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WO1990003399A1 - Proteines de transport d'acides amines, analogues d'acides amines, appareil d'analyse et utilisations de ces elements pour le traitement et le diagnostic du cancer - Google Patents

Proteines de transport d'acides amines, analogues d'acides amines, appareil d'analyse et utilisations de ces elements pour le traitement et le diagnostic du cancer Download PDF

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Publication number
WO1990003399A1
WO1990003399A1 PCT/AU1989/000427 AU8900427W WO9003399A1 WO 1990003399 A1 WO1990003399 A1 WO 1990003399A1 AU 8900427 W AU8900427 W AU 8900427W WO 9003399 A1 WO9003399 A1 WO 9003399A1
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WO
WIPO (PCT)
Prior art keywords
transporter
glutamine
fragment
subunit
cells
Prior art date
Application number
PCT/AU1989/000427
Other languages
English (en)
Inventor
Clive Frederick Palmer
Edward Mc Evoy-Bowe
George Victor Meehan
Terrence Piva
Donna Rigano
Paula Favot
Michael West
Michael Grenville Peter Mccabe
David John Miller
Original Assignee
Australian Commercial Research & Development Limited
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Publication date
Application filed by Australian Commercial Research & Development Limited filed Critical Australian Commercial Research & Development Limited
Priority to JP1510278A priority Critical patent/JPH06504031A/ja
Publication of WO1990003399A1 publication Critical patent/WO1990003399A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/40—Acylated substituent nitrogen atom
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/24—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one carboxyl group bound to the carbon skeleton, e.g. aspartic acid
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    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
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    • C07C243/26—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C243/34—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of a carbon skeleton further substituted by nitrogen atoms
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    • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
    • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/63—Esters of sulfonic acids
    • C07C309/64—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to acyclic carbon atoms
    • C07C309/65—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to acyclic carbon atoms of a saturated carbon skeleton
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    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/25—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
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    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
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    • C07D263/10—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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Definitions

  • This invention relates to compounds and methods for the treatment and diagnosis of cancer in animals such as humans. More particularly, this invention relates to identification and isolation of human amino acid transporters, including transporters such as glutamine transporters that are common to tumour cells but which are generally not found, or are less active, or are present in lower quantities in most non-tumour cells.
  • transporters such as glutamine transporters that are common to tumour cells but which are generally not found, or are less active, or are present in lower quantities in most non-tumour cells.
  • This invention also relates to recombinant products and methods useful for preparing such transporters and other biological products such as antibodies, to diagnostic products and assays useful for detecting the presence of such transporters in animals, and to compositions of matter such as antibodies to the transporter, anti-glutamine compounds and glutamine analogues which can successfully inhibit the passage of amino acids such as glutamine through such transporters, or which interfere with the glutamine metabolism in the cells, and various therapeutic compositions comprising such inhibitors.
  • This invention further relates to methods for treating cancer by administering such compositions, to vaccines comprising such transporters, fragments or subunits thereof, and to methods for immunization using such vaccines.
  • chemotherapeutic compounds include analogues of glutamine such as 6-diazo-5-oxo-L-norleucine (DON), O-diazo-acetyl-L-serine (Azaserine), and ⁇ -amino-3- chloro-4,5- dihydro-5-isoxazoleacetic acid (Acivicin), which interfere with intracellular biosynthesis.
  • DON 6-diazo-5-oxo-L-norleucine
  • Azaserine O-diazo-acetyl-L-serine
  • Acivicin ⁇ -amino-3- chloro-4,5- dihydro-5-isoxazoleacetic acid
  • Glutamine is transported into tumour cells by glutamine transporter proteins.
  • the capacity of such glutamine transporters to move glutamine across the plasma membranes of tumour cells is thus vital to their growth and development.
  • glutamine transporters in tumour cells are considerably more active than the glutamine transporters of normal cells.
  • glutamine in the bloodstream can possibly weaken the cancer cells and thereby render them more susceptible to conventional chemotherapy or radiation therapy. Again, however, this therapy can ultimately result in the indiscriminate destruction of both tumour and normal cells.
  • tumour cells require an increased uptake of glutamine from surrounding blood and tissues
  • no significant progress has been made in utilizing this increased uptake to design compounds or methods for selectively acting on tumour cells.
  • this difference has not been exploited in the area of cancer diagnosis, where reliable assays for diagnosing the presence of cancer are clearly desireable.
  • tumour glutamine transporters as well as other human and mammalian amino acid transporters, have thus made possible a wide new range of diagnostic, therapeutic and other related compositions of matter and methods which exploit the difference in glutamine uptake between tumour and non-tumour cells, and which can be used to detect tumours, or selectively act on tumour cells while producing little or no effect on normal cells.
  • a first embodiment of this invention provides substantially purified human amino acid transporter, including glutamine and alanine transporters found in tumour cells
  • Other embodiments of this invention provide methods for screening compounds to determine their ability to bind to binding sites on a tumour glutamine transporter, or the ability of compounds to inhibit the transport of glutamine into tumour cells.
  • Another embodiment of this invention provides laboratory equipment for carrying out the binding and transport inhibition assays of this invention.
  • kits for detecting tumour cells in vivo or in vitro. Also provided is a method for monitoring the progress of cancer by repeated, periodic assays in accordance with this invention.
  • inventions provide biological products which are useful for producing transporter proteins of this invention by recombinant DNA methods, and for producing monoclonal antibodies which specifically bind to the transporters.
  • inventions provide anti-glutamine compounds and glutamine analogues, and methods for using such compounds and analogues in the treatment of cancer.
  • inventions of this invention provide methods for treating cancer, including methods in which the transport of glutamine into tumour cells is inhibited, using anti- glutamine compounds, glutamine analogues, or antibody compositions. Such methods may be used in combination with other conventional anti-cancer therapy such as chemotherapy or radiation therapy.
  • This invention also provides vaccines comprising at least one antigenic human amino acid transporter or fragment or subunit thereof.
  • amino acid transporters including tumour glutamine transporters, which are useful in the diagnosis and treatment of cancer. It is another object of this invention to provide screening methods which can identify compounds useful in the treatment of cancer.
  • a further object is to provide diagnostic products and methods for diagnosing and monitoring the progression of cancer.
  • a still further object of this invention is to provide biological products for diagnosing and treating cancer, and for preparing quantities of purified transporters or antibodies which bind to such transporters.
  • Yet another object of this invention is to provide compounds and compositions for use in treating cancer, and methods for treating cancer comprising administering such compounds and compositions.
  • a further object of this invention is to provide methods for treating cancer by inhibiting transport of glutamine into tumour cells, or by using the transport mechanisms of tumour cells to selectively transport cytotoxic compounds into the cells. Methods are included wherein the cell is weakened by the inhibition of glutamine uptake into the cell, and thereafter killed by conventional methods such as chemotherapy, radiation therapy, or any other therapy directed against the cell.
  • a still further object is to provide vaccines which can be used for the prevention of cancer.
  • a further object is to provide compounds and methods which effect the proliferation of lymphocytes.
  • FIG. 1 illustrates a polyacrylamide gel showing purification of the glutamine transport proteins, lane 1 containing molecular weight standards, lane 2 containing eluted protein, and lanes 3-8 containing whole membrane samples;
  • FIG. 2 is a graph illustrating glutamine uptake into HeLa cells
  • FIG. 3 is a graph illustrating titration of glutamine uptake into HeLa cells with NEM
  • FIG. 4 is a graph illustrating the effect of different NEM incubation times on glutamine uptake into HeLa cells
  • FIG. 5 is a graph illustrating the effect of 1 mM NEM on glutamine uptake into HeLa cells
  • FIG. 6 is a graph illustrating the effect of antiserum on glutamine transport into HeLa cells
  • FIGS. 7a-7j are graphs illustrating glutamine transport into red blood cells (RBC) and various cell lines;
  • FIG. 8 is a graph illustrating a binding of 3 H-NEM to HeLa cell plasma membranes
  • FIG. 9 is a graph illustrating the binding of 3 H- NEM to material obtained from peaks A-E of HeLa cell plasma membranes defined in FIG. 8;
  • FIG. 10 is a graph illustrating titration of glutamine transport with 3 H-NEM binding to material obtained from peaks A-D defined in FIG. 8;
  • FIG. 11 is a graph illustrating the theoretical curve for binding of 3 H-NEM to material obtained from peak C defined in FIG. 8;
  • FIG. 12 is a graph illustrating protection of 3 H- NEM binding with excess substrate
  • FIG. 13 is a graph illustrating the effect of different NEM incubation times on glutamine uptake into HeLa cells
  • FIG. 14 is a graph illustrating the differential labelling of HeLa cell plasma membranes with 3 H-NEM
  • FIG. 15 is an autoradiograph of 3 H-NEM labelled HeLa cell plasma membrane proteins, lanes 1 and 2 show separated proteins labelled with 1 mM 3 H-NEM, and lanes 3 and 4 show separated proteins labelled with 1 mM 3 H-NEM in the presence of 100 mM glutamine;
  • FIG. 16 is a graph illustrating protection of
  • FIG. 17 illustrates a polyacrylamide gel of HeLa cells membrane proteins extracted into four fractions, lanes 1 and 2 containing fraction 1, 3 and 4 containing fraction 2, lanes 5 and 6 containing fraction 3, and lanes 7 and 8 containing fraction 4;
  • FIG. 18 illustrates a polyacrylamide gel of HeLa cell membrane proteins stained for carbohydrate by Schiff- Periodate staining
  • FIG. 19 illustrates a polyacrylamide gel of HeLa cell membrane proteins under reducing and non-reducing conditions, lane 1 containing molecular weight standards, lane 2 containing membrane protein under non-reducing conditions, and lanes 3 and 4 containing membrane protein under reducing conditions;
  • FIG. 20 is a graph illustrating the effect of sodium on 3 H-NEM binding to HeLa cell plasma membranes
  • FIG. 21 is a graph illustrating the binding of 3 H- NEM to outer plasma membrane sites (UPM).
  • FIG. 22 is a graph illustrating the binding of 3 H- NEM to inner plasma membrane sites (LPM).
  • FIG. 23 is a graph illustrating the effect of serum on 3 H-NEM binding to HeLa cell plasma membranes
  • FIG. 24 is a graph illustrating a Lineweaver-Burk plot for binding of representative compounds
  • FIG. 25 illustrates silver stained 4% - 30% SDS- polyacrylamide gradient gel showing the position of glutamine transport proteins
  • FIG. 26 illustrates a representative immunoblot showing the response to anti-serum by various cell lines, lane 1 containing molecular weight standards, lane 2 containing HeLa cells, lane 3 containing Detroit 562 cells, lane 4 containing Molt-3 cells, and lane 5 containing Molt-4 cells;
  • FIG. 27 is a graph illustrating protection of 3 H- NEM binding to protein bands with excess alanine, or without alanine, prior to and during exposure to 3 H-NEM;
  • FIG. 28 is a graph illustrating inhibition of alanine uptake into HeLa cells by NEM correlated with binding of 3 H-NEM to peaks A-E;
  • FIG. 29 is a graph illustrating corrected values for inhibition of alanine uptake into HeLa cells by NEM correlated with binding of 3 H-NEM to peak D;
  • FIG. 30 is a graph illustrating a comparison of the concentration dependent glutamine uptake curve in HeLa cells and bovine lymphocytes under zero-trans conditions
  • FIG. 31 is a graph illustrating the time course for 14 C-glutamine uptake by normal lymphocytes subsequent to stimulation with concanavalin A;
  • FIG. 32 is a graph illustrating the initial rate of glutamine uptake into bovine lymphocytes in the presence and absence of concanavalin A;
  • FIGS. 33-38 are graphs illustrating the survival of lymphocytes measured by the dye exclusion method and relative mitosis measured by the MTT assay plotted against a range of concentrations of glutamine analogues;
  • FIG. 39 is an inverse velocity plot of label uptake against substrate concentration for one single degree system
  • FIG. 40a is an inverse velocity plot of label uptake using two single degree systems
  • FIG. 40b is an inverse velocity of label uptake plot of three single degree systems
  • FIG. 41 is an inverse velocity of label uptake plot of three single degree systems
  • FIG. 42 illustrates the inverse velocity of label uptake of a multidegree system followed by a first degree system
  • FIG. 43 is a plot of a single degree system followed by a multi-degree system
  • FIG. 44 is a plot of two multi-degree systems
  • FIG. 45 is a plot of inverse velocity of uptake of three systems, two of which are multi-degree
  • FIG. 46 is a plot of uptake velocity of glutamine in HeLa cells
  • FIG. 47 is a plot of 1/v t * against concentration
  • FIG. 48 is a final curve calculated from the final equation
  • FIG. 49 is a plot illustrating glutamine uptake in HeLa cells under conditions of Na + -deficiency
  • FIG. 50 is a plot of inverse velocity of label uptake against concentration
  • FIGS. 51-59 comprise Reaction Schemes 1-9, respectively, which illustrate syntheses useful for making antiglutamine compounds and glutamine analogues in accordance with this invention
  • FIG. 61 is a graph illustrating alanine uptake into HeLa cells
  • FIG. 62 is a graph illustrating a Lineweaver-Burk analysis of alanine uptake into HeLa cells, the HeLa cells having been pre-incubated with 1 mM NEM, or without NEM;
  • FIG. 63 is a graph illustrating titration of alanine uptake into HeLa cells with varying NEM
  • FIG. 64 is a graph illustrating the effect of 1 mM NEM on alanine uptake into HeLa cells which were preincubated with, or without NEM prior to radioactive
  • FIG. 65 is a flow diagram illustrating methods for preparing upper plasma membranes (UPM) and lower plasma membranes (LPM) in accordance with this invention.
  • FIG. 66 is a schematic illustration of a six-well assay plate which is placed upside down on an inverted assay plate cover in accordance with this invention
  • FIG. 67 is a schematic view of the assembly of FIG. 66 inverted so that the contents of the lid are transferred to wells in the plate;
  • FIG. 68 illustrates an inverted 6 well cover plate in accordance with this invention
  • FIG. 69 illustrates the manner in which a 6 well bottom plate is inverted and placed over an inverted cover plate which contains solution
  • FIG. 70 illustrates a 6 well plate and cover plate assembled for transfer of solution
  • FIG. 71 illustrates a 96 well cover plate in accordance with this invention
  • FIG. 72 illustrates the 96 well cover plate showing channels into which solution is placed
  • FIG. 73 illustrates the manner in which a 96 well plate is inverted and placed over an inverted cover plate containing solution
  • FIG. 74 illustrates a 96 well plate and cover assembled for transfer of solution
  • FIGS. 75-86 illustrate Tables 4-15, respectively, of this invention.
  • Anti-glutamine compound - refers to any compound that enters the cell via the glutamine transporters or by any other route, and/or acts on glutamine uptake, and/or metabolism, biosynthesis, intracellular degradation (e.g., enzymatic), etc., and/or prevents the binding of glutamine to the transporter of any other substance which binds glutamine.
  • Anti-receptor - refers to agents which bind selectively to the receptor of a ligand-receptor pair.
  • the ligand is an antigen and the receptor is an antibody, for example, a mouse IgG antibody
  • the anti- receptor may be an antibody against IgG.
  • the anti-receptor may also be a substance which binds selectively with a moiety conjugated to the receptor.
  • the moiety may be a hapten and the anti-receptor an antibody against the hapten. Fragments and derived products are also encompassed.
  • Glutamine analogue - refers to any compound which bears a structural similarity to glutamine.
  • Human Amino Acid Transporter - refers to a molecule located within the membrane of a cell derived from human tissue which transports amino acids into and out of the cell.
  • Ligand - refers to a target analyte being assayed. This term includes any member of an immunological binding pair such as an antibody or an antigen.
  • a ligand can be, for example, an amino acid or its transporter, an enzyme or a nucleic acid sequence such as RNA or DNA probe.
  • Receptor - refers to a molecule which binds
  • the receptor may be an antibody, preferably a monoclonal antibody. If the target ligand is an antibody, the receptor may be an antigen or anti-antibody. If the ligand is an enzyme, the receptor may be substrate for the enzyme. If the ligand is a nucleic acid sequence, the receptor may be a complementary sequence. Fragments and derived products are also encompassed.
  • Tumour Glutamine Transporter (TGT) - refers to a molecule located within the membrane of a tumour cell which is capable of transporting the amino acid glutamine across the membrane, from external medium into the cell's cytoplasm and vice versa.
  • the first embodiment of this invention comprises human amino acid transporter proteins.
  • transporters which are present in tumour cells, but which are not present, or are not active, or are present in lower quantities in non-tumour cells.
  • Three such transporters have been discovered, namely TGT I, TGT II and TGT
  • TGT I - III which are present in tumour cells but do not appear to be present or active in non-tumour cells.
  • TGT I - III may be present in normal cells, but only in low quantities.
  • the method developed for isolating TGT I, TGT II and TGT III is illustrative of the method by which other transporters, especially those which are specific to tumour cells, can be identified and isolated. This method is based on a set of procedures (Hayes et al., Biochem. J., 214,
  • tumour glutamine transporters of this invention were isolated from HeLa cells, which are derived from an established human tumour cell line.
  • the cell line originated from a biopsy of a cervical carcinoma from Henrietta Lacks in 1951.
  • Procedures for Separating HeLa Cell Proteins Procedures used for initially preparing a monolayer culture for HeLa cells and other adherent cell lines, and for preparing microcarrier culture cells were as follows.
  • Adhesive cells were grown on the surface of glass beads (Sigma Chemical Company) by an adaptation of the methods described by Pharmacia (1981). Beads (0.5 g) were rinsed three times with fresh medium containing 10% (v/v) FBS and then mixed with approximately 10 6 cells which were harvested from monolayer cultures with ATV and diluted to 200 ml with fresh medium containing 10% (v/v) FBS. The inoculum was then transferred to a microcarrier vessel (500 ml
  • Membrane proteins were selectively extracted into four groups by a procedure which is based on the unique properties of the detergent Triton X-114 (Thomspon et al., Biochemistry. 26, (1987) 743-750). Monolayer cultures were rinsed three times with 10 ml of phosphate buffered saline (PBS A) and the first group of membrane proteins was
  • HeLa cells extracted from the surface of intact HeLa cells by incubating with 2 ml of 1 mM EDTA, 0.15 mM NaCl and 10 mM 4-( 2- hydroxyethyl ) -1-piperazine-ethanesulphonic acid (HEPES) pH 7.4 (fraction 1).
  • the cells were scraped from the surface of the flask with a rubber policeman and collected by centrifugation at 500 g for 5 minutes in a conical polypropylene tube.
  • the cell pellet was treated with 100 ⁇ l of 2% (v/v) Triton X-114 in 10 mM HEPES, pH 7.4 and insoluble material was removed by centrifugation at 11600 g for 10 minutes.
  • the upper detergent-free phase contained hydrophobic proteins (fraction 2) and the lower phase contained hydrophillic proteins (fraction 3).
  • the insoluble pe.let was solubilized in SDS solubilization buffer (fraction 4).
  • the proteins of fractions 1-3 were precipitated by adding acetone to a final concentration of 90% (v/v) and pelleted at 11600 g in an Eppendorf ultracentrifuge (model 5414) for 10 minutes. The acetone precipitates were dried under vacuum before solubilizing in 100 ⁇ Z of SDS solubilization buffer and separating by SDS-PAGE.
  • Membrane preparations (approximately 1 mg protein) were solubilized in 1 ml of SDS solubilization buffer (25 mM tris-HCl, 2% (w/v) SDS, 2% (w/v) ficoll, 1% (v/v) 2-mercaptoethanol, 2 mM EDTA, 5 mM PMSF, pH 6.8) by vortexing vigorously three times for 1 minute and then placing at -20°C overnight. Proteins were separated on 12% polyacrylamide gels using the Bio-Rad Mini-Protean II apparatus (see
  • Solubilized membranes (1 mg protein/ml) were mixed with sample buffer (62.5 mM tris-HCl pH 6.8, 2% (w/v) SDS, 2% (w/v) ficoll, 5% (v/v) mercaptoethanol, 0.00125% bromophenol blue, 25 mM EDTA) at a ratio of 1:2 and heated at 70°C for 10 minutes. Samples of 30 ⁇ l were applied to the wells of 0.75 mm gels and a
  • the gels were removed from the electrophoresis apparatus and placed in a solution of 70% (w/v) polyethylene glycol (PEG) 2000 overnight. The gel which had become opaque was then stained in a solution of 1% (w/v) coomassie blue for 15 minutes followed by destaining in several changes of 10% acetic acid.
  • the gels were shrunk in 70% (w/v) polyethylene glycol 2000 overnight and then transferred to a silver nitrate solution (20% (w/v)AgNO 3 , 35% (v/v) ammonia, 4% (w/ v) NaOH, which was used within five minutes) for 20 minutes with gentle agitation.
  • the gel was rinsed three times in distilled water for 1 minute and then transferred to developer solution (0.002% (v/v) formaldehyde, 0.005% (w/v) citric acid) until bands became visible. Development could be stopped with 10% (v/v) acetic acid and the gel could be stored in the PEG solution.
  • the stained gels were placed on a clear plastic cutting surface which was placed on a transilluminator
  • Plasma membrane preparations were solubilized in buffer containing 5% (v/v) mercaptoethanol or in buffer lacking mercaptoethanol. (Allore et al., Mol. Immunol., 20, (1983) 383-395.) The solubilized membranes were mixed with sample buffer with or without mercaptoethanol and the
  • Membrane proteins were separated by SDS-PAGE as described above and then stained for carbohydrate.
  • the gel was destained overnight in 2-3 changes of 10% (v/v) acetic acid.
  • Electroelutions were carried out in the Bio-Rad Electroeluter model 422. Polyacrylamide gels of membrane proteins were lightly stained (5 minutes) in 0.1% (w/v) coomassie blue and destained in several washes of 10% (v/v) acetic acid. The stained band corresponding to the glutamine transport protein was sliced out of the gel using a scalpel and placed into the glass tube connected to a dialysis membrane. The tank was filled with volatile elution buffer (0.05 M NH 4 HCO 3 1% (w/v) SDS) and a constant current of 6 mAmps/tube was applied for 24 hours.
  • volatile elution buffer 0.05 M NH 4 HCO 3 1% (w/v) SDS
  • the eluted protein was collected from the surface of the dialysis membrane and stored in elution buffer containing 50 ⁇ l of 0.1 M PMSF at -20°C.
  • the eluted protein samples were concentrated using Centricon microconcentrators with a molecular weight cutoff of 10000.
  • the microconcentrators were centrifuged in a 45° fixed angle rotor (IEC/Damon model B-20A centrifuge) at 5000 g for 30 minutes to give a final volume of 50 - 100 ⁇ l.
  • a silver stained polyacrylamide gel showing purificatin of the eluted protein band with M 42000 is shown in FIG. 1.
  • the microconcentrators could also be used to dialyse the protein solution against several washes of PBS A in order to remove salts and SDS . Purity of the eluted protein was detected by equilibrating 50 ⁇ l of the concentrated protein with 200 ⁇ l of SDS sample buffer followed by SDS-PAGE and staining with silver. The amount of protein present in each sample was determined by an adaptation of the MicroLowry method using 200 ⁇ l of protein solution and reading the absorbance at 660 nm. (Markwell et al ., Anal. Biochem., 87, (1978) 206.)
  • NEM solutions ranging from 0.25 to 10 mM in 10 ml HBSS were prepared, with each containing 0.1 ml 3 H-NEM stock solution.
  • Plasma membrane preparations (0.2 mg protein/ml) were incubated with 200 ⁇ l of the NEM solutions for 1 hour at 37°C: then labelling was stopped by washing in ice-cold 0.9% NaCl.
  • Membranes were pelleted at 11600 g, and then solubilized in 200 ⁇ l of solubilization buffer as described above and stored at -20°C.
  • Plasma membrane preparations (0.2 mg protein/ml) were pre-incubated with the D or L isomer of 100 mM glutamine in HBSS for 20 minutes at 37°C before labelling with 1 mM 3 H-NEM containing 100 mM glutamine for a further 20 minutes. Labelling was stopped by washing in ice-cold 0.9% NaCl and these membranes were then solubilized in solubilization buffer.
  • Plasma membrane preparations (0.2 mg protein/ml) were incubated with 100 ⁇ l of various glutamine concentrations ranging from 0.5 to 10 mM for 5 minutes at 37°C.
  • NEM 100 ⁇ i
  • the membrane suspension was incubated for a further 10 minutes at 37°C
  • the membranes were centrifuged for 1 minute at 11600 g rpm and the pellet was washed with Hanks balanced salt solution (HBSS).
  • HBSS Hanks balanced salt solution
  • the plasma membranes were then incubated with 1 mM 3 H-NEM for 1 hour at 37°C. The labelling was stopped by washing with ice-cold 0.9% NaCl.
  • Membranes were solubilized in solubilization buffer and stored at -20°C.
  • the flow diagram in FIG. 65 describes the methods for preparing upper plasma membrane (UPM) and lower plasma membrane (LPM) fractions and labelling with 3 H-NEM. This procedure can be applied to any adherent cell line in addition to HeLa, and can be used for any type of assay where the inner and outer faces of the same cell membrane are required.
  • a radioactive solution of 1 mM NEM was prepared by adding 100 ⁇ l of 3 H-NEM stock solution (0.5 mCi/ml) to 10 ml of 1 mM NEM in HBSS, pH 7.4.
  • Membranes were pre-incubated with 10 mM glutamine for 5 minutes in the presence of either 100 mM NaCl or 100 mM choline chloride at 37°C. A solution of NEM (containing glutamine and sodium or choline at the appropriate concentrations) was added to the incubating membranes to a final NEM concentration of 1 mM for a further 10 minutes at 37°C. The membranes were then pelleted at 11600 g in an Eppendorf ultracentrifuge for 5, minutes then resuspended in 1 mM 3 H- NEM (containing 100 mM sodium or choline) for 1 hour at 37°C.
  • NEM containing glutamine and sodium or choline at the appropriate concentrations
  • the membranes were washed with HBSS and then solubilized in 100 ⁇ l of SDS solubilization buffer.
  • the solubilized membrane samples were applied to a 12% polyacrylamide gel and subjected to 50 mAmps of constant current for 1 hour.
  • the separated proteins were stained with coomassie blue then sliced out of the gel and counted for radioactivity.
  • HeLa cells Prior to the isolation of membrane fractions, the HeLa cells were treated in a variety of ways in order to induce migration of the glutamine transporters into the membrane. HeLa cells were incubated in fresh Medium 199 containing 20% (v/v) FBS or lacking FBS 2 hours prior to
  • Membranes were isolated in the usual way and then labelled with 3 H-NEM. All solutions were prepared in HBSS and radioactive NEM was prepared by adding 100 ⁇ l of a stock solution of 3 H-NEM (0.5 mCi/ml) to 1 ml of 1 mM NEM in HBSS. The membranes were preincubated in 10 mM glutamine for 5 minutes at 37°C and then NEM was added to a final concentration of 1 mM for a further 10 minutes. The membranes were pelleted at 11600 g for five minutes in an Eppendorf ultracentrifuge and then resuspended in 3 H-NEM for 1 hour at 37°C. The membranes were washed in 0.9% NaCl and solubilized in 100 ⁇ l of SDS solubilization buffer for SDS- PAGE analysis. The separated proteins were sliced out of the gel and counted for radioactivity.
  • HeLa cell plasma membranes were incubated with 3 H- NEM, dissolved in SDS detergent and the proteins were separated by polyacrylamide gel electrophoresis. A number of proteins were found to bind 3 H-NEM as shown in FIG. 8 and they were labelled A-E. This pattern of binding was consistently observed in several membrane preparations.
  • Table 1 shows the approximate molecular weights of the peaks A- E and the binding of 3 H-NEM to each peak which was calculated knowing that the specific activity for the 3 H-NEM solution was 2.22 x 10 6 dpm/nmole. Molecular weights were determined relative to standard markers and values are the mean ⁇ S. E. of at least three determinations. g
  • FIG. 10 A plot of NEM binding to peaks A-E at various NEM concentrations against glutamine transport at the same NEM concentrations is shown in FIG. 10.
  • Binding of 3 H-NEM to peaks B-E does not show one to one correlation with inhibition of glutamine transport by NEM.
  • a similar plot for peak C alone is given in FIG. 11 where the rate of glutamine uptake at 100% saturation of 3 H- NEM binding was assumed to be equal to the non-saturable component of total glutamine uptake and was deducted from the uptake rates measured at each NEM concentration.
  • the line drawn is the theoretical line for agreement between percent saturation of the carrier protein with 3 H-NEM and percent control for glutamine uptake. There is good agreement between the observed values and the theoretical line which supports the implication of peak C in glutamine transport into HeLa cells. Further evidence for the involvement of peak C in the NEM-sensitive transport of glutamine was provided by showing that the transport substrate could protect the transport protein from binding NEM. Membranes were preincubated with a large excess of L-glutamine or D-glutamine before incubating with unlabelled NEM. The excess glutamine and NEM were removed and the proteins separated by gel electrophoresis. FIG.
  • Peak B On the other hand, does not become labelled with 3 H-NEM until 5 mM glutamine and above, suggesting that this protein with M r 53000 is involved with the low affinity transport of glutamine.
  • the position of peaks B and C in a polyacrylamide gel is shown in FIG. 1.
  • HeLa cell plasma membranes were incubated with 14 C-NEM in the presence or absence of excess transport substrate.
  • the membrane proteins were resolved by SDS-gel electrophoresis and the gel was then exposed to X-ray film.
  • the autoradiograph in FIG. 15 shows that the band at M r 42000 is radioactively labelled with NEM in the absence of the protecting substrate glutamine (lanes 1 and 2) but when glutamine is present in the incubation medium the band is protected from becoming radioactively labelled (lanes 4 and 5).
  • Membrane proteins were selectively extracted into four fractions depending on their hydrophobic nature and location within the cell membrane.
  • the stained gel in FIG. 17 demonstrates that there was little overlap of proteins between the groups with fraction 1 containing the surface or extrinsic membrane proteins.
  • Fraction 2 contained the hydrophillic membrane proteins and fraction 3 contained the hydrophobic membrane proteins.
  • the final fraction 4 contained integral membrane proteins which could only be solubilized in the presence of the ionic detergent SDS.
  • the stained gel shows that the high affinity glutamine transport protein with M r 42000 appeared mainly in the fourth fraction with slight overlap into the hydrophobic protein fraction.
  • the low affinity transport protein with M r 53000 appears predominantly in the third fraction indicating that it is a
  • hydrophobic membrane protein hydrophobic membrane protein
  • HeLa cell membrane proteins which had been separated by electrophoresis were stained for carbohydrate using the Schiff method.
  • the gel in FIG. 18 indicates that a number of proteins were glycosylated including the glutamine transport protein with M r 42000.
  • Membrane proteins were separated by SDS-PAGE under reducing and non-reducing conditions in order to detect the presence of intra- and inter- molecular disulphide bonds.
  • the binding of 3 H-NEM to HeLa cell plasma membranes was investigated in the presence or absence of sodium ions in order to establish if binding of NEM to the glutamine transport protein is sodium dependent.
  • the graph in FIG. 20 shows that the specific binding of NEM to the high affinity glutamine transport protein with M r 42000 following protection of the glutamine binding site with excess substrate was decreased by 30% in the absence of sodium ions.
  • the inhibition of NEM binding in the absence of sodium ions suggests that binding is dependent on the presence of sodium ions at or near the active site on the glutamine transport protein.
  • Binding of NEM to the low affinity glutamine transport protein with M r 53000 was not affected by the presence or absence of sodium ions.
  • a method was developed for the selective labelling of glutamine transport sites on the inner and outer faces of the plasma membrane (described above in Section H.A.6.).
  • Fig 21 The binding of 3 H-NEM to the outer face of the plasma membrane is shown in Fig 21 and indicates that the peaks with M r 42000 and 53000 are specifically labelled with the isotope following protection of the glutamine binding site with excess glutamine. As expected, binding to these peaks was proportional to the concentration of glutamine used for protection of the binding site.
  • Inner plasma membranes were also specifically labelled with 3 H-NEM and the binding pattern shown in FIG. 22 is markedly different from that obtained for outer plasma membranes.
  • the magnitude of the binding to peaks with M r 42000 and 53000 was still comparable to that for the outer plasma membranes, there was no apparent correlation between the amount of binding and the concentration of glutamine used for protection.
  • the most significant difference was the appearance of another band (designated peak F) which had not been shown to bind 3 H-NEM in previous experiments but apparently showed a good positive correlation between binding and glutamine concentration when the inner face of the membrane was exclusively labelled.
  • peak E which had previously given an inverse correlation with glutamine concentration, now showed a direct correlation between binding of 3 H-NEM and glutamine concentration.
  • the reason for the significant difference in binding patterns between the inner and outer faces of the plasma membrane is unclear but may be related to the regulation of glutamine transport by sites containing reactive sulphydryl groups.
  • FBS contains growth factors and hormones which potentiate the active proliferation of cells in culture.
  • HeLa cells were incubated in the presence or absence of FBS prior to the preparation of plasma membranes which were subsequently labelled with 3 H-NEM after protection of the glutamine active site of the transport protein with excess substrate.
  • the graph in FIG. 23 shows the binding activity of HeLa cell membrane proteins prepared from normal cells and serum-depleted cells. Binding of NEM to the high affinity glutamine transporter was decreased by 50% in the absence of serum. A number of other proteins which normally show significant binding of NEM, namely peaks A, B, D, and E, also exhibited a decreased binding of NEM in the absence of serum. Re-addition of serum to the medium following incubation with serum-free medium resulted in a small 10% increase in the binding of 3 H-NEM to the glutamine transport protein.
  • the K m should be adjusted downwards if the higher concentration is giving the lower h value, or upwards if the reverse is true.
  • the K m adjustment is continued until the h values are within 1 unit of one another, which then provides a new estimate of K m(2 ) .
  • the mean of the two h values is then taken as an estimate of h 2 .
  • the estimation of the approximate V m for the separate transporters in multi-variate systems can also be directly derived from the plot gradients.
  • the plot of 1/v* against substrate concentration provides an initial indication of the number of transporters present, the range of concentration over which they operate, and whether the type of uptake is represented by single or multi-degree
  • Table 11 shows the parameters of the models chosen for testing the plotting procedure. In this analysis each K m is assumed to represent a single transporter.
  • Figure 39 shows the plot for model 1 (one single degree transporter).
  • the axes intercepts are:
  • Model 2 shows the plot of model 2 (two single degree transporters). This plot is non-linear and is a composite of two linear relationships separated by a single inflexion, the first linear relationship being the initial gradient which approaches 1/L 1 * and the second being the final gradient which is equal to 1/L t * .
  • Figure 40 shows that there is a marked departure from linearity (relative to the final gradient as S ⁇ ⁇ with the downward inflexion starting near K m(2 ) and interceptmg the 1/v* axis at a point which is 30% below the extrapolated final slope (S ⁇ ⁇ ), a difference which should be very obvious in most transport experiments.
  • Model 2 the departure from linearity is negligible and can be observed only between 1/K m(2 ) and 0, and at 1/32K m(2 ) the divergence is only 9% of the extrapolated line which would be difficult to distinguish from experimental error in most experiments, since a very small adjustment in the slope of the line would be sufficient to include the points below 1/K m(2 ) .
  • the departure from linearity is even more marked than that in Figure 40, and it is possible to obtain values for V m(t) and V m(1) / m(1) + V m(2) /K m(2) from the extrapolated intercepts at the v and v/S axes respectively.
  • Model 3 the departure from linearity is negligible and can be observed only between 1/K m(2 ) and 0, and at 1/32K m(2 ) the divergence is only 9% of the extrapolated line which would be difficult to distinguish from experimental error in most experiments, since a very small adjustment in the slope of the line would be sufficient to
  • Figure 41 shows the inverse label uptake plot for a multiple transport mechanism which consists of three single degree transporters. This curve is similar to that obtained in Figure 40, except that there are three linear relationships linked by two inflexions.
  • the gradient of the first linear relationship (where S is close to 0) intercepts at the 1/v* axis to give the relationship shown in equation 3. This is usually an accurate intercept and can be used to determine K m(2) /V m(2) once K m(1) /V m(1) has been estimated.
  • the curve is first analyzed by obtaining the slopes ⁇ (1/V*)/ ⁇ S from one lower point to the next. In multi-component single degree systems this will reveal portions of the plot where the change in the slopes from one point to the next are at a minimum.
  • These sections of the plot which are near linear alternate with more entensive sections which extend from each near linear section up to the next K m value.
  • the near linear sections start at or just above the successive K m values (see
  • the lowest one on the concentration scale to be seen being that of the K m(1 ) .
  • the first gradient is an underestimate of 1/L 1 * (overestimate of V m(1) ), and the intercept at the S axis as 1/v* ⁇ 0 is an overestimate of
  • each V m can be obtained by subtraction of the previous estimate moving along the plot from the lowest concentration.
  • Figure 42 shows the characteristic curve obtained when there are two transporters present with transporter 1 being multi-degree relationship (all the multi-degree relationships considered here are of the Hill type) and transporter 2 a single degree relationship.
  • This plot displays a negative gradient starting from zero concentration with a sharp minimum near K m(1 ) , as previously indicated by the application of equations 2 and 17 in the main text.
  • Figure 43 shows the type of curve given by a single degree transporter followed by a multi-degree transporter. In this case there is a positive gradient followed by a sharp inflexion towards the final gradient.
  • the first gradient provides an estimate of 1/L 1 * and the second gradient provides an estimate of 1/L t * .
  • Line 1 depicts the plot for model 6, and this repeats the essential features of model 4 over the first segment of the curve: these being a negative gradient followed by a sharp minimum just above the K m(1) value.
  • This curve then follows a different course to that of model 4 in that there is a sharp increase in the gradient which provides an estimate of 1/L 1 * followed by an inflection over towards the final gradient which as before gives an estimate of 1/L t * .
  • the curve shown as line 2 demonstrates one type of curve which is very difficult to analyze and quantify.
  • Figure 45 shows the 1/v* plot for a mixed three component system.
  • the diagnostic analysis of the previous plots should be sufficient to indicate the nature of the transport mechanism indicated by this plot.
  • the three gradients representing the three transporters are indicated in the Figure.
  • Transporter 1 is a single degree relationship
  • the plot shows a minimum gradient between transporters 1 and 2 indicating that transporter 2 is a multidegree relationship.
  • the roll-over from transporter 2 to transporter 3 is an indication that transporter 3 is also a multi-degree relationship. The roll-over is very gradual, so that the estimation of the minimum by eye is very approximate.
  • the transporters are always numbered 1 to n commencing at the lowest K m . All plots were tested as regression lines on the H-P 15C. The following data is prepared before commencing the analysis: velocity against concentration, 1/v* against concentration and ⁇ (1/V*)/ ⁇ S. In the examples considered below the maximum number of transporters present is never greater than three. In general the middle transporter provides the model for the treatment of all intermediate transports irrespective of number.
  • successive K m values should be greater that four-fold (in the case of multi-degree systems this can be close to two-fold).
  • V m values should not differ from one another more than five-fold, and that as a general rule, -
  • V mL the lower of the two V m values
  • V mH higher V m value
  • K m(x+1) the upper K m value
  • K m(x) the lower K m value
  • That plot points must go at least as high as 2K m(n) (the highest K m ), with a minimum of at least three points above K m(n) .
  • Transporter I is a single degree relationship.
  • Transporter I is a multi-degree relationship.
  • concentration at which the initial gradient reaches its minimum value gives the first estimate of K m(1)
  • slope at the top of the first positive gradient gives an estimate of 1/L 1 * and hence V m(1) .
  • transporter I If transporter I is a single degree relationship, estimate the net v 2 values using the transporter I parameters estimated as part of .the structural hypotheses by subtracting the estimated v 1 from v obs . Plot 1/v 2 * against S from 0.5K m(2) to 2K m(2) . A negative gradient indicates that transporter II is a multi-degree system. The concentration when 1/v* is at a minimum then indicates the approximate K m(2) (equation 17).
  • K m(2) and h 2 by calulate the v 2 ratios at 0.75K m(2) to K m(2) , and K m(2) to 1.25K m(2) , or 0.5K m(2) to K m(2) and K m(2) to 1.51K m(2) , and use Table 12 to obtain the estimates of h 2 .
  • the values of h obtained from the paired readings should not differ by more than 1. If the difference between the paired readings is significant then the K m should be adjusted downwards if the higher concentra-tion is giving the lower h value, or upwards if the reverse is true. The K m adjustment is continued until the h values are within 1 unit of one another, which then provides a new estimate of K m(2) .
  • the mean of the two h values is then taken as an estimate of h 2 .
  • Table 13 (using equation 17) then indicates the correction required from the first esti-mate using the plot minimum.
  • the two adjusted K m values should agree to within about 20% (it should be noted that the Table 13 correction is based on a single plot point esti-mate).
  • Carry out a 1/ h v* plot against S h The transporter II values are then used to obtain net v 1 values and the 1/v 1 * is plotted against S in the range below K m(1) .
  • This plot provides a reestimation of the transporter I parameters, which are then used to reestimate the transporter II parameters, and the process above is repeated until constant values of one set of the transporters is obtained.
  • the final plot of net 1/ h v 2 * against S h in the range of S from below K m(2) to 0.2 K m(3) provides an estimate of the transporter II parameters.
  • Fine tune Check observed and calculated velocities at the K m values, and at selected below the lowest, above the highest, and between.
  • Figures 46 and 47 show the uptake curve and the inverse label plot respectively.
  • Estimted K m(3) 2 .0.
  • v(1) are results from calculated parameters.
  • v(2) adjustments are: K m(1) to 0.03mM, V m(1) to 6.8, V m(2) to 25 and V m(3) to 22.
  • Figure 48 shows the calculated curve in relation to the means of the plot points.
  • Figures 49 and 50 show the uptake curve and the inverse label plots respectively for normal uptake and Na + - deficient uptake.
  • Figure 502 shows the calculated curve in relation to the means of the plot points.
  • K m values are in mM and velocities are in nmol . min -1.mg -1 protein.
  • the procedure consisted of a double-labelling experiment in which the cells were first exposed to 2 mM 3 H- glutamine (50 ⁇ Ci/ml) for 10 minutes to measure uptake of label into protein (subsequently used in determination of cell number) and were then exposed to 1 4 C-glutamine (50 ⁇ Ci/ml) for 1 minute to determine uptake rates.
  • the same labelling solutions were used and therefore a 50 ⁇ l sample was removed after each incubation and counted to correct for label dilution. Concentration dilution did not occur because each labelled, incubation was preceded by a 3 minute incubation with unlabelled glutamine at the same test concentration as that of the labelled
  • the cells were removed from the surface of the culture bottle using a spatula and acetone and the protein was precipitated by coagulation at 100°C (McEvoy-Bowe et al., J. Chromatog., 347, (1985) 199-208).
  • the upper aqueous layer was removed by pasteur pipette and kept. This fraction contained cytoplasmic amino acids and a 0.2 ml sample was taken to which 0.3 ml of H 2 O and 4 ml of ACS (Amersham) were added for radioactive counting.
  • the lower layer was re-extracted with another 0.5 ml of ammonium acetate and the upper layer was this time discarded.
  • the chloroform was evaporated and the protein precipitate was washed in 2 ml of 5% (w/v) sulphosalicylate and then two times in 2 ml of H 2 O. After each wash, the tubes were centrifuged at 600 g for 10 minutes. The protein pellet was dried at 56°C overnight then 1 ml of 0.1 M NaOH was added to the dried protein and the tubes were tightly capped then placed at 100°C overnight to digest the protein. Protein content was determined using BSA as a standard and reading absorbancies at 500 nm (Lowry, et al., J . Biol . Chem . , 193 , ( 1951 ) 265-275 ) . Radioactive samples were counted on the LKB Wallac 1215 Rackbeta Liquid Scintillation counter, which was programmed for double label counting of ACS quenched
  • Another embodiment of this invention relates to transport assay plate assemblies useful in carrying out the assays of this invention. Another embodiment comprises a procedure for using such assemblies.
  • a conventional multi-well plate may take the form of a one piece plastic moulding shaped to provide anything from six to ninety six wells positioned in a rectangular array.
  • the plate is often provided with a flat cover which simultaneously closes off the top of all the wells. Individually removable covers may also be provided to allow separate access to each well.
  • U.S. Patent 4,599,314 there is a disclosure of this type of multiple well plate and lid structure.
  • Tissue culture cells which grow as a monolayer on the surface of culture plates provide a population of homogeneous cells which may be exposed to changes in culture conditions instantaneously and simultaneously thereby allowing physiological conditions to be approximated.
  • Multi-well plates provide the opportunity to provide transport experiments on a large number of samples at the same time, but there is a need for a method of transferring pre-incubation and radioactive solutions into each of the wells at the same time.
  • This invention provides a cover for a transport multi-well assay plate comprising a lid with an array of downwardly projecting open mouthed enclosures arranged to be coincident with the wells in the assay plate, whereby when the cover is placed onto the assay plate and the lid abuts the plate, the mouths of the enclosures extend into the well cavities of the plate.
  • This invention also provides a transport assay plate assembly comprising an assay plate with a plurality of spaced apart wells therein and a removable cover adapted to be free standing on a flat support surface when inverted, the cover having an array of downwardly projecting open mouthed enclosures coincident with the wells in the plate whereby, when the cover is placed over and in abutting contact with the plate, the mouths of the enclosures project into the well cavities of the assay plate.
  • This invention also provides a transport assay procedure comprising growing cells on the base of a plurality of wells in an assay plate, inverting a cover comprising an array of a plurality of enclosures coincident with the wells in the assay plate, adding an incubation medium to each enclosure in the cover, inverting the assay plate and placing it over the cover, and inverting the assembly to ensure that the incubation medium in each enclosure is transferred to the adjacent well in the assay plate.
  • the transport assay cover described herein is adapted for use with a variety of sizes of multi-well assay plates.
  • the covers are adapted for use with a six well plate and a ninety six well plate.
  • this invention includes other covers that would be equally suitable for a variety of multi-well specimen plates.
  • manufacture of the cover from readily available laboratory equipment it is understood that the covers could be moulded from suitable sterilizable plastics.
  • FIGS 66 and 67 illustrate an assembly of a cover 10 and a transport assay plate 20 comprising a rectangular base structure 21 with six flat bottomed wells 22 spaced in two rows of three.
  • the cover 10 comprises a substantially rectangular lid 11 of the same cross section as the plate with downwardly extending peripheral flanges 12.
  • a plurality of cylindrical enclosures 13 are formed to extend downwardly from the lid in an array corresponding with the array of wells 22 in the plate 20.
  • Each enclosure 13 is in the form of an inverted cylinder closed at one end 14 and with a mouth 15 at the other end that projects downwardly from the plane of the lid 11.
  • cells are grown on the bottom of the culture plate to a confluency of 75% to 90% over a time period of two to three days.
  • the culture medium is discarded by tipping the medium from the assay plate and a new incubation medium is added to the wells by using the cover described above.
  • Any number of different solutions can be placed into the enclosures of an upturned cover prior to the commencement of the experiment.
  • the solution may be kept at the required temperature by placing the inverted cover in a water bath at a predetermined temperature. A few seconds prior to the required time, the culture plate containing cells that have adhered to the base of the flat bottomed wells, is inverted and fitted over the corresponding solutions in the enclosures defined by the cover (see FIGS.
  • the cross section of the enclosures 13 in the cover 10 are considerably smaller than the cross section of the flat bottomed wells 22 in the assay plate 20. While it is not essential that the cross section of the enclosures be considerably smaller, it is advantageous because it reduces the likelihood of spilling or sloshing of the contents of the cover when the transfer takes place.
  • the cross section of the enclosures be considerably smaller, it is advantageous because it reduces the likelihood of spilling or sloshing of the contents of the cover when the transfer takes place.
  • enclosures can be virtually identical with the cross section of the wells.
  • a second embodiment embraces a ninety six enclosure cover for use with a ninety six well assay plate.
  • the cross section of the enclosures can be smaller or virtually
  • Suitable clamping means such as clips may be used to improve the close fit between the cover and the plate.
  • the six well cover is designed so that the enclosures accommodate between 1 and 3 ml of solution, although the design can be modified to accommodate more solution. It is important that the mouths of the enclosures project from the underside of the lid by at least about 5 mm to thereby protrude into the well cavity.
  • the ninety six well cover can be designed with enclosures that accommodate between 100 and 300 ⁇ l or more of solution. Both the six well and ninety six well cover are designed with flat bottomed enclosures to ensure that the cover is free standing when inverted on a flat surface.
  • Glutamine transport into HeLa cells was concentration dependent and saturable. The uptake rates are expressed per mg protein which reflects the number of viable cells present in a harvested sample as opposed to cell number.
  • FIG. 2 shows that the uptake of glutamine into HeLa cells is divided between two high affinity transport systems (transporters I and II) and a low affinity system (transporter III).
  • Uptake of glutamine below 0.2 mM (transporter I) showed a small degree of cooperativity whilst uptake via transporter II obeyed Michaelis-Menten kinetics between the concentration range of 0.2 mM - 1 mM.
  • Transporter III exhibited cooperative kinetics for 1.5 - 3.0 mM and the nonsaturable component was 3.3 nmol/min/mg protein, which was deducted from all velocity determinations.
  • transporter II was very high at 167 ml/min/mg protein whereas the specificity of transporter III is very low, 12.2 ml/min/mg protein. As expected, the specificity of transporter I is extremely high at 227 ml/min/mg protein indicating that this transporter is highly specific for the substrate glutamine.
  • Table 4 (FIG. 75) classifies glutamine transporters according to Na + -dependency, the velocity of uptake at 0.75 mM glutamine concentration, the nature of the major known inhibitor, and the capacity to be derepressed in glutamine deficient medium.
  • FIG. 3 Titration of glutamine transport at 1 mM glutamine with NEM ranging from 0.25 to 10.0 mM is shown in FIG. 3.
  • the pre-incubation time used was twenty minutes which was found to be the optimum incubation time for maximum effect of 1 mM NEM on glutamine transport at 1 mM, as shown in FIG. 4.
  • the effect of the sulphydryl reagent N- ethylmaleimide on glutamine transport into HeLa cells over the concentration range of 0.25 - 4.0 mM glutamine is shown in Fig 5.
  • the monolayers were exposed to radioactive glutamine and uptake measured for 1 minute. Uptake via all transporters I, II, and III was inhibited by 1 mM NEM.
  • the inhibitory action of NEM was found to be by direct competition with glutamine for a site on the carrier by including a large excess of the substrate in the pre-incubation mixture which should protect the carrier from binding NEM.
  • Table 3 shows that an excess of L-glutamine partially protected glutamine transport at 1 mM from inhibition by 1 mM NEM. D-glutamine however exerted no protective effect.
  • the isolated receptors may be subunits.
  • the conditions used to isolate the transporters particularly using SDS (which breaks non- covalent bonds) and, in combination with 2 mecaptoethanol (which breaks disulphide bonds), has the capacity of reducing large functional molecules to their lowest molecular weight.
  • the transporter exists as many subunits of size M r 42000 bonded either by disulphide bonds or by non-covalent forces. It may also be bonded to other smaller or larger molecules that are undetected in this system.
  • the combination of such subunits, which together make up the function of the glutamine transporter is also encompassed by this invention.
  • tumour glutamine transporters can be isolated in a manner similar to that described above for tumour glutamine transporters.
  • transporters which are responsible for the uptake of amino acids into tumour cells, especially if such transporters are not present or are not active in non-tumour cells.
  • an alanine transporter found in HeLa cells was also isolated using the following procedure.
  • FIG. 63 The titration of alanine transport at 1 mM alanine with NEM concentrations ranging from 0.25 mM - 10.0 mM is shown in FIG. 63.
  • HeLa cells were pre-incubated with varying NEM concentrations prior to exposure to 14 C- alanine to measure the rate of alanine uptake at 1 mM.
  • Inhibition of alanine transport reached a maximum after 2 mM NEM where the rate of alanine transport was 40% of the control value.
  • the effect of 1 mM NEM on alanine transport into HeLa cells that were pre-incubated for 15 minutes with 1 mM NEM is shown in FIG. 64.
  • the mode of action of the sulphydryl reagent was shown to be by direct competition with alanine for a site on the carrier by including a large excess of substrate in the pre-incubation mixture.
  • Table 14 (FIG. 85) shows that 100 mM alanine partially protected the inhibition of alanine uptake by 1 mM NEM while the non-specific stereoisomer D-alanine failed to protect the carrier from NEM inhibiton.
  • NEM Lineweaver-Burk analysis of the inhibition of alanine uptake by NEM (shown in FIG. 62) indicated that, although NEM is an irreversible inhibitor, its mode of action appears to be manifested in a lowering of the K m suggesting it is a competitive inhibitor.
  • alanine transport protein of HeLa cells was based on the same principles used for identification of the glutamine transport proteins.
  • the use of NEM to label the alanine carrier was a likely possibility because alanine transport has been reported to be highly sensitive to NEM inhibition.
  • HeLa cells plasma membranes were incubated with 3 H-NEM in the presence or absence of excess transport substrate alanine and the membrane proteins were resolved by SDS gel electrophoresis. A number of proteins were found to bind 3 H-NEM as shown in FIG. 27, but only peaks C and D were found to be protected by the presence of L-alanine. In FIG.
  • HeLa cell plasma membranes were pre-incubated with 100 mM alanine, or without alanine, prior to and during incubation with 3 H-NEM.
  • the non-transported stereoisomer D-alanine failed to protect either of these peaks from binding the label.
  • Alanine transport into HeLa cells appears to have distinct kinetic characteristics which distinguishes it from glutamine transport in these cells.
  • the uptake curve conforms to simple Michaelis-Menten kinetics, reaching maximum velocity at 15 nmol/min/mg protein with a Km of 5.0 mM.
  • the presence of 1 mM NEM in the pre-incubation medium resulted in a 50% inhibition of alanine uptake at 1 mM alanine.
  • Correlation of transport inhibition with binding of NEM to membrane proteins as well as substrate protection of binding indicated that a protein band of M r 31000 is involved in alanine transport into HeLa cells.
  • the protein with M r 31000 could likely be isolated in the same manner as described above for TGT II.
  • tumour amino acid transporters
  • tumour glutamine transporter isolated from HeLa cells it would be desirable to know whether the same transporter is found in other tumour cells.
  • TGT II is present in other solid-type tumour cell lines and lymphoma cell lines. The obvious benefit from this knowledge is that it will now be possible to diagnose and selectively treat many different types of cancer using the same diagnostic and therapeutic compositions.
  • the following experimental procedure was used to determine whether antibodies raised against TGT II would react with proteins present in other solid-type tumour cell lines, lymphoma cell lines, leukaemia cell lines and normal human cell lines.
  • mice Male Balb/c mice were used to raise antiserum by injecting 500 ⁇ l of antigen (250 ⁇ l purified glutamine transport protein (250 ⁇ g/ml) and 250 ⁇ l of Freunds complete adjuvant) intramuscularly. The mice were boosted every month with antigen made up in Freunds incomplete adjuvant for three months before being bled through the tail to collect serum. The blood was allowed to clot overnight at room temperature and the serum was transferred to a microfuge tube and centrifuged at 1200 g (Eppendorf ultracentrifuge) for 10 seconds to remove red blood cells. The antiserum was stored at -20°C. Control serum from uninjected mice was also obtained.
  • antigen 250 ⁇ l purified glutamine transport protein (250 ⁇ g/ml) and 250 ⁇ l of Freunds complete adjuvant
  • the mice were boosted every month with antigen made up in Freunds incomplete adjuvant for three months before being bled through the tail to collect serum. The blood was allowed to clot overnight at
  • Antiserum producting in rabbits Male rabbits were used to raise antiserum by injecting 2 ml of antigen (250 ⁇ g/ml mixed 1:1 with Freund's complete adjuvant) intramuscularly followed by booster shots subcutaneously every two weeks using Freund's incomplete adjuvant over a period of three months. Blood was collected by bleeding the rabbits through the peripheral ear vein and allowing the blood to clot overnight at 4°C. The serum was collected and centrifuged at 400 g for 10 minutes to remove red blood cells. The serum was stored at -20°C in 500 ⁇ l aliquots. Control serum was collected from the rabbits prior to the first injection.
  • a 96-well microtitre plate (Flow Laboratories) was coated with 50 ⁇ l of a doubling dilution series of antigen (50 ⁇ g/ml) solutions in coating buffer (35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6) into the first five rows of 12 wells. PBS A (50 ⁇ l ) was added to the wells of the final three rows. The plate was incubated for 2 hours at room temperature
  • the antigen/PBS solution was flicked out of the wells and the plate was washed three times in washing buffer (0.05% (v/v) Tween 20 in PBS A).
  • the plates were dried by slapping them face down on wads of paper towels.
  • the wells were blocked by incubating with 0.2% (w/v) casein in TEN buffer (0.5 M Tris-HCl, 0.01 M EDTA, 1.5 M NaCl, pH 9.0) for 2 hours at room temperature followed by washing three times in washing buffer as before.
  • a screening assay was developed for further screening of the antisera.
  • plates were coated with a 1/50 dilution of antigen (50 g/ml) and probed with a 1/100 dilution of antiserum.
  • the conjugate dilution used was 1/1000 and plates were read as described above.
  • Nitrocellulose cut to size was wetted slowly in transfer buffer and a sandwich was made with the gel at the cathode and the nitrocellulose on the anode side. Transfer was carried out at a constant voltage of 60 volts for 3 hours.
  • the nitrocellulose was removed and placed onto a piece of filter paper wetted with tris-buffered saline (10 mM tris- HCl, 150 mM NaCl, pH 8.0) containing 0.05% (v/v) Tween 20 (TBST) and 4% (v/v) FBS and incubated overnight at 4°C.
  • the nitrocellulose was then transferred to a solution of
  • Plasma Membrane Preparation Plasma membranes from adhesive cell lines were prepared as described above for HeLa cell plasma membranes. Suspension cultures were grown to a confluence of approximately 10 8 cells/ml and then a plasma membrane fraction was prepared by sucrose gradient centrifugation (see Segal et al., J. Cell. Phvsiol., 100, (1979) 109-118). All manipulations were carried out at 4°C where the cells were first pelleted at 200 g for 5 minutes (Clements 2000 benchtop centrifuge) and then washed two times with 0.9% (w/v) sodium chloride.
  • the pellet was resuspended in 10 ml of lysis solution (1 mM NaHCO 3 , 0.5 mM CaCl 2 , pH 7.4) and homogenized with 30 strokes of a tight fitting Dounce homogenizer (maximum setting).
  • the cell homogenate was centrifuged at 500 g for 20 minutes in a swinging arm rotor (SW 25.1) of a Beckman centrifuge and the supernatant was collected and kept.
  • the pellet was re-homogenized in another 10 ml of lysis solution and pelleted as described above. The supernatants were pooled and then sedimented at 12800 g for 20 minutes.
  • the membrane pellet was resuspended in 5 ml of lysis buffer and mixed with 15 ml of 40% (w/v) sucrose (made up in lysis solution) to make a 30% solution.
  • a 20 ml syringe fitted with a long piece of tubing was used to carefully layer 15 ml of 40% (w/v) sucrose underneath the 30% sucrose membrane solution in the centrifuge tube.
  • the tube was then centrifuged at 54450 g for 4 hours after which time a white membrane layer was visible at the interface of the gradient.
  • the membrane layer was collected with a pipette and sedimented at 45000 g for 1 hour.
  • the pellet was resuspended in 10 mM tris-HCl, pH 7.4 and stored at -20°C.
  • Purified plasma membranes from a variety of cell lines (Table 9 (FIG. 80) were solubilized in SDS-solubilization buffer and applied to separated wells of a 12% (w/v) polyacrylamide gel. The separated proteins were blotted onto nitrocellulose and probed for reaction with glutamine transporter anti-serum as described above.
  • the rate of glutamine uptake into the other cell lines was also determined using the following procedure.
  • a new procedure for measuring uptake into adhesive cells was developed for investigating the effects of glutamine analogues on the rate of glutamine uptake into HeLa cells and for measuring the rate of glutamine uptake into other adhesive cell types.
  • a separate procedure is described for measuring glutamine uptake into suspension cultures.
  • Adhesive cells were grown in 6-well culture plates (Flow) until confluence was reached. All incubations were carried out at 37°C and each new solution was transferred into the wells using a modified culture plate lid (described above) which had 6 x 4 ml scintillation vials inserted into the lid directly above each well. Normal procedure was to pour out the old solution from the culture wells, invert the plate and clamp it to the lid with the new solution in place in the scintillation vials. The clamped assembly is then inverted, and the culture wells are simultaneously supplied with the new solution. Two hours before each experiment the cell cultures were incubated with fresh medium 199 containing 10% (v/v) FBS.
  • the medium was then exchanged for unlabelled 2 mM glutamine in medium 199 and incubated for 20 minutes. This medium was removed and replaced with glutamine in HBSS at the test concentration (0.25 - 5.0 mM) for three minutes followed by 14 C-glutamine (62.5 ⁇ Ci/ml) at the test concentration in HBSS for exactly 1 minute. Uptake was stopped by washing the wells three times with ice-cold 0.9% NaCl. Soluble amino acids were removed by incubation with 0.5 ml of 5% (w/v) trichloroacetic acid and counted for radioactivity on the scintillation counter. The remaining protein was solubilized in 1 M NaOH for 120 hours and the amount of protein present was determined. Results were expressed as nmoles of glutamine incorporated per minute per mg protein.
  • microfuge tubes were spun for a minute and then placed in the freezer overnight.
  • the bottoms of the microfuge tubes were amputated into scintillation vials which contained 200 ⁇ l of PBS A and were shken vigorously. Scintillation fluid was added, the vials were again shaken, and then counted for radioactivity on a Liquid Scintillation Counter. Inhibitor/ analogue was added during the 20 minute incubation with the HBSS/2 mM glutamine for transport inhibition experiments.
  • the rate of glutamine uptake into a variety of cell lines was investigated over the concentration range of 0.25 - 4.0 mM glutamine.
  • the uptake curves for the different cell lines are given in FIGS. 7a-g.
  • a summary of the results for uptake rates at 0.75 mM glutamine (physiological concentration) is given in Table 6 (FIG. 77) with the inclusion of the results from the literature for a variety of tissues. All of the solid-type tumour cell lines investigated had high uptake rates. Most of the normal and lymphocyte-derived cell lines (both normal and cancer) exhibited low uptake rates with the exception of the normal cell line MRC5 and the lymphoblastic leukaemic cell line MOLT 3.
  • Table 9 (FIG. 80) lists the immunoblot analysis of the cell lines with either mouse or rabbit anti-serum raised against the glutamine transport protein of HeLa cells. A positive antibody response was indicated by a coloured band appearing on the blot in a position analogous the molecular weight of 42000. A representative immunoblot is given in FIG. 26 showing both positive and negative results. A number of other bands reacted non-specifically with the anti-serum which suggests some degree of cross reactivity was occurring.
  • Table 10 (FIG. 81) lists the cell lines with their response to immunoblot analysis with either mouse or rabbit anti-serum as well as their glutamine uptake rates at 0.75 mM. Only cell lines with complete data are listed. Comparison of the results given in Table 9 (FIG.
  • inventions comprise monoclonal and polyclonal antibodies which recognize human amino acid transporters in accordance with this invention.
  • the procedure for preparing polyclonal antibodies to TGT II can also be followed for preparing polyclonal antibodies to other human amino acid transporters.
  • monoclonal antibodies that are specific to epitopes on a human amino acid transporter.
  • monoclonal antibodies can also be used in recombinant cloning procedures to isolate the gene which codes for TGT II.
  • Monoclonals for any transporter can be prepared using a procedure similar to the following procedure for preparing monoclonal antibodies which recognize TGT.
  • mice, rats or other animals are immunized against the glutamine transport protein using, for example, either purified dissociated TGT, purified native TGT, a preparation of crude membrane proteins in native form, or peptides made from the amino acid sequence of the
  • the spleen of an immunized mouse is removed and fused with cultured myeloma cells in fusion experiments.
  • the fused cells are seeded into culture dishes under conditions which select for hybridoma cell growth. 4. Once the hybridoma colonies have begun
  • One screening procedure is an ELISA assay comprising the following steps:
  • Plates e.g., 96 well are coated with
  • IgG conjugate anti-mouse antibody which is conjugated to an enzyme or other reporter molecule
  • Positive clones may be grown in bulk by injecting into ascites fluid of mice.
  • the hybridomas can be screened using a glutamine transport inhibition assay.
  • Monoclonal antibodies which bind to the transporter will inhibit transport of glutamine and, in this way, can also be identified. The screening procedure
  • Plates e.g., 96 well are inocculated with HeLa cells which are grown until 75% confluent.
  • the HeLa cells are pre-loaded with a high concentration of glutamine (2-3 mM).
  • Radioactive uptake of glutamine is stopped by washing with ice-cold 0.9% (w/v) NaCl.
  • cytoplasm is quantitated by radioactive counting (the soluble components are extracted using trichloracetic acid).
  • Positive clones are grown in large numbers either by culturing or by injecting into ascites fluid of mice.
  • antibodies to regions of the glutamine transporter which are distant from the glutamine binding sites. While such antibodies are equally valuable in diagnosing the presence of tumour cells, they cannot be screened for by the inhibition assay described above. Rather, they can be identified in other ways. First, if the receptor has been isolated, then antibodies will bind but will not block the binding of glutamine. Second, they could be examined by using cells which are high in the glutamine receptor, and the results compared with other cells which have absent or low amounts of receptor. A differential reactivity will indicate the likelihood that the antibodies are to the glutamine receptor.
  • the antibodies that are obtained will depend upon the protein that is used to immunize the mouse or other animal.
  • TGT II is obtained from SDS- PAGE
  • the protein will be in its "dissociated” or “denatured” form.
  • Antibodies which depend upon the three dimensional configuration of the transporter protein will not be formed.
  • an animal in order to obtain monoclonal (and polyclonal) antibodies which depend upon the three dimensional configuration of, for example, TGT II, an animal must be immunized with the native form of the protein. This can be accomplished in at least two ways.
  • the animal can be immunized against portions of the tumour cell membranes which contain TGT II. Monoclonal antibodies to the native form can then be screened for in the same manner as described above. Alternatively, the animal can be immunized with purified native protein obtained from recombinant methods as described below.
  • the type of monoclonal antibody obtained will depend not only on the amino acid transporter used, but also whether the transporter is in its native or dissociated form. Similarly, the antibodies obtained may depend upon whether the entire protein or only a fragment or subunit is used. Monoclonal antibodies can be produced to the glutamine transporter, and these can be produced in mice, rats, other species and eventually in humans. All of these varieties of monoclonal antibodies are within the scope of this invention if they can recognize parts of the glutamine transporter or subunits thereof. Additionally, it is now recognized that antibodies can be used whole, in part, (e.g., Fab'2 or Fab), or, indeed, single chain antibodies are now being described wherein the specificity is due to the retention of the hypervariable region.
  • monoclonal antibodies can be genetically engineered so that segments of the sequence from one species can be transferred to the genes of the same or another species to provide hybrid chimeric molecules. All such antibodies which have the capacity of binding to the glutamine transporter (in part or in whole) are within the scope of this invention.
  • the N-terminal and partial amino acid sequence of the transporter (or its subunit) can be obtained, oligonucleotide probes can be synthesized, and both cDNA and genomic clones can be obtained by standard procedures involving the probing of both cDNA and genomic libraries of different sources and different species.
  • the isolation of the glutamine transporter described herein will naturally lead to the cloning of both cDNA and genomic clones in one species (human), but should also lead to the isolation of the transporter in other species using cross-species hybridization.
  • the methods used for gene cloning are numerous and usually several methods are tried which lead to the isolation of the gene.
  • polyclonal antisera or monoclonal antisera could include the use of polyclonal antisera or monoclonal antisera, and an expression system; the isolation in cells where the antigens expressed on the cell surface lead to the isolation of cDNA; transfection methods; messenger RNA selection procedures; identification of restriction fragment length polymorphisms leading to the cloning of the gene or translocations.
  • polyclonal antisera or monoclonal antisera and an expression system
  • the isolation in cells where the antigens expressed on the cell surface lead to the isolation of cDNA
  • transfection methods messenger RNA selection procedures
  • identification of restriction fragment length polymorphisms leading to the cloning of the gene or translocations are now standard and used in many laboratories throughout the world and need not be described in detail.
  • An important feature is the nucleotide sequence obtained from the cDNA and in genomic clones and variations thereof due to genetic polymorphism, tissue polymorphism, gene rearrangement or alternative splicing, or any
  • E. coli cells, other bacteria, yeast and higher eucaryotic cells may be made to synthesize foreign proteins, such as the glutamine transport protein, by cloning cDNA into expression vectors. This is achieved by first isolating (total) mRNA from tissue known to be expressing the protein of interest and generating cDNA from the mRNA, using reverse transcriptase to generate the first strand, degrading the RNA template with RNase H, and second strand synthesis with DNA polymerase. The double-stranded cDNAs are then cloned into suitable expression vectors, either plasmids or phage.
  • suitable expression vectors either plasmids or phage.
  • the cloning sites in these vectors are either (1) in the 5' coding region of a regulated gene, resulting in synthesis of a fusion protein, or (2) insertion immediately downstream of a suitable promoter/ribosome binding site, resulting in synthesis of non-fused protein.
  • Colonies plasmid vectors
  • plaques phage vectors
  • cDNA clones capable of expressing the glutamine transport protein (or parts of it) in prokaryotic systems offers the potential for production of large quantities of the protein.
  • the pure material can be used in diagnostic tests or for therapeutic purposes. Additionally, it can be used for raising antibodies (both monoclonal and polyclonal), as well as making the screening of new chemical compounds (or new monoclonals) for anti- glutamine uptake activity much simpler than at present.
  • mRNA source HeLa derived D98-AH2 cells (HPRT in phenotype)
  • Screening criteria (recombinants versus non-recombinants): clear plaques versus blue plaques.
  • Average insert size 0.86 kb (range: 0.48 to 3.1 kb).
  • the basis of one screening method which can be used is as follows. Cloning cDNAs into the EcoRI site of lambda gtll results not only in insertional inactivation of the (phage-encoded) ⁇ -galactosidase but, if the insert is in the correct orientation and reading frame, the cDNA insert can be expressed as a fusion protein, which can be detected with antibodies specific for the cDNA-encoded protein(s). Phage are induced using isopropyl b-D-thiogalactopyranoside (IPTG) and fusion proteins bound to a nitrocellulose filter lift.
  • IPTG isopropyl b-D-thiogalactopyranoside
  • the filters are then screened by soaking briefly in dilute primary antibody (in this case the polyclonal antiserum raised to purified transport protein in mouse hosts), unbound primary antibody is washed off, and then bound primary antibody is detected using a secondary antibody to the primary host, which has been conjugated to an enzyme.
  • the secondary antibody conjugate can be affinity purified using goat anti-mouse IgG (H+L) alkaline phosphatase conjugate.
  • Phosphatase conjugated secondary antibody bound is then detected using the coloured phosphatase substrate cocktail 5-bromo-4-chloro-3-indolyl phosphate (BCIP) plus nitro blue tetrazolium (NBT).
  • nitrocellulose filter Place plates in a 37°C incubator. Pre-soak a nitrocellulose filter disc in 10 mM (aqueous) isopropyl ⁇ -D-galactopyranoside (IPTG). Overlay the plates with dried IPTG-treated filters and incubate for 3.5 hours at 37°C.
  • IPTG isopropyl ⁇ -D-galactopyranoside
  • Process filter for screening Remove plates to room temperature, apply marks to filter to allow subsequent alignment of filter with plate, and carefully remove filter from the agar. Wash the filters in 100 mM Tris-HCl, pH 8.0, containing 150 mM NaCl and 0.05% (v/v) Tween 20 (TBST solution) to remove any adherent agar. To saturate nonspecific protein binding sites incubate the filters in TBST solution containing 1% bovine serum albumin for 30 minutes, using 7.5 ml of solution for each filter.
  • Primary antibody solution ie. mouse anti-transport protein
  • E. coli extract immediately prior to use of the serum for screening nitrocellulose filters.
  • 100 ⁇ g/ml of E . coli extract can be used, and the incubation can be for 30 minutes.
  • Stop colour development When plaques are visible and before the background becomes dark, stop the phosphatase reaction by discarding the substrate solution and transferring the filters to 20 mM Tris-HCl, pH 8.0, containing 5 mM EDTA. Then store the filters either dry or in this solution.
  • Primary screening generally results in an average of about 2 positives per plate, i.e., the overall frequency of positives on the primary screen is about 1 per 1.5 x 10 4 plaques.
  • Putative positive clones from the primary screening plates are picked and transferred to secondary screening plates of the host bacterium E . coli Y1090r. Positive plaques are aligned on a grid pattern template, and negative (control) plaques are included on each plate. The plates are treated as in the primary screen (steps 4-12). Approximately 75% of the putative positives from the primary screen are positive on the secondary screen. This purification process is repeated twice; on the tertiary screening plates the frequency of positives is about 80-100%, and on the quaternary screen all plaques can be positives. Positives from the quaternary screen can be grown in bulk and from them DNA prepared by standard methods (Davis et al., 1986, Basic
  • the cloned, native glutamine transporter molecule or fragments thereof can then be used to produce both mono- clonal and polyclonal antibodies.
  • the dissociated protein obtained from SDS-PAGE could be sequenced to obtain an amino acid sequence of the transporter.
  • a DNA probe can be prepared to probe the cDNA library for complimentary strands of DNA.
  • genomic clones can be isolated from genomic libraries.
  • homologous cDNAs or genes in humans or other species can be isolated using these reagents. In this way, the gene which codes for production of the native protein can be obtained. Expression is carried out as previously described.
  • the nucleotide sequence encoding the transporter can be variable for the reasons described below.
  • the degeneracy of the genetic code nucleotide change does not necessarily bring about a change in the amino acid encoding, e.g., the codon GUU specifies a valine residue as do the codons GUC, GUA, GUG, each being different by a single nucleotide.
  • Two or three nucleotide changes can give rise to the same amino acid, e.g., codons UUA, UUG, CUU, CUC, CUA and CUG all encode Leucine. Codons AGU, UCC, UCU, UCA and UCG encode serine.
  • allelic variations Variations in nucleotide sequence and amino acid sequences of the encoded protein as well as resultant may occur between individual members of the same species. These variations arise from changes in the nucleotide sequences encoding the protein. Thus, different forms of the same gene (called alleles) give rise to protein of slightly different amino acid sequence but still have the same function.
  • Variation can occur as the result of differential mRNA splicing where one gene composed of many different segments (exons) of coding sequence - DNA encoding the mature protein - gives rise to a RNA that is spliced such that the portion of the RNA derived from certain exons are removed. Selection of exons may be different in different cell types.
  • Proteins having the same function e.g., major histocompatibility proteins may arise from related genes.
  • immunoglobulms which have nucleotide and amino acid sequence variation but retain their primary function of antigen binding.
  • homologous proteins are encoded by homologous genes. These genes arise by duplication of one original gene or by gene conversion.
  • Variation may be intentionally introduced by:
  • Such mutated (variant) clones can be used to generate variant proteins or peptides which in the context of this specification may have glutamine transport function.
  • the present invention also includes the use of segments of the glutamine transporters (peptides) and variant peptides synthesised or genetically engineered.
  • Other transporters are within and between species that are homologous at the nucleic acid, protein and functional levels. Because of substantial sequence homologies, the cDNA clones described herein would enable the isolation of related sequences.
  • Another embodiment of this invention provides anti-glutamine compounds and glutamine analogues suitable for the treatment of cancer, which have the general Formula (I): (I)
  • X is selected from CR 5 R 6 , NH, NOH and O;
  • W is selected from
  • V is -
  • R 1 is selected from OCH 2 Ph, OR 13 , NH 2 and NHNH 2 , wherein R 13 is selected from H and a C 1-5 substituted or unsubstituted, cyclic or acyclic, saturated or unsaturated hydrocarbon group;
  • R 3 is selected from groups defined by R 13 ;
  • R 5 and R 6 can be independently selected from groups defined by halogen and R 13 ;
  • R 7 is selected from groups defined by OR 14 and NR 15 R 16 , wherein R 14 , R 15 and R 16 are independently
  • R 8 is selected from groups defined by R 14 , OR 14 , amino, ami do NO, and NO 2 ;
  • R 9 is selected from groups defined by R 14 ;
  • R 8 and R 9 taken together with the nitrogen atom represent a 3- to 8-membered, substituted or unsubstituted, saturated or unsaturated heterocycle
  • R 10 is selected from groups defined by R 14 ,
  • R 11 and R 12 are independently selected from
  • V — W — X — CH 2 can represent the group
  • R 17 is selected from groups defined by R 14 ,
  • R 18 and R 19 are independently selected from
  • R 14 groups defined by R 14 , halogen and R 14 groups substituted at the carbon ⁇ to the 4- membered ring by a leaving group (exemplified by, but not limited to, OMs, OTs and halogen);
  • V— w— X represents the group
  • R 20 is selected from groups defined by R 14 ,
  • Z is selected from O, S and NR 14 ;
  • Classes of compounds defined by Formula (I) which are of interest include, but are not limited to compounds of the general Formula (II):
  • X is selected from CH 2 , NH, NOH or O, and wherein
  • R 1 , R 2 , R 8 , and R 9 are as defined above.
  • Classes of compounds defined by Formula (II) which are of interest include, but are not limited to compounds of the general Formula (III): (HI)
  • X is CH 2 , NH, NOH or O
  • R 23 is OH when R 8 is selected from groups defined by
  • R 23 and R 8 are independently selected from groups previously defined by R 14 ;
  • R 23 and R 8 taken together with the nitrogen atom form a 5-or 6-membered heterocyclic ring which may be saturated or unsaturated, and substituted or unsubstituted.
  • Classes of compounds defined by Formula (III) which are of interest include, but are not limited to:
  • R 26 is H, OH or NO
  • R 24 and R 25 are independently selected from H, OR 13 , SR 13 , NR 1 32 , NO 2 , CHO, CO 2 H or halogen,
  • R 13 is as previously defined.
  • R 27 and R 28 are independently selected from H and
  • R 29 is selected from:
  • R 30 and R 31 are independently selected from groups as previously defined by R 13 .
  • C 1-5 substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic hydrocarbon group includes alkyl, alkenyl or alkynyl, inclusive of straight and branched chain groups and cyclic groups of one to five carbon atoms, which may be substituted with substituents which assist the binding of such compounds to the glutamine transporter protein of tumour plasma cell membranes, or which modify the action once the compounds have been transported.
  • 3- to 8-membered cyclic hydrocarbons includes 3- to 8-membered saturated and unsaturated hydrocarbon rings, which hydrocarbon rings may contain 1 to 3 heteroatoms (N, O oo S) and may be substituted with substituents which assist the binding of such compounds to the glutamine transporter protein of tumour plasma cell membranes or which modify the action once the compounds have been transported.
  • the "3- to 8-membered cyclic hydrocarbons are selected from C 5-6 aromatic or heteroaromatic hydrocarbons substituted by one or more polar groups such as OH, SH, NHR 13 , NO 2 , CHO, COOR 13 , or halogen, wherein
  • R 13 is as previously defined.
  • amino includes primary secondary and tertiary amino groups, NR 8 R 9 , wherein R 8 and R 9 are as previously defined.
  • Possible salts of compounds of the general formulas (I), (II) and (III) include, for example, all of the acid and base addition salts.
  • Physiologically acceptable salts may be generally derived from inorganic or organic acids or bases.
  • Physiologically unacceptable salts for example, which may initially be obtained as process products, for example in the preparation of the compounds according to the invention on an industrial scale, can be converted into physiologically acceptable salts by known processes.
  • physiologically acceptable salts are water-soluble and water- insoluble acid or base addition salts, such as the
  • hydrochloride hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, citrate, gluconate, benzoate, butyrate, sulfosalicylate, maleate, laurate, malate, fumarate,
  • succinate oxalate, tartrate, stearate, tosylate, mesylate, salicylate, sodium, potassium and ammonium.
  • Compounds of the general Formula I - III may be synthesised by a number of different methods as outlined below.
  • the method of synthesis will generally depend upon the nature of X, W and V.
  • Protecting groups will generally be required, and the choice of protecting groups for precusors will depend upon the functional groups desired in the final product.
  • the use of protecting groups, as well as the methods for protection and deprotection, are well known to those skilled in the art. (For example, see T.W.Greene,
  • R 1 to R 4 are also well known and can be introduced using reported syntheses or simply by exercise of skill following this disclosure, together with reported syntheses.
  • the order of substitution and the time of placement in the synthesis may change depending upon the nature of R 1 to R 4 and the other substituents.
  • one of ordinary skill will recognize various ways to prepare the compounds of this invention, and will have to tailor the synthesis depending upon the particular groups chosen for X, W, V and R 1 to R 4 .
  • derivatives include the pyroglutamic acid derivatives 2 and 3 as well as the glutamic acid half-esters, 6 and 7 . While several of these compounds are commercially available, it may be less expensive to synthesise them as needed.
  • activation of compounds 6 or 7 to render them suitable for nucleophilic substitution may be achieved by two main methods, viz., by forming a mixed anhydride, e.g. 8 , or an activated ester, e.g. 9 .
  • Both methods are reported in the literature (for example, see Goodman et al. (1962) J. Am. Chem. Soc, 84, 1279; Anderson et al. (1967) J. Am. Chem. Soc, 89, 5012; Dutta et al. (1971) J. Chem. Soc. (Perkins C), 2896; Kim et al. (1975) J. Chem. Soc. Chem. Commun., 473) and are
  • compounds of Formula I can likely be synthesised by reacting a suitable sulphonic acid or phosphonic acid or activated derivatives (e.g., the acid halides) with a serine derivative.
  • compounds of the Formula I may be synthesised by procedures similar to those shown in Scheme 5.
  • R 17 , R 18 , R 19 are as previously defined, may be synthesised from a suitably protected alkenyl glycine 22 by "[2 + 2] cycloaddition" with a substituted isocyanate 23 under thermolytic conditions, as exemplified in Scheme 7 (FIG. 57).
  • nitrile 25 may be synthesised by reaction of a suitably protected form of glutamine e.g. 24 with tosyl chloride and pyridine under dehydrating conditions to form the nitrile 25.
  • the nitrile may then be subject to partial solvolysis with an alcohol, R 21 OH, to yield the imino ether 26, as exemplified in Scheme 8 (FIG. 58). See, for example, Hirotsu et al.
  • the imino ether may be further elaborated by alkylation or acylation of the imine nitrogen.
  • a substituted amide 27 may be acylated or alkylated at the amide oxygen to yield the imino ether or imino ester 28.
  • reacting 25 or 26 (R 21 Me or Et) with thiols (R 21 SH) or amines (R 21 R 14 NH), wherein R 21
  • the program of chemical synthesis should be highly integrated with the biochemistry program and can be guided by computer-aided molecular modelling.
  • biological assays viz. binding parameters (B m , K s '), together with glutamine transport inhibition and tumour growth measurements, have enabled identification of at least three classes of anti-glutamine compounds and glutamine analogues which have potential in cancer therapy.
  • Preliminary molecular modelling studies can make gross structural comparisons as a means of identifying potential synthetic target molecules.
  • quantitative structure-activity relationships for each of the above-mentioned classes can be developed. Such relationships correlate observed biological activities with a variety of physical and chemical properties of the compounds, e.g., minimum energy conformations, atom juxtapositions, critical dipole moments, electrostatic potentials etc., which are available from molecular modelling and/or molecular orbital calculations undertaken within the program.

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Abstract

La présente invention se rapporte à des composés et à des procédés servant au traitement et au diagnostic du cancer, ainsi qu'à des procédés permettant d'isoler des transporteurs d'acides aminés humains ou des sous-unités de ces transporteurs, ainsi que des transporteurs ou des sous-unités sensiblement purifiés. La présente invention décrit en particulier des transporteurs de glutamine qui sont communs aux tumeurs, mais qui ne se trouvent généralement pas ou sont moins actifs ou sont présents en quantité moins grande dans la plupart des cellules non tumorales. Des produits et des procédés diagnostiques, ainsi que des produits biologiques se rapportant aux transporteurs d'acides aminés sont décrits. Des produits thérapeutiques contenant des composés antiglutamine, des analogues de glutamine, des compositions d'anticorps, des compositions pharmaceutiques et des vaccins, ainsi que des procédés de traitement du cancer chez les animaux et chez l'homme sont prévus. Des procédés et des appareils de criblage, qui servent au criblage de composés inhibant l'absorption de glutamine dans les cellules tumorales, sont également décrits.
PCT/AU1989/000427 1988-09-30 1989-10-02 Proteines de transport d'acides amines, analogues d'acides amines, appareil d'analyse et utilisations de ces elements pour le traitement et le diagnostic du cancer WO1990003399A1 (fr)

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WO2002010196A2 (fr) * 2000-07-28 2002-02-07 Merck Patent Gmbh Identification d'un nouveau transporteur de glutamine
US6541453B2 (en) * 1996-06-07 2003-04-01 Syngenta Limited Peptide derivatives
US20120101163A1 (en) * 2010-10-25 2012-04-26 Taipei Medical University Composition for inhibiting cancer metastasis
RU2527349C1 (ru) * 2013-05-30 2014-08-27 Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Новгородский государственный университет имени Ярослава Мудрого" Способ исследования скорости всасывания аминокислот в пищеварительном тракте
WO2016040527A1 (fr) * 2014-09-09 2016-03-17 Vanderbilt University Sondes du métabolisme pour la thérapie et le diagnostic
EP4074694A1 (fr) * 2011-10-27 2022-10-19 Massachusetts Institute Of Technology Lipides d'acides aminés, de peptides et polypeptides, isomères, compositions, et leurs utilisations

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CHEMICAL ABSTRACTS, Volume 96, No. 15, 1982 (Columbus, Ohio, U.S.A.), MARCIA WHEELER et al, "Agaridoxin: A Fungal Catecholamine which Acrs as an Alph-1, Agonist of Mamalian Hypothalamic Adenylate Cyclase". see page 65, column 2, Abstract No. 115866m, Brain Res. 1982, 231(2), 387-98. *
CHEMICAL ABSTRACTS, Volume 96, WO. 21, 1982 (Columbus, Ohio, U.S.A.), SZEWCZUK et al, "GGT-Test Diagnostic Kit for Determination of Y-Glutamyl Transferase Activity in Serum". see page 301, column 1, Abstract No. 176648p, Diagn. Lab. 1981, 17(4-5), 143-50. *
CHEMICAL ABSTRACTS, Volume 97, No. 23, 1982 (Columbus, Ohio, U.S.A.), MAGNAN et al, "Drug Latentiation by Y-Glutamyl Transpeptidase". see page 6, column 1, Abstract No. 192660n, J. Med. Chem. 1982 25(9) 1018-21. *
CHEMICAL ABSTRACTS, Volume 97, No. 3, 1982 (Columbus, Ohio, U.S.A.), UEKI et al, "Synthesis of Some Y-Glutamyl Amides". see page 342, column 1, Abstract No. 19678b, Stud. Univ. Babes-Bolyai, (Ser.) Chem. 1981, 26(2), 43-6. *
CHEMICAL ABSTRACTS, Volume 98, WO. 11, 1982 (Columbus, Ohio, U.S.A.), HAWKINS et al, "Helix Initiation and Propagation by (Hydroxy Ethyl=) -L-Glutaminyl Residues in Water". see page 607, column 1, Abstract No. 898962r, Macromolecules 1983, 16(2), 158-61. *
DERWENT ABSTRACT Accession No. 88-272879/39 Class B05, EP, A, 284461 (ROUSSEL UCLAF) 28 September 1988 (28.09.88). *
DERWENT ABSTRACTS Accession No. 84-057566/10, Class S03, DE, A, 3331588 (Toyo Jozo K.K.) 1 March 1984 (01.03.84). *
PATENT ABSTACTS OF JAPAN, C-349, page 19 JP, A, 61-1651 (Toyama Kagaku Kogyo K.K.) 7 January 1986 (07.01.86). *
PATENT ABSTRACTS OF JAPAN, C-121, page 135, JP, A, 57-82352 (Mitsubishi Kasei Kogyo K.K.) 22 May 1982 (22.05.82). *
PATENTS ABSTRACTS OF JAPAN, C-121, page 136, JP, A, 57-82353 (Mitsubishi Kaseikogyo K.K.) 22 May 1982 (22.05.82). *
PATENTS ABSTRACTS OF JAPAN, C-252, page 113, JP, A, 59-130253 (Fujisawa Yakuhin Kogyo K.K.) 26 July 1984 (26.07.84). *
PATENTS ABSTRACTS OF JAPAN, C-36, page 42, JP, A, 52-137910 (Mitsubishi Kasei Kogyo K.K.) 12 January 1978 (12.01.78) *
PATENTS ABSTRACTS OF JAPAN, C-57, page 89, JP, A, 56-30956 (Wako Junyaku Kogyo K.K.) 28 March 1981 (28.03.81) *
PATENTS ABSTRACTS OF JAPAN, C-76, page 39, JP, A, 56-92845 (Toyo Boseki K.K.) 27 July 1981 (27.07.81) *
PATENTS ABSTRACTS OF JAPAN, C-77, page 4834, JP, A, 52-148030 (Sanyo K.K.) 12 August 1977 (12.08.77) *
PATENTS ABSTRACTS OF JAPAN, C-87, page 87 JP, A, 56-131554 (Kaken Kagaku K.K.) 15 October 1981 (15.10.81). *
PATENTS ABSTRACTS OF JAPAN, C-95, page 10, JP, A, 56-158745 (Daiichi Kagaku Yakuhin K.K.) 7 December 1981 (07.12.81). *
See also references of EP0436612A4 *
The Merck Index, Tenth Edition (Merck and Co. Inc.) !983, see page 641 Monograph No. 4332. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020454A1 (fr) * 1993-03-10 1994-09-15 Rotta Research Laboratorium S.P.A. Nouveaux derives des acides glutamique et aspartique, leur procede de preparation et leur utilisation comme medicaments ameliorant la memoire et l'apprentissage
AU685438B2 (en) * 1993-03-10 1998-01-22 Rottapharm S.P.A. New derivatives of glutamic and aspartic acids, a method of preparing them, and their use as drugs for enhancing memory and learning
US6541453B2 (en) * 1996-06-07 2003-04-01 Syngenta Limited Peptide derivatives
WO2002010196A2 (fr) * 2000-07-28 2002-02-07 Merck Patent Gmbh Identification d'un nouveau transporteur de glutamine
WO2002010196A3 (fr) * 2000-07-28 2002-07-25 Merck Patent Gmbh Identification d'un nouveau transporteur de glutamine
US20120101163A1 (en) * 2010-10-25 2012-04-26 Taipei Medical University Composition for inhibiting cancer metastasis
EP4074694A1 (fr) * 2011-10-27 2022-10-19 Massachusetts Institute Of Technology Lipides d'acides aminés, de peptides et polypeptides, isomères, compositions, et leurs utilisations
RU2527349C1 (ru) * 2013-05-30 2014-08-27 Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Новгородский государственный университет имени Ярослава Мудрого" Способ исследования скорости всасывания аминокислот в пищеварительном тракте
WO2016040527A1 (fr) * 2014-09-09 2016-03-17 Vanderbilt University Sondes du métabolisme pour la thérapie et le diagnostic
US10189805B2 (en) 2014-09-09 2019-01-29 Vanderbilt University Metabolism probes for therapy and diagnosis

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EP0436612A1 (fr) 1991-07-17
JPH06504031A (ja) 1994-05-12
EP0436612A4 (en) 1992-05-20

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