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WO1999064847A1 - Sondes electrochimiques aux fins de la detection d'interactions moleculaires et de la mise au point de medicaments - Google Patents

Sondes electrochimiques aux fins de la detection d'interactions moleculaires et de la mise au point de medicaments Download PDF

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Publication number
WO1999064847A1
WO1999064847A1 PCT/US1999/011848 US9911848W WO9964847A1 WO 1999064847 A1 WO1999064847 A1 WO 1999064847A1 US 9911848 W US9911848 W US 9911848W WO 9964847 A1 WO9964847 A1 WO 9964847A1
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WO
WIPO (PCT)
Prior art keywords
binding pair
biological binding
electrochemical
reaction chamber
biological
Prior art date
Application number
PCT/US1999/011848
Other languages
English (en)
Inventor
Thomas W. Welch
Original Assignee
Xanthon, Inc.
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 Xanthon, Inc. filed Critical Xanthon, Inc.
Priority to AU43185/99A priority Critical patent/AU4318599A/en
Publication of WO1999064847A1 publication Critical patent/WO1999064847A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • the present invention relates to methods and apparatus for performing electrochemical analyses that depend on specific binding between members of a biological binding pair.
  • the invention provides an electrochemical analysis apparatus for performing amperometric, coulometric and potentiometric or voltammetric analyses for detecting specific binding between a first member of a biological binding pair in solution with a second member of a biological binding pair that is electrochemically labeled.
  • the second member of the biological binding pair is linked to an electrochemical catalyst, preferably an enzyme and most preferably a redox enzyme, in the presence of a substrate for the electrochemical catalyst.
  • apparatus for performing voltammetric analyses of current produced over a range of applied voltages in the presence of electrochemically labeled biologically active binding species are provided by the invention.
  • the invention also provides methods for performing high throughput screening assays for detecting inhibition of specific binding between the members of the biological binding pair for use in drug development, biochemical analysis and protein purification assays.
  • surrogate ligands for the biological binding species of the invention and methods for identifying surrogate ligands from complex biological mixtures including biologically-derived fluids.
  • U.S. Patent No. 5,198,367, issued March 30, 1993 to Aizawa et al. disclosed an amperometric immunoassay.
  • U.S. Patent No. 5,346,832, issued September 13, 1994 to Aizawa et al. disclosed an amperometric immunoassay.
  • Embree et al., 1990, J. Chromatogr. 526: 439-446 describes electrochemical detection of an electrochemically active derivative of digoxin.
  • Devlin et al., 1990, Science 249: 404-406 describes random peptide libraries.
  • Hirai & Varmus, 1990, Molec. Cell. Biol. K): 1307-1318 describes site-directed mutagenesis of src homology domain 3.
  • Lam et al., 1991 , Nature (London) 354: 82-84 describes random peptide libraries. Burrows et al., 1991 , Eur. J. Biochem. 202: 543-549 describes direct electrochemistry of proteins.
  • Wanatabe-Fukunaga et al., 1992, Nature (London) 356: 314-317 describes fas as an apoptotic factor.
  • Phizicky & Fields 1995, Microbiol Rev 59 94-123 describe methods for detecting and analyzing protein-protein interactions.
  • the present invention provides methods and apparatus for performing electrochemical analysis for detecting binding between a biological binding pair. These methods and apparatus are useful for performing direct binding and competition binding experiments for detecting and analyzing compounds capable of inhibiting binding between the biological binding pair, thereby identifying compounds capable of interacting with biologically-active portions of the species comprising the biological binding pair. The methods of the invention are useful for performing rapid, high throughput screening of biologically active compounds for use as drugs that interact with one of the members of the biological binding pair and thereby interfere with or affect its biological function.
  • the invention provides an apparatus for performing an electrochemical assay for detecting binding between members of a biological binding pair.
  • the apparatus comprises a first electrode, the electrode comprising a conducting or semiconducting material.
  • the apparatus also comprises a second, reference electrode, and a third, auxiliary electrode.
  • Each of the electrodes is electrically connected to a potentiostat, and the electrodes are all in electrical contact with a reaction chamber containing a solution of an electrochemically reactive liquid.
  • This electrochemically- reactive solution contains a first member of a biological binding pair.
  • the solution further comprises a second member of the biological binding pair, wherein said second member is electrochemically labeled with a chemical species capable of participating in a reduction or oxidation reaction with the electrode under conditions whereby an electrical potential is applied to the electrodes.
  • a current is produced in the apparatus when an electrical potential is applied to the electrodes; this current is reduced upon binding of the second member of the biological binding pair to the first member of the biological binding pair.
  • either the first or the second member of the biological binding pair is present in the reaction chamber of the apparatus in concentration excess to the other member of the pair.
  • the first member of the biological binding pair is linked to a particle, most preferably a bead.
  • the electrochemical assay is a voltammetric, amperometric or coulometric assay, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.
  • the first member of the biological binding pair is a receptor protein or ligand binding fragment thereof.
  • the first member of the biological binding pair is an antibody protein or antigen binding fragment thereof
  • the first member of the biological binding pair is a first protein or fragment thereof that specifically binds to a second protein
  • the second member of the biological bingmg pair is a hgand, and antigen or a protein that binds to the first member of the biological binding pair
  • the second member of the biological binding pair is a surrogate hgand for the first member of the biological binding pair, having a binding affinity of from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar
  • the apparatus of the invention also includes embodiments wherein the apparatus further comprises a multiplicity of each of the electrodes and reaction chambers of the invention, wherein each reaction chamber contains an electrolyte and is m electrochemical contact with one each of the three electrodes among the multiplicity of electrodes in the apparatus, and each of the electrodes in electrochemical contact with each reaction chamber is elect ⁇ cally connected to a potentiostat
  • the apparatus of the invention also includes embodiments wherein the apparatus further comprises a multiplicity of each of the first and second members of the biological binding pair, wherein cun-ent is produced in the apparatus by participation in a reduction/oxidation reaction at the surface of the first electrode by each of the electrochemically labeled second members of the biological binding pairs at a particular elect ⁇ cal potential applied between the electrodes, wherein the current produced at each particular elect ⁇ cal potential is reduced upon binding of the second member of the biological binding pair to the first member of the biological binding pair
  • the second member of the biological binding pair is electrochemically labeled with an organic or inorganic complex comp ⁇ sing a transition metal cation
  • the electrochemical label is a ruthenium complex, preferably a pentaamineruthenium compound such as ⁇ Ru(NH 3 ) 5 Cl ⁇ Cl, Ru(NH 3 ) 6 ⁇ or Ru(NH 3 ) 5 (H 2 O) 2 ⁇ .
  • the electrochemical label is an iron complex, most preferably
  • the invention also provides a kit comprising at least one second member of the biological binding pair, preferably comprising a surrogate ligand having binding specificity for the first member of the biological binding pair characterized by a dissociation constant (K d ) of from about from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar ( ⁇ M), and most preferably from about 1 OnM to about 10 ⁇ M, thus comprising an electrochemical probe.
  • K d dissociation constant
  • said second member of the biological binding pair is provided in an electrochemically labeled embodiment.
  • said second member of the biological binding pair is provided with reagents including an electrochemical label for preparing the electrochemically labeled embodiment by the user.
  • the kit also preferably provides a first member of the biological binding pair specific for the second member of the biological binding pair provided therewith. Additional and optional components of the kits of the invention include buffers, reagents and electrodes as described herein.
  • Methods of using the apparatus of the invention are also provided.
  • a method for detecting binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus according to this aspect of the invention is provided.
  • the method comprises the steps of: a) providing a first reaction chamber in electrochemical contact with each of the electrodes of an apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, wherein each of the electrodes is electrically connected to a potentiostat; wherein the first reaction chamber contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, and wherein the second reaction chamber contains an electrochemically-labeled second member of the biological binding pair and an unrelated species that does not specifically bind to the second member of the biological binding pair, or the first member of the biological binding pair and an unrelated, electrochemically-labeled species that does not bind to the first member of the biological binding pair
  • the method further comp ⁇ ses the steps of.
  • the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulometric assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry
  • a method for identifying an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus of this aspect of the invention the method composing the steps of a) providing a first reaction chamber m electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, wherein each of the electrodes being elect ⁇ cally connected to a potentiostat; wherein each of the reaction chambers contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, and wherein the second reaction chamber further contains a compound to be tested to determine its ability to inhibit binding of the second member of the biological binding pair to the first member of the biological binding pair
  • the method further comprises the steps of: b) performing an electrochemical assay in each of the first and second reaction chamber
  • the method comprises the steps of a) providing a reaction chamber m electrochemical contact with each of the electrodes of the apparatus according to the invention, wherein each of the electrodes is electrically connected to a potentiostat and wherein the reaction chamber contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, b) performing an electrochemical assay in the reaction chamber to produce a cuoent in the electrodes of the apparatus in the presence and absence of an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair; and c) comparing the cuoent produced in the electrochemical assay in the presence of the inhibitor to the cuoent produced in the electrochemical assay in the absence of the inhibitor; wherein an inhibitor of
  • Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical cuoent produced in the reaction chamber when an electrical potential is applied between the electrodes in the reaction chamber.
  • the level and amount of cuoent produced in the electrochemical assay in the presence of an inhibitor of binding between the members of the biological binding pair is compared with the level and amount of cuoent produced in the electrochemical assay in the reaction chamber in the absence of the inhibitor of binding of the first and second members of the biological binding pair.
  • the inhibitor is added to the reaction chamber after the second member of the biological binding pair is added to the reaction chamber.
  • the inhibitor is added to the reaction chamber before the second member of the biological binding pair is added to the reaction chamber.
  • the inhibitor is added to the reaction chamber together with the second member of the biological binding pair.
  • the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulometric assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.
  • a method for screening a complex chemical mixture for an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus of this aspect of the invention comp ⁇ sing the steps of a) providing a first reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, wherein each of the electrodes being electncally connected to a potentiostat, wherein each of the reaction chambers contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, and wherein the second reaction chamber further contains a portion of the complex mixture comp ⁇ smg an inhibitor of binding of the second member of the biological binding pair, the method further comp ⁇ sing the steps of b) performing an electrochemical assay in each of the first
  • the method comp ⁇ ses the steps of a) providing a reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, wherein each of the electrodes is elect ⁇ cally connected to a potentiostat, and wherein the reaction chamber contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, b) performing an electrochemical assay in the reaction chamber to produce a cuoent in the electrodes of the apparatus in the presence and absence of a complex mixture comprising an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair, and c) comparing the cuoent produced in the electrochemical assay in the presence of the complex mixtuie to the cu
  • the complex mixture comprising the inhibitor is added to the reaction chamber after the second member of the biological binding pair is added to the reaction chamber.
  • the complex mixture comp ⁇ sing the inhibitor is added to the reaction chamber before the second member of the biological binding pair is added to the reaction chamber.
  • the complex mixture comp ⁇ sing the inhibitor is added to the reaction chamber together with the second member of the biological binding pair
  • the electrochemical assays performed using the methods of the invention are voltammet ⁇ c, amperometric or coulomet ⁇ c assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry
  • the method is used to isolate and identify an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair
  • the method comprises the additional steps of chemically fractionating the complex mixture having an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair, to produce fractionated submixtures, and performing the steps of the method on each of the fractionated submixtures to identify the submixtures that have an inhibitor of binding of the biological binding pair
  • the chemical fractionation includes chemical, biochemical, physical, and immunological methods for fractionation of chemical or biochemical species of inhibitor
  • the second member of a biological binding pair is an electrochemically labeled suoogate ligand characte ⁇ zed by a dissociation constant (K d ) for the first member of the biological binding pair of from about from about 50 picomolar (pM) to about 0 5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar ( ⁇ M), and most preferably from about lOnM to about 10 ⁇ M
  • the apparatus comprises a first electrode, wherein the electrode comp ⁇ ses a conducting or semiconducting surface, a second, reference electrode comp ⁇ sing a conducting metal in contact with an aqueous electrolyte solution, and a third, auxiliary electrode comp ⁇ smg a conducting metal, wherein each of the electrodes is elect ⁇ cally connected to a potentiostat
  • the apparatus further comp ⁇ ses a reaction chamber containing a solution of an electrolyte, wherein each of the electrodes is in electrochemical contact therewith
  • the solution further contains a first member of a biological binding pair, and a second member of the biological binding pair, wherein said second member is bound to an electrochemical catalyst capable of participating in a reduction/oxidation reaction at the surface of the first electrode in the presence of a substrate for the electrochemical catalyst under conditions whereby an elect ⁇ cal potential is applied to the electrodes, and wherein the electrochemical catalyst
  • either the first or the second member of the biological binding pair is present in the reaction chamber of the apparatus in concentration excess to the other member of the pair
  • the first member of the biological binding pair is linked to a particle, most preferably a bead
  • the electrochemical assay is a voltammet ⁇ c, amperomet ⁇ c or coulomet ⁇ c assay, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry
  • the first member of the biological binding pair is a receptor protein or ligand binding fragment thereof
  • the first member of the biological binding pair is an antibody protein or antigen binding fragment thereof
  • the first member of the biological binding pair is a first protein or fragment thereof that specifically binds to a second protein
  • the second member of the biological bingmg pair is a ligand, and antigen or a protein that binds to the first member of the biological binding pair
  • first and second members of the biological binding pair e g , receptor/hgand, antigen/antibody, etc
  • the second member of the biological binding pair is a suoogate ligand for the first member of the biological binding pair, having an affinity of binding of from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar ( ⁇ M), and most preferably from about l OnM to about 10 ⁇ M
  • said suoogate ligand is electrochemically labeled, more preferably with a ruthenium compound
  • the electrochemical catalvst bound to the second member of the biological binding pair is an enzyme, most preferably horse radish peroxidase
  • the substrate of the electrochemical catalyst produces a detectable product upon undergoing an oxidation/reduction reaction with the electrochemical catalyst at the surface of the first electrode of the apparatus, most preferably a colored product
  • the apparatus of this aspect of the invention also includes embodiments wherein the apparatus further comp ⁇ ses a multiplicity of each of the electrodes and reaction chambers of the invention wherein each reaction chamber contains an electrolyte and is m electrochemical contact with one each of the three electrodes among the multiplicity of electrodes in the apparatus, and each of the electrodes in electrochemical contact with each reaction chamber is elect ⁇ cally connected to a potentiostat
  • the apparatus of this aspect of the invention also includes embodiments wherein the apparatus further comp ⁇ ses a multiplicity of each of the first and second members of the biological binding pair, wherein cuoent is produced in the apparatus by participation in a reduction/oxidation reaction at the surface of the first electrode by each of the electrochemically labeled second members of the biological binding pairs at a particular electrical potential applied between the electrodes, wherein the cuoent produced at each particular elect ⁇ cal potential is reduced upon binding of the second member of the biological binding pair to the first member of the biological binding pair
  • the second member of the biological binding pair is labeled with an electrochemical catalyst.
  • the electrochemical catalyst is an enzyme, most preferably a redox enzyme capable of catalysis of its substrate to product by an oxidation/reduction mechanism wherein either functional groups on the enzyme or bound cofactors are involved in the oxidation/reduction cycle.
  • the electrochemical catalyst is a peroxidase, for example horse radish peroxidase.
  • said cofactor is provided in the reaction chamber of the apparatus.
  • the apparatus of the invention comprises a second member of the biological binding pair chemically linked with a species, preferably an enzyme, that is capable of being oxidized or reduced at the surface of the first electrode and to participate in cyclic oxidation/reduction reactions at the surface of the electrode m the presence of a third species present m the solution, in embodiments wherein the electrochemical catalyst is an enzyme, the third species is a substrate for the enzyme. As a consequence of these reactions, an observable cuoent is produced in the apparatus of the invention.
  • a species preferably an enzyme
  • This third species cannot be directly oxidized or reduced at the surface of the first electrode
  • specific binding interactions between the members of the biological binding pair are detected by observation of an electrical cuoent.
  • Said elect ⁇ cal cuoent is produced at an electrode potential sufficient to activate (oxidize or reduce) the electrochemical catalyst attached to the second member of the biological binding pair
  • the electrochemical catalyst is reduced by (oxidized by) the first electrode, thereby activating the catalyst for its substrate.
  • the electrode potential permits cycles of oxidation/reduction of the electrochemical catalyst pair, thereby producing a cuoent related to catalysis of the substrate by the electrochemical catalyst.
  • the amount of cuoent produced by specific binding of the members of the biological binding pair is compared to the amount of cuoent produced before addition of the first member of the biological binding pair, or to the amount of cuoent produced upon addition of a known non-bmding first member (thereby providing a negative control).
  • Specificity of binding is determined by compa ⁇ son of the cuoent to that generated in the presence of a known inhibitor of binding. Additional comparisons of the extent, capacity or rate of binding inhibition, activation or competition can be determined by analysis of the extent of produced cuoent m the presence of putative inhibitors, competitors, activators or drug lead candidates, wherein specific details of the performance of such compansons will be understood by those with skill in the art and are more fully disclosed below.
  • the invention also provides a kit comprising at least one second member of the biological binding pair, preferably comp ⁇ sing a suoogate ligand having binding specificity for the first member of the biological binding pair characte ⁇ zed by a dissociation constant (K d ) of from about from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar ( ⁇ M), and most preferably from about 1 OnM to about 10 wM, thus comprising an electrochemical probe
  • said second member of the biological binding pair is provided in an embodiment wherein it is linked to an electrochemical catalyst
  • said second member of the biological binding pair is provided with reagents including an electrochemical catalyst label for preparing the electrochemical catalyst-linked embodiment by the user
  • the kit also provides a first member of the biological binding pair specific for the second member of the biological binding pair provided therewith.
  • kits of the invention include buffers, reagents, substrates for the electrochemical catalyst and electrodes as described herein.
  • Methods of using this embodiment of the apparatus of the invention are also provided.
  • a method for detecting binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus according to this aspect of the invention is provided.
  • the method comp ⁇ ses the steps of: a) providing a first reaction chamber m electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, each of the electrodes of the apparatus being elect ⁇ cally connected to a potentiostat; wherein the each of the reaction chambers contains a substrate for the electrochemical catalyst and wherein the first reaction chamber comprises a first member of the biological binding pair and a second member of the biological binding pair that specifically binds to the first member of the biological binding pair, bound to an electrochemical catalyst, and wherein the second reaction chamber contains a second member of the biological binding pair that specifically binds to the first member of the biological binding pair, bound to an electrochemical catalyst, and a chemical species that does not specifically bind to the second member of the biological binding pair, or vv herein the second reaction chamber contains a first member of a biological binding pair and a chemical species that does not specifically bind to the second member of the biological binding pair,
  • the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a smaller amount of the detectable product in the first reaction chamber than is produced in the second reaction chamber
  • the method comp ⁇ ses the steps of a) providing a reaction chamber in electrochemical contact with each of the electrodes of an apparatus according to the invention, and wherein each of the electrodes is elect ⁇ cally connected to a potentiostat, and wherein the reaction chamber contains an electrochemical catalyst-labeled second member of the biological binding pair that specifically binds to a first member of the biological binding pair and a substrate for the electrochemical catalyst, and b) performing a
  • the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected bv the production of a smaller amount of the detectable product in the second electrochemical assay than is produced m the first electrochemical assay
  • the electrochemical assays performed using the methods of the invention are voltammet ⁇ c, amperometric or coulomet ⁇ c assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry
  • a method for identifying an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus according to the invention comp ⁇ ses the steps of a) providing a first reaction chambei in electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, each of the electrodes of the apparatus being elect ⁇ cally connected to a potentiostat, wherein each of the reaction chambers contains a first member of a biological binding pair, a second member of the biological binding pair that specifically binds to the first member of the biological binding pair, bound to an electrochemical catalyst, and a substrate for the electrochemical catalyst, and wherein the second reaction chamber further contains an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair, the method further comp ⁇ sing the steps of b) performing an electrochemical chambei in electrochemical contact with each of the electrodes of the apparatus of the invention,
  • the second member of the biological binding pair is a suoogate ligand for the first member of the biological binding pair
  • the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein inhibition of binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a larger amount of the detectable product in the second reaction chamber than is produced in the first reaction chamber
  • the method comprises the steps of a) providing a reaction chamber in electrochemical contact with each of the electrodes of the apparatus according to this aspect of the invention, and wherein each of the electrodes is elect ⁇ cally connected to a potentiostat and wherein the reaction chamber contains a first member of a biological binding pair and an electrochemical catalyst-labeled second member of the biological binding pair that specifically binds to a first member of the biological binding pair and a substrate for the electrochemical catalyst, and b) performing an electrochemical assay in the reaction chamber to produce a cuoent in the electrodes of the apparatus in the presence and absence of an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair, and c) comparing the cuoent produced in the electrochemical assay in the presence of the inhibitor to the cu
  • the inhibitor is added to the reaction chamber after the second member of the biological binding pair is added to the reaction chamber. In another prefeoed embodiment, the inhibitor is added to the reaction chamber before the second member of the biological binding pair is added to the reaction chamber. In yet another prefeoed embodiment, the inhibitor is added to the reaction chamber together with the second member of the biological binding pair
  • the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a larger amount of the detectable product in the electrochemical assay performed in the presence of the inhibitor than is produced in the electrochemical assay performed m the absence of the inhibitor
  • the electrochemical assays performed using the methods of the invention are voltammet ⁇ c, amperometric or coulomet ⁇ c assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry
  • a third embodiment of the methods of this aspect of the invention is provided a method for screening a complex chemical mixture for an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus of the invention.
  • These methods comprise the steps of: a) providing a first reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, each of the electrodes being electrically connected to a potentiostat; wherein each of the reaction chambers contains a first member of a biological binding pair, a second member of the biological binding pair that specifically binds to the first member of the biological binding pair, bound to an electrochemical catalyst, and a substrate for the electrochemical catalyst, and wherein the second reaction chamber further contains a portion of the complex mixture comprising an inhibitor of binding of the second member of the biological binding pair; the method further comprising the steps of: b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of this aspect of the invention to produce a cuoent in the electrodes of the apparatus; and c) comparing the cuoent produced in the electrochemical assay in the first reaction chamber to the cuoent produced in the electrochemical assay in
  • Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical cuoent produced in each reaction chamber when an electrical potential is applied between the electrodes in the chamber.
  • the level and amount of cuoent produced by specific binding of the first and second members of the biological binding pair in the first reaction chamber is compared with the level and amount of cuoent produced in the second reaction chamber in the presence of a complex chemical mixture comprising an inhibitor of specific binding.
  • the second member of the biological binding pair is a suoogate ligand for the first member of the biological binding pair
  • the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein inhibition of binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a larger amount of the detectable product in the second reaction chamber than is produced m the first reaction chamber
  • a method for screening a complex chemical mixture for an inhibitor of binding of a second member of a biological binding pair that is labeled with an electrochemical catalyst with a first member of a biological binding pair using an apparatus comp ⁇ sing the steps of a) providing a reaction chamber in electrochemical contact with each of the electrodes of the apparatus wherein each of the electrodes is electrically connected to a potentiostat, and wherein the reaction chamber contains a first member of a biological binding pair and an electrochemical catalyst-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, and a substrate for the electrochemical catalyst, b) performing an electrochemical assay in the reaction chamber to produce a cuoent in the electrodes of the apparatus in the presence and absence of a complex mixture comp ⁇ sing an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair, and c) compa ⁇ ng the cuoent produced
  • Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical cuoent produced in the reaction chamber when an elect ⁇ cal potential is applied between the electrodes in the reaction chamber.
  • the level and amount of cuoent produced in a first electrochemical assay in the presence of a complex chemical mixture comprising an inhibitor of specific binding between the members of the biological binding pair is compared with the level and amount of cuoent produced in a second electrochemical assay in the reaction chamber in the absence of a complex chemical mixture comprising an inhibitor of specific binding of the first and second members of the biological binding pair
  • the second member of the biological binding pair is a suoogate ligand for the first member of the biological binding pair
  • the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein inhibition of binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a larger amount of the detectable product in the electrochemical assay m the presence of a complex mixture comp ⁇ sing an inhibitor of specific binding than is produced in the electrochemical assay in the absence of the complex mixture comp ⁇ sing an inhibitor of specific binding between the members of the biological binding pair
  • the complex mixture comprising the inhibitor is added to the reaction chamber after the second member of the biological binding pair is added to the reaction chamber In another prefeoed embodiment, the complex mixture comp ⁇ sing the inhibitor is added to the reaction chamber before the second member of the biological binding pair is added to the reaction chamber In yet another prefeoed embodiment, the complex mixture comprising the inhibitor is added to the reaction chamber together with the second member of the biological binding pair
  • the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulomet ⁇ c assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry
  • the method is used to isolate and identify an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair
  • the method comp ⁇ ses the additional steps of d) chemically fractionating the complex mixture having an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair, to produce fractionated submixtures, and e) performing the steps of the method on each of the fractionated submixtures to identify the submixtures that have an inhibitor of binding of the biological binding pair
  • the chemical fractionation includes chemical, biochemical, physical, and immunological methods for fractionation of chemical or biochemical species of inhibitor
  • the second member of a biological binding pair is an electrochemically labeled suoogate ligand characterized by a dissociation constant (K d ) for the first member of the biological binding pair of from about fiom about 50 picomolar (pM) to about 0 5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolai ( ⁇ M), and most preferably from about 1 OnM to about 10 ⁇ M
  • an electron donor species contained in the reaction chambers of the apparatus of the invention wherein the electron donor species is oxidized with transfer of an electron to the electrochemical label at the surface of the first electrode
  • oxidation/reduction of the electrochemical label at the surface of the first electrode is accompanied by the production of a detectable amount of chemiluminescence that is diminished upon binding of the second member of the biological binding pair with the first member of the biological binding pair
  • Figure 1 is a schematic diagram illustrating the principle of diffusion limits on the rate of interfacial electrochemistry between an electrode surface and an electrochemically active species in solution.
  • Figure 2 is a graph showing cyclic voltammetric analysis of an electrochemically labeled peptide under the conditions described in Example 2.
  • Figure 3 is a graph showing the results of the experiment described in Example 3 and illustrating the decrease in collectable, Faradaic cuoent upon binding of a small, electrochemically labeled, biotinylated peptide to neutravidin.
  • Figure 4 is a graph showing the determination of the effective diffusion coefficent of a small, electochemically labeled, biotinylated peptide free in solution, in the presence of biotin-saturated neutravidin, and bound to a stoichiometric excess of neutravidin as described in Example 3.
  • Figure 5 is a graph illustrating the increase in collectable, Faradaic cuoent observed when a small electrochemically labeled species is displaced from a larger species by competitive binding of an unlabeled species as described in Example 4.
  • Figure 6 is a graph depicting the changes in peak anodic cuoent as a function of added competitor under the conditions described in Example 4.
  • the present invention provides apparatus and methods for detecting specific interactions, particularly including binding interactions, between members of a biological binding pair.
  • biological binding pair is intended to encompass any two biologically-derived or isolated molecules, or any chemical species that specifically interact therewith, that specifically bind with a chemical affinity measured by a dissociation constant of at least 50mM.
  • a biological binding pair proteins that interact with other proteins, including fragments thereof; proteins and peptides; proteins and ligands; proteins and co-factors; proteins and allosteric or cooperative regulators; proteins and nucleic acids; proteins and carbohydrates; antigens and antibodies; lipids, including fatty acids, triglycerides and polar lipids that interact with proteins or peptides; receptors and ligands, particularly cytokines; virus-receptor pairs; enzymes and substrates; and enzymes and inhibitors Also encompassed with this definition are any chemical compound or mixture that interacts with at least one member of a biological binding pair
  • the members of the biological binding pairs of the invention are intended to encompass molecules that are naturally-occur ⁇ ng, synthetic, or prepared by recombinant genetic means or biochemical isolation and extraction means Synthetic embodiments of a member of a biological binding pair will be understood to typically share structural simila ⁇ ty with at least a portion of any naturally-occur ⁇ ng analogue which they resemble
  • the apparatus of the invention comp ⁇ ses a first, conductive or semiconductive electrode
  • mate ⁇ als useful forprepa ⁇ ng the conductive or semiconductive electrodes of the invention include metallically-impregnated glass, such as tm-doped mdium oxide or fluorine-doped tin oxide glass, gold, carbon or platinum
  • the apparatus also includes an auxiliary electrode, comprised of any conducting metal, including for example, platinum (Pt)-w ⁇ re auxiliary electrodes, and a reference electrode, including silver wire electrodes, silver/sih er chloride (Ag/AgCl) reference electrodes, saturated calomel, and others know to those with skill m the art
  • the apparatus comp ⁇ ses one or a multiplicity of reaction chambers, each having one of each of these electrodes, and each of the electrodes being elect ⁇ cally connected to a potentiostat or other device that applies and maintains a constant potential under varying cuoent conditions
  • the term "electrically connected" is intended to mean, inter
  • Inorganic complexes such as Ru 2" 3+ -amme complexes, feoocenes, and osmium- or cobalt-polypy ⁇ dyl complexes are attached to the peptide via histidine or cysteine residues or at the amino terminus.
  • Redox-active organic molecules such as paraquat derivatives and quinones, are attached to peptides by conjugating the redox-active organic moiety via lysine or cysteine residues or at the amino terminus.
  • organic labels or the organic ligands of transition-metal complexes can include reactive substituents that allow direct incorporation of these complexes in the context of automated biopolymer synthesis.
  • prefeoed compounds comprising the second member of the biological binding pair are "suoogate" ligands to the first member of the specific binding pair.
  • the term "suoogate ligand" is intended to define a set of biologically-active compounds that specifically bind to any defined target comp ⁇ sing a first member of a biological binding pair
  • this definition is intended to encompass a variety of ligands of a target, particularly a target protein, comprising the first member of a biological binding pair, including a natural hgand.
  • the suoogate ligands of the invention preferably comprise those ligands that specifically bind to the target protein with a chemical affinity measured by a dissociation constant (K d ) of from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar ( ⁇ M), and most preferably from about lOnM to about 10 ⁇ M.
  • K d dissociation constant
  • suoogate ligands are prefeoed because they bind with sufficient affinity that the concentration of the electrochemical label at the surface of the first electrode of the apparatus of the invention is sufficient to produce an expenmentally-detectable cuoent, while at the same time the binding affinity is weak enough to be displaced by competitors and inhibitors at concentrations of these compounds that are economical and can be expe ⁇ mentally achieved.
  • Suoogate ligands therefore provide both the required degree of specificity and the required degree of easy dissociabihty to enable the methods and apparatus of the invention to detect binding inhibition by competitor species.
  • second members of the biological binding pair that are electrochemically-labeled suoogate ligands include, but are not limited to, peptides, nucleic acids, carbohydrates, and small molecules.
  • the peptides are preferably obtained from phage-displayed combinatorial peptide libraries (see co-owned and co-pending U.S. patent application, Serial No. 08/740,671, filed October 21, 1996, incorporated by reference herein) as well as other means, such as synthetic peptides prepared on pins or beads.
  • Peptides and proteins comprising the electrochemical probes and targets of the apparatus and methods of this invention can be prepared by synthetic methods, including solid phase peptide synthesis, biochemical isolation and modification techniques including partial proteolysis, and by recombinant genetic methods understood by those with skill in the art (see Sambrook et al, 1990, Molecular Cloning, 2d ed, Cold Spring Harbor Laboratory Press, N.Y.).
  • An example of a useful electrochemical labeling method is addition of a ruthenium group (Ru(NH 1 ) 5 (OH : ) 2 ⁇ ) to histidine residues within a peptide sequence.
  • Another example of a useful electrochemical labeling method is addition of a feoocene carboxylic acid to side chains of lysine residues via the use of selective deprotection strategies during automated peptide synthesis.
  • Feoocenes are also attractive labels because addition of substituents to the organic cyclopentadienyl- ligands can alter the redox potential by several hundred millivolts, thereby allowing observation of binding of multiple labels in one assay.
  • electrochemical labels can be added to the amino- or carboxyl termini of peptides or protein fragments post-synthetically, or to the reactive side chain thiol groups of a cysteine residue, the hydroxyl group of a serine or threonine residue (or on a carbohydrate moiety), or the amino group of a lysine residue of the peptide.
  • Fmoc derivatives of "unnatural" amino acids such as D- amino acids or amino acid analogues such as ⁇ -aminocaproic acid
  • cobalt or osmium complexes with polypyridyl ligands possessing carboxylic acid substituents can be coupled to the ⁇ -amine of lysine using standard peptide chemistry.
  • Table I Targets for which binding peptides have been identified from combinatorial libraries
  • Urokinase receptor 19 bFGF-R integrin Ilb/IIIa/avB 1 20-23
  • Hsc70 24 tissue factor Vila atrial naturiuretic peptide A receptor fibronectin 25
  • CD 1 -B2M complex 27 tissue-type plasminogen activator 28 core antigen of Hepatitis B virus 29
  • HIV-1 nucleocapsid protein NCp7 30 erythropoietin receptor 31 trypsin 32 chymotrypsin 33 interleukin- 1 receptor 34
  • nucleic acids e.g., RNA and DNA species, including poly- and oligonucleofides
  • aptamers can be electrochemically-labeled with a labeling group at either the 3 ' or 5' termini, or modified nucleotide triphosphate that binds an electrochemical labeling group can be incorporated into oligonucleofides by non-discriminating RNA or DNA polymerases during the in vitro generation of the aptamer.
  • cyclic AMP can be electrochemically-labeled without diminishing binding to protein kinase A, thereby providing a biological binding pair for electrochemical analysis of compounds that affect cAMP binding to protein kinase A.
  • Solutions of electrochemically-reactive liquids useful in the apparatus of the invention include any electrolyte solution at physiologically-relevant ionic strength (equivalent to about 0.15M NaCl) and neutral pH.
  • electrolyte solutions useful with the apparatus of the invention include phosphate buffered saline (PBS), HEPES buffered solutions, and sodium bicarbonate buffered ion solutions.
  • PBS phosphate buffered saline
  • HEPES buffered solutions sodium bicarbonate buffered ion solutions.
  • an electron donor species contained in the reaction chambers of the apparatus of the invention wherein the electron donor species is oxidized with transfer of an electron to the electrochemical label at the surface of the first electrode.
  • oxidation/reduction of the electrochemical label at the surface of the first electrode is accompanied by the production of detectable amount of chemilummescence that is diminished upon binding of the second member of the biological binding pair with the first member of the biological binding pair
  • the term "electron donor species” is intended to encompass any chemical species having a readily-oxidized electron pair, including but not limited to amines (such as t ⁇ ethylamine, tnpropylamme, t ⁇ ethanolamine), phosphmes, arsines and the like
  • the chemilummescence produced by oxidation/reduction of the electrochemical label in the presence of the electron donor species has a wavelength in the visible portion of the electromagnetic spectrum, although the invention encompasses detectable chemilummescence at any wavelength
  • the term "electrochemical assay” is intended to encompass any assay performed m an apparatus of the invention, wherein the oxidation/reduction of an electrochemical label attached to a second member of a biological binding pair is achieved at a first electrode on the invention upon application of an electric potential between the electrodes
  • the electrochemical assays of the invention include but are not limited a voltammetric, amperomet ⁇ c or coulomet ⁇ c assay, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry
  • high-throughput screens of natural product and combinatorial chemical libraries for antagonists of protein-protein interactions Such low molecular weight chemical antagonists of specific protem-protein interactions are of value to the pharmaceutical industry as potential drug leads for developing therapeutic agents
  • each of the reaction chambers or microtitre sample wells in a representative experiment contain discrete combinatorial compounds or purified natural products (such as polyketides or fermentation broth components). After incubating the compounds in the presence of the electrode, voltammetric analysis of the cuoent produced in the reaction chamber is performed.
  • results of these analyses are compared for wells containing the electrochemically-labeled suoogate ligand in the presence and absence of the compound or mixture of compounds to be tested.
  • Compounds that inhibit the binding of the electrochemically-labeled suoogate ligand to the target protein on the electrode surface yield an increased amount of cuoent compared with compounds that do not bind to the target, which show no effect on suoogate ligand binding.
  • appropriate controls for detecting increase in observed cuoent due to target protein denaturation are included in each determination.
  • These controls include testing the target protein in a reaction chamber freshly prepared with a solution comprising an electrolyte and electrochemically-labeled suoogate ligand in the absence of the compounds to be tested, and by testing these compounds with unrelated target proteins.
  • the methods of the invention are practiced on a minimal volume of solution, either on a 96-well microtitre plate whereby 96 electrodes are configured to be utilized simultaneously, or in the context of emerging microfluidic technology.
  • the competition binding assays are performed to detect compounds that affect specific binding between the target protein and the electrochemically-labeled suoogate ligand by causing a confo national change in the target protein.
  • the target protein is first incubated with the electrochemically-labeled suoogate ligand, and then placed in a reaction chamber containing the compound or compounds to be tested.
  • Compounds that bind to an available site on the target and induce a confoonational or allosteric change in the target cause release of the electrochemically-labeled suoogate ligand, and are detected by the production of an increase in the observed cuoent in the reaction chamber as detected, for example, by cyclic voltammetric analysis.
  • appropriate control reactions are performed to detect loss of suoogate ligand binding due to target protein denaturation.
  • the invention also provides methods for measuring the binding affinity of interaction between members of a biological binding pair, such as protein-peptide and protein-protein interactions. These measurements are useful for determining the dissociation constant (K d ) of the interaction between the components of the biological binding pair.
  • K d dissociation constant
  • These methods provide an alternative to existing methods for measuring binding affinities and dissociation constants, such as surface plasmon resonance instruments (e.g., BIAcore*, Pharmacia).
  • the methods of the present invention are advantageous compared with such previously-disclosed technologies because the present methods are more rapid, less costly and require less biological material.
  • the methods of the invention constitute a homogeneous assay whereas the methods known in the prior art require immobilization of one member of the biological binding pair.
  • the methods of the present invention can be practiced using electrochemical probes and electrochemical ligands having molecular weights of 300 daltons or more.
  • the methods known in the prior art require ligands that are at least about 5 kilodaltons (kD) in size, since the signal strength using prior art methods is proportional to the size of the binding ligand.
  • This limitation prevents analysis of binding interaction properties of molecules having molecular weights less than the cutoff threshold, 5kD. This limitation is important, since small molecular weight compounds form a large percentage of potential drug lead compounds.
  • assay conditions using the methods and apparatus of this invention are more permissible than the assay conditions required using the methods of the prior art, including but not limited to conditions of probe concentration, salt concentration and assay performance in the presence of organic solvents.
  • the invention also provides methods and apparatus for determining the binding affinity and chemical "strength" of the interactions between members of a biological binding pair. Knowing the strength of the interaction between two members of a biological binding pair is important for determining whether the interaction has potential as a good target for drug discovery. The ability to detect these interactions with a rapid, inexpensive and convenient assay can greatly accelerate both target validation and screening.
  • the methods of the present invention provide the ability to screen any two members of a biological binding pair for the capacity to specifically bind or otherwise specifically interact.
  • the invention also provides methods for mapping region(s) of interaction between the members of the pair, using various truncated or altered forms of either or both members of the binding pair. For protein-protein interactions, there are several cuoently of interest in drug discovery, that are listed in Table II.
  • protein.-protein interaction methods are provided. Such interactions are difficult to detect or characterize using existing technology.
  • a particular target protein is incubated with an electrochemically- active solution containing an electrochemically-labeled suoogate ligand and a cell extract comprising a protein(s) that specifically interacts with the target protein.
  • binding of the interacting protein instead of the electrochemically-labeled suoogate ligand results in an increased amount of cuoent produced during electrochemical analysis, e.g., cyclic voltammetry.
  • This inventive method for detecting protein-protein interactions is advantageous compared with cuoently-available methods, as illustrated by a comparison with cuoent methods for assaying column fractions during protein purification.
  • the cuoently- available techniques include enzymatic assays of chromatographic column fractions that generate a radioactive product and that are only applicable to proteins having known enzymatic activity.
  • ELIS A assays For proteins with unknown enzymatic activity, ELIS A assays, band shift assays using a radiolabeled target, or co-immunoprecipitations (that require antibodies to a radiolabeled target) are used.
  • ELIS A assays band shift assays using a radiolabeled target, or co-immunoprecipitations (that require antibodies to a radiolabeled target) are used.
  • Each of these methods is time-consuming and tedious, and frequently require the use of radiochemical detection methods that are disadvantageous in terms of safety and regulatory concerns.
  • the methods of the cuoent invention are rapid, specific, and inexpensive.
  • An additional advantage of the electrochemical screening methods of the present invention is that such screening methods are able to detect weak protein-protein interactions that cannot be detected by existing techniques.
  • the methods of the present invention are also applicable to a variety of alternative embodiments of protein purification techniques, including analysis of chromatographic fractions, tissue distribution surveys for the presence of the target binding protein in tissue samples from tumors, for example, and for cell-cycle specific interactions, for example, by using extracts from synchronized cells. Table II Interactions of Interest
  • K-channel src various proteins WW domain containing proteins ptyr proteins SH2 domains, PTB domains, phosphatases
  • RNA RNA binding proteins concanavalin A lectins lipids lipoproteins fatty acids (FA) FA binding proteins steroids steroid hormone receptors cytomegalovirus DNA polymerase polymerase accessory factor
  • the methods and apparatus of the invention are advantageous over the analytical techniques and equipment known in the art for the following reasons.
  • the sensitivity of the methods of the invention permit detection of specific binding interactions between the members of a biological binding pair over 4-5 orders of magnitude of concentration (i.e., 10,000- to 100,000-fold).
  • This invention provides detection methods having the sensitivity of radiochemical detection methods without the health, safety and regulatory concerns that accompany radiochemically-based methods.
  • the invention also affords detection of biological binding interactions with high sensitivity over a wide range of binding affinities.
  • the assays are rapid, inexpensive and are performed in vitro.
  • the reagents used in the practice of the invention i.
  • the electrodes and electrochemically-labeled suoogate ligands are stable and have a relatively long shelf-life compared with, for example, radiochemical reagents.
  • structure-activity relationships can be determined quantitatively, based on the deteonination of changes in binding kinetics observed using cyclic voltammetry, for example.
  • the analyses can be multiplexed, that is, each reaction can be performed in a reaction chamber comprising more than one biological binding pair, wherein the electrochemical label of each of the second members of the binding pair present in the reaction chamber has a different redox potential, so that one or a mixture of potential drug lead compounds can be analyzed for binding to a variety of potential targets.
  • the methods and apparatuses of the invention are amenable to automation, including but not limited to the use of multiwell (such as 96-well microtitre) assay plates and robotic control of electrodes and electrochemical components of the reaction chambers thereof.
  • the sensitivity of the electrochemical assays of the invention permit detection of small amounts of either suoogate ligand, inhibitory compounds, or both, thereby increasing the efficiency of performing assays such as drug screenings.
  • these increases in efficiency result in higher throughput screening, addressing a maj or obstacle to drug development.
  • the invention provides methods for determining dissociation constants for biological binding pair interactions that are more rapid, less expensive and require less sample than known methods (including, for example, equilibrium dialysis, analytical ultracentrifugation, analytical microcalorimetry and
  • the assays provided by the present invention can be performed in the absence of any information on the identity of the binding partner for any target protein or suoogate ligand. This advantage eliminates the requirement that the biological activity of a target protein be known before the protein can be characterized. Eleventh, the assays of the invention are flexible, and allow analysis of binding or competition binding for any biological binding pair. Moreover, either of the binding pairs can be electrochemically-labeled, and under appropriate assay conditions, the diffusion behavior of both members of the biological binding pair in the electrochemically active solution can be observed and characterized.
  • a second member of a biological binding pair preferably a peptide or nucleic-acid suoogate ligand as defined herein, coupled to an electrochemical catalyst comprising an electrochemically activated catalytic species.
  • electrochemical catalysts are enzymes such as glucose oxidase and horseradish peroxidase, which effect the oxidation or reduction of their substrates and are electrochemically reactivated at potentials that are insufficient to effect direct electrochemistry of the substrate.
  • Such enzymes are understood in the art to achieve catalysis by lowering an electrochemical barrier in the redox chemistry of the substrate, so that judicious choice of electrode potential allows selective electrochemical detection of the enzyme-catalyzed reaction in the vicinity of the electrode.
  • several synthetic transition-metal complexes such as those of oxoruthenium(IV), oxoosmium(IV), oxomolybdenum(IV), dioxomolybdenum(VI) and dioxorhenium(VI) are capable of oxidizing or reducing a variety of organic functional groups in a substrate at potentials at which direct electrochemistry is impossible. (For examples, see Stultz et al, 1995, J. Am Chem. Soc.
  • binding of the second member of the biological binding pair to the first member of the biological binding pair results in codiffusion of the electrochemical catalyst and the target protein. Redox reactions between the electrochemical label, the substrate and the electrode results in cuoent flow at the electrode, due to transfer of redox equivalents to the substrate.
  • binding of the suoogate with the first member of a biological binding pair causes the horse radish peroxidase enzyme to be reduced at a rate limited by codiffusion with the target.
  • This reduced form of the enzyme is the active form, which can therefore act catalytically to transfer electrons to hydrogen peroxide in the solution, producing oxygen and water.
  • the enzyme is constitutively reduced by the electrode after each catalytic cycle and, as the entire process is repeated, the binding of the suoogate ligand-enzyme conjugate is detected and quantitated as decrease in cuoent flowing through the electrode to the solution.
  • the amount of cuoent produced is related to the amount and extent of binding between members of the biological binding pair.
  • the substrate of the electrochemical catalyst produces a detectable product upon undergoing an oxidation/reduction reaction with the electrochemical catalyst at the surface of the first electrode of the apparatus.
  • the term "detectable product” is intended to encompass a product that can be differentially detected in a solution containing the biological binding pairs, the electrochemical catalyst, and the substrate therefore.
  • a detectable label but not limited thereto is a colored product, that is, a product that absorbs electromagnetic radiation, preferably within that portion of the spectrum comprising visible light, so that the extent of the reaction can be monitored colorimetrically.
  • detectable product any product that can be detected spectrophotometrically, including for example differences in the oxidation state of the NAD T /NADH or NADP7NADPH redox pair, as well as other cofactors including for example riboflavin.
  • the detection of the detectable product can be used as a control for stability of the electrochemical catalyst, the substrate therefor or both, or for depletion of the substrate by the catalyst during the reaction (i.e., saturation), and can be used as a suoogate for cuoent production or as an internal validating control for the observed cuoent produced in the reaction chamber of the apparatus.
  • binding or lack of binding of the conjugate is used to determine the occupancy of the available binding sites by an electrochemically inactive species.
  • this species will be a single drug candidate from a large library of either natural products or combinatorially synthesized molecules. Binding of the drug candidate can be ascertained by at least three related techniques.
  • the first member of the biological binding pair comprising a target can be preincubated with the drug candidate to allow all possible binding interactions between the candidate drug and the first member of the biological binding pair to occur prior to adding the electrochemically-labeled suoogate ligand.
  • the increase in cuoent upon addition of the electrochemically-labeled suoogate ligand when compared with cuoent produced in the absence of the drug candidate, is a measure of the extent of occupancy of the available binding sites of the electrode-immobilized first member of the biological binding pair by the drug.
  • a drug candidate is added concuoently with an electrochemically-labeled suoogate ligand conjugate at different concentrations, and the effect of the presence of the drug candidate on the produced cuoent used to deteonine the inhibition constant of the drug for suoogate ligand binding.
  • the target can be saturated with electrochemically-labeled suoogate ligand prior to the addition of the drug, whereby loss of observable cuoent indicates the capacity of the drug candidate to displace suoogate ligand binding.
  • Electrochemically labeled peptides were prepared using art-recognized techniques (see Yocom et al, 1982, Proc. Natl Acad. Sci. USA 79: 7052 - 7055; Nocera et al, 1984, J. Amer. Chem. Soc. 106: 5145 - 5150).
  • the derivatized peptide EC-1 having the amino acid sequence:
  • An example of such a voltammogram is shown in Figure 2 and was acquired under the following conditions.
  • the reaction chamber of the apparatus of the invention contained 100 ⁇ M ECl peptide as disclosed in Example 1 dissolved in a buffered aqueous solution containing 10 mM Tris, 150 mM NaCl, and 0.05% Tween 20. Cyclic voltammetry was performed where the applied voltage was scanned at 50 mV sX The cyclic voltammogram shown in Figure 2 illustrates the electrochemical behavior of the ECl peptide in the absence of a first member of a biological binding pair that specifically binds to the peptide.
  • the electrochemical analysis apparatus and methods of the invention were used to detect and analyze the specific binding interaction between neutravidin and the biotinylated ECl peptide as follows. Cyclic voltammetry was performed using a solution in the reaction chamber as disclosed in Example 2 at applied voltage scan rates of 25, 50, 100, 200, and 400 mV s "1 . The voltammogram acquired at 50 mV s "1 is shown in Figure 3 (the curve designated "A"). Voltammograms were then acquired under identical peptide and scan-rate conditions in the presence of stoichiometric excesses of neutravidin (shown in Figure 3, the curve designated as "B") and biotin-saturated neutravidin.
  • the effective diffusion coefficients of EC-1 alone was determined to be approximately 3.8 x 10 "7 cm 2 s "1
  • biotin-saturated neutravidin was determined to be approximately 3.4 x 10 "7 cm 2 s "1 , respectively.
  • Electrochemical Assay for Screening Combinatorial Libraries An electrochemical assay of the invention is used to screen combinatorial chemical libraries to detect samples that perturb the electrochemical signal in cyclic voltammetry resulting from the interaction between a target protein and electrochemically-labeled binding peptide.
  • the conditions of the screen must not be easily perturbed, or the voltammetry output diminished thereby.
  • such a screening assay will function over a wide range of pH and salt concentrations, and is not sensitive to common contaminants (such as coupling reagents) that are frequently encountered in combinatorial chemical libraries.
  • Cyclic voltammograms were calculated assuming protein and suoogate ligand concentrations of 100 ⁇ M under conditions of increasing concentrations of a competitor with a target-binding affinity of 50 nM, using a theoretical microfluidic cell with electrode area of 8.5 x 10 "6 cm 2 and using a potential scan rate of 50 mV s "1 .
  • the electrochemical analysis methods of the invention are also useful for determining the K d of the interaction between a protein and specific binding peptides therefor, for example, the Src-SH3 domain and a number of short, proline-rich specific binding peptides.
  • the interaction of the Src SH3 domain with short, proline-rich peptides such as Arg-Pro-Leu-Pro-Pro-Leu-Pro (SEQ ID No.: 2) and Ala-Pro-Pro-Val- Pro-Pro-Arg (SEQ ID No.: 3) have been intensively studied, and K d values have been determined by validated means such as BIAcore 8 (Pharmacia, Upsalla).
  • the K d value for the interaction of GST-Src SH3 and SH3 binding peptides is determined using the electrochemical analysis methods of the invention to provide a compa ⁇ son with a pharmacologically-validated method
  • Solutions of the GST-Src SH3 fusion protein are mixed with electrochemically-labeled species of the prohne- ⁇ ch SH3- domain specific binding peptides shown above
  • Electrochemical signal is generated using cyclic voltammetry as desc ⁇ bed in Examples 2-4 above, and the signal is monitored over time as desc ⁇ bed in Example 4 above
  • Electrochemical signal data are collected at va ⁇ ous concentrations of the peptide, and the electrochemical signal is then used to calculate a K d value for the peptide K d values are also determined using the BIAcore* method as a validated control, and a comparison of the results between the tw o analytical methods used to validate the values determined using the electrochemical analysis assays of the invention
  • the electrochemical analytic methods and apparatus of the invention are used to detect protein peptide interactions in a complex mixture A variety of different target molecules and sources of specific binding peptides are used in these expe ⁇ ments Although it is usually possible to find a suoogate ligand for a protein by using phage display or by screening combinatorial libraries (see co-owned and co-pendmg L S patent application, Se ⁇ al No 08/740,671 , filed on October 31, 1996, incorporated herein by reference), the natural ligand for a protein is more difficult to identify
  • the electrochemical screening assays of the invention provide a relatively simple means for identifying natural ligands
  • the natural ligand for the Src SH3 domain in cell extracts is detected GST-Src SH3 domain fusion protein solutions are incubated with electrochemically-labeled Src-SH3 binding peptide to specifically "load" the SH3 domain with the electrochemically-labeled peptide
  • the solutions are
  • fractions are analyzed for the presence of a compound(s) that can displace binding of the electrochemically-labeled specific binding peptide as detected by cyclic voltammetry. Only those fractions containing such activity are caoied through subsequent steps of the biochemical fractionation. After several such biochemical purification steps, active fractions are analyzed by SDS-polyacrylamide gel electrophoresis to determine the relative purity of the fractions. Microsequencing of homogeneous protein-containing
  • bands is then used to isolate and identify the active protein(s) comprising the fraction having specific peptide displacement activity.
  • the methods of the invention thereby provide a sensitive method for detecting protein-protein interactions from heterogeneous mixtures of biological compounds.
  • the electrochemical analysis assays of the invention are also used to determine functions for orphan receptors isolated and identified by recombinant genetic methods. Frequently, DNA sequences are discovered encoding regions resembling receptor coding domains. When these sequences are discovered, it is presently quite difficult to determine the biological function or activity of the encoded receptor or the natural ligand of these receptors.
  • the extracellular domain of the receptor is expressed and used as the target for screening phage displayed peptide libraries to identify a suoogate ligand.
  • the suoogate ligand is then used in a number of ways. Electrochemical screens of combinatorial libraries are conducted to identify antagonists of the assay. These compounds are then used in model biosystems (for example, mammalian cells transformed with a recombinant expression construct encoding the receptor that express the receptor at the cell surface) to decipher the biological role of the receptor (for example, to determine if the receptor is linked to adenylate cyclase).
  • the suoogate ligands are also used in an electrochemical screening assay to identify the natural ligand.
  • Cell lysates, supernatants or tissue extracts are fractionated and assayed by the electrochemical screening assays of the invention to identify fractions containing a molecule that displaces the labeled suoogate ligand from the electrode-bound target.
  • Protein or peptide ligands isolated thereby can be then identified by sequencing.
  • Small molecule ligands may be identified by mass spectral analysis and other analytical systems.
  • Such assays systems can also be applied to molecules found in biological fluids (such as mammalian brain extracts) to indicate the existence of the natural ligand in such biological extracts.
  • electrochemical analysis assays of the invention is the identification of ligands for the fas receptor (see Hahne et al , 1996, Science 274: 1363; Nagata et al , 1995, Science 267: 1449, Takahashi et al , 1994, Cell 76: 969; Wanatabe-Fukunaga et al , 1992. Nature 356 314).
  • the fas receptor which is expressed in almost all cell types, t ⁇ ggers the apoptotic (programmed cell death) pathway when it is bound by its ligand.
  • the expression of the ligand for the fas receptor is much more restricted Apoptosis is t ⁇ ggered when the ligand on one cell interacts with the receptor on another cell This is a therapeutically useful target since it has recently been demonstrated that the expression of the fas ligand on the surface of some melanoma cells t ⁇ ggers apoptosis m body's immune cells, thereby allowing cancer cells to evade the host immune response
  • the extracellular domain of the fas receptor is expressed for use as a target m phage display to identify a suoogate ligand (using, for example, the methods disclosed in co-owned and co-pending U S patent application. Serial No 08/740.671, filed on October 31 , 1996, incorporated herein by reference) Suoogate ligands so identified are then electrochemically-labeled and incubated with the fas receptor extracellular domain.
  • Plasma cell supernatants are fractionated, and the fractions assayed by cyclic voltammetry and electrochemical screening as described herein to detect those fractions that contain activity capable of displacing the labeled suoogate ligand from the electrode.
  • the fas receptor ligand is detected
  • the function of the fas receptor is identified using the electrochemical analytic methods of the invention. In these assays, the extracellular domain of the fas receptor is used to obtain a suoogate ligand via phage display library as desc ⁇ bed above.
  • the labeled suoogate ligand is then used in an electrochemical screen to identify compounds from a combinato ⁇ al chemical library that displace or compete the labeled hgand from the fas receptor protein.
  • the identified compound may either be an agonist or an antagonist of fas receptor activity.
  • the compound(s) identified in this screen are tested m a model biological system to study receptor function. For example, the compound is added to cells in culture that express the fas receptor and the biological response of the cells observed A receptor antagonist blocks the apoptotic pathway in the presence of the fas ligand.
  • apoptosis provides a sensitive assay that can be used in conjunction with the electrochemical analysis assays of the invention to analyze fas receptor function
  • the electrochemical assay of the invention is used to screen combinato ⁇ al chemical libra ⁇ es to detect samples that perturb a chemilummescence signal resulting from the interaction betw een a target protein and an electrochemically labeled binding peptide
  • a variety of different target molecules and sources of specific binding suoogate ligands are used in these experiments

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Abstract

Cette invention a trait à des techniques et à l'appareil correspondant permettant d'effectuer des analyses électrochimiques. Cet appareil permet d'effectuer des ampérométries, des analyses coulométriques ainsi que des potentiométries ou des analyses par voltampèremètre et ce, aux fins de la détection de liaisons spécifiques entre éléments d'une paire de liaison biologique dans laquelle un élément est marqué par voie électrochimique ou lié à un catalyseur électrochimique. L'invention concerne également des méthodes d'utilisation de l'appareil de l'invention aux fins de l'exécution d'analyses de liaisons et de liaisons compétitives. Elle porte, de surcroît, sur des techniques d'exécution d'analyses de dépistage à haut rendement permettant de détecter une inhibition de liaison spécifique entre les éléments de la paire de liaison biologique utilisable aux fins de la mise au point de médicaments, de l'analyse biochimique et de déterminations de purification de protéines.
PCT/US1999/011848 1998-06-08 1999-05-28 Sondes electrochimiques aux fins de la detection d'interactions moleculaires et de la mise au point de medicaments WO1999064847A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6261782B1 (en) 1999-04-06 2001-07-17 Yale University Fixed address analysis of sequence tags
US6383754B1 (en) 1999-08-13 2002-05-07 Yale University Binary encoded sequence tags
WO2004065951A1 (fr) * 2003-01-20 2004-08-05 Universal Biosensors Pty Limited Procede de detection electrochimique
WO2006043095A3 (fr) * 2004-10-22 2006-07-06 E2V Biosensors Ltd Controle de reaction substrat-enzyme

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4945045A (en) * 1984-07-06 1990-07-31 Serono Diagnostics Ltd. Electrochemical methods of assay
US5149630A (en) * 1984-01-26 1992-09-22 Forrest Gordon C Methods of assay
US5541113A (en) * 1993-09-22 1996-07-30 Beckman Instruments, Inc. Method for detecting an analyte using an electrochemical luminescent transition metal label

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5149630A (en) * 1984-01-26 1992-09-22 Forrest Gordon C Methods of assay
US4945045A (en) * 1984-07-06 1990-07-31 Serono Diagnostics Ltd. Electrochemical methods of assay
US5541113A (en) * 1993-09-22 1996-07-30 Beckman Instruments, Inc. Method for detecting an analyte using an electrochemical luminescent transition metal label

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6261782B1 (en) 1999-04-06 2001-07-17 Yale University Fixed address analysis of sequence tags
US6677121B2 (en) 1999-04-06 2004-01-13 Agilix Corporation Fixed address analysis of sequence tags
US6383754B1 (en) 1999-08-13 2002-05-07 Yale University Binary encoded sequence tags
US6773886B2 (en) 1999-08-13 2004-08-10 Yale University Binary encoded sequence tags
WO2004065951A1 (fr) * 2003-01-20 2004-08-05 Universal Biosensors Pty Limited Procede de detection electrochimique
US7403017B2 (en) 2003-01-20 2008-07-22 Universal Biosensors Pty Limited Methods of measuring barrier formation
CN100458430C (zh) * 2003-01-20 2009-02-04 环球生物传感器有限公司 电化学检测方法及设备
WO2006043095A3 (fr) * 2004-10-22 2006-07-06 E2V Biosensors Ltd Controle de reaction substrat-enzyme

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