9 amino acids.
3. The recombinant GPCR variant of claim 1 or 2, wherein the binding site or sequence for the fluorophore comprises a tetra-cysteine tag (TC tag), preferably an amino acid sequence comprising or consisting of the amino acid sequence
CC(X)nCC (SEQ ID NO. 1) wherein C is cysteine and X is any amino acid and n is 1 to 5,
more preferably an amino acid sequence selected from the group of
CCPGCC (SEQ ID NO. 2),
CCRECC (SEQ ID NO. 3),
CCACC (SEQ ID NO. 4),
CCGCC (SEQ ID NO, 5),
CCPCC (SEQ ID NO. 6),
CCAECC (SEQ ID NO. 7),
CCSECC (SEQ ID NO. 8),
CCDECC (SEQ ID NO. 9),
CCGPCC (SEQ ID NO. 10) CCDEACC (SEQ ID NO. 1 1),
CCKAEAACC (SEQ ID NO. 12).
4. The recombinant GPCR variant of claim 1 or 2, wherein the binding site or sequence for the fluorophore comprises a tag or fusion selected from the group of
Rhodococcus dehalogenase (DhaA) (HaloTag System),
SNAP tag,
CLIP tag,
fusion with 06-alkyl guanine- DNA alkyltransf erase (wildtype),
Lipoic acid ligase acceptor peptide (LAP).
5. The recombinant GPCR variant of claims 1 to 4, wherein the binding site or sequence for the acceptor or quencher comprises an amino acid sequence of multiple His (His6 tag), preferably amino acid sequence HHHHHH (SEQ ID NO. 13).
6. The recombinant GPCR variant of any one of claims 1 to 5, wherein the fluorophore is selected from
a fluorescein or derivative or biarsenical fluorophore,
preferably
FIAsH-EDT2,
ReAsH-EDT2,
sFlAsH-EDT2,
F2-FlAsH and F4-FlAsH,
Carboxy-FlAsH, or CrAsH-EDT2}
CHoX-AsH-EDT2,
Br2REAsH-EDT2,
ThAsH-EDT2,
AsCy3,
fluorescein,
FAM (Carboxyfluorescein),
AlexaFluor 488,
(cyclo-octyne-conjugated) AlexaFluor 568, or
Cy3,
a rhodamine or derivative such as cai'boxytetramethylrhodamine (TMR).
7. The recombinant GPCR variant of any one of claims 1 to 6, wherein the acceptor or quencher is a metal-ion-chelating nitrilotriacetate moiety, preferably nitrilotriacetic acid clrromophore (NTA-I), gold nanoparticles (Au NPs) that are conjugated to a nitrilotriacetic acid (NT A) motif, other ffuorophores that are conjugated to a NTA, such as Cy3.
8. The recombinant GPCR variant of any one of claims 1 to 7, wherein the GPCR is a 5HT2A serotonin receptor, a Wnt receptor, or an odorant receptor.
9. The recombinant GPCR variant of any one of claims 1 to 8, wherein the GPCR is (human) 5HT2A serotonin receptor (preferably encoded by nucleic acid sequence SEQ ID NO. 14 or having amino acid sequence of SEQ ID NO. 15 or 16), comprising
(1 ) the binding site or sequence for the fluorophore introduced in the proximity of residue Arg 173 at transmembrane helix 3 (H3),
(2) the binding site or sequence for the acceptor or quencher introduced in the proximity of Glu 318 at transmembrane helix 6 (H6).
10. The recombinant GPCR variant of claim 9, wherein
the binding site or sequence for the fluorophore (1) is a terra- cysteine tag (TC tag), preferably comprising or consisting of amino acid sequence CCPGCC (SEQ ID NO. 2) and wherein the fluorophore is FlAsH-EDT2
and/or
the binding site or sequence for the acceptor or quencher (2) is an amino acid sequence of multiple His (His6 tag), preferably comprising or consisting of amino acid sequence HHHHHH (SEQ ID NO. 13) and wherein the acceptor or quencher is nitrilotriacetic acid chromophore (NTA-I).
1 1 . The recombinant GPCR variant of claim 9 or 10, wherein
the binding site or sequence for the fluorophore (1) is introduced in the proximity of residue Arg 173 in the following way: by insertion C-tenninal of Arg 173 (preferably adjacent to Arg 173) or by replacing the wildtype amino acids C-tenninal of Arg 173 (preferably adjacent to Arg 173),
and/or the binding site or sequence for the acceptor or quencher (2) is introduced in the proximity of Glu 318 in the following way: by insertion N-terminal of Glu 318 (preferably adjacent to Glu 318 or 3 amino acids N-terminal of Glu 3 8) or by replacing the wildtype amino acids N-terminal of Glu 318 (preferably adjacent to Glu 318 or 10 amino acids N- terminal of Glu 318).
12. The recombinant GPCR variant of claim 11 , selected from the variants comprising an amino acid sequence selected from at least one of SEQ ID NOs. 20 to 29, preferably one of SEQ ID NOs. 19, 21 , 23, 25 and 27 and one of SEQ ID NOs. 20, 22, 24, 26 and 28.
13. A nucleic acid encoding a recombinant GPCR variant of any one of claims 1 to 12, preferably an expression vector or construct.
14. A mammalian cell or cell line comprising a nucleic acid of claim 13 and expressing a recombinant GPCR variant of any one of claims 1 to 12 on the cell membrane, preferably stably expressing the GPCR variant,
such as HEK293T.
15. A method of obtaining a crude membrane preparation comprising a recombinant GPCR variant of any one of claims 1 to 12, comprising the steps of
(a) providing a nucleic acid of claim 13, t ansfecting mammalian cells with said nucleic acid or providing a cell or cell line of claim 14,
(b) culturing the cells or cell line and thereby expressing the recombinant GPCR variant in said cell or cell line,
(c) harvesting and homogenizing the cells and centrifuging,
(d) resuspending the pellet to obtain the crude membrane preparation.
16. A recombinant GPCR variant of any one of claims 1 to 12 labeled with fluorophore and acceptor or quencher,
such as labeled with FIAsH-EDT2 and NTA-I,
such as human 5HT2A serotonin receptor labeled with FlAsH-EDT2 and NTA-I.
17. The method of claim 15, further comprising the steps of (e) adding a fluorophore and acceptor or quencher, either at the same time or subsequently, to the crude membrane preparation and incubating,
(f) obtaining a cmde membrane preparation comprising a labeled recombinant GPCR variant,
such as the labeled recombinant GPCR variant of claim 16.
18. A biosensor comprising a recombinant GPCR variant of any one of claims 1 to 12, a crude membrane preparation obtained in claim 15, a labeled recombinant GPCR variant of claim 16 or a cmde membrane preparation obtained in claim 17,
preferably comprising a 96-well plate, a solid substrate (such as nitrocellulose membrane) or a solid support (such as a glass plate, microanay or chip).
19. Use of a recombinant GPCR variant of any one of claims 1 to 12, a crude membrane preparation obtained in claim 15, a labeled recombinant GPCR variant of claim 16, a crude membrane preparation obtained in claim 17 or a biosensor of claim 18 in ligand binding assays, drug discovery and drug screening.
20. A recombinant GPCR variant of any one of claims 1 to 12, a cmde membrane preparation obtained in claim 15, a labeled recombinant GPCR variant of claim 16, a cmde membrane preparation obtained in claim 17 or a biosensor of claim 18 for use in the diagnosis of diseases related to the GPCR,
wherein a disease related to the GPCR is selected from
diseases associated or caused by high serotonin levels , such as brain injury due to high serotonin levels, violence and aggression, autism, colon cancer,
diseases associated or caused by low serotonin levels, such as schizophrenia, depression.
21. The recombinant GPCR variant of claim 19 or 20 or the biosensor of claim 18, wherein the GPCR is (human) 5HT2A serotonin receptor for use in the diagnosis of diseases related to serotonin activity, such as vascular smooth muscle contraction, platelet aggregation, perception, emotion, mental and behavioral disorders, autism, violent behavior, schizoplnenia, depression.
22. The recombinant GPCR variant of claim 1 or 20 or the biosensor of claim 18, wherein the GPCR is a Wnt receptor for use in the diagnosis of cancer, developmental dysregulations,
23. The recombinant GPCR variant of claim 19 or 20 or the biosensor of claim 18, wherein the GPCR is an odorant receptor for use in the diagnosis of dysfunctions in smelling, detection of specific odorants.
24. A method for detecting the presence and/or measuring the concentration of GPCR ligand(s) in a sample,
comprising the use of a recombinant GPCR variant of any one of claims 1 to 12, a crude membrane preparation obtained in claim 15, a labeled recombinant GPCR variant of claim 16, a crude membrane preparation obtained in claim 17 or a biosensor of claim 18.
25. The method of claim 24, wherein the GPCR ligands are neurotransmitters, like serotonin, an odorant or ligands binding to Wnt receptors, like FZD, amino acids, amines, fragrants.
26. The method of claim 24 or 25, comprising the steps of
(i) providing a sample,
(ii) providing a recombinant GPCR variant of any one of claims 1 to 12 or a crude membrane preparation obtained in claim 15 or a biosensor of claim 18, and adding a fluorophore and acceptor or quencher, either at the same time or subsequently, to the recombinant GPCR variant or the crude membrane preparation and incubating;
or
providing a labeled recombinant GPCR variant of claim 16 or a crude membrane preparation obtained in claim 17;
(iii) adding the sample to the labeled recombinant GPCR variant or crude membrane preparation comprising a labeled recombinant GPCR variant,
(iv) determining whether ligand is present in the sample by determining a fluorescence signal, wherein no fluorescence signal is indicative that no ligand is present and a fluorescence signal is indicative that ligand is present.
27. The method of any of claims 24 to 26, comprising using or establishing a calibration curve with a ligand, in order to allow quantification or measuring the concentration of said ligand in a sample,
28. The method of any of claims 24 to 27, comprising
adding the labeled recombinant GPCR variant or crude membrane preparation comprising a labeled recombinant GPCR variant of step (ii) to a 96-well plate, a solid substrate (such as nitrocellulose membrane) or a solid support (such as a glass plate, microarray or chip).
29. The method of any of claims 24 to 28, comprising the use of antagonists or agonists, such as in competitive ligand binding assays.
30. A method of drug discovery or screening, comprising the steps of
(i) providing a compound or drug to be tested;
(ii) providing a recombinant GPCR variant of any one of claims 1 to 12 or a crude membrane preparation obtained in claim 15 or a biosensor of claim 18, and adding a fluorophore and acceptor or quencher, either at the same time or sequentially, to the recombinant GPCR variant or the crude membrane preparation and incubating;
or
providing a labeled recombinant GPCR variant of claim 16 or a crude membrane preparation obtained in claim 17;
(iii) providing a ligand (or agonist) of the GPCR;
(iv) adding the ligand and compound or drug to be tested to the labeled
recombinant GPCR variant or crude membrane preparation comprising a labeled recombinant GPCR variant,
(v) determining whether the drug has an effect on ligand/agonist binding by determining the fluorescence signal,
wherein a decrease of the fluorescence signal (compared to the fluorescence signal of the ligand/agonist) is indicative that the compound or drug to be tested is an antagonist of the ligand, i.e. competes for the binding site;
and wherein an increase of the fluorescence signal (compared to the fluorescence signal of the Hgand/agonist) is indicative that the compound or drug to be tested is an agonist of the ligand, i.e. increases ligand binding; or that the compound or dmg to be tested is an agonist that is capable of activating the GPCR, by triggering the release of the ionic lock.
31. The method of claim 30, wherein the GPCR ligands are neurotransmitters, like serotonin, an odorant or ligands binding to Wnt receptors, like FZD, amino acids, amines, fragrants.
32. The method of claim 30 or 31 , comprising
adding the labeled recombinant GPCR variant or crude membrane preparation comprising a labeled recombinant GPCR variant of step (ii) to a 96-well plate, a solid substrate (such as nitrocellulose membrane) or a solid support (such as a glass plate, microarray or chip).
33. The method of any of claims 24 to 32, comprising the conversion of the fluorescence signal into an electrical signal, such as by the use of photodiode.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020124688A1 (en) * 2018-12-18 2020-06-25 中国科学院大连化学物理研究所 Full-spectrum high-brightness and high-stability fluorescent dyes, and synthesis and application thereof
KR20200095117A (en) * 2019-01-31 2020-08-10 한국과학기술연구원 Biosensor based on fluorescence resonance energy transfer for detecting of antipsychotic medicine and detection method for using same
US11009502B2 (en) 2017-08-16 2021-05-18 Aromyx Corporation Ectopic olfactory receptors and uses thereof
US11092599B2 (en) 2016-02-24 2021-08-17 Aromyx Corporation Biosensor for detecting smell, scent, and taste

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033102A1 (en) * 2000-10-16 2002-04-25 The Johns Hopkins University Receptor mediated activation of heterotrimeric g-proteins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033102A1 (en) * 2000-10-16 2002-04-25 The Johns Hopkins University Receptor mediated activation of heterotrimeric g-proteins

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HOFFMANN, C. ET AL.: "A FlAsH-based FRET Approach to Determine G Protein-Coupled Receptor Activation in Living Cells", NATURE METHODS., vol. 2, no. 3, 2005, pages 171 - 176 *
HOFFMANN, C. ET AL.: "Fluorescent Labeling of Tetracysteine-Tagged Proteins in Intact Cells", NAT PROTOC., vol. 5, no. 10, 2010, pages 1666 - 1677 *
XIE, X. ET AL.: "Advances in Methods to Characterize Ligand-Induced Ionic Lock and Rotamer Toggle Molecular Switch in G Protein-Coupled Receptors", METHODS ENZYMOL., vol. 520, 2013, pages 153 - 174 *
YAO, X. ET AL.: "Coupling Ligand Structure to Specific Conformational Switches in the beta2-Adrenoceptor", NATURE CHEMICAL BIOLOGY., vol. 2, no. 8, 2006, pages 417 - 422 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11092599B2 (en) 2016-02-24 2021-08-17 Aromyx Corporation Biosensor for detecting smell, scent, and taste
US11460469B2 (en) * 2016-02-24 2022-10-04 Aromyx Corporation Biosensor for detecting smell, scent, and taste
US11009502B2 (en) 2017-08-16 2021-05-18 Aromyx Corporation Ectopic olfactory receptors and uses thereof
WO2020124688A1 (en) * 2018-12-18 2020-06-25 中国科学院大连化学物理研究所 Full-spectrum high-brightness and high-stability fluorescent dyes, and synthesis and application thereof
KR20200095117A (en) * 2019-01-31 2020-08-10 한국과학기술연구원 Biosensor based on fluorescence resonance energy transfer for detecting of antipsychotic medicine and detection method for using same
KR102176538B1 (en) 2019-01-31 2020-11-09 한국과학기술연구원 Biosensor based on fluorescence resonance energy transfer for detecting of antipsychotic medicine and detection method for using same

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