WO2019118314A2 - Système de dosage de la scintillation par proximité comprenant une déshalogénase mutante - Google Patents

Système de dosage de la scintillation par proximité comprenant une déshalogénase mutante Download PDF

Info

Publication number
WO2019118314A2
WO2019118314A2 PCT/US2018/064650 US2018064650W WO2019118314A2 WO 2019118314 A2 WO2019118314 A2 WO 2019118314A2 US 2018064650 W US2018064650 W US 2018064650W WO 2019118314 A2 WO2019118314 A2 WO 2019118314A2
Authority
WO
WIPO (PCT)
Prior art keywords
moiety
captureprotein
radiolabeled
capture ligand
assay
Prior art date
Application number
PCT/US2018/064650
Other languages
English (en)
Other versions
WO2019118314A3 (fr
Inventor
Scott E. Wolkenberg
Raphaelle BERGER
Paul Tawa
Keith P. Moore
Xiaoqing Han
Marla L. WATT
Original Assignee
Merck Sharp & Dohme Corp.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Sharp & Dohme Corp. filed Critical Merck Sharp & Dohme Corp.
Publication of WO2019118314A2 publication Critical patent/WO2019118314A2/fr
Publication of WO2019118314A3 publication Critical patent/WO2019118314A3/fr

Links

Classifications

    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)

Definitions

  • Scintillation proximity assay technology is a sensitive and versatile approach to measuring molecular binding interactions. It achieves the sensitivity of other radiometric assay formats without the need for time-consuming phase separations, leading to streamlined protocols compatible with automation and miniaturization.
  • SPA Scintillation proximity assay
  • SPA provides a continuous luminescence signal and therefore provides a convenient and flexible way to perform kinetic studies as well.
  • SPA involves two principle components: a radioisotopically-labeled ligand and a solid support containing a scintillant.
  • Scintillation counting is a generally known and widely used technique for the measurement of low energy beta-emitting radionucleotides in addition to gamma and alpha emitters. Scintillation counting is utilized to make quantitative measurements of radioactivity by incorporating a radiolabelled analyte into a solution with a scintillator capable of producing photons resulting from the kinetic interaction of nuclear decay products.
  • Radioisotopes useful in SPA emit particles which dissipate their energy in aqueous solution through non-radiative processes and within a defined distance. For example, 3 H emits b-particles which dissipate within 4 pm in aqueous solution.
  • 3 H emits b-particles which dissipate within 4 pm in aqueous solution.
  • ligands containing these radioisotopes are bound to the solid support, their decay particles are sufficiently close to the scintillant to activate it and produce light; when unbound, the energy of these particles is dissipated without generating light. In this way, bound and unbound states can be differentiated without the need for phase separation.
  • Radioisotopes used in SPA assays include 3 H, 125 1, 33 P, 14 C, 35 S, 45 Ca, 86 Rb, 75 Se, and 65 Co. (Wu, S. et ak, BioDrugs 19, 383-392 (2005)).
  • Solid supports made of a variety of organic and inorganic substrates have been described, including polyvinyltoluene (PVT), polystyrene, yttrium silicate (YSi), and yttrium oxide. These substrates comprise hydrophobic organic compounds or inorganic salts as scintillants.
  • Matrices the component of a sample other than the analyte compatible with SPA assays are varied and can include highly complex mixtures.
  • SPA assays have been designed in a variety of configurations. In an early configuration, scintillant-containing solid supports were covalently modified to apply a coating of antibodies which specifically recognize certain ligands. (Hart, H. E., et al., Mol Immunol 16, 265-267 (1979); ETdenfriend, S., et al., Proc Natl Acad Sci EG S A 82, 8672-8676 (1985)).
  • Such modified solid supports were used to directly measure quantities of radioisotopically-labeled ligands or, in a competition format, to measure quantities of unlabeled ligands in a test matrix.
  • Ligands measured by these techniques include peptides and small molecules.
  • Protein A (a 42 kDa surface protein originally found in the cell wall of the bacteria staphylococcus aureus) exhibits affinity for the Fc domain of mammalian antibodies
  • the Protein A-coated solid support has been further derivatized with an antibody of choice through a simple mix, filter, and wash protocol without the need for additional reagents.
  • These Protein A- antibody supports have been used directly in SPA assays or further derivatized with a protein of interest, presenting an epitope recognized by the antibody. In the latter case, binding of radioisotopically-labeled ligands to the protein of interest have been assayed.
  • biotin serves as a capture ligand and streptavidin as a capture protein.
  • a peptide substrate for human cytomegalovirus EIL-80 protease was biotin-labeled at its N-terminus and 33 P-labeled toward its C-terminus prior to being loaded onto streptavidin-coated SPA beads. Because the UL-80 protease cleavage site in the peptide was located between the N-terminus and the 33 P label, cleavage caused a decrease in SPA signal.
  • the assay has been used to quantify inhibition of the UL-80 protease.
  • a biotinylated oligonucleotide (oligo(dT)i 6 ) has been used as a primer with a poly(A) template to measure HCV NS5B RNA polymerase activity. After incubation of the primer and template with the polymerase and 3 H-UTP, the reaction is stopped with addition of streptavidin-coated SPA beads and luminescence signal directly measured. (Zheng, W.
  • biotin-labeled chemical probes and biomolecules have been used to label cell-surface proteins selectively over intracellular proteins because of its inability to penetrate cells.
  • Biotinylated chemical probes for detecting isoprenoid post translationally modified proteins have been used in cell lysates but not intact cells. (Nguyen, U. T. et al., Nat. Chem. Biol., 5, 227-235, doi: l0.l038/nchembio. l49 (2009)).
  • biotin-labeled chemical probes alternative and more labor-intensive methods have been developed. For example, chemical probes based on isoprenoid pyrophosphates which are cell -penetrant when bearing an azide group have been used.
  • the azide is modified in a Staudinger ligation to append a biotin group to the chemical probe which is subsequently used to enrich for proteins bearing the isoprenoid group.
  • Staudinger ligation to append a biotin group to the chemical probe which is subsequently used to enrich for proteins bearing the isoprenoid group.
  • the present invention provides an assay system comprising a novel combination of Scintillation proximity assay (SPA) technology and a Capture Protein/Capture Ligand system and methods for its use.
  • SPA Scintillation proximity assay
  • Figure 1 illustrates the fluorescence intensity of HaloTag® SPA beads treated with HaloTag®-TMR ligand with and without pre-treatment with HaloTag®-biotin ligand, demonstrating that HaloTag® SPA beads specifically capture HaloTag® ligands.
  • Figure 2 illustrates a dose titration of a radiolabeled Capture Ligand with and without excess non-radiolab el ed Capture Ligand using HaloTag® SPA beads 7 and CBT SPA beads 5, providing a measurement of loading density of HaloTag® SPA beads.
  • Figure 3 illustrates a dose titration of radiolabeled Capture Ligand with subtraction of nonspecific signal contributed from CBT SPA beads 5. Saturation point was determined by linear regression, providing a measure of specific loading density of HaloTag® SPA beads.
  • Figure 4 illustrates the capture by HaloTag® SPA beads of radiolabeled Capture Ligands from cell lysates generated with a variety of lysis buffers. SPA signal is specifically blocked by pre-treatment with non-radiolabeled HaloTag®-biotin ligand.
  • alkyl means an aliphatic hydrocarbon group which may be straight
  • Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, or longer alkyl moieties are attached to a linear alkyl chain.
  • the number of carbon atoms comprising a specified alkyl moiety may be expressed as a range of the form -(C x -C y )alkyl, wherein x is the lower limit of the number of carbon atoms in the alkyl group and y is the upper limit of the number of carbon atoms in the alkyl group.
  • Alkylene means a difunctional group obtained by removal of a hydrogen atom from an alkyl group that is defined above.
  • alkylene include ethylene, propylene, butylene and the like. More generally, the suffix“ene” on alkyl, aryl, hetercycloalkyl, etc. indicates a divalent moiety, e.g., -CH 2 CH 2 - is ethylene, and
  • linker -L- is a moiety of the formula: , wherein L , L A , and L B2 are as defined herein.
  • the wavy line represents the point of attachment to a polar group on the surface of solid support material S; and the wavy line represents the point of attachment to the Coupling Moiety C.
  • Aryl means an aromatic monocyclic or multicyclic ring system comprising 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms.
  • the aryl group can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein.
  • suitable aryl groups include phenyl and naphthyl.
  • Heteroaryl means an aromatic monocyclic or multicyclic ring system comprising 5 to 14 ring atoms, preferably 5 to 10 ring atoms, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination.
  • Preferred heteroaryls contain 5 to 6 ring atoms.
  • a nitrogen atom of a heteroaryl can be optionally oxidized to the corresponding N-oxide.
  • Non-limiting examples of suitable heteroaryls include pyridyl, pyrazinyl, furanyl, thienyl (alternatively referred to as thiophenyl), pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, l,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[l,2-a]pyridinyl, imidazo[2, l-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinoliny
  • Cycloalkyl means a non-aromatic mono- or multicyclic ring system comprising 3 to 10 carbon atoms, preferably 5 to 10 carbon atoms. Preferred cycloalkyl rings contain 5 to 7 ring atoms.
  • suitable monocyclic cycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
  • suitable multicyclic cycloalkyls include l-decalinyl, norbornyl, adamantyl and the like.
  • Cycloalkenyl means a cycloalkyl group described above but which further comprises at least one carbon-carbon double bond. Suitable cycloalkenyl rings contain 5 to 7 ring atoms. Non limiting examples of suitable monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, cyclohepta-l,3-dienyl, and the like. Non-limiting example of a suitable multicyclic cycloalkenyl is norbomylenyl.
  • Heterocycloalkyl (or “heterocyclyl”) means a non-aromatic saturated monocyclic or multicyclic ring system comprising 3 to 10 ring atoms, alternatively 5 to 10 ring atoms, in which one or more of the atoms in the ring system is an atom other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. There are no adjacent oxygen and/or sulfur atoms present in the ring system. Exemplary heterocyclyls contain 5 to 6 ring atoms. The prefix aza, oxa or thia before the heterocyclyl root name means that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom.
  • heterocyclyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.
  • Heterocycloalkenyl (or “heterocyclenyl”) means a non-aromatic monocyclic or multi cyclic ring system comprising 3 to 10 ring atoms, preferably 5 to 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen, oxygen or sulfur atom, alone or in combination, and which is not aromatic but which further comprises at least one carbon-carbon double bond or carbon-nitrogen double bond.
  • heterocyclenyl root name means that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom.
  • the nitrogen or sulfur atom of the heterocyclenyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.
  • Non-limiting examples of suitable heterocyclenyl groups include 1,2, 3, 4- tetrahydropyridinyl, l,2-dihydropyridinyl, 1,4- dihydropyridinyl, l,2,3,6-tetrahydropyridinyl, l,4,5,6-tetrahydropyrimidinyl, 2-pyrrolinyl, 3- pyrrolinyl, 2-imidazolinyl, 2-pyrazolinyl, dihydroimidazolyl, dihydrooxazolyl,
  • Example of such moiety is pyrrolidenone (or pyrrolone).
  • Crabtree catalyst (l,5-Cyclooctadiene)(pyridine)(tricyclohexylphosphine)-iridium(I) hexafluorophosphate
  • DCM dichloromethane
  • HATEG 1 -[Bis(dimethyl amino)methylene]- l H- 1 ,2,3-triazolo[4,5-/>]pyridinium 3-oxid hexafluorophosphate
  • HT-TMR HaloTag® TMR ligand, Promega product # G8251
  • IGEPAL CA640 CAS: 9002-93-1
  • PBS Buffer HyCloneTM Phosphate Buffered Saline SH30256.01
  • PVT PEI SPA beads Perkin Elmer RPNQ0097
  • TX-100 Triton X-100, t-Octylphenoxypolyethoxy ethanol
  • YSi Poly-lysine SPA beads Perkin Elmer RPNQ0010
  • the present invention provides an assay system comprising:
  • S is a scintillant-containing solid support
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on L;
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein; and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant- containing solid support material S.
  • the term“scintillant-containing solid support” (S) refers to a solid material whose surface or coating comprises a multiplicity of polar functional groups capable of covalently bonding to linker moiety (L), described below, is insoluble in aqueous solutions and further comprises a scintillant.
  • Suitable solid supports may take the form of wells, tubes, slides, plates, resins, beads, or the like.
  • Suitable solid support materials include organic and inorganic substrates.
  • inorganic materials suitable for use as substrates include glass materials such as yttrium silicate and yttrium oxide.
  • suitable organic materials include polyvinyl toluene, acrylamide, acrylic acid, polymers of styrene, agar, agarose, polycarbonate, polypropylene, polystyrene, cellulose acetate, and latex.
  • suitable solid support materials include hydrophilic plastics.
  • the surfaces of such solid supports provide a surface comprising a multiplicity of polar functional groups capable of carrying a net negative charge due to the presence of oxygen-containing functional groups such as hydroxyl and carboxyl.
  • suitable solid support materials include plastics whose surfaces are coated with a cationic polymer, e.g., polylysine or polyethyleneimine, to provide a surface exhibiting a polar functional group having a net positive charge.
  • Non-limiting polar functional groups capable of covalently bonding to at least one linker (L) on such organic and inorganic solid support materials include: amines and carboxylic acids.
  • useful scintillant-containing solid support materials will comprise a multiplicity of polar functional groups on their surface capable of covalently bonding to linker moiety (L).
  • the number of such polar functional groups on a given amount of surface area of the solid support material S will be sufficient to allow the attachment of a sufficient number of -L(C-CaptureProtein)p units to carry out the measurements based on SPA signal, e.g., as described herein.
  • Scintillants are incorporated into the solid support materials described above according to methods known in the art or described below. Scintillants are any material that will emit detectable photons of light when excited by the kinetic interaction of nuclear decay particles as described herein. Scintillants suitable for use in the present invention may be organic or inorganic.
  • Suitable organic scintallants include optionally substituted aryl oxazoles, (such as diphenyloxazole, as described in US 4,568,649, 2-(4-tert-Butylphenyl)-5-(4-phenylphenyl)- l,3,4-oxadiazole, and 2,5-diphenyloxazole (McCairn, M.C., et al., Tetrahedron Lett., 45, 2163- 2166 (2004), Culliford, S.J., et al., Biochem. Biophys. Res. Commun ., 296, 857-863 (2002)).
  • aryl oxazoles such as diphenyloxazole, as described in US 4,568,649, 2-(4-tert-Butylphenyl)-5-(4-phenylphenyl)- l,3,4-oxadiazole, and 2,5-diphenyloxazole (McCairn, M.
  • Suitable inorganic scintillants include any metal or rare earth metal that will emit detectable photons of light when excited by the kinetic interaction of nuclear decay particles as described herein.
  • Non-limiting examples of such metals include Mn, Cu, Pb, Sn, Au, Ag, Sm, and Ce.
  • Examples of rare earth metals and their use as scintillants are described in, e.g., WO1991/08489.
  • inorganic scintillants may be incorporated into inorganic substrates (e.g., glass) by doping or other suitable activation processes.
  • organic scintillants may be incorporated into organic substrates (e.g., plastics) by following a procedure such as is provided in US Patent No. 4,568,649.
  • the support bodies are soaked in a solvent for the scintillant which is miscible in water to dehydrate the support bodies. Thereafter, the bodies are placed in a solution composed of the scintillant and solvent so that the scintillant is integrated and/or adsorbed into the bodies. Then, the bodies are removed from the solvent and then placed in an aqueous solution which causes precipitation of the scintillant within the bodies, thereby locking the scintillant therein.
  • the scintillant is integrated within the interior of the bodies so that the radiolabeled reactant is placed in very close proximity to the scintillant upon binding to the ligand, which is disposed on the exterior of the bodies.
  • Scintillant-containing solid supports (S) suitable for use in the present invention are commercially available in a variety of support shapes and materials.
  • suitable scintillant-containing solid supports (S) include CytoStar-T scintillating microplate® (Perkin Elmer, Waltham, MA, USA), Polylysine YSI beads® (Perkin Elmer, Waltham, MA, USA), and PVT PEI coupled beads® (Perkin Elmer, Waltham, MA, USA), and the like.
  • linker refers to a multivalent moiety capable of covalently linking one or more (e.g., one, two, three, or four or more) Coupling Moieties (C), described below, to a scintillant-containing solid support (S) described above.
  • Suitable linkers comprise a linear or branched carbon chain comprising from 2 to 40 carbon atoms or a group that comprises one or more rings, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo groups, and wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide -0-, -S- or - NH-.
  • Non-limiting examples of such include polyethylene glycol (PEG).
  • Additional non limiting examples of such linkers include the aforementioned linear or branched alkyl groups which optionally further comprise one or more rings, which rings are selected from aryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, and heteroaryl rings as described below.
  • Linkers having any suitable number of valences may be used in the invention, including but not limited to divalent, trivalent, tetravalent, and pentavalent and higher valent linker moieties.
  • one of the available valences of L will couple the linker (L) to the solid support (S) and a second available valence of L will couple the linker (L) to the Coupling Moiety (C).
  • the linker moiety (L) is divalent
  • one of the two available valences of the linker moiety will couple the linker (L) to the solid support (S) and the other will couple the linker (L) to the -C-CaptureProtein.
  • the additional valences can be used to attach additional - (C-CaptureProtein)p groups to the linker moiety, wherein p is 1 or more and wherein p represents the number of available valences of L.
  • L is divalent, trivalent, tetravalent or pentavalent and p is from 1 to 4.
  • L is divalent, trivalent, or tetravalent and p is from 1 to 3.
  • L is divalent or trivalent and p is from 1 to 2.
  • L is divalent and p is 1.
  • suitable multivalent linker moieties are described below. As will be appreciated by those of ordinary skill in the art, each of the units -L(C-
  • CaptureProtein)p of the multiplicity of such units covalently attached to each of a multiplicity of available polar functional groups on the solid support S may be chosen for uniformity or, alternatively, may be made to vary one from the other from the independent selection each of L, p, C, and CaptureProtein.
  • p is 1 and -L- is a divalent linker moiety, providing for the attachment of a single CaptureProtein moiety to an available polar functional group on the scintillant-containing solid support S.
  • -L- is a moiety according to the formula -L B1 -(L A -L B1 ) q -, wherein: q is an integer from 1 to 5;
  • each -L - is independently selected from
  • n is an integer from 0 to 35; and each -L B1 - is independently selected from
  • linkers and/or reagents for their construction are commercially available, e.g., from Broadpharm, 9380 Waples Street, Suite 101, San Diego, CA 92121 or MilliporeSigma, 3050 Spruce St., St. Louis, MO 63103.
  • q is 1 or 2. In another alternative of this embodiment, q is 1.
  • p is 2, 3, 4, or 5 and -L- is a trivalent, tetravalent, pentavalent or hexavalent linker moiety, allowing, respectively, for the attachment of two, three, four or five -C-CaptureProtein moieties to an available polar functional group on the scintillant-containing solid support S.
  • each -L B2 - is a trivalent moiety
  • n is an integer from 0 to 35; and each -L - is independently selected from wherein each R L is independently selected from H, -(Ci-C 6 )alkyl; and benzyl; and -C-CaptureProtein are as described below.
  • Coupling Moiety C refers to a divalent moiety capable of covalently connecting (or coupling) the linker moiety -L-, described above, to CaptureProtein, described below.
  • Suitable Coupling Moieties include bioconjugation products of an antibody, protein, or an oligonucleotide and the C-terminus, N-terminus or other point of attachment of the CaptureProtein protein. Bioconjugation is a chemical strategy known to those of ordinary skill in the art to form a stable link between two molecules, at least one of which is a biomolecule.
  • biomolecules include antibodies, proteins, and oligonucleotides.
  • Suitable Coupling Moieties can be made from chemical modification of proteins and other biomolecules according to methods known in the art. See, e.g., Justine N. deGruyter, et al., Biochemistry 2017, 56, 3863-3873.
  • the Coupling Moiety is attached to the N-terminus or the C-terminus of the CaptureProtein, wherein the N-terminus or the C-terminus of said CaptureProtein are cysteine or lysine residues.
  • the Coupling Moiety is attached to the N-terminus of
  • CaptureProtein wherein said N-terminus of said CaptureProtein is a cysteine residue or a lysine residue.
  • the Coupling Moiety is attached to the N-terminus of
  • CaptureProtein wherein said N-terminus of said CaptureProtein is a cysteine residue.
  • the Coupling Moiety is attached to the N-terminus of
  • CaptureProtein wherein said N-terminus of said CaptureProtein is a lysine residue.
  • the Coupling Moiety is attached to the C-terminus of
  • CaptureProtein wherein said C-terminus of said CaptureProtein is a cysteine residue or a lysine residue.
  • the Coupling Moiety is attached to the C-terminus of
  • CaptureProtein wherein said C-terminus of said CaptureProtein is a cysteine residue.
  • the Coupling Moiety is attached to the C-terminus of
  • CaptureProtein wherein said C-terminus of said CaptureProtein is a lysine residue.
  • the Coupling Moiety is attached to the CaptureProtein protein at a position other than the C-terminus or the N-terminus of the CaptureProtein sequence at a site compatible with the generation of said Coupling Moiety via a bioconjugation reaction.
  • suitable Coupling Moieties include 2-(6-aminobenzo[ ⁇ 7]thiazol-
  • 2-yl)-4,5-dihydrothiazole-4-carboxamide which can be formed by the coupling of an N- terminal cysteine and 7-amino-2-cyanobenzothiazole as described in PCT publication number W02010033647.
  • suitable Coupling Moieties (C) can be formed by reacting a suitable bioconjugation moiety with a suitable biomolecule using techniques known to those of ordinary skill in the art, e.g., as set forth in Justine N. deGruyter, et al., Biochemistry 2017, 56, 3863-3873, incorporated herein by reference. Examples of Coupling Moieties which can be made through such bioconjugation reactions and the corresponding bioconjugate reactants are set forth in the table below.
  • CaptureProtein refers to a capture protein derived from the bacterial haloalkane dehalogenase enzyme e.g., a haloalkane dehalogenase or a dehalogenase, that cleaves carbon-halogen bonds in an aliphatic or aromatic halogenated substrate, such as a substrate for Rhodococcus, Sphingomonas, Staphylococcus, Pseudomonas, Burkholderia,
  • Capture proteins suitable for use in the present invention include those in which at least one amino acid of the bacterial haloalkane dehalogenase enzyme is substituted relative to the wild-type hydrolase such that the resulting enzyme exhibits reduced catalytic activity compared to the corresponding wild-type hydrolase resulting in a stable dehalogenase-alkane complex.
  • the CaptureProtein thus includes a reactive site that specifically, rapidly, and irreversibly reacts with a haloalkane capture ligand (described below) under normal physiological and test conditions. (See, e.g., Los, G. V.
  • Capture proteins derived from suitable mutant bacterial hydrolase enzymes may be obtained by methods known in the art. See, e.g., U.S. Patent No. 7,425,436; U.S. Patent No. 7,429,472; U.S. Patent No. 7,867,726; U.S. Patent No.
  • said CaptureProtein comprises a hydrolase enzyme with at least 70% sequence identity with SEQ ID NO.: 1. Met Ala Glu lie Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu
  • Leu lie lie Asp Gin Asn Val Phe lie Glu Gly Thr Leu Pro Met Gly
  • Trp Leu Ser Thr Leu Glu Ile Ser Gly SEQ ID NO.: 1.
  • the CaptureProtein protein comprises a hydrolase enzyme with at least 75% sequence identity with SEQ ID NO.: 1. In another embodiment, the CaptureProtein protein comprises a hydrolase enzyme with at least 80% sequence identity with SEQ ID NO.: 1. In another embodiment, the CaptureProtein protein comprises a hydrolase enzyme with at least 85% sequence identity with SEQ ID NO.: 1. In another embodiment, the CaptureProtein protein comprises a hydrolase enzyme with at least 90% sequence identity with SEQ ID NO.: 1. In another embodiment, the CaptureProtein protein comprises a hydrolase enzyme with at least 95% sequence identity with SEQ ID NO.: 1. In another embodiment, the CaptureProtein protein comprises a hydrolase enzyme with at least 98% sequence identity with SEQ ID NO.: 1.
  • the CaptureProtein protein comprises a hydrolase enzyme with at least 99% sequence identity with SEQ ID NO.: 1. In another embodiment, the CaptureProtein protein comprises a hydrolase enzyme having an amino acid sequence according to SEQ ID NO.: 1. In each of these embodiments, the CaptureProtein includes a reactive site that specifically, rapidly, and irreversibly reacts with a haloalkane capture ligand (described below) under normal physiological and test conditions.
  • Capture protein suitable for use in the present invention is available for purchase under the tradename HaloTag® from Promega Corp. Madison WI, USA.
  • Capture protein according to any of the above described embodiments may be further modified using methods known in the art (non-limiting examples of which are described below) with additional amino acids appended at the C- or N-terminus.
  • the appended polypeptide sequences may be used to confer specific reactivity useful for bioconjugation to suitable coupling agents described below.
  • CaptureProteinp include the following, wherein represents any scintillant-containing solid support material described above:
  • the present invention further comprises a Capture Ligand for use with the S-L(C-CaptureProtein)p assay system described above.
  • Capture Ligands suitable for use in the present invention are comprised of a chemical moiety that undergoes capture by the
  • CaptureProtein portion of S-L(C-CaptureProtein)p system with rapid kinetics and substantial irreversibility while also exhibiting cell penetration and relatively low reactivity toward endogenous nucleophiles in mammalian cells, e.g., in cell matrices of high complexity including intact cells and cell lysates.
  • suitable Capture Ligands are comprised of a substrate for CaptureProtein.
  • the Capture Ligand comprises a haloalkane.
  • the Capture Ligand comprises a chloroalkane.
  • Capture Ligands comprising haloalkane, e.g., chloroalkane have been shown to exhibit excellent cell penetration.
  • haloalkane e.g., chloroalkane Capture Ligands
  • Specific suitable haloalkane Capture Ligands are described below.
  • the Capture Ligand according to this aspect of the invention further comprises at least one radiolabeled cargo moiety which comprises at least one radioactive isotope which, when the Capture Ligand is captured by the CaptureProtein portion of the S-L(C-CaptureProtein)p system is capable of stimulating scintillation by the chosen scintillant present on the solid support (S) suitable for measurement in SPA.
  • at least one radiolabeled cargo moiety which comprises at least one radioactive isotope which, when the Capture Ligand is captured by the CaptureProtein portion of the S-L(C-CaptureProtein)p system is capable of stimulating scintillation by the chosen scintillant present on the solid support (S) suitable for measurement in SPA.
  • Non-limiting examples of suitable cargo moieties include cellular metabolites (such as isoprenoids, purines, pyrimidines, steroids, porphyrins, lipids, catecholamines, amino acids, carbohydrates, and nucleic acids); photoactivatable groups (such as aryl azides, benzophenones, and diazirines); digoxigenin; nickel nitrilotriacetic acid (NT A); chromophores and fluorophores (such as AlexaFluor 350, 488, 546, 555, 568, 594, 647, fluorescein, and bodipy reagents); luminophores (such as luciferin and its derivatives); drugs, drug candidates, prodrugs; chemical probes; and biotin.
  • Non-limiting examples of radioactive isotopes suitable for use in R* include 3 ⁇ 4 125 1, 33 P, 14 C, 35 S, 45 Ca, 86 Rb, 75 Se, and 65 Co. Specific non-limiting examples of R
  • the Capture Ligand takes the form R*-L 2 -A-X, wherein: R* is a Cargo Moiety comprising one or more radiolabeled isotopes;
  • -A-X is a haloalkane wherein -A- is a -(C 2 -Cio)alkyl group, which may be straight or branched, and X is a halogen selected from Cl or Br.
  • suitable radiolabeled cargo moieties R* include cellular metabolites (such as isoprenoids, purines, pyrimidines, steroids, porphyrins, lipids,
  • catecholamines, amino acids, carbohydrates, and nucleic acids include photoactivatable groups (such as aryl azides, benzophenones, and diazirines); digoxigenin; nickel nitrilotriacetic acid (NT A); chromophores and fluorophores (such as AlexaFluor 350, 488, 546, 555, 568, 594, 647, fluorescein, and bodipy reagents); luminophores (such as luciferin and its derivatives); drugs, drug candidates, prodrugs; chemical probes and biotin.
  • radioactive isotopes suitable for use in R* include 3 ⁇ 4 125 1, 33 P, 14 C, 35 S, 45 Ca, 86 Rb, 75 Se, and 65 Co.
  • -L 2 - is a divalent moiety capable of covalently linking R* (cargo moiety) to the haloalkane moiety -A-X.
  • Suitable linkers comprise a linear or branched carbon chain comprising from 2 to 30 carbon atoms or a group that comprises one or more rings, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo groups, and wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide -0-, -S- or -NH-.
  • Non-limiting examples of such include polyethylene glycol (PEG).
  • linkers include the aforementioned linear or branched alkyl groups which optionally further comprise one or more rings, which rings are selected from aryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, and heteroaryl rings as described herein.
  • one of the available valences of L 2 will covalently attach -L2- to the cargo moiety (R*) and the other available valence of-L 2- will covalently attach -L 2- to an available carbon atom on the haloalkane moiety -A-X.
  • -L 2 - is a moiety according to the formula -L B2 -(L A2 - L B2 ) t -, wherein: t is an integer from 1 to 5; D 9
  • each -L - is independently selected from ?
  • n is an integer from 0 to 35;
  • each -L B2 - is independently selected from
  • linkers and/or reagents for their construction are commercially available, e.g., from Broadpharm, 9380 Waples Street, Suite 101, San Diego, CA 92121 or MilliporeSigma, 3050 Spruce St., St. Louis, MO 63103.
  • q is 1 to 3.
  • q is 1 or 2.
  • q is 1.
  • Non-limiting examples of-L 2- include:
  • the moiety -A-X is a haloalkane moiety wherein -A- is a -(C 2 -Ci 0 )alkyl group, which may be straight or branched, and X is a halogen selected from Cl or Br.
  • -A- is a -(C 2 -C 6 )alkyl group, which may be straight or branched, and X is Cl or BR. In another embodiment, -A- is a straight chain alkyl group comprising from two to six carbon atoms; and X is Cl.
  • the Capture Ligand comprises the above described radiolabeled capture ligand and further comprises one or more additional Capture Ligands comprising a non- radiolabeled cargo moiety for use in combination with one or more of the above described radiolabeled Capture Ligands.
  • the one or more additional non-radiolab el ed Capture Ligand take the form R- L 2 -A-X, wherein R is an analyte and -L 2- , and -A-X are as set forth in any of the embodiments described above.
  • Non-limiting examples of analytes include cellular metabolites (such as isoprenoids, purines, pyrimidines, steroids, porphyrins, lipids, catecholamines, amino acids, carbohydrates, and nucleic acids); drugs, drug candidates, prodrugs; and chemical probes.
  • cellular metabolites such as isoprenoids, purines, pyrimidines, steroids, porphyrins, lipids, catecholamines, amino acids, carbohydrates, and nucleic acids
  • drugs, drug candidates, prodrugs include cellular metabolites (such as isoprenoids, purines, pyrimidines, steroids, porphyrins, lipids, catecholamines, amino acids, carbohydrates, and nucleic acids); drugs, drug candidates, prodrugs; and chemical probes.
  • the present invention provides methods of using the assay system described herein in a wide variety of quantitative measurements, including but not limited to the measurement of analytes, biological processes, structure-activity relationships of small molecule drug candidates, cellular metabolic events, intracellular trafficking, effects of genetic and epigenetic changes, intracellular signaling events, gene transcription regulation, and others.
  • the radioisotope of the radiolabeled Cargo Moiety of the Capture Ligand is placed in sufficiently close physical proximity to the scintillant contained in the solid support material S to allow the radiation energy emitted from the isotope to activate the scintillant, thereby causing the scintillant to emit light energy.
  • the amount of light energy emitted is proportional to the quantity of radiolabeled Capture Ligand bound to CaptureProtein, and may be conveniently measured with a scintillation counter or other monitoring device. Any scintillation counter or suitable monitoring device, such as those employing a photomultiplier tube, will suffice.
  • the radiation energy emitted by the radiolabeled Capture Ligand has a very limited range of travel in aqueous solution. Thus, the Capture Ligand molecules that have not bound to the CaptureProtein are substantially too far from the
  • CaptureProtein to enable the radiation energy emitted from unbound Capture Ligand to reach the scintillant in the solid support S.
  • the Capture Ligand molecules that have not bound to the ligand need not be separated from the
  • CaptureProtein/Capture Ligand complexes prior to scintillation counting Prior to scintillation counting. Accordingly and advantageously, the traditional laborious and potentially hazardous procedures associated with such separation may be avoided where desired.
  • Cargo Moiety, solid support material S, scintillant, linker, Coupling Moiety, CaptureProtein, and Capture Ligand described herein can each be selected and configured independently to enable the quantitative
  • a method of measuring the enzymatic activity on a biological substrate by an enzyme of interest in a biological sample comprising: Providing a quantity of a biological sample comprising a quantity of an enzyme of interest;
  • Capture Ligand Adding to said sample a quantity of a radiolabeled Capture Ligand comprising said biological substrate, wherein said Capture Ligand has the formula (R*)-L 2 -A-X, wherein: R* is a Cargo Moiety comprising said biological substrate, wherein said biological substrate comprises a radiolabeled moiety which is capable of being enzymatically cleaved from the Cargo Moiety by action of said enzyme of interest;
  • -A-X is a haloalkane wherein -A- is a -(C 2 -Cio)alkyl group which may be straight or branched, and X is Cl or Br; wherein the enzymatic activity of said enzyme of interest is capable of cleaving the radiolabeled moiety of said biological substrate, and wherein such enzymatic activity results in a Capture Ligand comprising a non-radiolabeled Cargo Moiety of the formula (R)-L 2 -A-X, wherein R is a Cargo Moiety whose radiolabeled moiety has been enzymatically cleaved by said enzyme of interest, and -L 2 -, and -A-X are as defined above;
  • S is a scintillant-containing solid support
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on L;
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein; and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant- containing solid support material S; and
  • the Capture Ligand is not initially radiolabeled and the enzymatic activity of said enzyme of interest is capable of adding a radiolabeled moiety to the Cargo Moiety of the Capture Ligand, and wherein such enzymatic activity results in a Capture Ligand comprising a radiolabeled Cargo Moiety of the formula (R*)-L 2 -A-X, wherein R* is a Cargo Moiety whose radiolabeled moiety has been enzymatically added to the Cargo Moiety by the enzyme of interest, and -L 2 -, and -A-X are as defined above.
  • the measured quantity of light energy emitted from the assay system is proportional to the quantity of radiolabeled Capture Ligand synthesized by said enzymatic activity and thus a proportional measure of the enzymatic activity on said biological substrate in said biological sample.
  • the biological sample comprising the enzyme of interest is derived from cellular extracts, cellular lysates, whole cells, biological tissues, or body fluids. Non-limiting examples of such body fluids include blood, lymph, urine and the like.
  • the biological sample may be washed with aqueous solution as needed or desired. Where the biological sample is derived from intact cells or biological tissues, the sample may be lysed prior to the addition of radiolabeled substrate, or lysed later but prior to combining the sample with S-L(C- CaptureProtein) p .
  • the radiolabeled substrate is selected from a nutrient, a metabolite, a biosynthetic precursor, cellular metabolites, or any other biological substrate capable of being radiolabeled with a moiety that is enzymatically cleaved by the enzyme of interest.
  • the radiolabeled moiety cleavable by said enzymatic activity comprises a radioactive isotope selected
  • radiolabeled cellular metabolites are selected from isoprenoids, purines, pyrimidines, steroids, porphyrins, lipids, catecholamines, amino acids, carbohydrates, and nucleic acids.
  • the radiolabeled Cargo Moiety comprising the radiolabeled substrate may be substituted with a chromophore or fluorophore whose light-emitting moieties are capable of being enzymatically cleaved by the enzyme of interest.
  • Non-limiting examples of chromophores are selected from AlexaFluor 350, 488, 546, 555, 568, 594, 647, fluorescein, bodipy reagents.
  • Non-limiting examples of luminophores are selected from luciferin and luciferin derivatives, which are well known in the art.
  • the measurement of the quantity of light energy emitted from the assay system is taken at a selected time point, wherein the selected time point is the time at which the assay system S-L(C-CaptureProtein) p is combined with the sample and optionally at one or more additional selected points in time thereafter.
  • a method of measuring the effect of a test compound on the enzymatic activity of an enzyme of interest in a biological sample is carried out in the absence of the test compound, in the presence of a test compound, and optionally in the presence of each of different concentrations of test compound to determine a dose-response relationship.
  • the measured quantity of light energy emitted from said assay system in the presence and absence of test compound, or in the presence of various concentrations of test compound is a measure of the influence of test compound on said enzymatic activity on the biological substrate in the biological sample.
  • Test compounds suitable for use in the methods according to the invention include small molecules (such as small molecules undergoing structure-activity testing during drug discovery) and biologies (including but not limited to cellular components, antibodies, antigens, proteins, DNA, RNA, siRNA, recombinant proteins, tissues, genes, allergens, cells, blood components, blood, vaccines, viruses, inactivated viruses, hormones, substances that suppress or activate components of the immune system, and the like).
  • a method of measuring the effect of a test compound on the enzymatic activity of an enzyme of interest in on a biological substrate in a biological sample comprising:
  • Capture Ligand has the formula (R*)-L 2 -A-X, wherein:
  • R* is a Cargo Moiety comprising said biological substrate, wherein said biological substrate comprises a radiolabeled moiety which is capable of being enzymatically cleaved from the Cargo Moiety by action of said enzyme of interest;
  • -A-X is a haloalkane wherein -A- is a -(C 2 -Cio)alkyl group which may be straight or branched, and X is Cl or Br; wherein the enzymatic activity of said enzyme of interest is capable of cleaving the radiolabeled moiety of said biological substrate, and wherein such enzymatic activity results in a Capture Ligand comprising a non-radiolabeled Cargo Moiety of the formula (R)-L 2 -A-X, wherein R is a Cargo Moiety whose radiolabeled moiety has been enzymatically cleaved by said enzyme of interest, and -L 2 -, and -A-X are as defined above; Combining said biological sample with an assay system S-L(C-CaptureProtein) p , wherein:
  • S is a scintillant-containing solid support
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on L;
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein; and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant- containing solid support material S; and
  • the quantity of test compound is combined with said biological sample prior to the addition of said radiolabeled Capture Ligand.
  • the quantity of test compound is combined with said biological sample after the addition of said radiolabeled Capture Ligand but prior to the addition of said assay system S-L(C-CaptureProtein) p .
  • the process in the absence of test compound and the process in the presence of test compound are run sequentially, in any order, or, alternatively, in parallel.
  • the process is repeated a sufficient number of times, each time in the presence of a different concentration of test compound, to determine a dose- response relationship.
  • the assay system and Capture Ligands according to the invention may be adapted to quantitatively measure a wide variety of biological processes beyond enzymatic activity.
  • the assay system according to the invention may be used to directly measure each such process, or it may be adapted for use in measuring the effect of a test compound (e.g., small molecule and/or biologic) on each such biological process.
  • a test compound e.g., small molecule and/or biologic
  • the present invention may be used to measure a biochemical reaction occurring inside a cell.
  • a Capture Ligand containing a non- radiolabeled Cargo Moiety which is a substrate for a cellular enzyme is incubated with living cells in media containing a radiolabeled nutrient.
  • the nutrient is absorbed, resulting in metabolic labeling of components of the cell.
  • the Capture Ligand is similarly absorbed and susceptible to intracellular enzyme-catalyzed modification of the Cargo Moiety, including appendage of the moiety containing radiolabeled atoms incorporated via metabolic labeling.
  • the resulting enzymatic product is a modified Capture Ligand comprising a radioactive label.
  • a process for the measurement of a biological process in vivo comprising: Administering, in vivo, to an organism a non-radiolabeled Capture Ligand of the formula
  • R is a Cargo Moiety comprising a biological substrate which is susceptible of being altered by the biological process of interest;
  • -A-X is a haloalkane wherein -A- is a -(C 2 -C i 0 )alkyl group which may be straight or branched, and X is Cl or Br;
  • S is a scintillant-containing solid support
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on L;
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein; and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant- containing solid support material S; and
  • the Capture Ligand has a formula (R*)-L 2 -A-X, wherein R* is a radiolabeled Cargo Moiety comprising a biological substrate which is susceptible of being altered by the biological process of interest; -L 2 - is a divalent linker moiety; -A-X is a haloalkane wherein -A- is a -(C 2 -Cio)alkyl group which may be straight or branched, and X is Cl or Br; and the biological process of interest is capable of cleaving the radiolabeled moiety from the Cargo Moiety of the Capture Ligand.
  • the quantity of light energy emitted from said assay system measured using a scintillation counter is
  • the measured quantity of light energy emitted from said assay system in the presence and absence of test compound, or in the presence of various concentrations of test compound is a measure of the influence of test compound on said enzymatic activity on the biological substrate in the biological sample.
  • Test compounds suitable for use in the methods according to the invention include small molecules (such as small molecules undergoing structure-activity testing during drug discovery) and biologies (including but not limited to cellular components, antibodies, antigens, proteins, DNA, RNA, siRNA, recombinant proteins, tissues, genes, allergens, cells, blood components, blood, vaccines, viruses, inactivated viruses, hormones, substances that suppress or activate components of the immune system, and the like).
  • small molecules such as small molecules undergoing structure-activity testing during drug discovery
  • biologies including but not limited to cellular components, antibodies, antigens, proteins, DNA, RNA, siRNA, recombinant proteins, tissues, genes, allergens, cells, blood components, blood, vaccines, viruses, inactivated viruses, hormones, substances that suppress or activate components of the immune system, and the like.
  • a method of measuring the rate of penetration of an analyte through a cellular membrane comprising: Providing a quantity of a biological sample comprising a population of cells;
  • Capture Ligand has the formula (R*)-L 2 -A-X, wherein:
  • R* is a Cargo Moiety comprising said analyte, wherein said analyte comprises at least one radiolabeled moiety;
  • -A-X is a haloalkane wherein -A- is a -(C 2 -Ci 0 )alkyl group which may be straight or branched, and X is Cl or Br;
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein; and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant-containing solid support material S; and Measuring the quantity of light energy emitted from said assay system using a scintillation counter, wherein the quantity of said emitted light energy measured is proportional to the quantity of radiolabeled analyte that has penetrated said cells.
  • the analyte of interest is present as a Cargo Moiety on a Capture Ligand which is not radiolabeled.
  • a method of measuring the rate of penetration of an analyte through a cellular membrane comprising:
  • R is a Cargo Moiety comprising the analyte of interest
  • -A-X is a haloalkane wherein -A- is a -(C 2 -Ci 0 )alkyl group which may be straight or branched, and X is Cl or Br;
  • S is a scintillant-containing solid support
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on L;
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein; and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant- containing solid support material S; and
  • Cell types suitable for use in each of the above assays are readily envisaged by those of ordinary skill in the art and may be derived from any known cell type or from any tissue comprising such cell types.
  • the biological sample may be washed with aqueous solution before the radiolabeled substrate is added.
  • Analytes suitable for use in this assay are readily envisioned to those of ordinary skill in the art.
  • Non-limiting examples of such analytes include cellular metabolites (such as isoprenoids, purines, pyrimidines, steroids, porphyrins, lipids, catecholamines, amino acids, carbohydrates, and nucleic acids); drugs; drug candidates; prodrugs; and chemical probes.
  • the radiolabeled moiety on said radiolabeled Cargo Moiety comprising said analyte is chosen from one or more radioactive isotope selected from 3 H, 125 1, 33 P, 14 C, 35 S, 45 Ca, 86 Rb, 75 Se, and 65 Co.
  • the above described process is carried out in the absence of the test compound, in the presence of a test compound, and optionally in the presence of each of different concentrations of test compound to determine a dose-response relationship.
  • the measured quantity of light energy emitted from said assay system in the presence and absence of test compound, or in the presence of various concentrations of test compound is a measure of the influence of test compound on the rate of cellular penetration by the analyte.
  • Test compounds suitable for use in the methods according to the invention include compounds that inhibit (or are under investigation for their ability to inhibit) the rate of penetration of the analyte through the cellular membrane.
  • Suitable test compounds are readily envisaged by those of ordinary skill in the art and include, but are not limited to, small molecules (such as small molecules undergoing structure-activity testing during drug discovery) and biologies (including but not limited to cellular components, antibodies, antigens, proteins, DNA, RNA, siRNA, recombinant proteins, tissues, genes, allergens, cells, blood components, blood, vaccines, viruses, inactivated viruses, hormones, substances that suppress or activate components of the immune system, and the like).
  • the measurement of the quantity of light energy emitted from the assay system is taken at a selected time point, wherein the selected time point is the time at which the assay system S-L(C-CaptureProtein) p is combined with the lysate optionally at one or more additional selected points in time thereafter.
  • a method of measuring the distribution of an analyte among one or more tissues or organs of an organism comprising:
  • R* is a radiolabeled Cargo Moiety comprising an analyte of interest
  • -A-X is a haloalkane wherein -A- is a -(C 2 -Cio)alkyl group which may be straight or branched, and X is Cl or Br;
  • S is a scintillant-containing solid support
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein; and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant-containing solid support material S; and
  • a method of measuring the distribution of an analyte among one or more tissues or organs of an organism comprising: Administering, in vivo, to an organism a Capture Ligand of the formula (R)-L 2 -A-X, wherein:
  • R is a non-radiolab el ed Cargo Moiety comprising an analyte of interest
  • -A-X is a haloalkane wherein -A- is a -(C 2 -Ci 0 )alkyl group which may be straight or branched, and X is Cl or Br;
  • S is a scintillant-containing solid support
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein; and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant-containing solid support material S; and
  • the administration can be carried out according to any method known to those of ordinary skill in the art, including, e.g., orally, intravenously, subcutaneously, and intraperitoneally.
  • the Capture Ligand is administered orally, intravenously, subcutaneously, or intraperitoneally.
  • Analytes suitable for use in this assay are readily envisioned to those of ordinary skill in the art.
  • Non-limiting examples of such analytes include cellular metabolites (such as isoprenoids, purines, pyrimidines, steroids, porphyrins, lipids, catecholamines, amino acids, carbohydrates, and nucleic acids); drugs; drug candidates; prodrugs; and chemical probes.
  • the radiolabeled moiety on the radiolabeled Capture Ligand is chosen from one or more radioactive isotope selected from 3 ⁇ 4 125 1, 33 P, 14 C, 35 S, 45 Ca, 86 Rb, 75 Se, and 65 Co.
  • a method of measuring the effect of one or more test compounds on the distribution of an analyte among one or more tissues or organs of an organism is carried out in the absence of the test compound, in the presence of a test compound, and optionally in the presence of each of different concentrations of test compound to determine a dose-response relationship.
  • concentrations of test compound is a measure of the influence of test compound on the distribution of the analyte among the one or more tissues or organs on the organism.
  • Test compounds suitable for use in these methods are described above.
  • each of the above methods is adapted for use in a competitive assay method according to the invention.
  • a method for quantitatively measuring the amount of a non-radiolab el ed Cargo Moiety comprising an analyte of interest in a sample comprising:
  • R is a Cargo Moiety comprising the analyte of interest
  • -A-X is a haloalkane wherein -A- is a -(C 2 -Cio)alkyl group which may be straight or branched, and X is Cl or Br; Combining said sample with a system comprising S-L(C-CaptureProtein) p , wherein:
  • S is a scintillant-containing solid support
  • L is a multivalent linker moiety
  • p is an integer of 1 or more up to the maximum number of available valences on L
  • CaptureProtein is a mutant halophilic bacterial hydrolase capture protein, and wherein each of a multiplicity of units of -L(C-CaptureProtein)p are covalently attached to each of a multiplicity of available polar functional groups on the surface of the scintillant- containing solid support material S;
  • radiolabeled Capture Ligand comprises a radiolabeled Cargo Moiety
  • the structural features of the Capture Ligand which binds to CaptureProtein in the radiolabeled Capture Ligand are preferably the same or substantially the same as the structural features of the Capture Ligand in the non-radiolabeled Capture Ligand, except for the presence of the radiolabel in the former, such that the rate of binding of
  • CaptureProtein to the Capture Ligand is substantially similar in both the radiolabeled and the non-radiolabeled species.
  • the quantity of radiolabeled Capture Ligand bound to the CaptureProtein will be proportional to the quantity of unlabeled Capture Ligand present in the sample.
  • the emitted light energy will be inversely proportional to the quantity of non-radiolabeled capture ligand.
  • This proportionality together with the use of a standard curve, can be used to measure the quantity of unknown non-radiolabeled Capture Ligand in the sample.
  • a standard curve is prepared in connection with the assaying of a particular sample. Suitable standard curves are prepared mixing varying known amounts of non-radiolab el ed Capture Ligand with constant amounts of radiolabeled Capture Ligand and with a fixed quantity of CaptureProtein coated solid supports in the system S-L(C-CaptureProtein)p. The quantity of light energy generated by excitation of the scintillant from the radiolabeled Capture Ligand that has bound to the CaptureProtein is measured for each sample containing a known amount of the non-radiolab el ed Capture Ligand.
  • a standard curve is prepared depicting the level of light energy measured per quantity of non-radiolab el ed Capture Ligand present. Then, when a particular sample containing an unknown amount of non-radiolab el ed Capture Ligand is assayed, the concentration of the non-radiolab el ed Capture Ligand in the sample may be determined from the standard curve once the level of light energy being emitted is measured.
  • radiolabeled Capture Ligand that is not bound to CaptureProtein are substantially too distant from scintillant to cause the emission of light energy.
  • the detected amount of light energy is a measure of the proportion of radiolabeled Capture Ligand that is actually bound to CaptureProtein. Consequently, and advantageously, there is no need to wash the solid support or otherwise remove the unbound radiolabeled Capture Ligand from the CaptureProtein; instead, the quantity of light energy may be measured with all of the components of the assay still present in the sample.
  • the time required to complete the assay is limited only by the CaotureProtein/Capture Ligand binding reaction rate of the system under investigation.
  • Example 1 S-L(C-HaloTag®)p, wherein S, L, C, and HaloTag® are as described above, were prepared in examples 1, 2, 3 A, and 3B as follows.
  • Example 1 S-L(C-HaloTag®)p, wherein S, L, C, and HaloTag® are as described above, were prepared in examples 1, 2, 3 A, and 3B as follows. Example 1.
  • the following provides an example of a process for the attachment of the linker- bioconjugation moiety produced by Example 1 to a YSi-SPA bead to produce an immobilized cyanobenzothiazole YSi-SPA bead 5.
  • Polylysine Coated Yttrium Silicate SPA beads (RPNQ0010, PerkinElmer, Inc. Boston, MA, EiSA) (1000 mg) were then added to the mixture and the tube was placed on a tube rotator overnight. The phases were separated using a centrifuge and the liquid was removed and replaced by 12 mL of PBS buffer. The beads were washed 4 times with l2mL PBS buffer. Finally the beads were stored in lOmL of PBS buffer to obtain a lg of Immobilized cyanobenzothiazole YSi-SPA beads 5 per mL.
  • the immobilized cyanobenzothiazole PVT-SPA beads 6 were prepared using the same protocol as for the preparation of the YSI-SPA beads 5 using PVT PEI coupled SPA Beads (RPNQ0097, PerkinElmer, Inc. Boston, MA, ETSA) as starting material.
  • HaloTag®- YSi-SPA beads 7 were equilibrated in assay buffer (HEPES, pH 7.5 (50 mM), NaCl (150 mM), IGEPAL CA640 (0.01 %)) and dispensed in 4 Eppendorf tubes (0.5 mg per tubes) and the residual buffer was removed.
  • Assay buffer HPES, pH 7.5 (50 mM), NaCl (150 mM), IGEPAL CA640 (0.01 %)
  • the four tubes were places on a tube rotator for 1 hat RT before the unbound fraction was removed and the beads washed 3 times with assay buffer. The remaining buffer was removed of all tubes and replaced by (10 pL, 0.5 nmol) of a 50 pM solution of HaloTag® TMR ligand (Promega Corp. Madison WI, ETSA) in assay buffer. The four tubes were places on a tube rotator for 1 h at RT before the aqueous fraction was removed and the beads washed 3 times with assay buffer. The fluorescence intensities were measured on a multi-mode plate reader PHERAstar plus (BMG Labtech). The resulting measurements are shown in FIG. 1. Example 7
  • the following example demonstrates the presence of active HaloTag® protein on the SPA beads and the ability of the SPA beads to emit a SPA signal when the coated HaloTag® reacts with a radiolabled probe. This example also allows the determination of the reactive HaloTag® density on the beads. Quantification of Active HaloTag® - Coupled to HaloTag®- YSi-SPA beads 7
  • the radiolabeled chloroalkane ligand 3 was used to quantify the amount of HaloTag® binding sites on the HaloTag®- YSi-SPA beads 7 by saturation binding.
  • the measurements were conducted in a buffer composed of HEPES, pH 7.5 (50 mM), NaCl (150 mM), IGEPAL CA640 (0.01 %) at RT.
  • An equal volume of a 3 (8.0 Ci/mmol) serial dilution in buffer at twice the final target concentration was added to 25 pl of buffer containing 50 pg of HaloTag®- YSi- SPA beads 7 in white clear-bottom plates.
  • HaloTag®- YSi-SPA beads 7 and 3 were allowed to incubate overnight at 4 °C, re-equilibrate to RT for lh, and the scintillation counts measured on a Perkin Elmer TopCount reader. The resulting measurements are shown in FIG. 2. All measurements were performed in duplicate. The minimum amount of 3 required to saturate the HaloTag®- YSi-SPA beads 7, equivalent to the number of active HaloTag® binding sites, was determined by the intercept of linear regressions fit to the rising and plateaued regions of the binding curve. Non-specific binding was assessed by two control binding conditions. In the first, the binding reactions were assessed in the presence of a 1000- fold excess of competitor compound 2.
  • the amount of 3 binding was assessed using the immobilized cyanobenzothiazole YSi-SPA beads 5 lacking the HaloTag® protein. Specific binding was calculated by subtracting the non-specific binding counts obtained with cyanobenzothiazole YSi-SPA beads 5 from the total counts measured using the HaloTag®- YSi- SPA beads 7 without excess of competitor compound 2. Saturation is observed at a concentration of 120 nM of compound 3. This correspond to 6 pmoles of compound 3 per well (FIG. 3). Each well contains 50 ug HaloTag®- YSi-SPA beads 7. The loading density was determined to be 120 pmoles of HaloTag® per mg of beads.
  • HaloTag® SPA beads can be sucessfully used to capture HaloTag® ligands form cell lysate.
  • a combination of cold and radioactively labels HaloTag® ligands can be used to asses capture efficiency.
  • Cell lysates were prepared from human dermal fibroblasts grown in T175 flasks. For each lysis condition (1% Triton X-100, 0.1% Triton X-100, 1% ddm, 0.1 %ddm , 1% CHAPS) the experiment was conducted as following. Cells in one T175 flask were washed twice with 20 mL PBS before addition of 6 ml of lysis buffer containing lx mammalian protease inhibitor cocktail (Promega, lOOx in ethanol). Lysis was allowed to proceed for 30 min at 4 °C before the lysate was collected and clarified by centrifugation at 1500 g for 5 min at 4 °C.
  • lx mammalian protease inhibitor cocktail Promega, lOOx in ethanol
  • HaloTag® biotin ligand Promega Corp. Madison WI, EISA
  • DMSO control DMSO control
  • Pre- incubated ligands were captured from the cell lysates by dispensing 25 pl of the lysates to white clear-bottom plates followed by addition of an equal volume buffer containing 50 pg HaloTag®- YSi-SPA beads 7. After brief mixing, capture was allowed to proceed for 2 h at RT.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un système de dosage comprenant une nouvelle combinaison de technologie de dosage de la scintillation par proximité (SPA) et de système de ligand de capture/protéine de capture et des procédés d'utilisation de celui-ci.
PCT/US2018/064650 2017-12-13 2018-12-10 Système de dosage de la scintillation par proximité comprenant une déshalogénase mutante WO2019118314A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762598079P 2017-12-13 2017-12-13
US62/598,079 2017-12-13

Publications (2)

Publication Number Publication Date
WO2019118314A2 true WO2019118314A2 (fr) 2019-06-20
WO2019118314A3 WO2019118314A3 (fr) 2020-03-26

Family

ID=66819476

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/064650 WO2019118314A2 (fr) 2017-12-13 2018-12-10 Système de dosage de la scintillation par proximité comprenant une déshalogénase mutante

Country Status (1)

Country Link
WO (1) WO2019118314A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225369A1 (fr) * 2022-05-19 2023-11-23 Duke University Composés, compositions et méthodes de pharmacologie spécifique à une cellule

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19533092A1 (de) * 1995-09-07 1997-03-13 Basf Ag Vorrichtung zur parallelisierten Zweiphotonen-Fluoreszenz-Korrelations-Spektroskopie (TPA-FCS) und deren Verwendung zum Wirkstoff-Screening
CA2476571A1 (fr) * 2002-02-28 2003-09-04 Intercell Ag Methode d'isolation de ligands
US7429472B2 (en) * 2003-01-31 2008-09-30 Promega Corporation Method of immobilizing a protein or molecule via a mutant dehalogenase that is bound to an immobilized dehalogenase substrate and linked directly or indirectly to the protein or molecule
EP1925940A1 (fr) * 2006-11-23 2008-05-28 INSERM (Institut National de la Santé et de la Recherche Médicale) VE-cadhérine circulante en tant que marqueur prédictif de la sensibilité ou de la résistance à un traitement anti-tumoral et procédé amélioré de détection de protéines solubles
CN104530415B (zh) * 2014-10-01 2017-09-01 厦门赛诺邦格生物科技股份有限公司 一种异官能化y型聚乙二醇衍生物、制备方法及其生物相关物质
CN114656483A (zh) * 2014-12-04 2022-06-24 细胞基因公司 生物分子共轭物

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225369A1 (fr) * 2022-05-19 2023-11-23 Duke University Composés, compositions et méthodes de pharmacologie spécifique à une cellule

Also Published As

Publication number Publication date
WO2019118314A3 (fr) 2020-03-26

Similar Documents

Publication Publication Date Title
Zhou et al. Aptamer-based biosensors for biomedical diagnostics
Mayer et al. Luminescent labels—more than just an alternative to radioisotopes?
Xie et al. Single-molecule observation of the catalytic subunit of cAMP-dependent protein kinase binding to an inhibitor peptide
Amaike et al. Recognition-driven chemical labeling of endogenous proteins in multi-molecular crowding in live cells
US20040161737A1 (en) Novel luminescent metal chelates and methods for their detection
CN101273096B (zh) 生物分子用标记色素、标记试剂盒以及生物分子的检测方法
EP1813948A1 (fr) Procédé de détection de substances cibles
KR20150093222A (ko) 생체활성 제제의 세포 표적 포획을 위한 조성물 및 방법
US20210041359A1 (en) Method for detecting biomaterial using linear upconversion fluorescent property
Tombelli et al. Aptamers biosensors for pharmaceutical compounds
CZ201520A3 (cs) Makromolekulární konjugáty pro vizualizaci a separaci proteinů a buněk
CN108893106B (zh) 检测高浓度γ-谷氨酰转肽酶的荧光探针及其制备方法
CN108698047A (zh) 电化学发光检测中的支链胺
Lou et al. Detection of adenine-rich ssDNA based on thymine-substituted tetraphenylethene with aggregation-induced emission characteristics
KR20070003827A (ko) 생체분자의 검출방법 및 이에 사용하는 표지색소와표지키트
Li et al. Drug screening strategies using metal-based luminescent probes
Ying et al. Purification of tetracysteine-tagged proteins by affinity chromatography using a non-fluorescent, photochemically stable bisarsenical affinity ligand
WO2019118314A2 (fr) Système de dosage de la scintillation par proximité comprenant une déshalogénase mutante
EP3137898B1 (fr) Capteur moléculaire fluorescent permettant de cibler des modifications affectant la surface de protéines et ses procédés d'utilisation
Liu et al. Protein modulation of electrochemical signals: application to immunobiosensing
JP3340715B2 (ja) 被検体、その相互作用又は反応動力学を定量的又は定性的に測定する方法及びサンプルキャリア
Huang et al. A graphene oxide-based multiplexed fluorescence assay for the detection of protein kinase activity in cell lysates and the evaluation of protein kinase inhibition
van der Wal et al. Bioorthogonally applicable fluorescence deactivation strategy for receptor kinetics study and theranostic pretargeting approaches
Cywinski et al. Cyclic GMP recognition using ratiometric QD-fluorophore conjugate nanosensors
Hennig et al. Cucurbituril-based sensors and assays

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18887720

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18887720

Country of ref document: EP

Kind code of ref document: A2