WO1994004185A2 - Procede pour inhiber la reduction de ponts disulfure - Google Patents

Procede pour inhiber la reduction de ponts disulfure Download PDF

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Publication number
WO1994004185A2
WO1994004185A2 PCT/US1993/007805 US9307805W WO9404185A2 WO 1994004185 A2 WO1994004185 A2 WO 1994004185A2 US 9307805 W US9307805 W US 9307805W WO 9404185 A2 WO9404185 A2 WO 9404185A2
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cell
membrane
virus
pdi
disulfide
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PCT/US1993/007805
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WO1994004185A3 (fr
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Hughes J.-P. Ryser
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Trustees Of Boston University
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Publication of WO1994004185A3 publication Critical patent/WO1994004185A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/305—Mercury compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines

Definitions

  • Cleavage of disulfide bonds is a necessary step in the activation of a variety of endocytosed macromolecules, such as the tcxins diphtheria toxin, ricin, abrin, modeccin, pseudomonas endotoxin, cholera toxin and tetanus toxin, which are made of two chains held together by disulfide bonds.
  • endocytosed macromolecules such as the tcxins diphtheria toxin, ricin, abrin, modeccin, pseudomonas endotoxin, cholera toxin and tetanus toxin, which are made of two chains held together by disulfide bonds.
  • a well-characterized example of this process is the translocation of the diphtheria toxin, secreted by the Corynebacterium Diphtheriae, across the membrane of early endosomes.
  • Diphtheria toxin which kills cells by irreversibly inhibiting protein synthesis, is secreted as a single-chain precursor comprised of 2 polypeptide chains, A and B, which are linked by a disulfide bond. It is currently believed both chains of the diphtheria toxin are internalized within the cell by receptor-mediated endocytosis. It is also believed that the B chain of nicked diphtheria toxin undergoes conformational changes in acidic endosomes and inserts itself into the endosomal membrane to facilitate translocation of the A chain into the cytoplasm, where inhibition of protein synthesis occurs. It is assumed that prior to translocation of the A chain, reduction of the interchain disulfide bond occurs, which allows the two chains to separate. However, little is known about the site or mechanism of this reductive cleavage, except that in the case of diptheria toxin it must occur very soon after binding of the toxin to its surface receptor.
  • alpha-2 macroglobulins are known to act as carriers of endocytosis for other proteins, such as enzymes and peptide hormones and they do so by linking themselves by disulfide bond(s) to the macromolecule to be carried. Such linkage would also be expected to be cleaved by a cell-surface associated reductive mechanism.
  • This invention pertains to a method of altering (inhibiting or enhancing) , directly or indirectly, reduction of disulfide bonds in the binding area of specific macromolecular ligand ⁇ , such as membrane-bound molecules or proteins, and, as a result, inhibiting (to ⁇ tally or partially) or enhancing the cellular penetration and the respective effects of all or part of these macromolecules.
  • the present invention relates to a method of inhibiting, directly or indirectly, the reductive function of cell membranes, particularly the cell surface (plasma) membrane, which is capable of cleaving disulfide bonds in the binding area of specific protein ligands which must be cleaved for all or part of the proteins to enter cells and produce their respective effects on cells they have entered.
  • the present invention is a method of inhibiting, directly or indirectly, the function of protein disulfide isomerase (PDI) , which catalyzes cleavage of disulfide bonds in membrane-bound macro ⁇ molecules which must be cleaved for passage of all or part of the protein across the cell membrane and, thus, for the protein to have its effect on the cell.
  • PDI protein disulfide isomerase
  • the reductive function of the cell membrane is inhibited, directly, by inhibiting the reductive function of PDI and thus decreasing the rate at which disulfide bonds in membrane-bound macromolecules are cleaved, or indirectly, by altering the configuration or structure of the membrane-bound macromolecule to render the disulfide bonds which must be reductively cleaved for uptake of the macromolecule less available to the reductive function of the cell-surface membrane.
  • the macromolecule is therefore taken up into the cell to a lesser extent than would occur in the absence of the present method.
  • This embodiment is particularly useful for preventing passage of any macromolecule containing disulfide bonds which musr be cleaved in order for all or a component of the macromolecule to pass across a cell membrane, particularly a cell surface membrane.
  • a cell membrane particularly a cell surface membrane.
  • passage of a toxin, such as diphtheria toxin, across a cell membrane is totally or partially inhibited and, thus, its cytotoxicity is reduced.
  • Passage of the toxin is reduced by preventing the reductive cleavage of the toxin's disulfide bonds by inhibiting the reductive function of cell membranes, which is capable of cleaving disulfide bonds in membrane-bound macromolecules, including toxins (e.g., bacterial and plant toxins).
  • toxins e.g., bacterial and plant toxins.
  • activation of a toxin, such as diphtheria toxin is inhibited by inhibiting reductive cleavage catalyzed by the enzyme protein disulfide isomerase.
  • cytotoxicity of the toxin such as diphtheria toxin, is less than would be the case in the absence of the present method.
  • HIV human immuno-deficiency virus
  • retroviruses containing disulfide bonds in their outer shell
  • entry of the HIV, and other virus, particularly retroviruses, containing disulfide bond(s) in their outer shell is reduced by inhibiting reductive cleavage of the disulfide bond(s) of the membrane-bound virus at the cell surface. This is effected by inhibiting, directly or indirectly, the reductive function of cell membranes, which Applicant has shown to be catalyzed by PDI.
  • the uptake of macromolecules e.g., hormones, growth factor
  • macromolecules e.g., hormones, growth factor
  • the reductive function of the cell membrane is increased, directly, such as by increasing the amount of surface-associated PDI, and thus increasing the rate at which disulfides of surface-bound effectors are cleaved, increasing the release of effectors (hormones, growth factors) at the cell surface, or indirectly, by altering the configuration or structure of the macromolecule to render the disulfide bonds which must be reductively cleaved for uptake of the macromolecule more readily available to the reductive function of the cell surface membrane.
  • a preferred embodiment is alteration (reduction or increase) of reductive cleavage at the cell surface of disulfide bonds which link the macromolecule to the carrier.
  • reductive cleavage of disulfide bonds in a macromolecule bound to the cell membrane is altered (inhibited or enhanced) , directly or indirectly.
  • direct alteration of reductive cleavage is effected through the use of agents which act upon the reductive function (e.g., act upon PDI to reduce or increase it activity) .
  • agents include direct inhibitors of the enzyme PDI, such as those that change the activity of the enzyme (e.g.
  • bacitracin or block the access of the enzyme to its substrate (anti- enzymes antibodies) and direct enhancers of PDI (e.g. Brefeldin A) .
  • direct enhancers of PDI e.g. Brefeldin A
  • indirect alteration is effected through the use of agents which act to render disulfide bonds in the macromolecule more or less accessible to the reductive function (e.g., more or less accessible to PDI) .
  • agents include those which block the disulfide-containing domains of the macromolecule ordinarily accessible to PDI (e.g.
  • antibodies against such domains change the conformation of the macromolecule (e.g., by mutations, mild pretreatment with protease or detergent) , and those which change the conditions (e.g., pH) at the cell membrane and, thus, the ability of PDI to catalyze reductive cleavage.
  • the method of the present invention is useful to prevent adverse effects of toxins, such as diphtheria toxin, on cells. It is also useful in preventing infection (e.g., viral or bacterial) or the spread of an established infection. The method may also be useful in enhancing uptake of macromolecules whose presence within a cell is desired.
  • toxins such as diphtheria toxin
  • Figure l is a graphic representation of the results of three experiments showing that bacitracin protects CHO cells from DT cytotoxicity. Concentration of bacitracin versus percent inhibition of diphtheria toxin cytotoxicity is graphed as a dose dependent curve and results demonstrate protection from the diphtheria toxin by bacitracin. Data from 3 separate, identical experiments were averaged to produce the curve.
  • Figure 2 is a graphic representation showing jLn vitro inhibition of PDI-activity by bacitracin and by three membrane-impermeant sulfhydryl blockers. The concentration of each of these inhibitors of PDI-activity versus relative inhibition of PDI is shown and graphed and provides a comparison of the inhibitors' effects. The slope of the bacitracin curve differs from the slopes of the three membrane-impermeant sulfhydryl blockers, which are identical, indicating that the inhibition of PDI by bacitracin works by a different mechanism than that of the three membrane-impermeant sulfhydryl blockers.
  • Figure 3 is a graphic representation of the effect of anti-PDI antibodies on PDI activity using antibodies directed against either rat or human PDI. This dose dependent curve demonstrates the inhibition of PDI as the antibody dose is increased.
  • Figure 4 is a graphic representation of bacitracin- induced inhibition of disulfide cleavage of membrane bound 125 I-tyn-SS-PDL.
  • Figure 6 is a graphic representation of inhibition of DT cytotoxicity by the anti-PDI antibody, RL77.
  • Figure 8 is a graphic representation comparing cells treated with Brefeldin A (upper curve) with untreated cells (lower curve) .
  • Figure 9 is a graphic representation demonstrating inhibition of HIV inf ⁇ ctivity by virus treatment with DTT.
  • Figure 10 is a schematic representation of the amino acid sequence (SEQ ID NO. : 1) of the conserved region of gpl20 (C3 and C4) which contain the binding site of CD4, the V3 loop and the 3 disulfide bonds that are in the proximity of the CD4 binding domains and of the loops they form.
  • An initial, critical step for activation and/or passage of several biologically important macromolecules across an eukaryote cell membrane is cleavage of disulfide bonds present in the macromolecule.
  • Enzymes known to carry out cleavage of disulfide bonds in mammalian cells include protein disulfide isomerase (PDI) , thioredoxin and glutaredoxin.
  • PDI protein disulfide isomerase
  • thioredoxin thioredoxin
  • glutaredoxin the critical sulfhydryls which are present at cell membranes, particularly cell surface membranes, and involved in or responsible for the reductive function capable of cleaving disulfide bonds of membrane-bound macromolecules are cysteine residues of PDI.
  • cysteine residues enable PDI to initiate a disulfide interchange in which the disulfide of the incoming protein is cleaved, thus creating two sulfhydryls on that protein, one of which simultaneously forms a new disulfide with the enzyme, and a new disulfide is formed between that protein and PDI, resulting in a free sulfhydryl on the incoming protein.
  • PDI is generally accepted to be the major cellular catalyst of native disulfide formation and can catalyze net oxidation, reduction or isomerization of disulfide bonds in proteins.
  • thioredoxin is not involved in the reductive function of cleaving disulfide bonds of mem- brane-bound macromolecules at the cell membrane, since it is not inhibited by specific inhibitors of PDI, such as bacitracin, which effectively inhibit the reductive function of the plasma membrane.
  • PDI activity can be inhibited by agents other than sulfhydryl blockers, such as bacitracin and anti-PDI antibodies, as well as agents which block the disulfide-containing domains of the macromolecule ordinarily accessible to PDI (e.g. antibodies directed against such domains; reducing agents) .
  • agents other than sulfhydryl blockers such as bacitracin and anti-PDI antibodies
  • agents which block the disulfide-containing domains of the macromolecule ordinarily accessible to PDI e.g. antibodies directed against such domains; reducing agents
  • This invention pertains to a method of altering (i.e., inhibiting or enhancing) reductive cleavage of disulfide bonds in the binding area of specific macromolecular ligands (such as membrane-bound macromolecules or proteins) , directly or indirectly, including the naturally occurring disulfide bonds of proteins and peptides, the intermolecular disulfide bonds of naturally assembled proteins and any newly formed or chemically synthesized intra- or intermolecular disulfide bonds.
  • specific macromolecular ligands such as membrane-bound macromolecules or proteins
  • activation or passage is eliminated or occurs to a lesser extent in the presence of a direct or indirect inhibitor of the cell membrane reductive function.
  • inhibition of PDI directly or indirectly, results in decreased (partial or total) reductive function of cell membranes, and consequently reduced activation or translocation of surface-bound macromolecules, such as toxins, HIV and other viruses, particularly retroviruses, which have disulfide bonds in their outermost proteins.
  • activation or passage is elimated or occurs to a lesser extent in the presence of inhibitors of the reductive process that act directly on PDI (e.g., by effects on PDI, such as degrading PDI).
  • Inhibitors of PDI function which inhibit PDI directly include membrane impermeant sulfhydryl blockers (such as 5,5'-dithiobis-(2- nitrobenzoic acid) , DTNB; p-chloromercuriphenysulfonic acid, p-CMBS; monobromotrimethylammoniobimane, thiolyte MQ and others) ; inhibitors of PDI acting by unknown echanism ⁇ , other than blocking PDI's sulfhydryl groups (e.g., bacitracin), and anti-PDI antibodies, preferably monoclonal antibodies to human PDI.
  • membrane impermeant sulfhydryl blockers such as 5,5'-dithiobis-
  • activation or passage is eliminated or occurs to a lesser extent in the presence of inhibitors of the reductive process that do not act directly on PDI (e.g., by effects on the membrane-bound macromolecule, such as by blocking disulfide bonds or altering the conformation of the disulfide bond(s)- containing region(s) of a protein, (e.g. a viral protein) rendering the bond(s) less available to the activity of PDI) .
  • inhibitors of the reductive process that do not act directly on PDI (e.g., by effects on the membrane-bound macromolecule, such as by blocking disulfide bonds or altering the conformation of the disulfide bond(s)- containing region(s) of a protein, (e.g. a viral protein) rendering the bond(s) less available to the activity of PDI) .
  • Such inhibitors include agents or procedures that modify the incoming macromolecule prior to the interaction with the cell surface so as to cleave the disulfide bond(s) of the macromolecule (such as membrane impermeant reducing ⁇ •agents, e.g., dithiothreitol, B-mercaptoethanol GSH) , block the disulfide bond(s) of the macromolecule from interaction with membrane-associated PDI (such as antibodies, e.g., monoclonal antibodies directed against a critical disulfide bond-containing domain of a virus) or make disulfide bond(s) of the macromolecule less accessible to membrane-associated PDI, and other agents which inhibit (reduce or prevent) reductive cleavage of disulfide bonds in membrane bound proteins.
  • membrane impermeant reducing ⁇ •agents e.g., dithiothreitol, B-mercaptoethanol GSH
  • membrane-associated PDI such as antibodies, e.g., monoclonal antibodies
  • reductive cleavage of disulfide bonds which are present in macromolecules and must be cleaved for the macromolecule to pass across a cell membrane and produce its effect on a cell is enhanced, resulting in greater activation of all or a part of the membrane-bound macromolecule than would occur otherwise.
  • Enhancers of reductive cleavage of disulfide bonds of membrane-associated macromolecules can act directly or indirectly on PDI by increasing the catalytic function of PDI (such as by optimizing the presence of cofactors) , or by increasing the presence of PDI at the cell surface (such as by influencing its routing from the endoplasmic reticulu (ER) to the plasma membrane) .
  • Enhancers of reductive cleavage can act on the incoming macromolecule by making its disulfide bonds more accessible or more susceptible to surface-associated PDI (such as by biochemically, immunologically, genetically or microenvironmentally induced changes in conformation) .
  • surface-associated PDI such as by biochemically, immunologically, genetically or microenvironmentally induced changes in conformation
  • Example 10 and Figure 8 provide evidence that Brefeldin A enhances PDI catalyzed reductive cleavage of disulfide bonds by shifting PDI from the endoplasmic reticulum to the cell surface and as a result, enhances the cleavage of membrane-bound molecules.
  • the present invention also relates to a method of assaying for agents which inhibit or enhance PDI function, either by direct effect on PDI in an .in vitro enzyme assay or by indirect effect on the membrane-bound substrate (e.g., toxin, virus, other disulfide-bond containing model compound) .
  • a compound or molecule referred to as an agent
  • an agent to be assessed for its ability to inhibit or enhance PDI function (and, thus, inhibit or enhance activation of a macromolecule referred to as a macromolecule of interest) is made to interact either with the macromolecule of interest or with membrane-associated PDI.
  • a compound or molecule to be assessed for its ability to inhibit or enhance PDI function (and, thus, inhibit or enhance activation of a macromolecule referred to as a macromolecule of interest) is made to interact either with the macromolecule of interest or with membrane-associated PDI.
  • a compound or molecule to be assessed for its ability to inhibit or enhance PDI function (and, thus, inhibit or
  • an agent to be assessed for its ability to inhibit or enhance PDI function (and, thus, inhibit or enhance activation of a macromolecule, referred to as a macromolecule of interest) is combined with the macro- molecule of interest or the recipient cells (e.g. , cells in which the macromolecule of interest normally crosses a membrane and has an effect) in an appropriate test system.
  • the resulting assay combination is maintained under conditions appropriate for entry of the macromolecule into recipient cells and its effect on cells it enters; these conditions are sufficient for reductive cleavage of disulfide bonds in membrane-associated macromolecules.
  • Uptake of the macromolecule in the presence of the agent being assessed is compared with its uptake and effect on recipient cells under the same conditions but in the absence of the agent being assessed (i.e., with control values) .
  • Control values can be determined at the time the agent is being assayed or can be previously determined values (standard values determined under control conditions which are the same as those used for assessing the agent, except that the agent is not present) . If the effect of the macromolecule of interest is less in the presence of the agent being assessed than in its absence, the agent is an inhibitor of activation of the molecule (presumably through inhibition of reductive cleavage of disulfide bonds in the macromolecule) . If the effect of the macromolecule of interest is greater in the presence of the agent being assessed, the agent is an enhancer of activation of the macromolecule (presumably through enhancement of reductive cleavage of disulfide bonds in the macromolecule) .
  • the assay consists of measuring the cleavage of a model disulfide compound ( 12S I-tyramine- SS-poly(D-lysine) ) , as a way of measuring a biologic function associated with disulfide cleavage (diphtheria toxin cytotoxicity, HIV infection of H9 cells) in the presence or absence of the agent, to assess whether the agent is capable of inhibiting (or enhancing) the reductive process. If the measured cleavage of the biologic functions is less in the presence of the agent being assessed than in its absence, the agent is an inhibitor of the membrane-associated reductive process.
  • a model disulfide compound 12S I-tyramine- SS-poly(D-lysine)
  • Agents assayed can be existing compounds or molecules, such as those in chemical libraries or available through commercial sources, or can be compounds or molecules designed to inhibit or enhance reductive cleavage of disulfide bonds in membrane-associated macromolecules by inhibiting or enhancing PDI function.
  • the present invention further relates to molecules, particularly those identified by the assay method described above and in the examples, which act as inhibitors or enhancers of activation of macromolecules by inhibiting or enhancing reductive cleavage of disulfide bonds.
  • the oxidoreductive activity of membrane-associated PDI serves many important functions with different biological endpoint ⁇ , and therefore the inhibition or enhancement of membrane-associated PDI activity has many areas of application. Examples of such applications are: a) The released functional moiety of a heterodimeric protein has biological effects independent of its uptake or translocation, such as a "signal transduction effect" or an enzymatic effect. The first instance is illustrated by the postulated reductive cleavage of insulin following insulin-receptor interaction. The second instance is illustrated by the reductive cleavage of tetanus toxin which releases an activated light chain endowed with endopeptidase activity.
  • a cascade of thiol:disulfide interchanges may be initiated by the bonding of an external soluble protein (SH-Y) with membrane-associated PDI (Y-SS-PDI) , leading to the subsequent disulfide bonding of PDI with a membrane protein SH-P' (Y-SH + PDI-SS-P'), followed by the formation of a new disulfide bond between P' and P", another membrane protein, (PDI-SH + P'-SS-P”) catalyzed by the oxidative function of PDI.
  • SH-Y external soluble protein
  • Y-SS-PDI membrane-associated PDI
  • Y-SH + PDI-SS-P' membrane protein
  • PDI-SS-X oxidized PDI
  • X is a membrane protein with a cysteine residue on cell B.
  • PDI would act in this case as an oxidase catalyzing the formation of a new disulfide bond between A-SH of cell A and PDI-SS-X of cell B, resulting in A-SS-X-B + SH-PDI.
  • Such a covalent cell-cell interaction could similarly result from the cleavage of an exposed disulfide on cell A (Y-SS-W) and an exposed SH-PDI on Cell B leading to Y-SS- PDI or W-SS-PDI.
  • thiol:disulfide interchanges could be associated with biologically important cell-cell interactions, such as contact inhibition of normal cells, aggregation of platelets or other blood cell, adhesion of platelets or other blood cells to endothelial cells, adhesion of aging erthyrocytes to spleen cells.
  • Decreased cytotoxicity of the toxin and decreased infectivity of HIV virus are both related to decreased cleavage of critical disulfide bonds by the reductive function of cell surface membranes that normally occurs upon attachment of the toxin or the virus to its cell surface receptors. Also described is identification of the reductive function of cell surface membranes as protein disulfide isomerase (PDI) and application of this finding to altering PDI function (activity and/or levels) at cell membranes, both cell surface membranes and inter ⁇ cellular membranes (e.g., in the endoplasmic reticulum and the Golgi apparatus) .
  • PDI protein disulfide isomerase
  • cleavage of the disulfide contained in a membrane-bound model compound was shown to be inhibited through the use of membrane impermeant inhibitors of surface sulfhydryls. These inhibitors prevent cleavage of disulfide bonds of that model compound at the plasma membrane.
  • a radioiodinated tyramine, ([ ,25 I]tyn) derivatised with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was reacted with an undegradable carrier, 3-thiopropionyl poly(D-lysine) (PDL-SH) to produce the model disulfide compound [ 125 I]tyn-SS-PDL.
  • CHO cells were exposed to the membrane-impermeant sulfhydryl inhibitor 5,5'-dithiobis- (2-nitrobenzioc acid) (DTNB) or p- chloromercuriphenylsulfonic acid (pCMBS) .
  • DTNB 5,5'-dithiobis- (2-nitrobenzioc acid
  • pCMBS p- chloromercuriphenylsulfonic acid
  • diphtheria toxin containing a critical disulfide bond
  • the diphtheria toxin produced by Corynebacterium diphtheriae, kills cells by irreversibly inhibiting protein synthesis of exposed cells.
  • This toxin is secreted as a single chain precursor comprised of two polypeptide chains, A and B, linked by a disulfide bond.
  • both chains of the diphtheria toxin enter cells by receptor-mediated endocytosis, in which a specific receptor protein on the plasma membrane surface recognizes the B chain of the diphtheria toxin, and firmly binds it.
  • the segment of the plasma membrane containing the protein-receptor- diphtheria toxin complex then invaginates, forming an endosome, which is a new intracellular membrane-bound vesicle completely surrounded by cell cytoplasm.
  • the endosome then undergoes acidification to around pH 5.0.
  • the B-chain of the nicked diphtheria toxin undergoes conformational changes due to the acidic environment of the endosome.
  • the B-chain inserts itself into the endosomal membrane which facilitates the passage of the A-chain into the cell's cytoplasm where irreversible inhibition of protein synthesis occurs.
  • sulfhydryl blockers used for that purpose were 5, 5'-dithiobis(2- nitrobenzoic acid) (DTNB) , and p-chloromercuriphenyl- sulfonic acid (pCMBS) ) . They were present in the buffer used to wash the cell monolayer prior to addition of the toxin, as well as in the toxin-containing medium.
  • DTNB 5'-dithiobis(2- nitrobenzoic acid)
  • pCMBS p-chloromercuriphenyl- sulfonic acid
  • impermeant inhibitors of disulfide bond cleavage present in the medium become part of the fluid volume of the endosomes and remain membrane- impermeant in newly-formed endosomes.
  • these membrane-impermeant inhibitors can inhibit entry of macromolecules, such as the cytotoxic chain of diphtheria toxin (Chain A) , which require passage through endosomal membrane and which require cleavage of a critical interchain disulfide bond as an initial step for its translocation across the endosomal membrane.
  • Choin A cytotoxic chain of diphtheria toxin
  • Disulfide bonds play a central role in the generation and maintenance of the 3-dimensional conformation of proteins such as those found in the outer lattice of many viruses. It was anticipated, therefore, that cleavage of intramolecular disulfides in other functional membrane- bound proteins, particularly the envelope proteins of membranebound viruses, might be associated with important biological functions.
  • the gpl20 glycoprotein of HIV I specifically interacts with CD , the virus receptor on the surface of human lymphoid cells. This protein is known to contain 9 disulfide bonds, two of which are situated in the binding domain of gpl20. See Figure 10.
  • PDI is a well characterized multi-functional enzyme which plays a critical role in the formation of disulfide bonds in nascent proteins. Consistent with this role, the major subcellular localization of PDI is the endoplasmic reticulum, the first compartment reached by newly synthesized proteins. There have been controversial findings regarding the possibility that PDI might be present also at the cell surface. For instance, Varandani presented evidence that the insulin disulfide transhydrogenase (another appellation for PDI) was detectable at the surface of pancreatic cells. Varandani, P.T. , et al.. Biochem. Biophys. Acta.
  • Kaetzel found no evidence that PDI was present at the surface of human placenta and rat liver cells. (Kaetzel, C. S. et al.. Biochem. J.. 241:39-47 (1987).
  • bacitracin does not contain functional groups that are known to block free sulfhydryls and is likely, therefore, to act by a different, albeit unknown, mechanism. This view is consistent with the fact that the slope of its dose-inhibition curve differs from that of three sulfhydryl reagents (Fig.
  • monoclonal antibodies directed against rat or human PDI inhibited the activity of calf liver PDI in vitro at the surface of CHO cells. This crossreactivity is consistent with the extensive homology of human, bovine and rat PDI (Parkkonen, T. , et al.. Biochem. J.. 256:1005-1011 (1988)). Crossreactivity between human, bovine and rat PDI was noted with polyclonal antibodies against human and rat PDI (Kaetzel, C.S., et al.. Biochem. J..
  • T3BP triiodothyronine binding protein
  • anti-PDI antibodies bind to PDI and reduce or abolish its ability to reductively cleave disulfide bonds at the cell surface membrane, PDI will be unable to cleave the critical disulfide bonds present in the gpi20 glycoprotein of HIV, a cleavage that is required to initiate virus penetration into the cells.
  • the same reductive process plays a role in the penetration of membrane-bound HIV and HIV infection of H9 cells is markedly reduced by anti-PDI antibodies and bacitracin, an inhibitor of PDI.
  • HIV alone was treated with the reducing agent dithiothreitol (DTT) prior to virus-cell interaction. This inactivated the virus in a dose- dependent fashion up to a 100% inhibition or infectivity. See Figure 9.
  • Viral infections can also be inhibited with antibodies directed against the discrete gpl20 domains that contain the critical disulfide bonds which are illustrated in Figure 10. Of the nine disulfide bonds of gpl20, two are situated in the binding domain that interacts with CD4 receptor, and one disulfide is in an adjacent fusogenic domain.
  • CYS378 is close to a sequence in C3 (364-370) believed to participate in CD4 binding.
  • Larsen, C. et al. Membrane Interactions of HIV, eds. Alola, R.C. et al. (Wiley-Liss, Inc., New York, 1992) 143- 146.
  • CYS418 is at the start of the primary CD4 binding site in C4 (418-437). Larsen, C. et a_l. , Membrane Interactions of HIV, eds.
  • the primary CD4 binding site is thus framed by two disulfide bonds which are plausible targets for a PDI- ediated interchange reactions.
  • a third disulfide bond (CYS296-331) forms the V3 loop, which is believed to have fusogenic properties. Larsen, C. et al. , Membrane Interactions of HIV, eds. Alola, R.C. et al., (Wiley-Liss, Inc., New York, 1992) 143-146.
  • V 3 loop where this disulfide is located although distant in the sequence by some 50 to 100 amino acids from important elements of the CD4 binding site, is believed to be situated in close proximity of the binding site in the tertiary structure of the native gpl20.
  • the variable regions V3 and V4 are thought to include amino acids 297-328, and 394-413, respectively.
  • the conserved region C3 and C4 follow V3 and V4, repectively.
  • the method of administering an effective dose of inhibitors of disulfide bond cleavage in vivo is by any means presently available to one skilled in the art.
  • cytotoxicity of a toxin which contains disulfide bonds which must be cleaved for the toxin to enter cells and exert cytotoxic effect is reduced by blocking the reductive function of the eukaryotic cell membrane.
  • Inhibition of the cytotoxic effect of the toxin, specifically diphtheria toxin results from the use of sulfhydryl blockers and from the use of a PDI-specific inhibitor. That is, DTNB (a non-specific sulfhydryl blocker) and bacitracin (a specific PDI inhibitor) have been shown to reduce diphtheria toxin cytotoxicity in eukaryotic, including mammalian, cells.
  • cytotoxicity of other toxins which must undergo reductive cleavage or processing at a cell membrane, particularly the cell surface membrane, and the membrane of nascent endosomes can be inhibited.
  • This can be done by contacting cells in which cytotoxicity is to be inhibited (totally or partially) with a sulfhydryl blocker or a PDI- specific inhibitor.
  • Sulfhydryl blockers include, but are not limited to, DTNB, pCMBS and thiolyte MQ.
  • PDI-specific inhibitors include, but are not limited to, bacitracin and anti-PDI antibodies, which can be polyclonal or monoclonal.
  • a sulfhydryl blocker or a PDI-specific inhibitor can be administered to (contacted with) cells individually or in various combinations (e.g., combinations of two or more sulfhydryl blockers; two or more PDI-specific inhibitors; sulfhydryl blocker(s) and PDI-specific inhibitor(s) ) .
  • Example 2 and Example 5 show the effectiveness of the present method in inhibiting the cytotoxicity of diphtheria toxin by an inhibitory effect at the cell surface membrane.
  • other toxins e.g., pseudomonas toxin E, gelonin, toxic ribonucleases, modeccin pseudomonas endotoxin, choleratoxin, tetanus toxin
  • toxins e.g., pseudomonas toxin E, gelonin, toxic ribonucleases, modeccin pseudomonas endotoxin, choleratoxin, tetanus toxin
  • the cytotoxic effects of ricin were not inhibited by the same cell membrane impermeant inhibitors (See Ryser, H. J.-P. et al.. J. Biol. Chem..
  • the surface associated reductive mechanism initially revealed by the cleavage of 125 I-tyn-SS-PDL may serve functions other than the reductive activation of DT.
  • alpha 2-macroglobulin a well characterized macromolecular carrier that interacts with a specific surface receptor, has been shown to form disulfide bonds with platelet derived growth factor (Huang, J.S., et al.. Proc. Natl. Acad. Sci. USA. 8_:342-346 (1988), interleukin- 1-beta (Borth, W. , et al.. J. Immunol.. 145:3747-3754
  • a sulfhydryl blocker in which cytotoxicity is to be reduced or prevented are contacted with a sulfhydryl blocker, a PDI-specific inhibitor or both in an amount sufficient to inhibit (totally or partially) reductive cleavage of the toxin macromolecule.
  • the sulfhydryl blocker(s) used will preferably be membrane-impermeant.
  • the sulfhydryl blocker( ⁇ ) used will preferably be membrane permeant.
  • the sulfhydryl blocker(s) , PDI-specific inhibitor(s) (e.g., bacitracin, anti-PDI antibodie ⁇ ) or a combination thereof (referred to as drug(s)) can be administered to an animal, particularly a mammal, including humans, using known methods.
  • the amount of each necessary to reduce cytotoxicity of the toxin will be determined empirically, taking into consideration, for example, the particular toxin, the drug (e.g., sulfhydryl blocker(s) , bacitracin, anti-PDI antibodies being administered) , the drug' ⁇ toxicity, the ⁇ ize and age of the individual and the severity of the condition being treated or prevented.
  • the drugs can be obtained from commercial sources or can be produced using known methods.
  • the drug(s) can be included in a formulation which i ⁇ administered to an individual being treated; such a formulation can also include a physiologically compatible carrier (e.g., a physiological buffer) , stabilizers, flavorant ⁇ , adjuvants and other components.
  • a physiologically compatible carrier e.g., a physiological buffer
  • stabilizers e.g., a physiological buffer
  • flavorant ⁇ e.g., aqueratives
  • adjuvants e.g., a physiological buffer
  • the drug(s) can be administered by a variety of routes (e.g., topically, parenterally, intravenously, intraperitoneally) , and the components of the formulation will be selected accordingly.
  • infectivity of a virus is inhibited by blocking the reductive function of the cell surface membrane.
  • Membrane impermeant sulfhydryl blockers, bacitracin and anti-PDI antibodies inhibited HIV infection of mammalian (human) cells.
  • infectivity of HIV and other viruses, particularly retroviruses can be inhibited by the pre ⁇ ent method.
  • cells to be protected against HIV (or other viral) infection are contacted with drug or drugs, which are sulfhydryl blocker(s), a PDI-specific inhibitor(s) or both in sufficient quantity and by an appropriate route to result in reduced infection of the cells by the HIV or other virus (i.e., infection is totally inhibited or occurs to a lesser extent than occurs in the absence of the present method) .
  • drug or drugs which are sulfhydryl blocker(s), a PDI-specific inhibitor(s) or both in sufficient quantity and by an appropriate route to result in reduced infection of the cells by the HIV or other virus (i.e., infection is totally inhibited or occurs to a lesser extent than occurs in the absence of the present method) .
  • infectivity of a virus containing critical disulfides bond domains in the outer proteins, particulary HIV is inhibited with reducing agents.
  • the drug(s) administered can be in a formulation suitable for the route of administration used (topical, parenteral, intravenous, intraperitoneal) .
  • the amount to be administered and the frequency of administration can be determined empirically and will take into consideration the age and size of the person being treated, the stage of infection by HIV or other virus (e.g., prior to infection, soon after infection occurs or in later stages of infection) and the particular virus.
  • macromolecules e.g. , growth hormone, interleukines, lymphokines, hematopoietic stimulating factors, specific epithelial, endothelial and fibroblast stimulating factors, transforming growth factors
  • present at or delivered to a cell surface receptor via a carrier to which it is linked by a disulfide bond can be inhibited or enhanced by the present method.
  • pCMBS pCMBS
  • DTNB N-ethylmalamide
  • poly(D-lysine) 60 kDa, (PDL,) were purchased from Sigma.
  • [ 125 I]PDL was prepared by iodination of Bolton-Hunter reagent-modified PDL, as described.
  • [ 125 I]-tyramine-SS-poly(D-Lysine) [ 125 I]tyn-SS-PDL) was prepared by disulfide exchange of 3- thiopropionyl PDL with 3-(2-pyridyldithio)propionyl [ 125 I]tyramine, i.e. with SPDP-[ 125 I ⁇ -tyramine.
  • DTT dithiothreitol
  • ricin ricin.
  • DT was from List Biological Laboratories, Inc., Campbell, CA.
  • Tissue culture products were from GIBCO.
  • Tissue culture flasks and microwell plates were from Falcon, Oxnard, CA.
  • CHO cells were from the American Tissue Culture Collection. Inhibition of Protein Synthesis bv Toxins. CHO cells were grown to confluence in 35mm dishes in ⁇ -minimal essential medium supplemented with fetal bovine serum. They were briefly washed with prewarmed serum-free Eagle's medium and incubated for 2 h at 37°C in serum-free Eagle's medium containing DT or ricin.
  • the postnuclear supernatant from a centrifuga- tion at 800 x g for 10 min was layered on a 17% Percoll gradient in 0.25 M sucrose and centrifuged for 1 h at 34,500 x g in a Sorvall SV288 vertical head rotor. Frac ⁇ tions of 1.5 ml were monitored for radioactivity. Most radioactivity was associated with the peak of lightest buoyant density (1.03 g/ml) . After a 15 min labeling, radioactivity is associated with endosomes and plasma membrane fragments. Fractions around 1.03 g/ml density were pooled and used for acidification studies. The centrifugation profile was identical for controls and cells pretreated with DTNB or pCMBS.
  • Monolayers of CHO cells were exposed to concentrations of 0, 100, and 500 ng/ml DT for 2 h at 37°C in the presence or absence of the sulfhydryl inhibitors. After washing, they were reincubated in a medium containing radioactive amino acids to measure the toxin's effect on protein biosynthesis. In the absence of inhibitors, 100 and 500 ng of DT caused a marked, dose-dependent decrease of amino acid incorporation (Table I). Addition of 1.0 mM DTNB almost totally prevented this inhibition (93.5 and 85.9%, respectively), and 0.1 mM pCMBS reduced the toxin's effect by 68 to 63%, respectively.
  • Cytotoxicity was measured as decrease in 14 C-labeled amino acid incorporation into cellular proteins.
  • DT Diphtheria Toxin
  • Diphtheria Toxin is not due to an inhibitory effect on the receptor-mediated endocytosis process itself.
  • DTNB and pCMBS might inhibit receptor-mediated endocytosi ⁇ wa ⁇ tested by measuring the cellular uptake of [ 35 S]Man-6-P enzymes) mediated by the Man-6-P receptor.
  • the two inhibitors failed to inhibit the endocytic uptake of this specific ligand (see Ryser et al.. 1991) , also indicating that they failed to inhibit its binding.
  • DTNB and pCMBS diphtheria Toxin
  • DTNB and pCMBS might protect cells from DT by interfering with endosome acidification was tested in two ways.
  • the pH of endosomes was increased by treating cells with NH 4 C1 prior to measuring the uptake of [ 3S S]Man- 6-P enzymes.
  • the internalized enzyme-receptor complex dissociate ⁇ at the acid pH of endosomes, allowing the Man-6-P receptor to recycle to the cell surface and the ligand to be carried to lysosomes.
  • H9 and C8166 cell lines as well as HIV/HTLV IIIB were obtained from Dr. Martin Hirsch, Massachusetts General Hospital, Boston, with permission of Dr. Robert Gallo, NIH.
  • H9 cells grown in R20 medium were resuspended in serum free medium and exposed for 2 h at 37°C to approximately 1 x 10 7 TCID J0 unit ⁇ per 10 6 cells. Unbound virus was then removed following centrifugation and cells were plated in 24-well culture plates (8 x 10 s cells/well) and incubated for 7 d at 37°C in R20 growth medium. Cell- free culture supernatants containing 0.5% Triton X-100 were then assayed for their content in p24 viral protein using the HIV-1 p24 Core Profile ELISA (DuPont-NEN Research Products, Boston) .
  • Human lymphoid H9 cells were exposed for 30 min at 37 ⁇ C to 2.5 mM DTNB in serum-free medium to which HIV was added for 2 h at 37°C. After removal of both DTNB and unbound virus and incubation for 7 days in growth medium, the cells were harve ⁇ ted and their supernatant tested for its content of p24 viral protein. As shown in figure 5(B) , no measurable level of p24 was detected in the supernatant of cells treated with 2.5 mM DTNB, in either of the five experiments performed.
  • the supernatants of one experiment represented in Figure 5(B) were tested in a viral titer assay to compare the level of HIV p24 antigen and the titer of infective HIV released in the growth medium.
  • the virus titer wa ⁇ determined by infecting C8166 cell ⁇ with supernatants of known P24 contents. DTNB was present half an hour prior and during the 2-hour exposure to the virus.
  • the p24 values and viral titers were as follows:
  • a virus excess was exposed to 5.0 mM DTNB in serum-free medium for 2.5 h at 37 ⁇ C and the virus- DTNB mixture was diluted to bring DTNB to a concentration known to have no effect on di ⁇ ulfide reduction.
  • Virus treated in this way was only moderately less infective to H9 cells than untreated virus, as determined by the release of p24 antigen (Fig. 5(C)).
  • DTNB is membrane-impermeant, the possibility that it might indirectly inhibit virus growth in virus infected cells was tested by exposing H9 cells first to HIV virus for 2 h, then to 5 mM DTNB for 2.5 h before transfer to regular growth medium for 7 d.
  • DTNB ha ⁇ a dramatic inhibitory effect on the HIV infection of H9 cell ⁇ as measured either by the release of p24 or of infectious virus by infected cells.
  • the cellular sulfhydryls engaged in the reductive mechanism of cell-bound viral proteins must be located in the immediate vicinity of the cellular receptor for HIV, known as the CD4 receptor.
  • the inhibition of viral P24 synthesis induced by 1.0 mM DTNB in the viral system is quantitatively very similar to the inhibition of diphtheria toxin cytotoxicity by the same DTNB concentration in CHO cells (85 to 93%) .
  • the latter inhibition can be attributed to the blocking, by DTNB, of the cleavage of the disulfide bond that holds together the two chains of the surface-bound diphtheria toxin (DT) .
  • DTNB diphtheria toxin
  • pCMBS which, like DTNB, i ⁇ a membrane impermeant disulfide blocker, was used as a positive control for cytotoxicity, since we found it to he cytotoxic to human cell ⁇ (HeLa cells in culture) . It is worth noting that in this control experiment, the DTNB concentration was four times the concentration used in the viral experiment, and present over a longer period of time (120 min vs. 90 min) .
  • the lack of visible and measurable cytotoxicity of DTNB support the data of Fig 5D in excluding the possibility that the marked inhibition of P24 synthesis seen in the HIV work would be due to DTNB cytotoxicity to H9 cells.
  • a further control experiment was the demonstration that the DTNB concentrations used did not inactivate the virus .in vitro and was carried out in the following manner. HIV at 100-fold the multiplicity used for infecting H9 cells was exposed to 2.5 mM DTNB for 90 min at 37° C. and was then diluted 100-fold to bring the concentration of DTNB below infection-inhibiting concentrations. After that dilution, DTNB-treated HIV remained fully infective. Inactivation of the virus by DTNB would inhibit infectivity and might indeed cause an inhibition of P24 synthesis.
  • the table gives values of insulin reduction, measured in the conventional assay for PDI activity, using insulin as substrate.
  • the PDI induced cleavage was comparable using either substrate.
  • bacitracin inhibits the membrane-associated disulfide cleavage of the surface- bound conjugate in a dose-dependent manner. Between 0.3 and 3.0 mM, the inhibition increased from 40 to 76%, an increment comparable to the one seen in the inhibition of PDI activity in vitro (60 to 95%, Fig. 2). Surprisingly, we found that acid-soluble cell-bound radioactivity was generated during the 60 min labeling at 0°C and that this cleavage was also decreased in dose-dependent fashion by bacitracin present during labeling. This bacitracin- induced inhibition of cleavage is represented in the upper curve of Fig. 4.
  • a bacitracin leakage of intracellular PDI into the medium would, if anything, increase the reductive cleavage of membrane- bound 125 I-tyn-SS-PDL.
  • the bacitracin-induced inhibitions seen in Fig. 4 must therefore be due to a specific inhibition of surface-associated PDI. That the inhibition of reduction occurs at the cell surface is further demonstrated by the fact that the zero-values measured separately for each bacitracin concentration also showed a dose-dependent decrease in cleavage under conditions that abolish endocytosis (See Fig. 4, upper curve).
  • EXAMPLE 5 Inhibition of the diphtheria toxin bv addition of bacitracin. a known inhibitor of PDI
  • Bacitracin purchased from Sigma, was used instead of DTNB or pCMBS in experiments identical to those described in Example 2. Bacitracin caused a dose-dependent inhibition of DT cytotoxicity in the range of 0.3 to 3.0 mM, concentrations that were non-toxic to CHO cells. Fig. 1 expressed the data as percent protection from DT cytotoxicity. At 3.0 mM, bacitracin afforded 80% protection from DT cytotoxicity. As shown in Example 6 (Fig. 2) 3.0 mM bacitracin caused a 95% inhibition of PDI in the in vitro insulin reduction assay.
  • bacitracin did not reduce the cytotoxicity of ricin, and in fact, slightly increased it. As discussed elsewhere, this observation is consistent with the currently accepted view that reductive cleavage of ricin's interchain disulfide occurs in the trans-Golgi network or the Golgi apparatus, i.e., at a site not accessible to membrane impermeant PDI inhibitors. HeLa cells, the only cell type other than CHO tested in this fashion, were also consistently protected by bacitracin against DT cytotoxicity.
  • PDI activity was measured by the glutathione: insulin transhydrogenase assay described by Carmichael, D.F., et al.. J. Biol. Chem.. 252: 7163-7167, (1977), as modified by Kaetzel C. S.-, et al.. Biochem. J.. 241: 39-47 (1987) .
  • the assay was further modified by reducing the total volume to 0.5 ml, extending the reaction time to 30 min, and stopping the reaction with 10 mM NEM, prior to TCA-precipitation.
  • the amount and radioactivity of 125 I- insulin were 50 ⁇ g and 10 5 CPM/0.5 ml.
  • PDI had a specific activity of 5,200 U/mg in the standard 5 min assay.
  • PDI activity was calculated as the difference between acid-soluble radioactivity generated in a 30 min incubation at 37°C in presence or absence of PDI.
  • Controls were run to determine acid soluble radioactive contamination of the 125 I-insulin preparations (usually 2 to 6%) .
  • Assays were carried out in the presence and absence of aprotinin to monitor possible proteolytic cleavage of ,25 I-insulin. They showed that insulin proteolysis was not a factor in our assay condition.
  • DTNB and pCMBS these inhibitors were added to the reaction mixture a few minutes prior to PDI addition.
  • thiorexdoxin The activity of thiorexdoxin was determined in the turbidi etric assay described by Holmgren, A. , J. Biol. Chem.. 254: 9627- 9632. The activity of thiorexdoxin was not inhibited by bacitracin.
  • Example 6 The same antibodies used in Example 6, as well as a third antibody (RL90) , were used to test their ability to inhibit cleavage of 125 I-tyn-SS-PDL.
  • I-tyn-SS-TDL (lug/ml, 1.8xl0 5 cpm/well) was then added to each well to label the surface of the cells for 30 min at 0°C.
  • the cells were washed twice and reincubated in prewarmed medium (37°C) containing antibodies or irrelevant IgG for a 30 min incubation.
  • prewarmed medium 37°C
  • bovine serum albumin from the incubation medium.
  • all three antibodies had marked inhibitory effects.
  • the affinity purified antibodies RL77 and HP-13 at concentrations of 25 ug/well caused inhibitions of 43% and 51%, respectively.
  • Example 7 The same antibodies used in Example 7 were tested for an inhibitory effect on DT toxicity in 4 experiments, similar to those carried out in Example 2, in which DT alone depressed amino acid incorporation into proteins to 47% control values.
  • Cells were preexposed to medium containing antibodies as described in Example 7, except that the antibodies were given as aliquots of reconstituted solutions of lyophilized ascites fluid containing 10 mg protein/ml (0.84 mg/well) .
  • Controls contained the same protein amount as a mixture of irrelevant IgG and bovine serum albumin at the ratio found in ascites fluid (1:9).
  • Bacitracin was tested at a concentration (3.0 mM) that had been found to inhibit the cytotoxicity of DT in CHO cell ⁇ .
  • concentration 3.0 mM
  • bacitracin expo ⁇ ed to bacitracin for 30 min prior to and during the 2 h infection period
  • the subsequent release of p24 proteins into their culture medium was significantly decreased (Fig. 7C) .
  • a corresponding viral titer assay was performed as in Example 3 and the viral titer was reduced to 0.13% of quadruplicate controls as shown below: HIV-Titer r ⁇ iD tn )
  • Bacitracin is transported poorly into cells and evidence was provided that its effect on the cleavage of membrane- bound [ 12S I]-tyramine-SS-poly-D(lysine) does not require cellular uptake. It can be as ⁇ umed therefore that, like DTNB and anti-PDI antibodie ⁇ , bacitracin exerts its effect on the plasma membrane of H9 target cell ⁇ .
  • DTNB and bacitracin would interfere with the attachment of HIV to its cellular receptor has not been experimentally excluded, but appears unlikely, since it would require that two molecules as similar as DTNB and bacitracin would have similar effects on virus binding, and would decrease in similar fashion the attachment of HIV and of DT to their respective cell surface receptors.
  • DTNB is a more powerful inhibitor than either bacitracin or anti-PDI antibodies.
  • a similar trend can be seen in the relative effect of these inhibitors on 125 I-tyn-Ss-PDL cleavage and DT cytotoxicity.
  • the monoclonal antibodies are the most specific.
  • the fact that DTNB is more powerful may be due to its ability to block membrane thiols required to regenerate the oxidized PDI that results from the PDI- catalyzed thiol:disulfide interchange.
  • gpl20 the viral protein involved in the binding of HIV to its cell receptor, is known to contain 18 conserved cysteine residues forming 9 disulfide bonds. Three of them are situated in, or close to, the domain that binds to CD4 and are plausible substrates for PDI-catalyzed interchanges following HIV-CD4 interaction. 4) Cleavage of protein disulfides is capable of initiating conformational changes comparable in importance to the pH-induced conformational changes associated with the penetration of other viruses. 5) The three classes of agents which in our experiments inhibit HIV infection, also inhibit the activity of PDI in vitro at comparable concentrations. 6) The multi ⁇ functional enzyme PDI, although predominantly located in the endoplasmic reticulum, is also exposed at the surface of mammalian cells where it is capable of initiating thiol-disulfide interchanges.
  • EXAMPLE 10 Kinetics of 12i I-tvn-SS-PDL reduction bv CHO cells following 16 hrs pretreatment with BFA (solid line and untreated controls (dotted line ⁇ .
  • CHO cells were preincubated for 16 hours in growth medium containing 1.0 ⁇ g/ml Brefeldin A (BFA). They were cooled to 0°C and pulse-labeled for 30 minutes at 0 ⁇ C with 125 I-tyn-SS-PDL in presence of BFA, washed to remove unbound label, conjugate, and reincubated for 2 hours at 37°C in absence of labeled conjugate and in presence of BFA, at 37°C. The generation of 12S I-tyn-SH was measured after 30, 60 and 120 min of chase, and expressed as reduction of cell-bound I25 I-tyn-SS-PDL in percent of the cell-bound radioactivity measured at time 0.
  • BFA Brefeldin A
  • Figure 8 compares cells treated with BFA (upper curve) with untreated cells (lower curve) . The largest difference in reductive cleavage is seen after 30 min, indicating that BFA influences the first phase of reduction occurring at the cell surface.
  • Figure 8 represents an average of 3 experiments. When expressed in percent of the initial cell-bound radioactivity, the reductive cleavage measured between 0 and 30 min was 2.29 10.14 and 4.8 ⁇ 1.7 for controls and BFA treated cell ⁇ , respectively. These results indicate that BFA pretreatment increases the reductive function of the plasma membrane.
  • EXAMPLE 11 Inactivation of HIV bv DTT. HIV/HTLV III B of very high titer (5 X 10 12 TCID units/ml) was exposed for 2.5 hours at 37°C to increasing concentrations of the reducing agent dithiotreitol (DTT) in serum free minimal essential medium (MEM) . The virus /DTT solution was serially diluted with MEM and virus aliquots were added to wells (sixtuplets) containing C8166 cells (See Example 3) in R20 growth medium, inoculating six wells for each concentration of DTT pretreatment. The multiwell plates were incubated for 7 days at 37°C to allow for virus expression and formation of syncytia.
  • DTT reducing agent dithiotreitol
  • MEM serum free minimal essential medium
  • HIV infection was then scored by determining the virus titer at which heterokaryons were detected in 3 out of 6 wells as described by Hartshorn K.L. et al., Antimicrob. Agents Che other.. 31: 168-172 (1987).
  • Serial dilution of the virus had brought the residual concentration of DTT to a level known to be non toxic to C8166 cells in all groups of 6 wells of determining virus titer.
  • the points of Figure 9 give the percent of DTT-induced inhibition of HIV infectivity, based on the averages of 2-6 experiments. The results demonstrate that DTT inactivated HIV in a dose-dependent fashion up to a 100% inhibition of infectivity.

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Abstract

La présente invention concerne un procécé pour modifier (inhiber ou stimuler), directement ou indirectement, la réduction des ponts disulfure de macromolecules à liaison membranaire, notamment des protéines, et, par suite, inhiber (totalement ou partiellement) ou stimuler la pénétration cellulaire et les effets respectifs de ces macromolécules. Plus particulièrement, la présente invention concerne un procédé pour inhiber, directement ou indirectement, la fonction réductive des membranes cellulaires, notamment la membrane cellulaire de surface (plasma) qui est susceptible de cliver, dans des protéines à liaison membranaire, des ponts disulfure qui doivent être clivés pour que les protéines pénètrent dans les cellules et y produisent leurs effets respectifs. Le clivage des ponts disulfure ainsi réalisé est une étape métabolique nécessaire à la fonction ultime de la protéine ou de la macromolécule. Selon la présente description, le déposant a démontré que la fonction réductive des membranes cellulaires de surface est catalysée par la disulfure-isomérase protéique. Le procédé décrit est utile pour prévenir les effets néfastes des toxines, telles que la toxine diphtérique, sur les cellules. Il est également utile pour prévenir l'infection (virale ou bactérienne) ou la propagation d'une infection établie. Ce procédé peut d'autre part être utile pour stimuler la fixation de macromolécules dont la présence au sein d'une cellule est souhaitée.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0607252A1 (fr) * 1991-09-30 1994-07-27 THOENE, Jess G. Procede de traitement d'infections a vih
WO1995025788A1 (fr) * 1994-03-21 1995-09-28 Research Development Foundation Nouveaux virus mutes, composes antiviraux et nouveaux procedes de production de vaccins
US5554655A (en) * 1991-09-30 1996-09-10 Jess G. Thoene Method of treating HIV infection
US5646189A (en) * 1993-10-15 1997-07-08 Thoene; Jess G. Prevention of HIV infection
US5725870A (en) * 1993-10-15 1998-03-10 Thoene; Jess G. Methods, composites and articles for contraception
US6977142B2 (en) * 2003-09-23 2005-12-20 Agy Therapeutics, Inc. High-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity
WO2013042093A1 (fr) 2011-09-23 2013-03-28 Wroclawskie Centrum Badan Eit Sp Z O O Utilisation de l'antibiotique bacitracine dans la dégradation hydrolytique d'arn

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY vol. 27, no. 13 , 28 June 1988 pages 4894 - 4902 CHIACCHIA, K.B. 'REOXIDATION OF THE CLASS I DISULFIDES OF THE RAT ADIPOCYTE INSULIN RECEPTOR IS DEPENDENT UPON THE PRESCENCE OF INSULIN: THE CLASS I DISULFIDE OF THE INSULIN RECEPTOR IS EXTRACELLULAR' *
CELL vol. 50 , 1987 pages 975 - 985 LASKY, L.A. ET AL 'DELINEATION OF A REGION OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 GP120 GLYCOPROTEIN CRITICAL FOR INTERACTION WITH THE CD4 RECEPTOR' *
DISSERTATION ABSTRACTS INTERNATIONAL B vol. 51, no. 1 , July 1990 page 182B FEENER, EDWARD P. 'THE ROLE OF THE PLASMA MEMBRANE AND A NON-LYSOSOMAL COMPARTMENT IN THE DISULFIDE CLEAVAGE OF ENDOCYTOSED MACROMOLECULES' *
JOURNAL OF VIROLOGY vol. 66, no. 6 , June 1992 pages 3504 - 3513 MEYER, W.J. ET AL 'CONFORMATIONAL ALTERATION OF SINDBIS VIRION GLYCOPROTEINS INDUCED BY HEAT, REDUCING AGENTS OR LOW PH' cited in the application *
POLYMER PREPRINTS vol. 27, no. 1 , April 1986 pages 9 - 10 SHEN, W-C. ET AL 'DISULFIDE AND OTHER SPACERS FOR THE INTRACELLULAR RELEASE OF DRUGS FROM POLYMERIC CARRIERS' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES vol. 90, no. 9 , 1 May 1993 pages 4112 - 4116 MANDEL, R. ET AL 'INHIBITION OF A REDUCTIVE FUNCTION OF THE PLASMA MEMBRANE BY BACITRACIN AND ANTIBODIES AGAINST PROTEIN DISULFIDE-ISOMERASE' *
THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 265, no. 26 , 15 September 1990 pages 15984 - 90 YOSHIMORI, T. ET AL 'PROTEIN DISULFIDE-ISOMERASE IN RAT EXOCRINE PANCREATIC CELLS IS EXPORTED FROM THE ENDOPLASMIC RETICULUM DESPITE POSSESSING THE RETENTION SIGNAL' cited in the application *
THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 265, no. 31 , 5 November 1990 pages 18780 - 18785 FEENER, E.P. ET AL 'CLEAVAGE OF DISULFIDE BONDS IN ENDOCYTOSED MACROMOLECULES' cited in the application *
THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 266, no. 28 , 5 October 1991 pages 18439 - 18442 RYSER, H. J.-P. ET AL 'CELL SURFACE SULFHYDRYLS ARE REQUIRED FOR THE TOXICITY OF DIPHTHERIA TOXIN BUT NOT OF RICIN IN CHINESE HAMSTER OVARY CELLS' cited in the application *
VIRUS RESEARCH vol. 10, no. 2,3 , 1988 pages 225 - 240 GIDWITZ, S. ET AL 'DIFFERENCES IN VIRION STABILITY AMONG SINDBIS VIRUS PATHOGENESIS MUTANTS' *

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EP0607252A1 (fr) * 1991-09-30 1994-07-27 THOENE, Jess G. Procede de traitement d'infections a vih
EP0607252A4 (en) * 1991-09-30 1996-03-13 Jess G Thoene Method of treating hiv infection.
US5554655A (en) * 1991-09-30 1996-09-10 Jess G. Thoene Method of treating HIV infection
US5646189A (en) * 1993-10-15 1997-07-08 Thoene; Jess G. Prevention of HIV infection
US5725870A (en) * 1993-10-15 1998-03-10 Thoene; Jess G. Methods, composites and articles for contraception
WO1995025788A1 (fr) * 1994-03-21 1995-09-28 Research Development Foundation Nouveaux virus mutes, composes antiviraux et nouveaux procedes de production de vaccins
CN1122105C (zh) * 1994-03-21 2003-09-24 研究发展基金会 新的突变病毒、抗病毒化合物和新的制备疫苗的方法
US6977142B2 (en) * 2003-09-23 2005-12-20 Agy Therapeutics, Inc. High-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity
WO2013042093A1 (fr) 2011-09-23 2013-03-28 Wroclawskie Centrum Badan Eit Sp Z O O Utilisation de l'antibiotique bacitracine dans la dégradation hydrolytique d'arn

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