EP1180244A2 - Systemes et procedes de dosage ne dependant pas de la cible - Google Patents

Systemes et procedes de dosage ne dependant pas de la cible

Info

Publication number
EP1180244A2
EP1180244A2 EP00932407A EP00932407A EP1180244A2 EP 1180244 A2 EP1180244 A2 EP 1180244A2 EP 00932407 A EP00932407 A EP 00932407A EP 00932407 A EP00932407 A EP 00932407A EP 1180244 A2 EP1180244 A2 EP 1180244A2
Authority
EP
European Patent Office
Prior art keywords
library
assay
target independent
screened
modulation
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00932407A
Other languages
German (de)
English (en)
Inventor
C. Nicholas Hodge
Michael R. Knapp
Anne R. Kopf-Sill
Theo T. Nikiforov
J. Wallace Parce
Calvin Y. H. Chow
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Caliper Life Sciences Inc
Original Assignee
Caliper Technologies 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 Caliper Technologies Corp filed Critical Caliper Technologies Corp
Publication of EP1180244A2 publication Critical patent/EP1180244A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA
    • 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
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • 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
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/80—Cytochromes
    • 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
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/9116—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • G01N2333/91165—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1)
    • G01N2333/91171—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1) with definite EC number (2.5.1.-)
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • 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)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)

Definitions

  • a compound can be rescreened to determine the precise nature of its activity, and to screen it for secondary effects, e.g., toxicological effects, bioavailability, and the like.
  • a variety of assays are typically performed subsequent to an initial activity-screening assay (or target dependent assay) in pharmaceutical research to assess whether an active compound will be useful in vivo.
  • a number of in vivo activities can affect whether a potential drug might ultimately be effective, including the toxicity of the compound, the non-specific binding of the compound to other elements, the passage of the compound through various biological barriers, and the like. These parameters are evaluated in secondary or "target independent" assays.
  • target independent assays are performed in pharmaceutical research following identification of a potential lead compound. This is typically due to the high cost of reagents for performing these assays. In order to conserve costs and expedite drug development, it would be desirable to be able to perform both the primary screening assay and the secondary, or target independent assays, as rapidly, inexpensively and as early as possible.
  • the present invention addresses these and a variety of other needs.
  • the present invention provides methods and assays for generating or designating large chemical composition libraries that are substantially devoid of compounds that have a undesirable effect in one or more target independent assay.
  • Methods for evaluating large numbers of chemical compositions (including individual compounds, mixes of compounds, etc.) in cost-effective, high throughput assay systems suitable for screening libraries prior to performing target dependent assays are provided.
  • the assays of the invention are performed sequentially, or simultaneously, on "parental” or “master” libraries which are typically in excess of 1000, 10,000, 100,000, or 1,000,000 compositions, or on successively screened libraries depending therefrom.
  • the pre-screened libraries can be physically separated from the master libraries, thereby generating pre-screened libraries, or can simply constitute designated members of the master library.
  • Pre-screened libraries evaluated in one or more target independent assay by the methods of the invention are an aspect of the invention.
  • the pre-screened libraries provided comprise in excess of 100, 1000, 10,000, 100,000, or 1,000,000 compositions that exhibit a desired activity in one or more target independent assay, and/or are substantially devoid of compositions that exhibit an undesired effect in the target independent assays.
  • the libraries are arrayed in multiwell plates or other array formats.
  • the libraries are represented by one or more data sets that correlate results of one or more target independent assay with locations in the master library array. Generally, such data sets are stored in a computer readable medium.
  • the invention provides assays that evaluate effects correlated with target independent parameters such as serum half-life, cellular uptake, oral availability, cellular or organismal viability, cellular or organismal toxicity, apoptosis, cellular adhesion, target independent receptor binding or activation, target independent enzyme modulation activity, target independent protein modulation activity, target independent nucleic acid modulation activity, glucuronide conjugation modulation, sulfate conjugation modulation, amino acid conjugation modulation, acylation modulation, methylation modulation, transcription modulation, translation modulation, protein folding modulation, modulation of cell growth, membrane permeability, membrane integrity, metabolic stability, thermal stability, solubility, solution viscosity, solution turbidity, acidity, basicity, RedOx state, superoxide secretion, lipid peroxidation, octanol/water partitioning, precipitation in one or more buffers, or the like.
  • target independent parameters such as serum half-life, cellular uptake, oral availability, cellular or organismal vi
  • HSA Human Serum Albumin
  • a composition of interest can have a considerable effects, e.g., on composition availability in vivo.
  • one can monitor composition binding to HSA e.g., by determining the composition's ability to inhibit binding of a labeled material to HSA in vitro.
  • the ability of a selected compound to bind HSA is measured as a function of its inhibition of binding of a fluorescent dye such as dansyl-1-sulfonamide or dansyl sarcosine to HSA.
  • a fluorescent dye such as dansyl-1-sulfonamide or dansyl sarcosine to HSA.
  • the invention also provides target independent assays that evaluate the inhibition of an enzyme by components of a chemical composition library.
  • Target independent assays of the invention measure the effect of a composition on enzymes including: glutathione-s-transferases, proteases, peptidases, kinases, phosphatases, G proteins, ATPases, cytochrome P450s, ligases, carboxylesterases, epoxide hydrolases, aza- or nitro-reductases, carbonyl reductases, disulfide reductases, sulfoxide reductases, quinone reductases, alcohol dehydrogenases, aldehyde dehydrogenases, aldehyde oxidases, xanthine oxidases, monoamine or diamine oxidases, prostaglandin sythases, flavin-monooxygenases, and dehalogenases.
  • enzymes including: glutathione-s-transferases, proteases, peptidases, kinases, phosphatases, G proteins, ATPases
  • the target independent assays are performed in a microfluidic device.
  • the microfluidic device comprises a pipettor element.
  • the target independent assay is performed by flow cytometry in a microfluidic cell-focusing device. In one embodiment, the flow cytometry distinguishes between live cells and dead cells.
  • Flow cytometry methods that distinguish between live cells and dead cells are based on differential permeability or binding to a fluorescent dye or dye conjugate, such as calcein AM, BCECF AM, ethidium bromide, propidium iodide, a cationic dye, a cationic membrane permeable dye, a neutral dye, a membrane permeable neutral dye, an anionic dye, an anionic membrane permeable dye.
  • a fluorescent dye or dye conjugate such as calcein AM, BCECF AM, ethidium bromide, propidium iodide, a cationic dye, a cationic membrane permeable dye, a neutral dye, a membrane permeable neutral dye, an anionic dye, an anionic membrane permeable dye.
  • the target independent assay evaluates the effect of a composition on calcium flux across the membrane of a cell preloaded with a fluorescent calcium indicator.
  • the master library is arrayed in one or more multiwell plate(s), on a solid substrate such as a membrane or card, or in a bead matrix.
  • Data sets correlating the results of one or more target independent assay with one or more members of the master library array are compiled in some embodiments. In some cases, the data set correlates results of multiple target independent assays with members of the master library array.
  • the invention further provides methods and devices for performing target independent assays in a multi-module workstation.
  • the multi-module workstations of the invention include screening modules for performing the target independent assays, substrates corresponding to composition libraries, robotic mechanisms connecting the screening modules, and, e.g., a computer assisted control system for controlling the movement of substrates by the robotic mechanism, or alternatively of the robotic mechanism between or within the substrates.
  • the screening modules are configured to perform target independent assays such as flow cytometry assays, assays which evaluate the effect of a selected composition on a biochemical system, assays which measure a calcium flux across a cell membrane, assays which evaluate binding of a serum protein, assays which evaluate binding to Human Serum Albumin, assays which evaluate the effect of a selected composition on an enzyme, and the like.
  • the screening modules comprise microfluidic devices.
  • Substrates include, e.g., multiwell plates, solid substrates or bead arrays.
  • the robotic mechanism includes, for example, a conveyor belt, a slide mechanism, rollers, a cable and pulley mechanism, a robotic armature, or any combination thereof.
  • the computer optionally includes instructions sets for movement of substrates, movement of the robotic mechanism, movement of the screening modules and/or selection of library members, etc., as well as, e.g., data sets correlating the results of the target independent assays with members of the library array, and a user interface for programming inputs into the system.
  • the invention additionally provides for use of any of the pre-screened libraries, or component compositions, in a target dependent assay.
  • Kits for performing one or more target independent and/or target dependent assay to produce the pre-screened libraries of the invention are also a feature of the invention.
  • FIG. 1 Panels A and B provide microfluidic channel configurations useful for performing target independent assays.
  • Figure 2 A microfluidic channel configuration for performing target independent assays.
  • FIG. 3 Panel A provides an HSA-drug binding plot.
  • Panel B provides a plot of inhibition vs. phenylbutazone concentration.
  • Panel C provides data of HSA binding to six drugs.
  • FIG. 4 Panel A provides an overview of glutathione S-transferase (GST) reactions. Panel B illustrates useful substrates for GST assays and Panel C provides results of such assays performed in microfluidic devices.
  • Figure 5 Protease assay results.
  • Figure 6 Panel A illustrates mobility shifts in protein kinase assays. Panel B provides protein kinase mobility shift data. Panel C provides a Lineweaver-Burk plot for determination of kinetic data in a PKA assay. Panel D provides PKA inhibitor data and Panel E provides data obtained in a microfluidic sipper device.
  • Panel A provides phosphatase assay data from a microfluidic assay.
  • Panel B provides raw data and
  • Panel C illustrates determination of K, from the raw data.
  • FIG. 8 Panel A is a schematic illustration of a G-protein coupled receptor cell based assay which is optionally performed in a microfluidic device as illustrated in Panel B. Panels C through F provide data from a G-protein coupled receptor cell based assay performed in a microfluidic device.
  • Figure 9 A multi-module workstation useful in performing multiple target independent assays, e.g., to produce a pre-screened library. DETAILED DESCRIPTION
  • the present invention provides methods for producing large chemical composition libraries which are pre-screened for secondary activities of interest, providing large libraries which meet selected criteria. These pre-screened libraries are then screened for activities of interest.
  • the present invention provides methods of pre- screening libraries, the pre-screened libraries resulting from practicing the methods, integrated systems for practicing the methods and databases of pre-screened compositions.
  • the chemical composition libraries of the invention are pre- screened in assays predictive of biologically relevant characteristics prior to their evaluation as potential drug leads.
  • pre-screened indicates that a chemical composition library, or collection of chemical compounds, is evaluated in one or more assay which evaluates properties correlated with, e.g., clinical performance or other activity criteria, rather than with properties specific to the therapeutic target of interest.
  • an assay is a "target independent assay,” that is, an assay that screens for a general property other than the activity of primary interest for a particular compound.
  • a kinase assay can be a target dependent assay when the subject kinase of the assay is the target of, e.g., modulation by the compound or components of the library.
  • a kinase assay used to determine, e.g., effects on global phosphorylation status, unrelated to the target (which, in some cases, is itself a kinase) is a target independent assay.
  • the methods of the invention provide the significant benefit that a particular target independent assay is performed once for a given library, rather than after each and every target dependent assay.
  • Basic methods and systems for performing these assays are described in U.S. Patent No. 5,942,443, issued August 24, 1999 and U.S. Patent Application Nos. 08/883,638, filed June 27, 1997; 09/173,469, filed October 14, 1998, and 09/093,489, filed June 8, 1998, which are inco ⁇ orated herein by reference for all purposes.
  • Chemical composition libraries are composed of a collection of chemical compositions or compounds. Such libraries can include a wide variety of different compounds, including chemical compounds, mixtures of chemical compounds, e.g., polysaccharides, small organic or inorganic molecules, biological macromolecules, e.g., peptides, proteins, nucleic acids, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, or naturally occurring or synthetic compositions.
  • a "parental library” or a "master library” refers to a library, the members of which are to be evaluated for their ability to affect a particular biochemical system. By pre- screening these libraries, one can effectively prioritize further screening of those members of the library that have positive or adverse effects in these target independent assays.
  • the pre-screened libraries can be selected for compounds that have positive effects in these target independent assays.
  • the master libraries will have in excess of 1000, more typically in excess of 10,000, and often in excess of 100,000 or even 1,000,000 or more members prior to the pre-screening assay, with the number of members being reduced, e.g., by typically at least 10%, often 20 %, in some cases 50%, and sometimes 90% or more.
  • target dependent assays can focus only on 90%, 80%, 50%, and potentially after several target independent assays, as little as 10% or less of the initial or master library population.
  • Master or pre-screened libraries can include arrays having one or more compound type per location in the array, e.g., the libraries can be arrayed as sets of single compounds or as arrays of mixtures of compounds.
  • mixtures can include about 1,000 compound types or less, typically about 100 compound types or less, generally about 10 compound types or less, and often about 5 or fewer compound types.
  • secondary screening assays can also be used as a basis to add additional members for consideration into the pre-screened library. For example, where compounds of a given general structure are active in the secondary screen, additional structurally related library members can be generated, tested and inco ⁇ orated into the pre-screened library.
  • entire master libraries are screened regardless of the activity of compounds in secondary screening assays. Positive hits which are also active in secondary assays are, of course, potential lead compounds. However, compounds which are negative in secondary screening assays, but positive in target dependent assays can be converted into potential lead compounds. For example, in some cases, hits in a target dependent assay can be modified for improved secondary properties, without eliminating activity in the target dependent assay (e.g., by chemical modification of the relevant composition). Information from target independent library screens can be used in modifying such potential lead compounds, and, of course, libraries can include such modified compounds for additional secondary screening assays.
  • a library of chemical compositions is evaluated in a target dependent assay to determine its potential as a drug lead.
  • Promising lead candidates are then evaluated for such parameters as serum half-life, membrane permeability, toxicity, enzyme inhibition, and the like.
  • This approach maximizes the number of potential leads identified, but is accompanied by a relatively high level of attrition and, therefore, high cost per potential lead. It is also extremely expensive in terms of reagent usage, as many leads do not meet the criteria of secondary assay screens.
  • the present invention provides libraries which have been pre-screened in target independent assays, and are therefore, substantially devoid of compounds which have undesirable properties in target independent assays. Accordingly, a high proportion of the compositions which are identified have the characteristics of successful drug candidates, e.g., high bioavailability, low toxicity, stability, etc.
  • pre-screened libraries of the invention downstream costs are minimized, as fewer identified leads are eliminated during the later stages of development, which are associated with higher development costs.
  • the libraries of the invention are produced by evaluating large collections of chemical compositions in target independent assays in a high throughput format.
  • Such high throughput formats can be multiwell plate based, e.g., in 96-well, 384-well, 1,536-well plates, or solid support based such as on pin arrays.
  • the high throughput format includes performing one or more assay in one or more microfluidic device.
  • the chemical compositions are preferably arranged in a logically accessible set, i.e., an "array.”
  • the logical organization of arrays can be based on spatial relationships of array members, (i.e., where the arrays are in a gridded arrangement), or can be in an arbitrary arrangement that can be corresponded to a look-up table to provide equivalency information.
  • Such arrays include master library arrays, as well as arrays of libraries that have been pre-screened in one or more than one target independent assay. Library arrays further include pre-screened libraries which have also been evaluated in one or more target dependent assay.
  • array members can alternatively have mixtures of compounds.
  • the member can be further deconvoluted to identify which of the constituents of the mixture are relevant for activity. For example, if the components of the array member are known, they can be separately screened following identification of activity of the mixture.
  • the library arrays of the invention can include any material structure in which the physical substance comprising the chemical compositions can be maintained and accessed, e.g., multi-well plates, pin arrays, bead arrays, test tube arrays, etc.
  • samples are arrayed and accessed by microfluidic systems as described, e.g., in "MICROFLUIDIC SAMPLING SYSTEM AND METHODS," U.S. Patent No. 6,042,709 to Parce et al.
  • a number of additional useful library-microfluidic systems are set forth in "CLOSED-LOOP BIOCHEMICAL ANALYZERS" WO98/45481.
  • the data set is typically stored in a computer readable medium and is optionally produced and/or accessed via an integrated system that performs the assays in question. In alternate embodiments, data storage and assay performance are separately maintained.
  • the data set can correlate the results of a single target independent assay, multiple target independent assays, and/or one or more target dependent assays with positional information identifying individual members of a chemical composition library array.
  • the array can be physically arranged in one multiwell plate, pin array or bead array or the like; in a plurality of multiwell plates, pin arrays or bead arrays or the like; or in a combination thereof.
  • One, or more than one, library member can be maintained in each position of the array.
  • the data set or sets corresponding to a master library, and any number of pre- screened libraries can coincide with a single physical library array, or with any number of distinct and separate library arrays.
  • a chemical composition library in excess of 100,000 members constituting a master library can be arrayed in a set of multiwell plates, e.g., about 66 x 1536 well plates, about 261 x 384 well plates, or about 1004 x 96 well plates, or various combinations thereof.
  • a data set that correlates the composition of each library member with its position within the array is established, typically via the integrated system of the invention.
  • the library is then evaluated in a target independent assay of the invention, and the results are correlated with positions within the array. Additional target independent and or target dependent assays are performed, sequentially or simultaneously, and the results correlated with positions within the library array.
  • one or more pre-screened library of the invention coincides physically with one or more master library of the invention.
  • selected library members can be transferred and or replicated in a physically and spatially distinct library array.
  • Target independent assays are, in general, in vitro model systems for measuring the effect of a chemical compound on a biological or biochemical system or molecule, regardless of the specific activity for which the compound is sought.
  • target independent assays are used to evaluate potential lead compounds, regardless of whether the lead compounds are chemical compositions or biochemical compositions or whether they are naturally occurring, isolated or synthetic compositions.
  • the present invention provides methods for producing chemical composition libraries that have been evaluated for their effects in one or more target independent assay.
  • the libraries produced by the methods herein fit designated criteria reflecting their effects in biochemical systems.
  • Target independent assays that measure such parameters as serum half-life, cellular uptake, oral availability, cellular or organismal viability, cellular or organismal toxicity, apoptosis, cellular adhesion, target independent receptor binding or activation, a target independent enzyme modulation activity, a target independent protein modulation activity, a target independent nucleic acid modulation activity, glucuronide conjugation modulation, sulfate conjugation modulation, amino acid conjugation modulation, acylation modulation, methylation modulation, transcription modulation, translation modulation, protein folding modulation, modulation of cell growth, membrane permeability, membrane integrity, metabolic stability, thermal stability, solubility, solution viscosity, solution turbidity, acidity, basicity, RedOx state, superoxide secretion, lipid peroxid
  • cellular toxicity can be evaluated by assays such as differential binding of vital or fluorescent dyes, evaluated by flow cytometry, that distinguish between live and dead cells.
  • Cultured cells such as the human hepatocellular carcinoma cell line, Hep G2, or other suitable cells, are exposed to compositions of a chemical compound library, and simultaneously or subsequently, incubated with a dye, e.g., propidium iodide, that demonstrates differential staining of dead versus live cells. (Dead cells are PI bright and live cells are PI dim). The staining is then evaluated and the proportion of dead cells is calculated to determine compositions that exhibit an acceptable level of toxicity, that is, which result in acceptable ratios of dead to live cells.
  • a dye e.g., propidium iodide
  • Bioavailablilty of a therapeutic composition is influenced by, among other things, solubility, stability in solution, water/lipid partition coefficients, intestinal degradation via brush border enzymes and proteases (e.g., as occurs in the gut in vivo) and binding to serum proteins. These parameters are the subject of target independent assays.
  • the methods of the invention measure the binding of members of a chemical composition library to a serum protein, such as human serum albumin (HSA). Binding is, for example, measured as a function of the ability of a composition to interfere or inhibit binding of HSA by one or more fluorescent dyes (e.g., dansyl-1-sulfonamide, dansyl sarcosine).
  • the target independent assays of the invention also evaluate binding and/or activation of cellular or reconstituted receptors.
  • assays include cellular and in vitro assays.
  • Cellular assays measure binding as a function of, e.g., cellular calcium flux.
  • In vitro assays can be based on competitive binding with a labeled ligand or other marker molecule, e.g., a dye. Examples of microfluidic cellular assays utilizing dyes are found, e.g., in U.S. Patent Application Nos. USSN 09/416,288, filed October 12, 1999; and USSN 60/158,323, filed October 8, 1999.
  • Enzymes are typically selected either because they play an essential or important cellular housekeeping role, or because they represent classes of enzymes that play a significant role in cellular homeostasis, growth, differentiation, or division.
  • Such enzymes include: glutathione-s-transferases, proteases, peptidases, kinases, phosphatases, G proteins, ATPases, cytochrome P450s, ligases, carboxylesterases, epoxide hydrolases, aza- or nitro-reductases, carbonyl reductases, disulfide reductases, sulfoxide reductases, quinone reductases, alcohol dehydrogenases, aldehyde dehydrogenases, aldehyde oxidases, xanthine oxidases, monoamine or diamine oxidases, prostaglandin sythases, flavin-monooxygenases, and dehalogenases. Inhibition, or conversely, enhancement, of enzymatic activity by a chemical composition of these and other enzymes can be evaluated by numerous well-established assays known to those of skill in the art. TARGET DEPENDENT ASSAYS
  • target dependent assays evaluate the effects of a chemical composition, or library of compositions on a particular pharmaceutical screening target, e.g., where an effect on that target is the desired result of the drug that is being sought.
  • a target can be any biochemical molecule that plays a role in a biochemical pathway mediating a process within an organism. As such, a target influences, any, or many, biological process involved in the organism's health or disease.
  • a therapeutic reagent is selected to have a desired effect on the target, or on the pathway or process that involves the target.
  • target dependent assays are employed.
  • target dependent assay Any in vitro, cellular, or in vivo assay that is used to determine the effect of a chemical composition on a potential therapeutic target is considered a target dependent assay for the pu ⁇ oses of the invention. Furthermore, in vitro or cellular assays that measure an aspect of the in vivo effect of a composition on a target are also considered target dependent assays. Target dependent assays are distinguished from target independent assays, not strictly on the basis of the type of assay, or biochemical molecule (or pathway) that they evaluate, but rather on the relationship of the biochemical molecule to the potential therapeutic use of the chemical compositions being evaluated.
  • target dependent assay evaluates the biochemical effect of the molecule, e.g., on a disease target
  • independent assay evaluates the effect on secondary considerations which affect, e.g., efficacy, bioavailability, and/or toxicity, etc.
  • target independent assays are generally not tied to any particular disease pathology, but instead can include systems or assays that are operative in normal healthy systems.
  • the subject of a target dependent assay can be, e.g., a receptor, an enzyme, a signaling molecule, a membrane protein, a blood protein, a structural protein, a ligand, a substrate, cells, nucleic acids (DNAs, RNAs, etc.), membrane fractions or the like.
  • Both target independent assays, and target dependent assays can be performed in high throughput formats such as multi-well plates and microfluidic devices and combinations thereof.
  • target dependent assays optionally are performed on the pre-screened libraries. It will be apparent that whether the target dependent assay is performed before or after conducting one or more target independent assays is not of critical importance. However, in general, it will be most efficient and therefore least costly, to generate pre-screened libraries that have been evaluated for several target independent parameters, and then repeatedly using this valuable resource as the basis for subsequent target dependent assay screening projects.
  • the production of a pre-screened library evaluated in multiple target independent assays prior to identification of potential drug leads was prohibitively expensive. While those of skill in the art have long known the basic assays, the capacity to so reduce the scale of the assays as to make them economically desirable as pre-screening tools has been lacking.
  • the chemical composition libraries can be provided, e.g., injected, free in solution, or can be attached to a carrier, or a solid support, e.g., beads.
  • the chemical composition libraries (which can be single compounds or compound mixtures) are arranged in an organized matrix or array.
  • An array can be of compositions in solution, e.g., microtiter plates, on a solid membrane, in a bead matrix or other existing formats.
  • solid supports are suitable for the immobilization of the chemical compositions, including polymers, plastics, agarose, cellulose, dextran, carboxymethyl cellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resins, plastic films, glass beads, polyaminemethylvinylether maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • microtiter plates have become a standard format for the performance of high throughput screening assays.
  • Standard microtiter plates are available with 96, 384 or 1536 wells, although other commonly used number of wells include 3456 and 9600. Well volumes range between 500 nanoliters to over 200 microliters, depending on well depth and cross sectional area. Microtiter plates are typically selected for compatibility with current automated loading and robotic handling systems, with an 86 mm by 129 mm dimension being an industry standard. Alternative formats have been proposed, e.g., U.S. Patent No. 5,989,835, which are equally suitable for the methods of the present invention. Other applicable systems are described, e.g., in "MICROFLUIDIC SAMPLING SYSTEM AND METHODS," U.S. Patent No. 6,042,709 to Parce et al. A number of additional useful library-microfluidic systems are set forth in "CLOSED-LOOP
  • BIOCHEMICAL ANALYZERS BIOCHEMICAL ANALYZERS
  • WO98/45481 Additional details regarding library systems and microfluidic systems are set forth in "ULTRA HIGH THROUGHPUT SAMPLING AND ANALYSIS SYSTEMS AND METHODS" to Wolk et al. USSN 60/042,709, filed January 6, 2000.
  • Some assays are preferably performed by transferring one or more assay component to a filter medium adapted for use in a multiwell, e.g., microtiter format (see e.g., U.S. Patent No. 5,939,024).
  • the assays of the present invention can be performed in the microtiter plate format described above.
  • appropriate selection of well dimensions, and reagents makes the microliter scale required in such formats feasible for generating the pre-screened libraries of the invention.
  • the cost remains prohibitive.
  • the target independent assays (and/or target dependent assays) of the present invention are carried out in microfluidic devices or "microlaboratory systems," which allow for integration of the elements required for performing the assay, automation, and minimal environmental effects on the assay system, e.g., evaporation, contamination, operator error.
  • microfluidic devices provide for integratability with e.g., microwell plates, solid substrates, bead arrays and the like.
  • PCT/US 98/06723 filed April 3, 1998, provides examples of formats for integrating microfluidic devices with microtiter plates, solid substrates and the like.
  • the specific configuration of these devices will generally vary depending upon the type of assay and/or assay orientation desired.
  • the screening methods of the invention can be carried out using a microfluidic device having two intersecting channels, or microchannels.
  • multichannel/intersection devices may be employed. The small scale, integratability and self-contained nature of these devices allows for virtually any assay orientation to be realized within the context of the microlaboratory system.
  • cells, enzymes, receptors, substrates, library members and other elements can be flowed in a microscale system by electrokinetic (including either electroosmotic or electrophoretic) techniques, or using pressure-based flow mechanisms, or combinations thereof.
  • Cells in particular are desirably flowed using pressure-based flow mechanisms.
  • Pressure forces can be applied to microscale elements to achieve fluid movement using any of a variety of techniques.
  • Fluid flow (and flow of materials suspended or solubilized within the fluid, including cells or other particles) is optionally regulated by pressure based mechanisms such as those based upon fluid displacement, e.g., using a piston, pressure diaphragm, vacuum pump, probe or the like to displace liquid and raise or lower the pressure at a site in the microfluidic system.
  • the pressure is optionally pneumatic, e.g., a pressurized gas, or uses hydraulic forces, e.g., pressurized liquid, or alternatively, uses a positive displacement mechanism, i.e., a plunger fitted into a material reservoir, for forcing material through a channel or other conduit, or is a combination of such forces.
  • pneumatic e.g., a pressurized gas
  • hydraulic forces e.g., pressurized liquid
  • a positive displacement mechanism i.e., a plunger fitted into a material reservoir, for forcing material through a channel or other conduit, or is a combination of such forces.
  • a vacuum source is applied to a reservoir or well at one end of a channel to draw the suspension through the channel.
  • Pressure or vacuum sources are optionally supplied external to the device or system, e.g., external vacuum or pressure pumps sealably fitted to the inlet or outlet of the channel, or they are internal to the device, e.g., microfabricated pumps integrated into the device and operably linked to the channel. Examples of microfabricated pumps have been widely described in the art. See, e.g., published International Application No. WO 97/02357. Hydrostatic, wicking and capillary forces can also be used to provide pressure for fluid flow of materials such as cells.
  • microfluidic systems can be inco ⁇ orated into centrifuge rotor devices, which are spun in a centrifuge. Fluids and particles travel through the device due to gravitational and centripetal/centrifugal pressure forces.
  • One method of achieving transport or movement of substrates, ligands, library members, and even cells (particularly substrates and library members) through microfluidic channels is by electrokinetic material transport.
  • Electrokinetic material transport systems include systems that transport and direct materials within a microchannel and/or chamber containing structure, through the application of electrical fields to the materials, thereby causing material movement through and among the channel and or chambers, i.e., cations will move toward a negative electrode, while anions will move toward a positive electrode.
  • movement of fluids toward or away from a cathode or anode can cause movement of receptors, enzymes, serum proteins, cells, library members, etc. suspended within the fluid.
  • the fluid can be immobile or flowing and can comprise a matrix as in electrophoresis.
  • electrokinetic material transport and direction systems also include those systems that rely upon the electrophoretic mobility of charged species within the electric field applied to the structure. Such systems are more particularly referred to as electrophoretic material transport systems.
  • electrophoretic material transport systems For electrophoretic applications, the walls of interior channels of the electrokinetic transport system are optionally charged or uncharged. Typical electrokinetic transport systems are made of glass, charged polymers, and uncharged polymers. The interior channels are optionally coated with a material which alters the surface charge of the channel.
  • electrokinetic controllers and systems are described, e.g., in Ramsey WO 96/04547, Parce et al. WO 98/46438 and Dubrow et al., WO 98/49548, as well as a variety of other references noted herein.
  • Modulating voltages are concomitantly applied to the various reservoirs to affect a desired fluid flow characteristic, e.g., continuous or discontinuous (e.g., a regularly pulsed field causing the sample to oscillate direction of travel) flow of labeled components toward a waste reservoir.
  • a desired fluid flow characteristic e.g., continuous or discontinuous (e.g., a regularly pulsed field causing the sample to oscillate direction of travel) flow of labeled components toward a waste reservoir.
  • modulation of the voltages applied at the various reservoirs can move and direct fluid flow through the interconnected channel structure of the device.
  • Figure 1A shows an example of a microfluidic device particularly suited to performing the target independent assays used to produce pre-screened libraries of the present invention.
  • Target independent and/or target dependent assays that are fluorogenic assays, including protease assays, kinase assays, phosphatase assays, among many others are readily adapted for performance in the microfluidic device of Figure 1 A.
  • a chemical composition comprising a member (or members) of a master library is drawn from e.g., a master library array, e.g., a multiwell microtiter plate, through pipettor element 101, into main channel 102, where it contacts a continuous stream of enzyme flowed from reservoir 103, through side channel 104, into main channel 102.
  • a master library array e.g., a multiwell microtiter plate
  • the library member interacts with the flowing enzyme stream.
  • the mixed enzyme/library members are flowed along main channel 102 past the intersection with channel 105.
  • a continuous stream of fluorogenic (or other labeled) substrate which is contained in reservoir 106, is flowed along side channel 105 into main channel 102, whereupon it contacts and mixes with the continuous stream of enzyme/library member.
  • Action of the enzyme upon the substrate produces an increasing level of the fluorescent signal.
  • This increasing signal is indicated by the increasing fluorescence within the main channel as it approaches detection window 107.
  • This increase in signal occurs in the absence of added library members, or in the presence of a library member with no effect on the enzyme/substrate interaction. Where a library member has an effect on the interaction of the enzyme and substrate, a variation appears in the signal produced. For example, assuming a fluorogenic substrate, a library member which inhibits the interaction of the enzyme with its substrate will result in less fluorescent product being produced. This will result in a non-fluorescent, or detectably less fluorescent region within the flowing stream as it passes detection window 107, which corresponds to the subject material region. After passage through the detection window, the flowing stream continues to waste well 108.
  • a similar configuration is adapted by the addition of voltage channels 109 and 111 fluidly connected to independent sources of high/low voltage 110 and 112, illustrated in Figure IB.
  • the microfluidic device includes main fluid channel 204 which runs longitudinally down the central portion of the body of the device.
  • Main channel 204 originates in and is in fluid communication with buffer channel 206, and terminates, and is in fluid communication with waste reservoir 208.
  • Buffer channel 206 is in fluid communication with buffer reservoirs 210 and 212.
  • the device is shown having a number of channels intersecting the main channel 204.
  • buffer channel 214 terminates in, and is in fluid communication with main channel 204 near its originating point, and is, at its other terminus, in fluid communication with buffer reservoir 216.
  • Sample introduction channel 218 also terminates in and is in fluid communication with main channel 204, and is, at its other terminus, in fluid communication with sample reservoir 220. Additional buffer/waste channels 222 and 224, and buffer/waste reservoirs 226 and 228 are also shown.
  • the descriptors for the various wells and channels are primarily offered for pu ⁇ oses of distinguishing the various channels and wells from each other. It will be appreciated that the various wells and channels can be used for a variety of different reagents depending upon the analysis to be performed.
  • Sources Of Assay Components And Integration With Microfluidic Formats Sources of assay components such as cells or isolated proteins, sources of substrates or ligands, and sources of potential modulators, including members of the chemical composition libraries of the invention, can be fluidly coupled to the microchannels noted herein in any of a variety of ways.
  • those systems comprising sources of materials set forth in Knapp et al. "Closed Loop Biochemical Analyzers” (WO 98/45481; PCTJUS98/06723) and Parce et al. "High Throughput Screening Assay Systems in
  • Microscale Fluidic Devices WO 98/00231 and, e.g., in 60/128,643 filed April 4, 1999, entitled “Manipulation of Microparticles In Microfluidic Systems," by Mehta et al. are applicable.
  • a "pipettor element” i.e., a channel in which components can be moved from a source to a microscale element such as a second channel or reservoir
  • a microscale element i.e., a channel in which components can be moved from a source to a microscale element such as a second channel or reservoir
  • the source can be internal or external to a microfluidic device comprising the pipettor element, e.g., a "sipper chip” (basically, a microfluidic device which includes a microscale channel, such as a capillary, which is external to the main body of the microscale device).
  • the device accesses external material sources such as microwell plates, membranes or other solid substrates comprising lyophilized components, bead matrices, wells or reservoirs in the body of the microscale device itself and others through the pipettor element.
  • the source of a cell type, assay component, or library member can be a microwell plate external to the body structure, having, e.g., at least one well with the selected cell type or reagent.
  • a well disposed on the surface of the body structure comprising the selected cell type, component, or reagent a reservoir disposed within the body structure comprising the selected cell type, component or reagent; a container external to the body structure comprising at least one compartment comprising the selected particle type or reagent, or a solid phase structure comprising the selected cell type or reagent in liquid, partially liquid, gel encapsulated, dried, partially dried, lyophilized or otherwise stabilized form.
  • Chemical compositions comprising a library, substrates, targets, reagents and other assay components- are typically supplied to the microfluidic device in liquid form, e.g., in solution.
  • chemical compositions, substrates, targets, reagents, and the like are provided attached to, or embedded in a particulate matrix such as a bead.
  • the particulate matrix can be essentially any discrete, non-soluble material and in some cases, can be flowed through a microscale system.
  • Example particles include beads and biological cells.
  • polymer beads e.g., polystyrene, polypropylene, latex, nylon and many others
  • silica or silicon beads e.g., silica or silicon beads
  • ceramic beads e.g., glass beads, magnetic beads, metallic beads and organic compound beads
  • organic compound beads can be used.
  • the definition for particles as intended herein includes both biological and non-biological particle material.
  • cells are included within the definition of particles for pu ⁇ oses of the present invention.
  • Array particles can have essentially any shape, e.g., spherical, helical, spheroid, rod-shaped, cone-shaped, cubic, polyhedral or a combination thereof (of course they can also be irregular, as is the case for some cell-based particles).
  • Cell based microfluidic assays are described in a variety of publications by the inventors and their co-workers, including, Parce et al. "High Throughput Screening Assay Systems in Microscale Fluidic Devices" WO 98/00231 and Knapp et al.
  • cell-based microfluidic assays are particularly preferred use for cell-based microfluidic assays to screen binding and/or internalization of cell ligands, e.g., cell receptor ligands, drugs, co- factors, etc.
  • Cells optionally exist as sets of particles in a variety of formats in the present invention.
  • cells can be fixed to solid supports such as beads or other microparticles.
  • arrays of the invention optionally include heterogeneous particles comprising solid supports and cells or other components of interest.
  • cells comprise surface proteins and other molecules, it is convenient to fix cell binding molecules (cell receptor ligands, cell wall binding molecules, antibodies, etc.) either to regions of the channels of the microfluidic device, or to particles which are then fixed or localized in position within the microfluidic device.
  • a loading channel region is optionally fluidly coupled to a pipettor channel with a port external to the body structure.
  • the loading channel can be coupled to an electropipettor channel with a port external to the body structure, a pressure -based pipettor channel with a port external to the body structure, a pipettor channel with a port internal to the body structure, an internal channel within the body structure fluidly coupled to a well on the surface of the body structure, an internal channel within the body structure fluidly coupled to a well within the body structure, or the like.
  • the integrated microfluidic system of the invention can include a very wide variety of storage elements for storing reagents to be assessed. These include well plates, matrices, membranes and the like.
  • the reagents are stored in liquids (e.g., in a well on a microtiter plate), or in lyophilized form (e.g., dried on a membrane or in a poious matrix such as a bead), and can be transported to an array component of the microfluidic device using conventional robotics, or using an electropipettor or pressure pipettor channel fluidly coupled to a reaction or reagent channel of the microfluidic system.
  • individual members of the library of chemical compositions are separately introduced into the assay systems described herein, or at least introduced in relatively manageable pools of library members.
  • the devices and systems specifically illustrated herein are generally described in terms of the performance of a few or one particular operation, it will be readily appreciated from this disclosure that the flexibility of these systems permits easy integration of additional operations into these devices.
  • the devices and systems described will optionally include structures, reagents and systems for performing virtually any number of operations both upstream and downstream from the operations specifically described herein.
  • upstream operations include sample handling and preparation operations, e.g., cell separation, extraction, purification, culture, amplification, cellular activation, labeling reactions, dilution, aliquotting, and the like.
  • downstream operations may include similar operations, including, e.g., separation of sample components, labeling of components, assays and detection operations, electrokinetic or pressure-based injection of components into contact with cells or other assay components, or materials released from cells or produced by the reactions of the assays of the invention, or the like.
  • the devices and systems optionally include structures for the storage of one or more master and/or pre-screened libraries, e.g., multiwell plates, pin arrays, bead arrays, etc.
  • the systems can include structures for cold storage, e.g., refrigeration or freezer units, storage of lyophilized materials, liquid storage, and the like.
  • Upstream and downstream assay and detection operations include, without limitation, cell fluorescence assays, cell activity assays, molecular activity assays, receptor/ligand assays, immunoassays, and the like. Any of these elements can be fixed to array members, or fixed, e.g., to channel walls, or the like.
  • An integrated system of the invention includes, for example, one or more multiwell plates, robotic elements (e.g., plate handling mechanisms, transport devices, etc.), controller, microfluidic device, detector, and a computer (e.g., including user input devices, data output devices, etc.).
  • robotic elements e.g., plate handling mechanisms, transport devices, etc.
  • controller e.g., microfluidic device, detector
  • computer e.g., including user input devices, data output devices, etc.
  • Instrumentation In general in the present invention, materials such as cells, fluorescently labeled substrates and/or products are optionally monitored and/or detected so that a function such as enzyme activity can be determined. Depending on the label signal measurements, decisions can be made regarding subsequent fluidic operations, e.g., whether to assay a particular modulator in detail to determine kinetic information.
  • the integrated systems described herein generally include microfluidic devices, as described above, in conjunction with additional instrumentation for controlling fluid transport, flow rate and direction within the devices, detection instrumentation for detecting or sensing results of the operations performed by the system, processors, e.g., computers, for instructing the controlling instrumentation in accordance with preprogrammed instructions, receiving data from the detection instrumentation, and for analyzing, storing and inte ⁇ reting the data, and providing the data and inte ⁇ retations in a readily accessible reporting format.
  • processors e.g., computers
  • an integrated system suitable for the performance of the target independent assays (and/or the target dependent assays) of the invention is the Labchip® microfluidic device high throughput screening system (HTS) by Caliper (Caliper Technologies Co ⁇ ., Mountain View, CA; www.calipertech.com.).
  • the device includes, e.g., robotics, fluidics, plate handler, detector, etc.
  • a plate handling station is used for the generation of common arrangements involving transfer of components to or from microtiter plates.
  • CCS Packard www.carlcreative.com
  • components for liquid handling e.g., dispensing, pipetting and the like
  • transport and plate handling via its PlateTrakTM, SideTrak®, and PlateStakTM elements. Controllers
  • a variety of controlling instrumentation is optionally utilized in conjunction with the microfluidic devices described above, for controlling the transport and direction of fluids and/or materials within the devices of the present invention, e.g., by pressure-based or electrokinetic control.
  • fluid transport and direction are controlled in whole or in part, using pressure based flow systems that inco ⁇ orate external or internal pressure sources to drive fluid flow.
  • Internal sources include microfabricated pumps, e.g., diaphragm pumps, thermal pumps, Lamb wave pumps and the like that have been described in the art. See, e.g., U.S. Patent Nos. 5,271,724, 5,277,556, and 5,375,979 and Published PCT Application Nos. WO 94/05414 and WO 97/02357.
  • the systems described herein can also utilize electrokinetic material direction and transport systems.
  • external pressure sources are used, and applied to ports at channel termini. These applied pressures, or vacuums, generate pressure differentials across the lengths of channels to drive fluid flow through them.
  • differential flow rates on volumes are optionally accomplished by applying different pressures or vacuums at multiple ports, or preferably, by applying a single vacuum at a common waste port and configuring the various channels with appropriate resistance to yield desired flow rates.
  • Example systems are described, e.g., in USSN 09/238,467 filed 1/28/99.
  • the controller systems are appropriately configured to receive or interface with a microfluidic device or system element as described herein.
  • the controller and/or detector optionally includes a stage upon which the device of the invention is mounted to facilitate appropriate interfacing between the controller and/or detector and the device.
  • the stage includes an appropriate mounting/alignment structural element, such as a nesting well, alignment pins and/or holes, asymmetric edge structures (to facilitate proper device alignment), and the like. Many such configurations are described in the references cited herein.
  • the controlling instrumentation discussed above is also used to provide for electrokinetic injection or withdrawal of material downstream of the region of interest to control an upstream flow rate.
  • the same instrumentation and techniques described above are also utilized to inject a fluid into a downstream port to function as a flow control element. Detector
  • the devices herein optionally include signal detectors, e.g., which detect atomic mass, fluorescence, phosphorescence, radioactivity, pH, charge, absorbance, luminescence, temperature, magnetism or the like. Fluorescent detection is especially preferred and generally used for detection of many compounds (however, as noted, upstream and downstream operations can be performed on cells, assay components, library members or the like, which can involve other detection methods). In addition to fluorescence, evaporative light scattering and mass spectrometry are two additional detection mechanisms that are useful in the context of the present invention.
  • the detector(s) optionally monitors one or a plurality of signals from downstream of an assay mixing point in which a labeled substrate and a cell or other assay component, are mixed with a library member.
  • the detector can monitor a plurality of optical signals which correspond to "real time" assay results.
  • Example detectors include photo multiplier tubes, spectrophotometers, mass spectrometers, CCD arrays, a scanning detector, a microscope, a galvo-scann or the like.
  • Cells, labels or other components which emit a detectable signal can be flowed past the detector, or, alternatively, the detector can move relative to the array to determine cell position (or, the detector can simultaneously monitor a number of spatial positions corresponding to channel regions, e.g., as in a CCD array).
  • the detectors used in the devices and systems of the present invention optionally include, e.g., one or more of: an optical detector, a microscope, a CCD array, a photomultiplier tube, a photodiode, an emission spectroscope, a fluorescence spectroscope, a phosphorescence spectroscope, a luminescence spectroscope, a spectrophotometer, a photometer, a nuclear magnetic resonance spectrometer, an electron paramagnetic resonance spectrometer, an electron spin resonance spectroscope, a turbidimeter, a nephelometer, a Raman spectroscope, a refractometer, an interferometer, an x-ray diffraction analyzer, an electron diffraction analyzer, a polarimeter, an optical rotary dispersion analyzer, a circular dichroism spectrometer, a potentiometer, a chronopotentiometer, a
  • detectors for use in the methods and devices of the invention include optical detectors and, e.g., electrospray ionization mass spectrometers, which can, e.g., be proximal to or coupled to one or more microscale channels of a device. Detector embodiments including mass spectrometer elements are discussed further below.
  • the detector can include or be operably linked to a computer, e.g., which has software for converting detector signal information into assay result information (e.g., kinetic data of modulator activity), or the like.
  • Signals are optionally calibrated, e.g., by calibrating the microfluidic system by monitoring a signal from a known source.
  • a microfluidic system can also employ multiple different detection systems for monitoring the output of the system.
  • Detection systems of the present invention are used to detect and monitor the materials in a particular channel region (or other reaction detection region). Once detected, the flow rate and velocity of cells in the channels is also optionally measured and controlled as described above. As described in PCTJUS98/11969, and 60/142,984, correction of kinetic information based upon flow velocity and other factors can be used to provide accurate kinetic information in flowing systems.
  • detection systems include optical sensors, temperature sensors, mass sensors, pressure sensors, pH sensors, conductivity sensors, and the like. Each of these types of sensors is readily inco ⁇ orated into the microfluidic systems described herein. In these systems, such detectors are placed either within or adjacent to the microfluidic device or one or more channels, chambers or conduits of the device, such that the detector is within sensory communication with the device, channel, or chamber.
  • within sensory communication generally refers to the placement of the detector in a position such that the detector is capable of detecting the property of the microfluidic device, a portion of the microfluidic device, or the contents of a portion of the microfluidic device, for which that detector was intended.
  • a pH sensor placed in sensory communication with a microscale channel is capable of determining the pH of a fluid disposed in that channel.
  • a temperature sensor placed in sensory communication with the body of a microfluidic device is capable of determining the temperature of the device itself.
  • Particularly preferred detection systems include optical detection systems for detecting an optical property of a material within the channels and/or chambers of the microfluidic devices that are inco ⁇ orated into the microfluidic systems described herein. Such optical detection systems are typically placed adjacent to a microscale channel of a microfluidic device, and are in sensory communication with the channel via an optical detection window that is disposed across the channel or chamber of the device. Optical detection systems include systems that are capable of measuring the light emitted from material within the channel, the transmissivity or absorbance of the material, as well as the materials spectral characteristics. In preferred aspects, the detector measures an amount of light emitted from a material, such as a fluorescent or chemiluminescent material.
  • a material such as a fluorescent or chemiluminescent material.
  • the detection system will typically include collection optics for gathering a light based signal transmitted through the detection window, and transmitting that signal to an appropriate light detector.
  • Microscope objectives of varying power, field diameter, and focal length are readily utilized as at least a portion of this optical train.
  • the light detectors are optionally spectrophotometers, photodiodes, avalanche photodiodes, photomultiplier tubes, diode arrays, or in some cases, imaging systems, such as charged coupled devices (CCDs) and the like.
  • the detection system is typically coupled to a computer (described in greater detail below), via an analog to digital or digital to analog converter, for transmitting detected light data to the computer for analysis, storage and data manipulation.
  • the detector typically includes a light source which produces light at an appropriate wavelength for activating the fluorescent material, as well as optics for directing the light source through the detection window to the material contained in the channel or chamber.
  • the light source can be any number of light sources that provides an appropriate wavelength, including lasers, laser diodes and LEDs. Other light sources are used in other detection systems. For example, broad band light sources are typically used in light scattering/transmissivity detection schemes, and the like. Typically, light selection parameters are well known to those of skill in the art.
  • mass spectrometry provides another preferred method for detecting the results of target dependent or target independent screening assays.
  • Mass spectrometry is a widely used analytical technique that can be used to provide information about, e.g., the isotopic ratios of atoms in samples, the structures of various molecules, including biologically important molecules, and the qualitative and quantitative composition of complex mixtures.
  • Common mass spectrometer systems include a system inlet, an ion source, a mass analyzer, and a detector which are typically under vacuum. The detector is typically operably connected to a signal processor and a computer.
  • Deso ⁇ tion ion sources for use in the present invention, include field deso ⁇ tion (FD), electrospray ionization (ESI), chemical ionization, matrix-assisted deso ⁇ tion/ionization (MALDI), plasma deso ⁇ tion (PD), fast atom bombardment (FAB), secondary ion mass spectrometry (SIMS), and thermospray ionization (TS).
  • ESI sources are especially preferred.
  • Mass spectrometry is well-known in the art. References specifically addressing the interfacing of mass spectrometers with microfluidic devices include, e.g., Karger, et al., U.S. Pat. No. 5,571,398, "PRECISE CAPILLARY ELECTROPHORETIC INTERFACE FOR SAMPLE COLLECTION OR ANALYSIS" and Karger, et al. U.S. Pat. No. 5,872,010 "MICROSCALE FLUID HANDLING SYSTEM.” General sources of information about mass spectrometry include, e.g., Skoog, et al. Principles of Instrumental Analysis (5 th Ed.) Hardcourt Brace & Company, Orlando (1998).
  • mass spectrometers are well suited to interface with microfluidic devices, because the usual input into a microfluidic system is a capillary channel. In the present invention, this mass spectrometry capillary is simply fluidly coupled to channel in the microscale system.
  • Methods of affixing external capillaries to microscale systems include various bonding and/or drilling operations, e.g., as described, e.g., in Parce, et al., U.S. Pat. No.
  • microfluidic device configurations which are specifically adapted to screening by mass spectrometry are described in "MICROSCALE ASSAYS AND MICROFLUIDIC DEVICES FOR TRANSPORTER, GRADIENT INDUCED, AND BINDING ACTIVITIES" by Parce et al., USSN 60/176,093, filed January 14, 2000.
  • FLIPR Fluorescence Imaging Plate Reader
  • This device can be adapted to the present invention, e.g., by using the assays of the invention in the indicated methods.
  • the detector can exist as a separate unit, but can also be integrated with the controller system, into a single instrument. Integration of these functions into a single unit facilitates connection of these instruments with the computer (described below), by permitting the use of few or a single communication port(s) for transmitting information between the controller, the detector and the computer.
  • either or both of the controller system and/or the detection system are coupled to an appropriately programmed processor or computer which functions to instruct the operation of these instruments in accordance with preprogrammed or user input instructions, receive data and information from these instruments, and inte ⁇ ret, manipulate and report this information to the user.
  • the computer is typically appropriately coupled to one or both of these instruments (e.g., including an analog to digital or digital to analog converter as needed).
  • the computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.
  • the software then converts these instructions to appropriate language for instructing the operation of the fluid direction and transport controller to carry out the desired operation.
  • the computer then receives the data from the one or more sensors/detectors included within the system, and inte ⁇ rets the data, either provides it in a user understood format, or uses that data to initiate further controller instructions, in accordance with the programming, e.g., such as in monitoring and control of flow rates, temperatures, applied voltages, and the like.
  • the computer typically includes software for the monitoring of materials in the channels. Additionally, the software is optionally used to control electrokinetic or pressure modulated injection or withdrawal of material. The injection or withdrawal is used to modulate the flow rate as described above, to mix components, and the like.
  • the target independent and/or target dependent assays of the invention are performed in an integrated system including a multi-module workstation.
  • the multi-module workstations of the invention typically include one or more microfluidic device, as well as optionally including one or more devices for performing high-throughput assays directly in microtiter plates, e.g., plate handlers such as the ORCATM and PlateTrakTM described above, plate readers, or from pin or bead arrays.
  • Screening modules comprising microfluidic or other microscale devices, multiwell plate readers, (e.g., fluorimetric, photometric, colorimetric plate readers such as the Elx800 from Bio-Tek Instruments, Inc: www.copalis.com; and the Ultramark and Benchmark Plate readers from Bio-Rad: www.bio-rad.com ), and/or devices for the automated, e.g., robotic, performance of target independent (and/or target dependent) assays, or any combination thereof, are arranged to provide access to the individual modules by a robotic mechanism.
  • multiwell plate readers e.g., fluorimetric, photometric, colorimetric plate readers such as the Elx800 from Bio-Tek Instruments, Inc: www.copalis.com; and the Ultramark and Benchmark Plate readers from Bio-Rad: www.bio-rad.com
  • devices for the automated e.g., robotic, performance of target independent (and/or target dependent) assays, or any combination thereof, are arranged to provide access to the individual modules by
  • Each module which can be operated singly, or in conjunction with one or more additional module in the multi-module workstation, includes the necessary means and components for carrying out a specified target independent or target dependent assay, or a subportion thereof (e.g., sample preparation, incubation, etc.), as described in further detail above.
  • the robotic mechanism can include an armature, conveyor belt, or other transport mechanism for obtaining and manipulating one or more assay component or library array, e.g., a chemical composition library arrayed in a stack of multiwell plates.
  • the armature or other transport mechanism can be movably deployed between modules of the multi-module workstation (or the modules can be movably deployed), and conveys one or more assay component, including but not limited to library arrays, reagents, cells, substrates, etc., to the individual modules (or, alternately, the modules can be moved to the library members, or both components can be moved to achieve operable coupling of the module and the library).
  • the armature or other transport mechanism can be transported between modules (or vice-versa) by a mechanism such as a track, a cable and pulley, a conveyor belt, rollers, robotic means or the like, e.g., positioned proximal to or between one or more screening modules, or between a screening module and one or more assay components.
  • a mechanism such as a track, a cable and pulley, a conveyor belt, rollers, robotic means or the like, e.g., positioned proximal to or between one or more screening modules, or between a screening module and one or more assay components.
  • Figure 9 illustrates an exemplary configuration of such a workstation.
  • Substrates comprising a master library array, e.g., in one or more multiwell plates, are arranged in an ordered manner in plate storage module 1012 (optionally including, e.g., refrigeration element, freezer element, or the like).
  • the user selects assay parameters via user input element 1015 (e.g., a keyboard, touch screen, mouse, or the like) which conveys an instruction set through controller element 1013 (e.g., a P.C. computer, drive mechanism, etc.) to track robot 1010.
  • controller element 1013 e.g., a P.C. computer, drive mechanism, etc.
  • Selected substrates are retrieved by robotic armature 1011 from plate storage module 1012 and transported by track robot 1010 to one of assay modules 1001 through 1008.
  • These assay modules will typically have the appropriate assay device elements (e.g., microfluidic chips and detection elements) for performing the relevant assay.
  • the substrate Upon completion of a first target independent assay, the substrate is optionally transported to a second assay module for evaluation in an additional target independent or target dependent assay. Results of one or more of the target independent and/or target dependent assays are displayed on output device 1014 (although depicted as a single output device, multiple output elements can be used in the system, e.g., each coupled to one or more of the assay modules).
  • Controller element 1013 optionally includes storage media for storing databases corresponding to the libraries of interest. The storage media can also be in separate elements such as computers or disk drives.
  • the storage media is operably coupled to the assay modules to store the results of the assays.
  • Databases which include the positions of assayed components and information for which assays the assayed components show desirable activity can also be included in controller element 1013 or in separate computers or other coupled data storage systems.
  • multiple pre- screening target independent assays and/or target dependent screening assays can be performed at each of the assay modules.
  • a single assay module running multiple target independent assays and/or target dependent screening assays can be used instead of multiple assay modules to achieve similar results.
  • One aspect of the present invention includes entering descriptors for members of a library of pre-screened compositions into a database.
  • descriptors can include any relevant information which describes any physical, logical or spatial characteristic or activity of any of the members of the pre-screened composition library (or any of the other libraries noted herein).
  • the database is embodied in a computer or computer readable medium for access by a user and/or integrated system, e.g., which acts on the library based upon information in the database (e.g., physically manipulates one or more element of the integrated system, e.g., plates, microfluidic components or the like in response to the information in the database).
  • a user and/or integrated system e.g., which acts on the library based upon information in the database (e.g., physically manipulates one or more element of the integrated system, e.g., plates, microfluidic components or the like in response to the information in the database).
  • Standard desktop applications such as word processing software (e.g., Microsoft WordTM or Corel WordPerfectTM), database software (e.g., spreadsheet software such as Microsoft ExcelTM, Corel Quattro ProTM, or database programs such as Microsoft AccessTM or ParadoxTM) can be adapted to the present invention by inputting a character string corresponding to the descriptor to be entered into the database.
  • the computer system embodying the database can include the foregoing software having the appropriate character string information, e.g., used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX system) to manipulate strings of characters.
  • Systems for analysis in the present invention can include a digital computer with software for tracking library entries, assay results, or the like and for using that information to manipulate the system (e.g., to select appropriately active members identified m pre-screening target independent assay steps or in target dependent assay screening steps
  • the computer can be, e.g., a PC (Intel x86 or Pentium chip- compatible computer, running any available operating system, e.g., DOSTM, OS2TM WINDOWSTM WINDOWS NTTM, WINDOWS95TM, WINDOWS98TM, LINUX, Apple OS, Power PC, UNIX) or other commercially common computer which is known to one of skill.
  • Software for manipulating information desc ⁇ ptor elements is available, or can easily be constructed by one of skill using a standard programming language such as Visualbasic, Fortran, Basic, Java, or the like.
  • kits for producing the bra ⁇ es of the invention typically include microfluidic devices, systems, modules and workstations for performing the assays of the invention.
  • a kit optionally contains additional components for the assembly and/or operation of a multimodule workstation of the invention including, but not rest ⁇ cted to robotic elements (e g., a track robot, a robotic armature, or the like), plate handling devices, fluid handling devices, and computers (including e.g., input devices, monitors, cp u., and the like).
  • the microfluidic devices desc ⁇ bed herein are optionally packaged to include reagents for performing the device's preferred function.
  • the kits optionally include any of microfluidic devices desc ⁇ bed along with assay components, e.g., buffers, reagents, enzymes, serum proteins, receptors, etc., for performing the target independent assays used to produce the pre-screened hbra ⁇ es of the invention.
  • kits optionally include pre-measured or pre-dosed reagents that are ready to inco ⁇ orate into the assay methods without measurement, e.g., pre-measured fluid aliquots, or pre-weighed or pre-measured solid reagents that may be easily reconstituted by the end-user of the kit
  • kits also typically include approp ⁇ ate instructions for using the devices and reagents, and in cases where reagents are not predisposed in the devices themselves, with approp ⁇ ate instructions for introducing the reagents into the channels and/or chambers of the device.
  • these k ts optionally include special ancillary devices for introducing mate ⁇ als into the microfluidic systems, e g., approp ⁇ ately configured sy ⁇ nges/pumps, or the like (in one preferred embodiment, the device itself comp ⁇ ses a pipettor element, such as an electropipettor for introducing mate ⁇ al into channels and chambers within the device)
  • kits typically include a microfluidic device with necessary reagents predisposed in the channels/chambers of the device.
  • such reagents are provided in a stabilized form, so as to prevent degradation or other loss during prolonged storage, e.g., from leakage.
  • a number of stabilizing processes are widely used for reagents that are to be stored, such as the inclusion of chemical stabilizers (i.e., enzymatic inhibitors, microcides/bacteriostats, anticoagulants), the physical stabilization of the material, e.g., through immobilization on a solid support, entrapment in a matrix (i.e., a bead, a gel, etc.), lyophilization, or the like.
  • chemical stabilizers i.e., enzymatic inhibitors, microcides/bacteriostats, anticoagulants
  • the physical stabilization of the material e.g., through immobilization on a solid support, entrapment in a matrix (i.e., a bead, a gel, etc.), lyophilization, or the like.
  • kits of the present invention are typically packaged together in a single package or set of related packages.
  • the package optionally includes written instructions for carrying out one or more target independent assay in accordance with the methods described herein.
  • Kits also optionally include packaging materials or containers for holding microfluidic device, system or reagent elements.
  • target independent assays performed in a microfluidic device are provided by way of illustration and not by way of limitation.
  • One of skill will recognize a variety of parameters which can be changed while still achieving substantially similar results. Any one or more of the following assays can be performed simultaneously or sequentially to provide the pre-screened libraries of the present invention.
  • HSA ASSAY Human serum albumin (HSA) is the most abundant protein in blood, and is bound by a wide variety of biological materials. Many drugs bind HSA reversibly and with high affinity. In drug discovery, the determination of drug binding to HSA is used to prioritize potential leads obtained from a target screening before proceeding to animal and/or clinical trials.
  • large libraries of chemical compounds are first evaluated for binding to HSA, to provide pre-screened libraries that are then evaluated in one or more target dependent, or screening assay.
  • the HSA assay protocol was adapted from published fluorogenic approaches to detect compounds interacting with human serum albumin in buffer (e.g., Epps et al. (1995) Analytical Biochemistry 227:342). In brief, this assay is a competitive binding assay that measures displacement of a dye binding to HSA by a library member.
  • Two major sites on albumin have been determined to be important for the binding of both dyes and potential drugs.
  • two different dyes were used that bind selectively to each of the major binding sites on albumin (Site I and II), respectively: dansyl- 1-sulfonamide (DNSA) and dansyl sarcosine (DS).
  • DNSA dansyl- 1-sulfonamide
  • DS dansyl sarcosine
  • DNSA dansyl- 1-sulfonamide
  • DS dansyl sarcosine
  • These dyes possess the property of greatly increased quantum yield or fluorescence upon binding to albumin.
  • a compound that binds to albumin through one of these two sites displaces the dye and result in a fluorescence decrease which is optionally used to calculate the Kd or rank order of binding to albumin.
  • Assays were run with only one dye at a time, but the dyes are spectrally compatible and could be run simultaneously.
  • the albumin-dye concentrations can be chosen to yield predetermined Kd values, and can be about ⁇ 4 ⁇ M or better; corresponding drug concentrations are then on the order of 20 ⁇ M.
  • the HSA assays were performed in a microfluidic device of the design illustrated in Figure 1 A, and previously described herein.
  • the buffer system is phosphate- buffered saline (PBS) at pH 7.4, and typical assay conditions require times of 10-60 sec/compound and compound amounts of less than 0.1 pmol/measurement at one compound concentration.
  • De-fatted and essentially globulin-free human serum albumin was used for the assay.
  • the assay is as follows:
  • the site I dye DNSA
  • the albumin concentration is 15 ⁇ M and the dye is the site I specific dye, DNSA at 30 ⁇ M.
  • the experiments were run at room temperature.
  • the fluorescence excitation is centered at 330 nm and the emission is at 475-525 nm.
  • the pressure applied to achieve flow was approximately 32 inches of water.
  • the duration of experiments ranged from 10 to 60 minutes.
  • the instrument used to collect the data is shown in Figure 1A.
  • albumin and DNSA were pre-mixed in the PBS buffer described above and added to wells 103 and 106 on the microfluidic device illustrated in Fig. 1A.
  • the albumin and dye can also be fed in separately to the main pipettor channel, dye from well 103, albumin from well 106).
  • the inhibitor in the HSA-drug binding plot ( Figure 3A) is phenylbutazone, a drug known to bind to site I on HSA.
  • the drug concentrations are ⁇ 7, 17, 35, 55, 75, 110, 150, and 180 ⁇ M.
  • the assay cycle time for each sample is 25 seconds.
  • the % inhibition values in the inhibition vs phenylbutazone plot ( Figure 3B) were extracted from data shown in Figure 3A (where two repetitions are shown). The assay was run for ⁇ 45 minutes repeating the same titration (8 different inhibitor concentrations) for 10 repetitions.
  • the different drugs are at 175 ⁇ M in 1% DMSO/PBS buffer.
  • the cycle time was 30 seconds/sample and the microfluidic device ( Figure 1) used was coated with a PEG (polyethylene glycol) coating.
  • the six different drugs used were: phenylbutazone, sulfinpyrazone, acetylsalicylic acid, tolbudamide, warfarin and ibuprofen.
  • HSA binding assay compilation the consumption was based on experimental conditions given above and within the range of concentrations given.
  • the cycle time was the current, non-optimized cycle time.
  • the percent DMSO that could be tolerated by the assay was tested in a cuvette format. Rank order of drug binding is maintained up to ⁇ 5% DMSO.
  • Assay Reagents 50-500 femtomoles/datapoint; 10 ⁇ l/8 hrs.
  • Glutathione-s-transferase plays a significant role in antioxidant defense and is part of the biochemical adaptation of animals to environmental stress.
  • GST catalyzes the conjugation of reduced glutathione to a wide variety of endogenous and exogenous lipid-soluble xenobiotics, including dimethylaniline analogs, aminopyrine, imipramine, chlo ⁇ romazine, and related antidepressants, and pesticides (Figure 4A).
  • (1.) indicates the structure of Glutathione ( ⁇ -Glu-Cys-Gly)
  • (2.) indicates the general scheme of reactions catalyzed by GST on an electrophillic substrate.
  • GST assays are utilized to obtain pre-screened libraries with desirable characteristics with respect to GST activity. It will be appreciated that a desirable level of GST activity varies with the intended application of the compounds being evaluated. GST Assays were performed in 100 mM bisTRIS at pH 6.5, 1M NDSB
  • FIG. 4 panel B illustrates the structure of example substrates for performing the GST assay, as well as the results of the assay (panel C).
  • (1.) is a colorimetric substrate (2,4-dinitro- chlorobenzene);
  • (2.) is a fluorescent substrate (a coumarin derivative),
  • (3.) is a fluorescein derivative.
  • Figure 4C shows data for the fluorescein derivative in the planar chip experiment, in lOOmM Bis-Tris, pH 6.5, 1M NDSB, 5mM NaCl.
  • EXAMPLE 3 PROTEASE ASSAYS
  • proteases are enzymes that degrade proteins by hydrolyzing peptide bonds between amino acid residues. They are also known as proteinases. Categories of proteases include thiol proteases, acid proteases, serine proteases, and the like. Example proteases include, but are not limited to, carboxypeptidase A, subtilisin, papain, and pepsin. Protease assays are described in greater detail in, e.g., U.S. Patent Application No. 09/093,542, filed June 8, 1998, which is inco ⁇ orated herein by reference.
  • a substrate e.g., a protein
  • a protease is mixed with or otherwise contacted by a protease.
  • a variety of fluorogenic protease substrates are commercially available, including, e.g., p-nitrophenolic compounds, and the like.
  • the protease catalyzes a reaction with the substrate, thus forming products, such as protease cleavage products.
  • protease assays are utilized to evaluate chemical compositions for their ability to modulate, e.g., inhibit or activate, the protease.
  • a protease inhibition assay is performed in a continuous-flow format in the microscale channels of a microfluidic device.
  • the reactants move through the channels due to the application of pressure or electrokinetic forces as described above.
  • a protease is optionally flowed through a microfluidic channel under pressure.
  • the protease is then contacted by or mixed with a substrate, e.g., a protein, peptide or the like.
  • substrate e.g., a protein, peptide or the like.
  • Substrates are typically labeled, e.g., fluorogenic substrates.
  • a protease catalyzes the hydrolysis of a substrate producing degraded protein products, e.g., cleaved protein fragments.
  • protease inhibition assay provides, e.g., information on the types of compounds that inhibit proteases and the level of inhibition they provide.
  • pepstatin acts as a protease inhibitor by inhibiting the activity of carboxyl proteases.
  • the present invention provides high throughput assay systems suitable for testing many compositions in protease inhibition assays in a short amount of time.
  • the chemical compositions of the master library are optionally added before, during, or after the enzyme and substrate are contacted, depending on the format of the assay.
  • a library member is introduced into the microscale channel in which the protease reaction is carried out.
  • the reaction proceeds in the presence of the potential modulator.
  • a potential modulator compound is optionally drawn from a microtiter plate containing a plurality of samples.
  • the plurality of samples optionally comprises potential modulators, inhibitors, and/or activators.
  • Protein kinases catalyze the phosphorylation of proteins by transferring the ⁇ - phosphate group from ATP onto the hydroxy group of a serine, threonine, or tyrosine residue of a target peptide or protein. It has been estimated that as many as 2-3% of all genes in eukaryotic organisms encode protein kinases (Cobb et al. (1991) Cell Regul 2:965). While many of these are potential targets for drug development in the treatment of a wide variety of diseases, including cancer, as a group, they are the subject of important target independent assays.
  • Kinase assays are generally described in U.S. Patent Application No. 60/108,628, inco ⁇ orated herein by reference. Kinase activity has traditionally been assayed using radioactive ATP and following the transfer of the isotopically labeled phosphate from ATP to a peptide or protein substrate.
  • a typical kinase assay of the present invention involves the use of a fluorescently tagged peptide, e.g., "KEMPtide.”
  • Phosphorylation is detected as a shift in the mobility of labeled KEMPtide.
  • Figure 6A schematically illustrates the kinase mobility shift assay. As depicted, binding results in a change in the mobility of bound species, resulting in a change in detectable signal. Assay results in the presence of varying levels of inhibiting compounds are illustrated in Figures 6B-E, including the calculation of kinetic constants ( Figure 6C).
  • Figure 6B shows an ATP Titration and detection by mobility shift for a protein kinase A assay.
  • the buffer conditions were lOOmM HEPES, pH7.5, 5mM MgCl 2 , 1M NDSB-195, 10 mM DTT, 0.01% Triton.
  • Figure 6C shows a lineweaver- Burk plot and ATP titration for a protein kinase A assay ( ⁇ ; ⁇ ; representing two series of assays).
  • 6D shows results of a protein kinase A assay with lO ⁇ M H-9 inhibitor injections.
  • the buffer conditions were 100 mM HEPES pH 7.5, 5 mM MgCl 2 , 1 M NDSB-195, 10 mM DTT, 0.01% Triton X-100, 0.05mM ATP, lO ⁇ M Fl-Kemptide substrate + 140 nM PKA catalytic subunit.
  • 6E shows the results of a Protein Kinase A assay performed in a "sipper chip" (a chip with an external capillary for sampling fluid from sources external to the chip, such as a microtiter dish). The results show titration of an inhibition, with detection by mobility shift.
  • the buffer conditions were 100 mM HEPES pH 7.5, 5 mM MgCl 2 , 1 M NDSB-195, 10 mM DTT, 0.01% Triton X-100, 0.05mM ATP, 100 nM PKA catalytic subunit 10 ⁇ M Fl-Kemptide substrate.
  • the phosphatase assay utilizes a fluorogenic substrate dFMU (6,8-difluoro-4-methylumbeliferyl phosphate), which produces a fluorescent signal upon dephosphorylation (e.g., upon incubation with a phosphate).
  • dFMU fluorogenic substrate
  • the reaction is described in detail in Kopf-Sill et al., U.S Patent Application No. 09/093,542 filed June 8, 1998.
  • Phosphatase reactions were performed in 1M NDSB-195 in 50 mM HEPES, pH7.9.
  • Reagent concentrations were 125 nM LAR and 50 ⁇ M dFMUP.
  • the system was programmed to run a series of controls followed by the enzyme plus substrate, optionally including a known inhibitor.
  • the total current flux remained constant in the main reaction channel with the proportion of overall flux from each reagent and buffer well being selected to provide the desired final reagent concentration in the main reaction channel.
  • the fluorescence response was monitored in each step.
  • Figure 7A shows typical data obtained from a phosphatase assay (a substrate titration) performed in a microfluidic device.
  • Raw data for the K m , V max , k cat and Ki are plotted in Figure 7B.
  • G-Protein coupled receptor assays are a target independent assay of the present invention.
  • G-Protein coupled receptor assays typically measure calcium flux across a cell membrane using a labeled calcium indicator, as illustrated schematically in Figure 8A.
  • WT3-CHO cells were obtained from ATCC and cultured in RPMI 1640 containing 10% FBS, 10 mM HEPES, Pen/Strep, 1 mM Sodium Pyruvate and 2 mM L-Glutamate, 100 ⁇ g/ml G418.
  • the cells (10 x 106 cells/5 ml) were then loaded with Fluo-3 (Molecular Probes Inc., Eugene, OR) by incubation with 4 ⁇ M Fluo-3-AM in Hank's balanced salt solution (HBSS) containing 1 mg/ml BSA, 20 mM HEPES, 0.04% pluronic acid, 2.5 mM probenecid for 40 min at 37° C.
  • HBSS Hank's balanced salt solution
  • the cells were counterstained with 1 ⁇ M Syto-62 (Molecular Probes Inc.) for 15 min at room temperature after Fluo-3 loading.
  • the dye loaded cells were washed twice by resuspending the pelleted cells in HBSS containing HEPES and 1 mg/ml BSA.
  • the cells were resuspended in Cell Analysis Buffer consisting of the same wash medium with the addition of a density agent, Hypaque (Pharmacia Biotech)or Optiprep (Nycomed), added to match the density of the cells and 8% (w/v) Ficoll-400 (Fluka) to adjust the buffer viscosity to 4.5 centipoise.
  • a density agent Hypaque (Pharmacia Biotech)or Optiprep (Nycomed)
  • the G-Protein coupled receptor assay was performed in a microfluidic device ( Figure 8B) with a pipettor element 801, etched with 90 ⁇ m wide by 20 ⁇ m deep channels and are configured as shown in Figure 8B.
  • the design of the microfluidic device is such that the given viscosity of buffers, the sample in HBSS + HEPES is diluted 1/3 with buffer + cells, and the agonist from the agonist well 802 is diluted 1/5 with the cell-sample mixture.
  • the channels were filled with cell analysis buffer and the wells were filled with 18 ⁇ l of assay medium.
  • Waste well 804 was connected to a vacuum source set at 0.8 lbs/sq. inch to start the flow of buffer to this empty well.
  • Cells were added into the cell well 806, and cells flowing through the main channel 803 were interrogated by the epifluoresence detector 805 down-stream of the agonist addition point.
  • the flow rate after agonist addition was 0.8 mm sec and the detector was set at a position that allowed an incubation time of 20 sec after agonist addition prior to detection. Samples were drawn from 96 well microplates at 30 second intervals.
  • the data stream was collected at 20 hertz in a Agilent 2100 Bioanalyzer using Caliper LabChip® Technology. Fluo-3 emission was collected at 520 nm using a green filter and Syto-62 emission was collected at 680 nm using a red filter: a blue LED at 488 nm excitation wavelength was used for both dyes. A software filter was used to segregate the fluorescent emission readings collected when cells were in the reading area of the detector. The ratio of the Fluo-3/Syto-62 cell readings were averaged during the 30 sec sampling period after background subtraction. Fluorescein dye injections were used to mark the start and end of groups of 20 sample injections. The data are illustrated in Figures 8C-8F. EXAMPLE 6: CYTOCHROME P450 ASSAY
  • Cytochrome P450 assays are important tools for assessing the metabolic and potentially toxic effects of a drug lead.
  • Human cytochrome P450 preparations are available commercially from companies such as Gentest Co ⁇ oration. These enzymes carry out metabolic oxidation of chemical compounds.
  • fluorogenic substrates available for monitoring P450 activity, e.g., the coumarin-derivatives, resorufin-derivative or fluorescein-derivatives obtainable from Molecular Probes.
  • fluorogenic substrates for monitoring the activity of these enzymes means that these assays can be carried out in a continuous-flow microfluidic device-based assay format.
  • the net consumption of reagents in the microfluidic device format was calculated from the assumed reaction conditions and compared to that calculated for the microplate assay protocol listed on the Gentest website (Table 5).
  • running the assay in the microfluidic device format yields a 280-fold reduction in enzyme consumption and a 16,500-fold reduction in substrate consumption, with an estimated throughput of approximately 2000 assay per 12 hour day.

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Abstract

La présente invention concerne un système et des procédés de génération au moyen de dosages ne dépendant pas de la cible de bibliothèques préalablement criblées, telles des bibliothèques de compositions chimiques préalablement criblées utiles pour le criblage de médicaments. Ces procédés comprennent, de manière caractéristique, un criblage de bibliothèques maîtresses dans des dispositifs microfluidiques pour des effets qui sont corrélés à un ou plusieurs paramètres indépendants pas de la cible. L'invention concerne également des postes de travail à pluralité de modules, tels des postes de travail microfluidiques, et des systèmes intégrés, pour effectuer des dosages indépendants de la cible.
EP00932407A 1999-05-13 2000-05-12 Systemes et procedes de dosage ne dependant pas de la cible Withdrawn EP1180244A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US13391999P 1999-05-13 1999-05-13
US133919P 1999-05-13
US16421899P 1999-11-09 1999-11-09
US164218P 1999-11-09
US19515900P 2000-04-06 2000-04-06
US195159P 2000-04-06
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WO2000070353A2 (fr) 2000-11-23

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