WO1999022729A1 - Utilisation d'inhibiteurs de proteasomes dans le traitement du cancer, de l'inflammation, de maladies autoimmunes, du rejet du greffon et du choc septique - Google Patents

Utilisation d'inhibiteurs de proteasomes dans le traitement du cancer, de l'inflammation, de maladies autoimmunes, du rejet du greffon et du choc septique Download PDF

Info

Publication number
WO1999022729A1
WO1999022729A1 PCT/CA1998/001010 CA9801010W WO9922729A1 WO 1999022729 A1 WO1999022729 A1 WO 1999022729A1 CA 9801010 W CA9801010 W CA 9801010W WO 9922729 A1 WO9922729 A1 WO 9922729A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
proteasome
lac
activity
composition
Prior art date
Application number
PCT/CA1998/001010
Other languages
English (en)
Inventor
Jiangping Wu
Xin Wang
Original Assignee
Centre De Recherche Du Centre Hospitalier De L'universite De Montreal
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre De Recherche Du Centre Hospitalier De L'universite De Montreal filed Critical Centre De Recherche Du Centre Hospitalier De L'universite De Montreal
Priority to JP52505499A priority Critical patent/JP2001508465A/ja
Priority to AU97318/98A priority patent/AU770798B2/en
Priority to EP98951135A priority patent/EP0967976A1/fr
Priority to CA002276013A priority patent/CA2276013A1/fr
Publication of WO1999022729A1 publication Critical patent/WO1999022729A1/fr

Links

Classifications

    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • 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
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • 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
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals

Definitions

  • the present invention relates to the use of proteasome inhibitors for targetting different cellular functions implicated in cancer, inflammation, autoimmune disease, graft rejection and septic shock.
  • the proteasome is a large protease complex. It is the main nonlysosomal proteolytic system in the cell, and resides in the cytoplasm as well as in the nucleus (Jentsch et al., 1995, Cell 32:881).
  • the proteasome possesses up to five different peptidase activities, in different catalytic domains (Ciechanover, 1994, Cell Z£:13), and the best characterized ones are chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing (PGPH) activities (Orlowski et al., 1981 , Biochem & Biophys. Res. Com. 101:814; Wilk et al., 1983, J.
  • the proteasome is responsible for the degradation of 70-90% of cellular proteins (Rock et al., 1994, Cell 7&761). Yet its activity is well controlled and only those destined to be destroyed are timely digested by the proteasome. It therefore plays a critical role in irreversibly removing short-lived regulatory proteins, and other types of proteins. Indeed, the degradation of some important regulators of cell proliferation such as cyclin 2, cyclin 3, cyclin B, p53 and p27 wp1 are mediated by the proteasome (Deshaies et al., 1995, EMBO J. 14303; Yaglom et al., 1995, Mol. & Cell. Biol. 15:731 ; Salama et al., 1994, Mol. & Cell. Biol. 14:7953; Seufert et al., 1995, Nature 37.3:78; Scheffner et al., 2729 r
  • NF- ⁇ B transacting nuclear factor NF- ⁇ B becomes active after the enzymatic cleavage of its precursor by the proteasome (Palombella et al., 1994, Cell 78:773); l ⁇ B ⁇ , the inhibitor of NF- ⁇ B, and c-JUN protein are degraded via the proteasome pathway (Palombella et al., 1994, supra; Treier et al., 1994, Cell 78:787).
  • the proteasome could be purified as 26S and 20S complexes.
  • the 20S proteasome is a cylindrical proteolytic core composed of multiple ⁇ and ⁇ subunits. Each subunit is coded by a different gene in high eukaryotic cells and the total number of subunits varies among different species (Groettrup et al., 1996, Immunol. Today 17:429).
  • the purified 20S proteasomes can digest small peptides in an ATP-independent fashion, but they are inactive on intact folded proteins (Peters, 1994, Trends in Biochem. Sci. 19:377).
  • the 20S proteasome can bind at its ends a 19S regulator and forms the 26S proteasome, which degrades ubiquitinated protein in an ATP-dependent fashion (Jentsch et al., 1995, supra).
  • the 20S proteasome can also complex with an 11S activator called PA28 (Groettrup et al., 1996, supra).
  • PA28 is a ring-like hexamer or heptamer composed of ⁇ and ⁇ subunits (PA28 ⁇ and PA28 ⁇ ), both of which are about 29KD in size (Realini et al., 1994, J. Biol. Chem. 269:20727; Ahn et al., 1995, FEBS Letters 366:37). It is not clear whether the 20S proteasome can associate both the 19S and 11S regulators at the same time.
  • the first is that of the substrate selection. This process is controlled by a cascade of enzymes called the ubiquitin-activating enzyme (E1), the ubiquitin- 2729
  • Certain peptide aldehydes such as N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL) and
  • N-carbobenzyoxyl-L-leucinyl-L-leucinyl-L-norvalinal are competitive inhibitors of chymotrypsin (Vinitsky et al., 1992, Biochem. 31:9421 ; Tsubuki et al., 1993, Biochem & Biophys. Res. Com.136:1195). These agents could effectively block the chymotrypsin-like activity, and to a lesser extent, the trypsin-like and PGPH activities of the proteasome (Rock et al., 1994, supra). They have been employed to study the function of the proteasome in various cellular processes.
  • Palombella et al. in WO 95/25533 teach a method for reducing the cellular content and activity of NF-kB, a transcriptional factor playing a central role in immune and inflammatory response, by using proteasome inhibitors, peptidyl aldehydes.
  • Stein et al. in WO 95/24914 teach a method for reducing the rate of intracellular protein breakdown by inhibiting proteasome activity.
  • the inhibitor MG 101 given as an example is shown to be an inhibitor of 26S proteasome. This inhibitory effect may result in inhibiting destruction of muscle proteins, antigen presentation and degradation of p53 .
  • LAC is a proteasome-specific protease inhibitor (Fenteany et al., 1995, Science 268:72). It inhibits the three major peptidase activities (i.e., chymotrypsin-like, trypsin-like, and PGPH activities) of the proteasome, and the inhibition of the first two is irreversible in in vitro assays. LAC does not affect other proteases such as calpain, cathepsin B, chymotrypsin, trypsin, and papain. Currently, LAC is the only proteasome-specific protease inhibitor available.
  • WO 96/32105 teaches lactacystin and various analogs to treat conditions that are mediated by the proteolytic function of the proteasome such as rapid elimination and post- translational processing of proteins involved in cellular regulation, intercellular communication and immune response, specifically antigen presentation.
  • Griscavage et al. (1996, PNAS 93:3308-3312) teach that proteasome activity is essential for the induction of nitric oxide synthase and that the proteasome peptidyl aldehyde inhibitors inhibit the induction of nitric oxide synthase.
  • Nitric oxide production is implicated in initiating and exacerbating symptoms of acute and chronic inflammation (Lundberg et al., 1997, Nature Medecine 3:30-31).
  • proteasome inhibitors by inhibiting nitric oxide induction have an anti- inflammatory activity.
  • LAC which is more specific to proteasome than peptidyl aldehydes.
  • T-cell hybridoma can be activated using dishes coated with anti-CD3. Once activated these cells die of apoptosis. It was demonstrated that lactacystin is an inhibitor of activation induced cell death (AICD) and, in these activated hybridoma T-cells, lactacystin must be administered within 2 hours of activation to efficiently block AICD. The same authors state that at higher doses LAC induces apoptosis in the artificial hybridoma T cells.
  • AICD activation induced cell death
  • proteasome inhibitors eliminate activated normal cells. There is no teachings in these references of the involvement of proteasome activity in mitochondrial function. In addition, these references do not describe in mammalian cells what proportion of the protease activity is derived from the proteasome and whether there are efficient and simple methods to screen for additional proteasome inhibitors.
  • LAC is the most specific inhibitor of proteasome available. It is mildly toxic and is unstable in aqueous solutions of high pH. LAC and some of its analogues binds directly to the proteasome and inhibits three peptidase activities of the proteasome. However, cellular events downstream of the proteasome are not totally clear. Knowledge of these down stream events related to proteasome activity will allow development of strategies and compounds capable of complementing, synergizing, or substituting the effect of proteasome inhibitors to maximize their effects and/or to minimize their side-effects.
  • the present invention seeks to meet these and other needs.
  • proteasome is essential for progression of T cells from G 0 to S phase. Taking advantage of LAC's specificity and potency, this compound was used to investigate the role of proteasomes in T lymphocyte activation and proliferation. It is demonstrated that the proteasome is essential for progression of T cells from the Go to S phase. Probably as a result of blockage of cycling, the activated but not resting T cells underwent apoptosis when treated with LAC. It is also shown that the proteasome controls the protein level of p21 Cip1 and p27 Wp1 as well as the CDK2 activity in the G t phase, and such control mechanism might be essential in the cell cycle progression. LAC can effectively inhibit T cell proliferation even if added at the G,/S boundary.
  • the present invention further relates to inducing apoptosis of activated T cells and T cell leukemia but not resting T cells with LAC or its analogues. Elimination of malignant cells by a proteasome inhibitor-induced apoptosis is useful in cancer therapy. In addition, normal T cells that become activated can be induced to undergo apoptosis with a proteasome inhibitor thus eliminating antigen specific T cells. This is useful in ameliorating autoimmune diseases and graft rejection by generating antigen specific tolerance.
  • the invention further uses the knowledge of the proteasome involvement in protein degradation and in the steps for the induction of nitric oxide synthase and the effect of LAC or its analogues on the expression of nitric oxide synthase and the production of nitric acid. This is useful in the prevention of septic shock and as an anti- inflammatory.
  • the present invention also relates to the inhibition of proteasome activity by LAC or its analogues such that the inhibition interferes with cell-cell interaction during lymphocyte activation in mammals and the up-regulation of the adhesion molecule ICAM-1 is repressed. This is useful to control undesirable immune responses during graft rejection, autoimmune diseases and inflammation.
  • the applicant is the first to show that the electron transport chain in mitochondria is dependent on the intact activity of the proteasome.
  • proteasome - specific inhibitor such as LAC reduces the electron transport at the complex IV of the respiratory chain.
  • exogenous cytochrome C reverses this effect.
  • the effect of LAC on mitochondria has potential applications for disorders that relate directly or indirectly to increased activity of mitochondrial function.
  • proliferating cells have a higher energy requirement, inhibition of mitochondrial respiration could effectively curb the proliferation of cancer cells and activated T cells by depriving the cells of energy, with minimal detriment to normal resting cells.
  • the applicant is further providing a method for screening proteasome inhibitors by assaying cellular proteinases activity with a tagged peptide substrate. It is understood that this assay protocol can be used in a large through-put screening procedure and that any means of tagging peptide substrates specific to different protease activities of the proteasome and any means for detection known to a person skilled in the art, can be used and incorporated into the large through-put procedure. All the elements comprising a method for screening proteasome inhibitors can be incorporated into a kit.
  • proteasome inhibitor to induce apoptosis in proliferating cells, wherein said proteasome inhibitor is lactacystin or an analogue thereof and said proliferating cells are cancerous cells and/or activated T cells, such that activated T cells are antigen induced.
  • the above cells are stopped from progressing from G 0 to G/M in a cell cycle as a consequence of proteasome inhibition.
  • CDK2 and the associated Cyclin E activities are substantially inhibited, whereby said cell cycle progression is substantially arrested. Additionally, CDK4 cell activity is not inhibited.
  • a proteasome inhibitor to reverse graft rejection in a patient in need for such a treatment comprising the step of administering to said patient an apoptotic amount of a proteasome inhibitor when said patient T cells are activated wherein said patient is in need of said treatment when an ongoing allograft rejection occurs or at least 24h after graft transplantation.
  • proteasome inhibitor in the making of a medicament to induce apoptosis in proliferating cells.
  • a method for screening a compound for proteasome inhibition activity which comprises: obtaining a mammalian cell lysate comprising proteasomes, a partially purified proteasomes preparation or a purified proteasomes preparation; tagging at least one peptide substrate specific to a known proteasome protease activity; combining said proteasomes and said at least one tagged peptide substrate; contacting the so combined proteasomes/tagged peptide substrate with said compound; said at least one tagged peptide substrate fails to release tag if said compound is a proteasome inhibitor, and detecting a decrease or absence of the released tag in the presence of said compound relating to the released tag in the absence of said compound as an indication of proteasome inhibition activity for said compound wherein said at least one tagged peptide substrate is a fluorogenic peptide and wherein said proteasome protease activity is trypsin-like chymotrypsin-like or peptidylglutamyl-peptide hydrolyzing activity.
  • said pathological condition is selected from the group consisting of: cancer, inflammation, undesirable immune responses and hyperthyroidism.
  • proteasome inhibitor to disrupt nitric oxide synthesis, wherein the proteasome inhibitor inhibits nitric oxide synthase gene expression.
  • An apoptotic composition comprising a therapeutically effective amount of a proteasome inhibitor and a pharmaceutically acceptable carrier which may additionally comprise a therapeutically effective amount of an inhibitor to CDK4 activity and/or a therapeutically effective amount of an inhibitor to CDK2 activity and more particularly to Cyclin E activity, a therapeutically effective amount of an inhibitor which prevents p21 Cip1 upregulation blocks the degradation of p27 ⁇ and a therapeutically effective amount of an inhibitor which prevents CD25 upregulation.
  • a composition for use in inhibiting graft rejection comprising a therapeutically effective amount of cyclosporin A, rapamycin or FK506 in combination with a therapeutically effective amount of a proteasome inhibitor and may be in combination with a therapeutically effective amount of an inhibitor of ICAM-1 expression.
  • a composition for use in inhibiting graft rejection comprising a therapeutically effective amount of an inhibitor which suppresses expression ICAM-1 in combination with a therapeutically effective amount of a proteasome inhibitor.
  • proteasome inhibitor to alleviate a disease or a disorder, wherein adhesion molecule ICAM-1 is upregulated and said disease or a disorder is graft rejection, autoimmune disease or inflammation.
  • proteasome inhibitor is to alleviate a desease or a disorder wherein at least one of CDK2, p21 Cip1 , CD25 is upregulated and/or p27 kip1 degraded, wherein said disease or disorder is graft rejection, autoimmune disease or cancer.
  • proteasome inhibitor to alleviate a disease or disorder, wherein nitric oxide synthase is upregulated and said disease or disorder is inflammation or septic shock.
  • the said proteasome inhibitor may be used alone or in combination with any drugs known in the art for use in treating cancer, inflammation, autoimmune disease, septic shock or inflammation.
  • proteasome inhibitor is lactacystin or an analogue thereof.
  • Figure 1 shows that LAC strongly inhibits T and B cell proliferation. Lymphocytes were stimulated with various mitogens as indicated, and LAC at different concentrations was added at the beginning of the cultures. The cells were pulsed with 3H-thymidine between 48h and 64h. Samples were in triplicates. All the experiments were performed at least three times and similar results were obtained. Representative results are shown.
  • A Peripheral blood T cells stimulated with PHA (2 ⁇ g/ml).
  • B Peripheral blood T cells stimulated with OKT3 (50ng/ml).
  • C Peripheral blood T cells stimulated with anti-CD28 (50ng/ml) plus ionomycin (1 ⁇ g/ml).
  • FIG. 2 shows that inhibition of the proteasome activity results in induction of apoptosis of activated normal cells and leukemic T cells but not resting normal T cells.
  • Tonsillar T cells A, B, and D
  • Jurkat cells C and E
  • LAC 10 ⁇ M for T cells and 6 ⁇ M for Jurkat cells.
  • LAC was added at the beginning of the culture or 40h after T cell activation as indicated.
  • the cells were harvested at the time points as shown. They were evaluated for their viability with trypan blue exclusion (A, B, and C), and for their mode of cell death according to DNA fragmentation (D and E).
  • Figure 3 shows by electron microscopy that the proteasome inhibitor induced apoptosis in activated T cells and Jurkat cells.
  • a and B Morphology of resting T cells treated with LAC. Tonsillar T cells were culture in the absence (A) or presence (B) of LAC (1 OmM) for 24h, and the cells were examined by EM.
  • C and D Morphology of activated T cells treated with LAC. Tonsillar T cells were first activated with PHA (2 ⁇ g/ml) for 40h. The cells were then cultured in the absence (C) or presence (D) of LAC (10 ⁇ M) for additional 24h, and were examined with EM.
  • E and F Morphology of Jurkat cell treated with LAC.
  • Jurkat cells were cultured in the absence (E) or presence (F) of LAC (6 ⁇ M) for 24h and were evaluated with EM. Arrows indicate condensed nuclei.
  • Figure 4 shows that the effect of LAC is rapid and reversible in cell culture.
  • LAC The activity of LAC in culture supernatants is short-lived LAC (6 ⁇ M) was added to Jurkat cell culture (0.5 x 10 6 cells/ml). The supernatants were harvested at 4h, 6h, 16h and 24h. These conditioned media were used to culture fresh Jurkat cells for 3h. The cells were then harvested and assayed for the proteasome activity as described in Fig. 4B. Samples were in duplicates.
  • Figure 5 shows that LAC inhibits CD25 upregulation during T cell activation.
  • Peripheral blood T cells were stimulated with PHA (2 ⁇ g/ml) for 48h in the presence or absence of LAC (10 ⁇ M, added at the beginning of the culture).
  • CD25 expression on T cells was evaluated by anti- CD25-PE/anti-CD3-FITC two-color flow cytometry. Similar results were obtained in two independent experiments, and a representative one is shown. The data are presented as two color histograms in forms of contours, as well as in an overlay histogram. Figure 6 shows the role of the proteasome in cell cycle progress.
  • LAC does not inhibit the progress from the G j /M phase to the G 1 phase in synchronized Jurkat cells
  • Jurkat cells were synchronized at the G 2 /M phase by 16h nocodazole treatment.
  • LAC (6 ⁇ M) was added to the cultures destined to be treated by LAC later.
  • the cells were then released by washing out nocodazole, and recultured in complete medium with or without 6 ⁇ M LAC.
  • the cells were sampled at Oh, 4h and 8h after the Gs/M release, stained with propidium iodide, and analyzed with flow cytometry.
  • B. LAC slows the cell cycle progress from the G S boundary to the
  • LAC blocks the S phase entry of the mitogen-stimulated peripheral blood T cells
  • Peripheral blood T cells were stimulated with PHA (2 ⁇ /ml) in the absence or presence of LAC (10 ⁇ M, added at Oh, 16h, 24h, or 40h, as indicated in the bottom of the panels).
  • LAC 10 ⁇ M, added at Oh, 16h, 24h, or 40h, as indicated in the bottom of the panels.
  • the cells were harvested at Oh, 16h, 40h and 64h as indicated on the top of the panels (Fig. 6C).
  • the triplicated cell samples were pulsed at 48h and harvested at 64h (Fig. 6D).
  • Figure 7 shows the results of the kinase assays for the effect of LAC on CDK activity.
  • Tonsillar T cells were activated with PHA (2 ⁇ g/ml) for a period as indicated in each graph.
  • LAC (10 ⁇ M) was added once at Oh.
  • the cells were harvested at 16h, 24h, or 40h as indicated.
  • An equal amount of lysate protein (40 ⁇ /sample) was precipitated with rabbit anti-CDK4, anti-CDK2 or anti-Cyclin E antisera (2.5 ⁇ g Ab/sample).
  • the immune complexes were assayed for their kinase activities using histone H1 as a substrate.
  • A CDK4 kinase activity.
  • B CDK2 kinase activity.
  • Cyclin E-associated CDK activity The membrane in (C) was subsequently hybridized with anti-Cyclin E (1 ⁇ g/ml) followed by 125 l-protein A for the evaluation of the protein level of Cyclin E.
  • Figure 8 shows the results of immunoblotting analysis of the effect of LAC on the protein levels of Cyclin E and Cyclin A. 729
  • Tonsillar T cells were stimulated with PHA (2 ⁇ g/ml) for 40h in the presence of hydroxyurea (1mM), and these cells were blocked at the G ⁇ S boundary (G ⁇ block).
  • the synchronization was released by washing out hydroxyurea, and the cells were recultured in medium containing 2 ⁇ g/ml PHA in the absence or presence of LAC (10nM, added once at the time of the release).
  • the cells were harvested at 6h and 22h post the G S block.
  • the cell lysates (40 ⁇ g/sample) were resolved in 10% SDS-PAGE, and transferred to PVDF membranes.
  • the membranes were hybridized with rabbit-anti-Cyclin E or anticyclin A antisera followed by 125 l-protein A.
  • the Cyclin E level (Fig. 8A) and cyclin A level (Fig. 8B) of representative experiments are shown. Similar results were obtained in a total of three independent experiments.
  • Figure 9 shows the results of immunoblotting analysis of the effect of LAC on the levels of CDK inhibitors p27 ⁇ ,p1 and p21 c,p1 .
  • Tonsillar T cells were stimulated with PHA (2 ⁇ g/ml) for 16h, 40 or 64h in the absence or presence of LAC (10 ⁇ M).
  • LAC was added once at Oh.
  • LAC was added once at 40h.
  • the cell lysates were resolved in 10% SDS-PAGE, and blotted onto PVDF membranes.
  • the membranes were hybridized with rabbit anti-p27 K ⁇ p1 antisera (Fig. 9A) or with anti-p21 C ⁇ p1 antisera (Fig. 9B) followed by 125 l-protein A.
  • the experiments were performed three times, and similar results were obtained. Representative data is shown.
  • Figure 10 shows human peripheral blood mononuclear cells that were cultured in medium (A), 2 ⁇ g/ml PHA (B), or PHA plus 10 ⁇ M lactacystin for 24h. Lactacystin could effectively block the aggregate formation.
  • Figure 11 shows mouse lymph node cells that were cultured in medium (A), 2 ⁇ g/ml Con A (B), or Con A plus 10 ⁇ M lactacystin for 24h. Lactacystin could effectively block the aggregate formation.
  • Figure 12 shows mouse lymph node cells from TCR transgenic mice named 2C that were cultured in medium (A), 2 ⁇ g/ml Con A (B), or Con A plus 10 ⁇ M lactacystin. After 24h and 48h, the cells were examined for ICAM-1 expression by flow cytometry, using FITC-anti- ICAM-1/ 1B2-PE. Monoclonal Ab 1B2 recognize a clonotypic determinant on the TCR of the transgenic T cells which are largely CD8 positive (>75%). Lactacystin could effectively block the upregulation of ICAM-1 on those CD8 positive T cells.
  • Figure 13 shows mouse peritoneal exudate macrophages that were stimulated with 2 ⁇ g/ml LPS in the presence of lactacystin at different concentrations. Nitric oxide production by the macrophages was measured according to the nitrate concentrations in the supernatants.
  • Figure 14 shows mouse peritoneal exudate macrophages that were stimulated with 2 ⁇ g/ml LPS in the presence or absence of lactacystin (10 ⁇ M). Nitric oxide synthase expression was measured with Northern blot analysis.
  • Figure 15 shows that Lactacystin blocks electron transport downstream of Complex I. Respiration of Jurkat cells (JC) or rat kidney mitochondria (RKM) was measured by O 2 consumption using an oxygen electrode. The function of Complex I of digitonin (Dig)-permeated Jurkat cells was blocked by rotenone (Rot), and the respiration was resumed by adding succinate (Sue), which provides electrons to Complex II directly and thus bypasses Complex I.
  • CCCP carbonyl cyanide m-chlorophenylhydrazone
  • Ant antimycin A
  • Curves 1 and 6 represent positive controls of rat kidney mitochondria.
  • Curves 2 and 5 represent normals untreated Jurkat cells.
  • Curves 3 and 4 represent Jurkat cells treated with lactacystin (6 ⁇ M) for 2h and 4h, respectively.
  • Figure 16 shows that Lactacystin blocks electron transport at Complex IV.
  • Complex III in the respiration chain was blocked at Complex III antimycin (Ant), and the electron flow was resumed by addiind ascorbate (Asc) and TMPD (tetramethyl-p-phenyl-enediamine).
  • the maximal respiration was triggered by CCCP, and was totally inhibited by potassium cyanide (KCN).
  • Figure 17 shows that Cytochrome completely corrects the defect at Complex IV caused by LAC.
  • the assay system is identical to that described in Figure 16.
  • Jurkat cells were treated with LAC for 4h (curve 3).
  • the decoupling reagent used in this experiment to achieve maximal respiration is FCCP (carbonylcyanide-p- trifluoromethoxyphenylhydrazone).
  • FIG. 18 shows that RAPA, FK506, and CsA inhibit PA28 expression at the mRNA level.
  • Tonsillar T cells (A) and B cells (B) were cultured in the presence of various reagents as indicated (PHA, 2 ⁇ g/ml, RAPA, 10 nM; FK506, 10 nM, CsA, 1 ⁇ M; SAC, 1 :10 000 dilution; II-2, 25 U/ml. After 6h, 20h or 40h, the cells were harvested and total RNA was analyzed by Northern blotting for PA28 ⁇ expression. The PA28 ⁇ message in T cells was also examined by Northern blotting using a similar condition as for PA28 ⁇ (C). The experiments were repeated more than three times, and representative ones are shown.
  • FIG. 19 shows that RAPA inhibits PA28 ⁇ and PA28 ⁇ protein in the activated T cells.
  • A An analysis of PA28 ⁇ protein by immunoblotting is shown. Tonsillar T cells were cultured with 2 ⁇ g/ml PHA or PHA plus 50 nM RAPA for 24h. The cells were harvested and lysed. Forty micrograms of cleared lysate protein per sample was analyzed by immunoblotting using rabbit anti-PA28 ⁇ antiserum.
  • B An analysis PA28 ⁇ and PA28 ⁇ protein by confocal immunofluorescence microscopy. Tonsillar T cells were cultured with 2 ⁇ g/ml PHA or PHA plus 50 nM RAPA for 24h. The cells were stained with antisera specific for PA28 ⁇ and PA28 ⁇ .
  • Figure 20 shows that effect of RAPA on proteasome activity in human PBMC.
  • Human PBMC were cultured in the absence or presence of 2 ⁇ g/ml PHA or 10 nM RAPA for 16h-70h as indicated. The cells were then harvested, and the chymotrypsin-like activity of whole cells lysates was assayed in the absence or presence of 20 ⁇ M proteasome inhibitor LAC. The data are presented as arbitrary units of fluorescence intensity per 20 ⁇ g lysate protein. The experiments were repeated three times and a representative one is shown. Samples are in duplicate and the mean ⁇ SD is shown.
  • the C57BL/6 spleen cells (H-2 b ) were stimulated with mitomycin c-treated BALB/c spleen cells (H-2 d ).
  • the mixed lymphocyte culture (MLR) was treated with lactacystin (LAC, 8 ⁇ M) for 3 h. After wash, the cells were put back in culture for additional 8 days, and then stimulated with either fresh BALB/c or C3H (H-2 k ) spleen cells.
  • MLR treated by LAC the C57B1J6 cells failed to respond to the BALB/c cells, but respond well to third party C3H (H-2 k ) cells. The difference is more pronounced in day three of the culture.
  • Figure 22 shows that the LAC-induced DNA fragmentation is inhibited by a broad spectrum caspase inhibitor zVAD.fmk.
  • Jurkat cells were treated with LAC (6 ⁇ M) in the absence or presence of different concentrations of zVAD.fms (0.4 ⁇ M to 33.3 ⁇ M) for 6 h. The cells were harvested and their DNA was analyzed by a DNA fragmentation assay according to DNA laddering.
  • Figure 23 shows that preventing the degradation of a pro-apoptotic Bcl-2 family member Bik is a mechanism for the proteasome inhibitor-induced apoptosis.
  • Jurkat cells were treated with lactacystin (6 ⁇ M) for 5 h (lanes 2 and 4 of panel A), 4 h (lane 2 of panel B) or 7h (lane 3 of panel B), lane 1 in panels A and B is untreated control samples.
  • the cells were separated into mitochondrial (mito in panel A and mitochondria in panel B) and cytosolic (cytosol in panel A) fractions, and the lysate of these two fractions analyzed by immunoblotting using goat anti- Bik, and rabbit anti-Bax, Bak and Bad Ab (all from Santa Cruz Biotech, Santa Cruz, CA) followed by enhanced chemiluminescence (ECL, kit from Amersham).
  • Figure 24 shows that overexpression of an anti- apoptotic Bcl-2 family member Bcl-xL in a B cell line could protect the cells from apoptosis caused by proteasome inhibition.
  • a human B cell line Namalwa was stably transfected with an anti-apoptotic Bcl-2 family member Bcl-xL, and its sensitivity to the proteasome inhibitor-induced apoptosis tested by the quantitative filter elution assay (Schmitt et al., Exp. Cell Res. 240:107, 1998), which detects DNA fragmentation during apoptosis.
  • the wild type Namalwa and transfected Namalwa cells overexpressing Bcl-xL were pulsed with 14 C- thymidine for 24 h, and then treated with different concentrations of lactacystin (0.75 ⁇ M, 1.5 ⁇ M, 3 ⁇ M, 6 ⁇ M and 10 ⁇ M). The cells were harvested at different time intervals (24-96 h), and DNA fragmentation measured.
  • Figure 25 shows that the wild type Namalwa cells have increased Bik level after treatment with lactacystin and that the Bcl-xL transfected Namalwa cells have overexpressed Bcl-xL.
  • Jurkat cells, wild type Namalwa cells and Bcl-xL transfected Namalwa cells were treated with medium (lanes 1), staurosporine (0.3 ⁇ M, lanes 2) and lactacystin (6 ⁇ M, lanes 3) for 6 H.
  • the proteins from the mitochondrial fraction of these cells were analyzed by immunoblotting and the amount of Bik, Bcl- xL, Bax, and Bak evaluated. The same membranes were used sequentially and probed with different antibodies against these factors.
  • the present invention relates to proteasome activities in cellular processes and any inhibitors of proteasome activities.
  • Proteasome Activity is Obligatory for Activation and Proliferation of T and B Cells
  • PBMC peripheral blood mononuclear cells
  • LAC proteasome-specific inhibitor
  • the PBMC were activated with various stimulants.
  • LAC was added to the cells in the beginning of the culture (Oh) along with the stimulants.
  • 3 H-thymidine uptake between 48h and 64h of 64h cultures was used as a parameter for cell proliferation.
  • Fig. 1 LAC strongly and dose-dependently inhibited the T cell proliferation induced by a T cell mitogen PHA (Fig. 1A), by crosslinking TCR with anti-CD3e (Fig. 1B), or by Ca ++ ionophore plus cross-linking of the T cell co-stimulating molecule CD28 (Fig. 1C).
  • LAC T cell-independent B cell proliferation induced with SAC plus IL-2 in tonsillar B cells was also potently inhibited by LAC (Fig. 1 D). In all the four systems employed, LAC at 5 ⁇ M could exert near-to-maximal inhibition. The results suggest that LAC's effect is not lymphocyte type(T or B cells)-specific nor stimulant- specific. Rather, it likely affects certain downstream events governing a more general process(es) in lymphocyte activation and proliferation.
  • LAC causes Apoptosis in Activated but not Resting T Cells
  • a compound is provided that induces activated and leukemic T cells to undergo apoptosis.
  • LAC has been reported to induce apoptosis in U937 cells (Chen et al., 1996, J. Immunol. 15Z:4297), it is crucial to examine whether the LAC-induced inhibition of cell proliferation is due cell death, be it apoptosis or necrosis.
  • T cells and Jurkat cells after LAC-treatment were first evaluated with trypan blue exclusion. Resting T cells (T cells in medium) or PHA-stimulated T cells were cultured with 10 ⁇ M LAC (LAC added at the beginning of the culture). As shown in Figure 2A, after 16h culture, the viability of the cells only had minor decreases ( ⁇ 12%) in LAC-treated cells compared with those without LAC (97% vs 92% for cells in medium, and 94% vs 83% for cells with PHA). After a prolonged culture for 64h, the decreases were more prominent although were still less than 27%(97% vs 79% for cells in medium, and 90% vs 63% for cells with PHA).
  • Electron microscopy was also employed to examine the mode of cell death induced by LAC.
  • the resting T cells (cells cultured in medium, figure 3A), activated T cells (40h after PHA activation, Figure 3C), and Jurkat cells (Figure 3E) were all healthy looking. Occasional condensed nuclei were observed in medium cultured T cells ( Figure 3A) and this is not unusual.
  • the resting T cells treated with LAC (10 ⁇ M)for 24h were still healthy ( Figure 3B). However, nuclear condensation, which is a hallmark of apoptosis, were frequently observed in activated T cells and Jurkat cells after they were exposed to LAC (10 ⁇ M and 6 ⁇ M, respectively) for 24h ( Figures 3D and F).
  • LAC's differential effect on the viability of resting versus cycling cells suggests that it is not simply nonspecific cytotoxicity, but relates to the status of the cell cycle.
  • the cell death without doubt contributes to but cannot solely account for the observed inhibition of proliferation by LAC, since there are still significant percentage ( about 60%) of live cells at the end of the culture according to trypan blue exclusion. Moreover, we will elaborate later that the cell death is a consequence of blockage of cell cycle progress.
  • Admittedly the trypan blue negative cells includes some early apoptotic cells, as evidenced by the fact that DNA laddering could be detected in a largely trypan blue negative population. However, it does not necessarily mean that the whole population is dead.
  • LAC could be used to study the role of proteasomes in lymphocyte activation and proliferation, as long as the compound is applied only once in the beginning of activation of the resting T cells and the experimentation is carried out in 24h-40h, or LAC is present for less than 8h in the case of cycling cells, since such treatments do not drastically affect the viability of the cells.
  • a specific embodiment of this invention is the ability of LAC to induce apoptosis mostly in activated and proliferating cells and not in normal resting cells. This has value in eliminating cancerous cells and antigen-specific T cells. The elimination of the latter will create a specific immune tolerance to alloantigens in transplantation, and to selfantigens in autoimmune diseases.
  • LAC (6 ⁇ M) was added to Jurkat cells culture for 4h, 6h, 16h or 24h.
  • the conditioned medium was harvested and used to treat fresh Jurkat cells for 3h, and then the proteasome activity in the lysates of the fresh Jurkat cells was assayed.
  • 4h to 24h conditioned media without LAC did not affect the proteasome activity of the fresh Jurkat cells.
  • the media conditioned with LAC up to 6h could still actively inhibit the enzymatic activity, but after 16h, the LAC-conditioned media lost their inhibitory effect.
  • LAC's capability to enter the cells to inhibit the proteasome activity rapidly (less than 3h), and its short active duration within the cell and in the culture media (about 16h) makes the compound a very useful reagent to evaluate the requirement of the proteasome activity in various events during cell activation and proliferation, since we could pinpoint the period when the proteasome activity is critical. It is an embodiment of this invention, the use of LAC can be regulated in a time course sequence to be most effective at the period when proteasome activity is critical to maximise the effect of LAC on cells.
  • Proteasome Activity is Required for IL-2R ⁇ Upregulation
  • the growth promoting activity of IL-2 is indirectly (for stimulation by PHA, anti-CD3, and anti-CD28 plus ionomycin), or directly (for SAC plus IL-2) involved.
  • PHA anti-CD3, and anti-CD28 plus ionomycin
  • SAC SAC plus IL-22
  • CD25 was upregulated in CD3 + T cells 40h after stimulation with PHA.
  • LAC (10 ⁇ M) was added in the beginning of the culture, the upregulation was significantly inhibited.
  • IL-2 production by PBMC 2 to 4 days after PHA stimulation in the absence or presence of LAC (10 ⁇ M, added at the beginning of the culture) was also examined, but no consistent difference was found (data not shown).
  • the viability of the LAC-treated cell was reasonable (>80% at 40h) as described in the previous section as LAC was added only once initially.
  • no consistent change of IL-2 production in LAC-treated cells was a functional indication that the cell viability was reasonable and is not of a concern in interpreting the data.
  • Jurkat cells Like normal T cells, the proliferation of Jurkat cells was also potently inhibited by LAC (data not shown).
  • Jurkat cells were first synchronized at the G2/M boundary by nocodazole (Fig. 6A). The cells were released from the blockage by washing out nocodazole. In the control sample, more than half the cells traversed through the M phase and arrived at the G: phase within 4h.
  • LAC (6 ⁇ M) was added to the culture 3h before the release, so the compound could have enough time to enter the cells. LAC was also added to the culture after the release.
  • the Jurkat treated with LAC traversed through the M phase to the G ⁇ phase at a similar pace as the control cells. Since the total duration of the assay was around 7h (3h preincubation plus 4h after the release), LAC was certainly active during this period. The fact that most of synchronized Jurkat cells could traverse through G2/M to G1 in the presence of LAC for 7h again suggests that the viability of the cells thus treated is not a matter of concern. This result shows that the G 2 to G ⁇ progression is not proteasome-dependent.
  • the Jurkat cells were synchronized at the G S boundary by HU blockage. The cells were then released by washing out HU.
  • the result from this part suggests that the proteasome activity is required for optimal progression from the G/S boundary to the G 2 /M phase, because the progression could still proceed albeit at a slower pace when the proteasome activity is inhibited.
  • the result also implies that the absolutely proteasome-dependent window during the cell cycle, as evidenced by the near-total inhibition of S phase entry in LAC-treated mitogen-stimulated lymphocytes according to the proliferation data, must be in the G1 phase before the target point of HU, which inhibits ribonucleotide reductase in the GJS boundary (Brown et al., 1996, Cell 86:517).
  • T cells from PBMC were at Go when isolated. After 16h stimulation with PHA, they remained before the S phase (Fig. 6C). At 40h, about 20% of the cells were in the S and G 2 /M phases.
  • the peak of 3 H-thymidine uptake according to a 16h pulse was between 48h and 64h (data not shown), although at 64h, the cells in the S and G 2 /M phases were still about 20% (Fig. 6C).
  • the lack of an increase in percentage of cells in the S and G 2 /M phases at 64h compared with that at 40h was likely due to the exit of the cells from the S and G 2 /M phase.
  • the cycling T cells in this system never reaches 100%, because about 15% of the cells were non T cells, and an additional 20% were non responsive T cells. Taken the cell proliferation and cell cycle analysis together, the G ⁇ S boundary of the cycling T cells should be between about 35h and 48h after the PHA stimulation.
  • LAC inhibition of proliferation by LAC was a combinatory effect of cell cycle progress and cell death, the latter possible being the consequence of the former.
  • the extensive cell death for the sample treated with LAC at 40h was not fully reflected in the flow cytometry (Fig. 6C) as cells with less than 2N DNA. This was due to that the histogram was gated on a region of largely viable cells.
  • LAC is used to reverse ongoing graft rejection during a rejection episode. Most immunosuppressive drugs do not have the capability to reverse rejection once it begun.
  • the use of LAC overcomes the prior art.
  • the Proteasome Activity is Essential for CDK2 but not for CDK4 Function
  • CDK4 Cyclin-dependent kinases
  • CDK2 is critical in the late G 1 as well as throughout the S phase for the cell cycle progression (Van der Heuvel et al., 1993, Science 262:2050).
  • LAC was added only once at the beginning of the culture. Consequently, the viability of the LAC-treated cells was good for the first 16h and was reasonable at 40h, and was not a factor that might interfere with the interpretation of the results.
  • the resting T cells had some CDK4 activity, and the activity reached a plateau within 16h of the activation. This was in agreement with the critical role of CDK4 in the early G phase. Inhibition of the proteasome activity by LAC from 0-16h (LAC added once at Oh) did not affect the CDK4 activity when examined at 16h and 40h (Fig. 7A). This indicates that the induction and maintenance of CDK4 activity during the G1 phase is not proteasome-dependent. In contrast to CDK4, the CDK2 activity was augmented at 16h but the augmentation was more prominent at a later stage close to 40h after the mitogen-stimulation (Fig.
  • CDK2 Cyclin E-associated CDK2 activity.
  • inhibitors of CDK2 can be used alone or in combination with proteasome inhibitors.
  • the aforementioned compositions are of a pharmaceutically effective amount to induce apoptosis or for any other cellular or physiological effect. Since CDK4 activity is important in G 0 to G 1 ⁇ progression and it is not affected by proteasome activity, it is conceivable that inhibitors for CDK4 can be used in combination with proteasome inhibitors of a pharamceutically effective amount to achieve additive effect in blocking cell proliferation and in any other relevant cell function.
  • Inhibitors in this application are defined as any element capable of silencing the activity of a protein at the level of gene transcription, translation, or post-translational modification of the protein as well as elements capable of interfering with the protein. These may include but are not limited to antibody or other ligands, anti-sense or antagonist molecules.
  • contacting LAC with CDK2 is inhibitory to CDK2 activity, more particularily it is the inhibitory effect of LAC on Cyclin E.
  • the inhibitory effect of LAC is the disruption of cell cycling.
  • Oscillation of cyclins during the cell cycle is a mode of regulation for the CDK activities. Since the CDK2 activity is proteasome-dependent, and CDK2 associates predominantly with Cyclin E and cyclin A at the G ⁇ S boundary and during the S phase respectively (Pagano et al., 1992, EMBO J. 11:961 ; Hall et al., 1995, Oncogene 11:1581), we studied the role of the proteasome in degradation of these two cyclins. As shown in Fig. 8A, the Cyclin E level was apparently increased around 40h after PHA stimulation of the T cells, which were then at the G S boundary.
  • LAC is capable of suppressing the up regulaion of the CDK inhibitor p21 c " p1 and in blocking the degradation of the CDK inhibitor p27 K ⁇ p1 .
  • the CDK activities are also controlled by several low molecular weight inhibitors.
  • the resting T cells had a high level of p27 K ⁇ p1 and the level decreased gradually when the cells moved to the G.,/S boundary 40h after the mitogen-stimulation.
  • Nitric oxide (NO) produced by macrophages is involved in inflammation and septic shock.
  • NO Nitric oxide
  • Fig. 13 The usefulness of proteasome inhibitors in inflammation and in septic shock is implicated.
  • Fig. 14 demonstrates that proteasome activity is required for NO synthase expression.
  • LAC decreases the expression of mRNA for NO synthase.
  • Mitochondria are pivotal organelles in the cells and their primary function is to produce ATP via the Krebs cycle coupled to the oxidative phosphorylation of the respiratory chain. An intact function of mitochondria is also required for proper cell viability. Damage of the mitochondrial membrane potential or release of cytochrome C or other apoptogenic factors from the mitochondria to the cytosol will induce cell death via apoptosis.
  • a proteasome-specific inhibitor lactacystin (LAC) could rapidly (within 4h) reduce the electron transport at the complex IV of the respiratory chain, and the effect could be reversed by adding back exogenous cytochrome C (cytoC).
  • the mitochondrial activiy is overactive due to the effect of the thyroid hormone. This results in many symptoms such as excessive body heat, and imbalance of energy uptake and consumption.
  • the proteasome inhibitors could reduce the rate of mitochondrial respiration and have therapeutic effect to this disease.
  • the mitochndria are more active than in normal cells in order to meet the energy requirement of a high metabolic activity of these cells. Consequently, inhibition of the mitochondrial respiration could curb the proliferation of the cancer cells or activated lymphocytes while have less detrimental effects to normal resting cells. In addition, apoptosis could be induced in the cycling cells but not resting cells. Thus, inhibition of the proteasome activity will have therapeutic effect in cancer and in diseases involving lymphocyte activation and proliferation, such as seen in graft rejection and autoimmune diseases. Rapid Assays for A High Through-Put Screening Procedure to Identify Additional Proteasome Inhibitors
  • proteasome inhibitors could be modified to use purified or partially purified 20S or 26S proteasome as a source of the proteasome enzymes. Since such assays are simple (only three components) and rapid (only several minutes of reaction period), they could be adapted for high through-put screenings, and included in a kit format.
  • Rapamycin is a potent immunosuppressive drug, and certain of its direct or indirect targets might be of vital importance to the regulation of an immune response.
  • Seven RAPA-sensitive genes are known and one of them encoded a protein with high homology to the ⁇ subunit of a proteasome activator (PA28 ⁇ ). This gene was later found to code for the ⁇ subunit of the proteasome activator (PA28 ⁇ ).
  • Activated T and B cells had upregulated PA28 ⁇ expression at the mRNA level. Such upregulation could be suppressed by RAPA, FK506, and cyclosporin A (CsA).
  • CsA cyclosporin A
  • RAPA and FK506 also repressed the upregulated PA28 ⁇ messages in PHA-stimulated T cells.
  • RAPA inhibited PA28 ⁇ and PA28 ⁇ in the activated T cells according to immunoblotting and confocal microscopy. Probably as a consequence, there was a fourfold increase of proteasome activities in the PBMC lysate after the PHA activation. RAPA could inhibit the enhanced part of the proteasome activity.
  • a proteasome activator is a relevant and important downstream target of rapamycin, and that the immune response could be modulated through the activity of the proteasome.
  • RAPA complexes with a 12KD FK506-binding protein (FKBP12) (Harding et al., 1989, Nature 341:371 ; Siekierka et al., 1989, Nature 341:755).
  • FKBP12 12KD FK506-binding protein
  • the RAPA-FKBP12 complex then binds to cytoplasmic proteins termed TOR1 and TOR2 (target of rapamycin) in yeast (Kunz et al., 1993, Cell 7.3:585; Helliwell et al., 1994, Mol. Biol. Cell.
  • PBMC lysates were assayed for their proteinase activity at pH 8.2 which favors the proteasome activity, using a chymotrypsin substrate as a representative parameter. Forty and seventy hours after stimulation by a T cell mitogen PHA, the chymotrypsin-like activity in the PBMC increased 2.1 fold and 3.8 fold, respectively (Fig. 20A). RAPA at 10nM repressed 23.1% and 41.1 % the activity in the PBMC, respectively, at these time points.
  • proteasome inhibitors can be administered when specific T cells are activated, thereby potentially eliminating the activity of specifically activated T cells while leaving non-activated T cells intact. It is therefore an embodiment of this invention to use proteasome inhibitors, particularly lactacystin in transplantation and autoimmune diseases where certain undesirable activated T cells can be repressed or eliminated and the rest of the T cell population is generally unaffected by such inhibitors.
  • Bax, Bak, and Bad are predominantly located in the mitochondrial fraction.
  • Treatment with lactacystin does not appear to have altered the amounts of Bax, Bak and Bad (Fig. 23 panels A and B).
  • the results shown in Fig. 23, suggests that under normal circumstances, Bik is degraded rapidly by the proteasome. Blocking of this degradation by a proteasome inhibitor, allows the pro-apoptotic Bcl-2 member to accumulate. The accumulation of Bik may possibly tip the balance between pro- and anti-apoptotic factors favoring apoptosis.
  • the elevated amount of Bik is likely a mechanism of lactacystin-induced apoptosis in the Jurkat and Namalwa cells.
  • the accumulation of Bik was only observed in the lactacystin-treated but not in staurosporine treated cells, eventhough staurosporine could equally induce apoptosis in these cells.
  • the balance between the pro- and anti-apoptotic factors in cells is crucial in deciding the fate of these cells.
  • Certain apoptosis-related factors have a short half life and their degradation is via the proteasome machinery. Therefore, modulating the proteasome activity with proteasome inhibitors is a useful way to control the balance between the pro- and anti-apoptotic factors. This control provides the means to induce cells into apoptosis or continued survival.
  • RPMI 1640, FCS, penicillin-streptomycin, and L-glutamine were purchased from Life Technologies (Burlington, Ontario, Canada). Lymphoprep was purchased from NYCOMED (Oslo, Norway).
  • PHA, hydroxyurea, nocodazole, and histone H1 were from Sigma (St. Louis, MO).
  • Staphylococcus aureus Cowan I (SAC) were obtained from Calbiochem (La Jolla, CA), and lactacystin from Dr. E.J. Corey (25).
  • Human rlL-2 was from La Roche (Nutley, NJ), and anti-CD3 mAb OKT3 was from ATCC (Rockville, MD).
  • FITC-conjugated anti-CD3 mAb(clone SFCIRW2-8C8) and PE-conjugated anti-CD25 mAb (clone IHT44H3) were from Coulter (Miami, FL).
  • Anti-CD28 mAb (clone 9.3) was a gift from Dr. P. Linsley (26).
  • [ ⁇ - 32 p]ATP (3000 ⁇ Ci/mmol) and [ 125 l] protein A (30mCi/mg protein) were ordered from Amersham (Oakville, Ontario, Canada), and [Methyl- 3 H] thymidine (2Ci/mmol) was from ICN (Irvine, CA).
  • PBMC Peripheral blood mononuclear cells
  • tonsillar T cells were prepared as described before (Luo et al., 1992, Transplantation 53:1071; Luo et al., 1993, Clin. & Exp. Immunol. 94:371).
  • the cells were cultured in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics.
  • 3 H-thymidine uptake was carried out as described previously (Luo et al., 1992, supra; Luo et al., 1993, supra).
  • DNA fragmentation assay DNA fragmentation assay
  • the assay was performed according to a protocol described by Liu et al (Liu et al., 1997, Cell. 89:175) with some modifications. Briefly, 2-6 million cells were re-suspended in 50 ⁇ l PBS followed by 300 ⁇ l lysis buffer (100 mM Tris-HCI, pH 8.0, 5 mM EDTA, 0.2 M NaCI. 0.2% w/v SDS, and 0.2 mg/ml proteinase K). After overnight incubation at 37°C, 350 ⁇ l of 3M NaCI was added to the mixture and cell debris was removed by centrifugation at 13000 g for 20 min at room temperature. DNA in the supernatant was precipitated with an equal volume of 100% ethanol.
  • the pellet was washed with cold 70% ethanol and then dissolved in 20 ⁇ l of TE containing 0.2 mg/ml RNase A. After incubation at 37°C for 2 h, the DNA was resolved on 2% agarose gel and visualized with ethidium bromide staining.
  • T cells and Jurkat cells were examined by electron microscopy as described by Tsao and Duguid (Tsao et al., 1987, Exp. Cell Res. 168:365).
  • Two-color staining with FITC-anti-CD3 and PE-anti-CD25 was performed on tonsillar T cells. The method was described before (Luo et al., 1993, supra).
  • Jurkat cells were cultured with various treatments and were harvested and sonicated in 300 ⁇ l PBS on ice for 40 sec. Twenty micrograms of protein per sample from the cleared lysates were supplemented to 100 ⁇ l with 0.1 M Tris buffer (pH 8.2). The fluorogenic chymotrypsin substrate sLLVY-MCA was added at a final concentration of 10nM. The samples were incubated at 37°C for 15 min and the reaction was terminated by adding 4 ⁇ l 2.5M HCI. The samples were then diluted to 2ml with 0.1 M Tris pH 8.2, and measured for their fluorescence intensity by a PTI fluorometer (Photo Technology International, South Brunswick, NJ). The excitation wavelength was 380nm, and the emission wavelength 440nm.
  • the Jurkat cells were starved in isoleucine deficient medium for 24h followed by 16h treatment with 2mM hydroxyurea (HU). Cells thus treated were synchronized at the G S boundary. For synchronization at the G 2 /M boundary, the G 1 /S synchronized cells were released from hydroxyurea and cultured in regular medium for 6h, and then treated with 0.1 ⁇ g/ml nocodazole for
  • Immunoblotting was employed to evaluate the levels of Cyclin E, cyclin A, p21 Cip1 and p27 Kip1 .
  • the general protocol was described in our previous publication (Chen et al., 1996, J. Immunol. 157:4297). Briefly, lymphocytes were lysed in the presence of proteinase inhibitors. The cleared lysates were quantitated for protein concentrations. An equal amount of lysate proteins (40 ⁇ g) of each sample was resolved by 10% SDS-PAGE and was transferred to PVDF membranes (Millipore, Bedford, MA).
  • the membranes were then blocked with 5% milk, and hybridized with rabbit antisera against Cyclin E, cyclin A, p27 Kip1 and p21 Cip1 at dilutions suggested by the manufacturer.
  • the signals on the membrane were detected by [ 125 l]-protein A followed by autoradiography.
  • Lymphocytes were lysed by a lysis buffer as used in the immunoblotting (Chen et al., 1996, supra), and cleared lysates were quantitated for their protein content.
  • 50 ⁇ l of rabbit antisera against CDK2, CDK4 or Cyclin E were added to the lysates equivalent to 20 or 40 ⁇ g protein depending on the experiment. After 2h incubation at 4°C, the immune complexes were recovered by protein A-conjugated Sepharose (Pharmacia Biotech, Montreal, Quebec, Canada).
  • the immune complexes bound to protein A-Sepharose were extensively washed in a lysis buffer without detergents or EDTA, and resuspended in 50 ⁇ l of kinase reaction buffer (100 ⁇ M NaCI, 20mM HEPES, pH7.S, 5mM MnCI 2 , 5mM MgCI 2 , 25 ⁇ M cold ATP, 2.5 ⁇ Ci [ ⁇ - 32 p] ATP, and 3 ⁇ g histone H1 as a substrate).
  • the reaction was carried out for 10 min at room temperature, and stopped by adding the SDS-PAGE loading buffer. After boiling for 3 min, the samples were subjected to 10% SDS-PAGE.
  • the proteins were then transferred to PVDF membranes and the signals were detected by autoradiography.
  • mice were injected i.p. with 3ml of 3% thioglycollate broth. Three days later, peritoneal exudate macrophages of the mice were harvested and washed at 170 g for 10 min at 4° C. The macrophages were cultured in Teflon vials (2cm in diameter) at 4x10 6 /2ml with various reagents (LPS, 2 ⁇ g/ml; IFN ⁇ , 100u/ml; LAC, 0.62-5 ⁇ M for the nitric oxide assay and 5 ⁇ M for the Northern blot assay).
  • LPS 2 ⁇ g/ml
  • IFN ⁇ 100u/ml
  • LAC 0.62-5 ⁇ M for the nitric oxide assay
  • 5 ⁇ M for the Northern blot assay 5 ⁇ M for the Northern blot assay.
  • nitrite concentration in the culture supernatant was measured as a way to indirectly reflect the nitric oxide level following a method described by Ding et al ( Ding et al., 1988, J. Immunology 141 :2407). Release of reactive nitrogen intermediates and reactive oxygen intermediates form mouse peritoneal macrophages: comparison of activation cytokines and evidence for independent production. Briefly, 100 ⁇ l of supernatants collected from 48h macrophage cultures was incubated with an equal volume of the Griess reagent (1% sulfanimide/ 0.1% naphthylethylene diamine dihydrochloride/ 2.5% H 3 PO 4 )at room temperature for 10 min in 96-well microtitration plates, the O.D. was measured at 550nm. Sodium nitrite of various concentrations were used to construct standard curves.
  • a 562-bp fragment corresponding to the mouse iNOS cDNA was obtained by reverse transcription/PCR using the mouse macrophages total RNA as templates. The fragment was labeled with 32 P with random primers and used as a probe for the Northern blot.
  • Rat liver of rat kidney proximal tubules mitochondria were isolated by differential centrifugation in a medium containing 250 mM sucrose, 1 mM HEPES-Tris, 250 ⁇ M EDTA (pH 7.5). The last washing of the mitochondria was performed in the same medium without EDTA. Protein concentration of the mitochondrial suspension was measured after solubilization of the membranes in 0.1% SDS with the Pierce-BCA (bicinchroninic acid) protein assay reagent (Pierce, Rockford, IL, USA), using bovine serum albumin as a standard.
  • Pierce-BCA bicinchroninic acid
  • the Jurkat Cells (JC) (30x10 6 /ml) or rat kidney proximal tubules mitochondria (RKM) (0.5 mg of protein/ml) were incubated in 1 ml measuring chamber at 37°C in a respiration buffer containing 200 mM sucrose, 5 mM MgCI 2 , 5 mM KH 2 PO 4 , and 30 mM HEPES-Tris (pH 7.5).
  • Digitonine Digitonine (Dig); 10 mM Succinate (Sue); 1 mM Ascorbate (Asc); 0.4 mM tetramethyl-p- phenylenediamine (TMPD); 1 ⁇ M CCCP, 1 ⁇ M FCCP; 0.1 ⁇ M Rotenone (Rot); 50 nM Antimycin A (Anti); 1 mM KCN; 100 ⁇ M Cytochrome C (Cyt C).
  • the respiration rate of the Jurkat Cells and mitochondria was measured polarographically with a Clarke oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH, USA) using 1 ml thermojacketed chamber.
  • Oxygen concentration was calibrated with air-saturated buffer using Hypoxanthine - Xanthine Oxidase - Catalase system ("chemical zero").
  • Oxygen consumption was continuously recorded using a "MacLab/8" (Analog Digital Instruments, USA) connected to a Macintosh SE computer and the MacLab Chart v.3.3.4 software. Rates of oxygen consumption are expressed as ng-atoms of oxygen/min.
  • PBMC peripheral blood mononuclear cells
  • Tonsillar T cells were prepared by one cycle of SRBC resetting and such preparation contained 80-85% CD3 + cells.
  • the remaining tonsillar cells were referred to as the tonsillar B cells, which were about 90% CD20 + cells.
  • Tissue or lymphocyte total RNA was extracted with the guanidine/CsCI method and used in the Northern blot analysis.
  • a 358-bp fragment corresponding to positions -14 to 314 of the PA28 ⁇ cDNA (Ahn et al., 1995, FEBS Lett. 366:37) from clone 5F2 was labeled with 32 p using random primers and was used as a probe for PA28 ⁇ messages.
  • a 400-bp fragment corresponding to positions between 267 and 666 of the PA28 ⁇ cDNA was obtained with RT-PCR and was used as a probe for PA28 ⁇ messages.
  • the 5' and 3' primers for the RT- PC R were GAAGAAGGGGGAGGATGA and
  • T cell lysates (40 ⁇ g protein/sample) were separated on 12% SDS-PAGE, and blotted onto PVDF membranes. The membranes were then hybridized with rabbit anti-PA28 ⁇ antiserum (Ahn et al. 1996, J. Biol. Chem. 271:18237) followed by 125 l-protein A. Detailed methods were described previously (Chen et al., 1996, J. Immunol. 157:4297).
  • PBMC peripheral blood mononuclear cells
  • PHA PHA 2 ⁇ g/ml
  • RAPA RAPA (10nM)
  • the cells were harvested and sonicated in 300 ⁇ l PBS on ice for 40 sec.
  • Twenty micrograms of protein per sample from the cleared lysates were supplemented to 100 ⁇ l with 0.1 M Tris buffer (pH 8.2).
  • a proteasome-specific inhibitor lactacystin (Omura et al., 1991 , J. Antibiot. (Tokyo) 44:113; Fentenay et al., 1995, Science 268:726) was added at a final concentration of 10nM in some samples as indicated.
  • the samples were incubated on ice for 15 min, and fluorogenic chymotrypsin substrate sLLVY-MCA was then added at a final concentration of 10nM.
  • the 20S proteasome which was prepared as previously described (Friguet et al, 1994, J. Biol. Chem. 269:21639), was used as a positive control in place of cell lysates.
  • the samples were incubated at 37°C for 15 min and the reaction was terminated by adding 4 ⁇ l 2.5M HCI.
  • the samples were then diluted to 2ml with 0.1M Tris pH8.2, and measured for their fluorescent intensity by a PTI fluorometer (Photo Technology International, South Brunswick, NJ).
  • the excitation wavelength was 380 nm, and the emission wavelength 440 nm.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Transplantation (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des compositions comprenant des inhibiteurs de protéasomes tels que la lactocystine et ses analogues. Ces compositions sont utilisées dans les cas suivants: (1) pour arrêter la fonction mytochondriale (utile contre le cancer, l'inflammation, les réactions immunes indésirables et l'hyperthyroïdisme), (2) pour interrompre la synthèse de l'oxyde nitrique (utile contre l'inflammation et le choc septique), et (3) pour inverser les réactions immunes indésirables en cours, telles que les maladies autoimmunes et le rejet du greffon. Dans le dernier cas, les compositions sont administrées une fois que les lymphocytes T des patients sont pour la plupart activés. On peut également combiner les inhibiteurs de protéasomes à des médicaments immunodépresseurs tels que la rapamycine, la cyclosporine A et FK506. Enfin, un procédé de criblage d'un composé présentant une activité d'inhibition des protéasomes est également décrit et revendiqué.
PCT/CA1998/001010 1997-10-31 1998-10-29 Utilisation d'inhibiteurs de proteasomes dans le traitement du cancer, de l'inflammation, de maladies autoimmunes, du rejet du greffon et du choc septique WO1999022729A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP52505499A JP2001508465A (ja) 1997-10-31 1998-10-29 ガン、炎症、自己免疫疾患、移植片拒絶反応および敗血症性ショックを処置するためのプロテアソーム阻害因子の使用
AU97318/98A AU770798B2 (en) 1997-10-31 1998-10-29 The use of proteasome inhibitors for treating cancer, inflammation, autoimmune disease, graft rejection and septic shock
EP98951135A EP0967976A1 (fr) 1997-10-31 1998-10-29 Utilisation d'inhibiteurs de proteasomes dans le traitement du cancer, de l'inflammation, de maladies autoimmunes, du rejet du greffon et du choc septique
CA002276013A CA2276013A1 (fr) 1997-10-31 1998-10-29 Utilisation d'inhibiteurs de pro-teasomes dans le traitement du cancer, de l'inflammation, de ma-ladies autoimmunes, du rejet du greffon et du choc septique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA002219867A CA2219867A1 (fr) 1997-10-31 1997-10-31 Utilisation d'inhibiteurs du proteasome dans le traitement du cancer, de l'inflammation, des maladies auto-immunes, du rejet de greffe et du choc septique
CA2,219,867 1997-10-31

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US34100999A Continuation-In-Part 1999-08-25 1999-08-25

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US09/904,251 A-371-Of-International US20020049157A1 (en) 1999-08-25 2001-07-12 Use of proteasome inhibitors for treating cancer, inflammation, autoimmune disease, graft rejection and septic shock

Publications (1)

Publication Number Publication Date
WO1999022729A1 true WO1999022729A1 (fr) 1999-05-14

Family

ID=4161712

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1998/001010 WO1999022729A1 (fr) 1997-10-31 1998-10-29 Utilisation d'inhibiteurs de proteasomes dans le traitement du cancer, de l'inflammation, de maladies autoimmunes, du rejet du greffon et du choc septique

Country Status (5)

Country Link
EP (1) EP0967976A1 (fr)
JP (1) JP2001508465A (fr)
AU (1) AU770798B2 (fr)
CA (1) CA2219867A1 (fr)
WO (1) WO1999022729A1 (fr)

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023614A1 (fr) * 1998-10-20 2000-04-27 Millennium Pharmaceuticals, Inc. Procede de controle de l'action medicamenteuse de l'inhibiteur de proteasome
WO2001067100A1 (fr) * 2000-03-03 2001-09-13 University Of South Florida Participation de la decomposition de bax a la survie et la progression des tumeurs
WO2002080907A1 (fr) * 2001-04-03 2002-10-17 Millennium Pharmaceuticals, Inc. Combinaison d'analogue de lactacystine et de medicament immunosuppresseur permettant la prolongation de la survie d'une allogreffe
WO2003084551A1 (fr) 2002-04-05 2003-10-16 Viromics Gmbh Agent de traitement d'infections par flaviviridae
US6740674B2 (en) 2001-05-21 2004-05-25 Alcon, Inc. Use of proteasome inhibitors to treat dry eye disorders
EP1565193A2 (fr) * 2002-11-06 2005-08-24 Dana-Farber Cancer Institute, Inc. Procedes et compositions de traitement du cancer au moyen d'inhibiteurs de la proteasome
US7112588B2 (en) 2001-05-21 2006-09-26 Alcon, Inc. Use of proteasome inhibitors to treat dry eye disorders
EP1720559A1 (fr) * 2004-02-06 2006-11-15 The University Of Chicago Facteur anti-inflammatoire, cytoprotecteur pouvant etre derive d'un organisme probiotique
WO2007138116A3 (fr) * 2006-06-01 2008-05-08 Virologik Gmbh Composition pharmaceutique pour traiter des infections virales et/ou des maladies tumorales par inhibition du repliement et de la décomposition des protéines
EP2088205A1 (fr) 2008-02-11 2009-08-12 Institut National De La Sante Et De La Recherche Medicale (Inserm) PSMB10 : marqueur de diagnostic et cible thérapeutique de rejet chronique
EP2152252A4 (fr) * 2006-11-13 2010-06-02 Univ Columbia Inhibiteurs de protéasome sélectifs pour traiter le diabète
US7759068B2 (en) 2002-04-15 2010-07-20 Proteosys Ag Use of substances for treating tumors
EP2301565A1 (fr) 2000-10-12 2011-03-30 ViroLogik GmbH Inhibiteurs du proteasome pour le traitement des infections virales
JP2011195590A (ja) * 1999-10-20 2011-10-06 Osteoscreen Inc 骨および毛成長を刺激するためのプロテアソーム活性のインヒビター
DE102010039631A1 (de) * 2010-08-22 2012-02-23 Ulrich Schubert Pharmazeutische Zusammensetzungen mit immunmodulatorischen Eigenschaften
US8168803B2 (en) 2003-06-20 2012-05-01 Nereus Pharmaceuticals, Inc. Methods of using [3.2.0] heterocyclic compounds and analogs thereof
US8217072B2 (en) * 2003-06-20 2012-07-10 The Regents Of The University Of California Salinosporamides and methods for use thereof
US8222289B2 (en) 2002-06-24 2012-07-17 The Regents Of The University Of California Salinosporamides and methods for use thereof
US8282967B2 (en) 2005-05-27 2012-10-09 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US8389564B2 (en) 2008-05-12 2013-03-05 Venkat Rami Reddy Macherla Proteasome inhibitors
WO2012164398A3 (fr) * 2011-06-01 2013-07-11 Netherlands Cancer Institute Modulation du système ubiquitine-protéasome (ups)
US8591876B2 (en) 2010-12-15 2013-11-26 Novan, Inc. Methods of decreasing sebum production in the skin
US8981139B2 (en) 2011-02-28 2015-03-17 The University Of North Carolina At Chapel Hill Tertiary S-nitrosothiol-modified nitric—oxide-releasing xerogels and methods of using the same
US9526738B2 (en) 2009-08-21 2016-12-27 Novan, Inc. Topical gels and methods of using the same
US9919072B2 (en) 2009-08-21 2018-03-20 Novan, Inc. Wound dressings, methods of using the same and methods of forming the same
AU2018247326B2 (en) * 2008-11-13 2020-04-09 Gilead Calistoga Llc Therapies for hematologic malignancies
WO2024193529A1 (fr) * 2023-03-20 2024-09-26 杭州天玑济世生物科技有限公司 Utilisation d'un composé à petites molécules ayant une structure de naphtylamine

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010004750A (ja) * 2008-06-24 2010-01-14 Institute For Rheumatic Diseases Co Ltd 自己免疫疾患による組織傷害の発症または発症可能性の診断方法、およびその利用
KR101757373B1 (ko) * 2015-07-24 2017-07-26 울산과학기술원 프로테아좀 억제제 스크리닝용 조성물 및 이를 이용한 프로테아좀 억제제 스크리닝 방법
KR102545392B1 (ko) 2016-03-28 2023-06-20 프레시지 바이오싸이언시스, 인크. 암 치료를 위한 제약학적 조합물

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017816A1 (fr) * 1993-02-10 1994-08-18 The President And Fellows Of Harvard College Role de la proteolyse dependant de l'ubiquitine atp dans la presentation de l'antigene restreint chm-1 et inhibiteurs utilises a cet effet
WO1995024914A1 (fr) * 1994-03-15 1995-09-21 Myogenics, Inc. Inhibiteurs du complexe proteolytique 26s et du proteasome 20s contenu dans ce dernier
WO1996013266A1 (fr) * 1994-10-28 1996-05-09 Proscript, Inc. Composes d'esters et d'acides boroniques, leur synthese et leurs utilisations
WO1996032105A1 (fr) * 1995-04-12 1996-10-17 President And Fellows Of Harvard College Analogues de lactacystine
WO1998013061A1 (fr) * 1996-09-24 1998-04-02 Cv Therapeutics, Inc. Inhibition de proteasome 26s et 20s au moyen d'indanones

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017816A1 (fr) * 1993-02-10 1994-08-18 The President And Fellows Of Harvard College Role de la proteolyse dependant de l'ubiquitine atp dans la presentation de l'antigene restreint chm-1 et inhibiteurs utilises a cet effet
WO1995024914A1 (fr) * 1994-03-15 1995-09-21 Myogenics, Inc. Inhibiteurs du complexe proteolytique 26s et du proteasome 20s contenu dans ce dernier
WO1996013266A1 (fr) * 1994-10-28 1996-05-09 Proscript, Inc. Composes d'esters et d'acides boroniques, leur synthese et leurs utilisations
WO1996032105A1 (fr) * 1995-04-12 1996-10-17 President And Fellows Of Harvard College Analogues de lactacystine
WO1998013061A1 (fr) * 1996-09-24 1998-04-02 Cv Therapeutics, Inc. Inhibition de proteasome 26s et 20s au moyen d'indanones

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
CONNER E M ET AL: "Proteasome inhibition attenuates nitric oxide synthase expression, VCAM-1 transcription and the development of chronic colitis.", JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, (1997 SEP) 282 (3) 1615-22. JOURNAL CODE: JP3. ISSN: 0022-3565., United States, XP002093378 *
CUI H ET AL: "Proteasome regulation of activation-induced T cell death.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (1997 JUL 8) 94 (14) 7515-20. JOURNAL CODE: PV3. ISSN: 0027-8424., United States, XP002093376 *
GRISCAVAGE J M ET AL: "Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (1996 APR 16) 93 (8) 3308-12. JOURNAL CODE: PV3. ISSN: 0027-8424., United States, XP002093377 *
HIRSCH T ET AL: "Proteasome activation occurs at an early, premitochondrial step of thymocyte apoptosis.", JOURNAL OF IMMUNOLOGY, (1998 JUL 1) 161 (1) 35-40. JOURNAL CODE: IFB. ISSN: 0022-1767., United States, XP002093373 *
IMAJOH-OHMI S ET AL: "Lactacystin, a specific inhibitor of the proteasome, induces apoptosis in human monoblast U937 cells.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, (1995 DEC 26) 217 (3 1070-7. JOURNAL CODE: 9Y8. ISSN: 0006-291X., United States, XP002093380 *
J.E.F. REYNOLDS: "MARTINDALE, THE EXTRA PHARMACOPOEIA", 1996, ROYAL PHARMACEUTICAL SOCIETY, LONDON, XP002093381 *
MANNING A.M. ET AL: "Transcription inhibitors in inflammation.", EXPERT OPINION ON INVESTIGATIONAL DRUGS, (1997) 6/5 (555-567). REFS: 86 ISSN: 1354-3784 CODEN: EOIDER, United Kingdom, XP002093379 *
MEYER S ET AL: "Cyclosporine A is an uncompetitive inhibitor of proteasome activity and prevents NF-kappaB activation.", FEBS LETTERS, (1997 AUG 18) 413 (2) 354-8. JOURNAL CODE: EUH. ISSN: 0014-5793., Netherlands, XP002093375 *
WANG X ET AL: "Role of proteasomes in T cell activation and proliferation.", JOURNAL OF IMMUNOLOGY, (1998 JAN 15) 160 (2) 788-801. JOURNAL CODE: IFB. ISSN: 0022-1767., United States, XP002093372 *
X. WANG ET AL.: "RAPAMYCIN INHIBITS PROTEASOME ACTIVATOR EXPRESSION AND PROTEASOME ACTIVITY", EUR. J. IMMUNOL., vol. 27, no. 11, November 1997 (1997-11-01), pages 2781 - 2786, XP002093374 *

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023614A1 (fr) * 1998-10-20 2000-04-27 Millennium Pharmaceuticals, Inc. Procede de controle de l'action medicamenteuse de l'inhibiteur de proteasome
US6613541B1 (en) 1998-10-20 2003-09-02 Millennium Pharmaceuticals, Inc. Method for monitoring proteasome inhibitor drug action
JP2011195590A (ja) * 1999-10-20 2011-10-06 Osteoscreen Inc 骨および毛成長を刺激するためのプロテアソーム活性のインヒビター
WO2001067100A1 (fr) * 2000-03-03 2001-09-13 University Of South Florida Participation de la decomposition de bax a la survie et la progression des tumeurs
EP2305291A1 (fr) 2000-10-12 2011-04-06 ViroLogik GmbH Inhibiteurs du proteasome pour le traitement des infections HIV
EP2301565A1 (fr) 2000-10-12 2011-03-30 ViroLogik GmbH Inhibiteurs du proteasome pour le traitement des infections virales
WO2002080907A1 (fr) * 2001-04-03 2002-10-17 Millennium Pharmaceuticals, Inc. Combinaison d'analogue de lactacystine et de medicament immunosuppresseur permettant la prolongation de la survie d'une allogreffe
US6740674B2 (en) 2001-05-21 2004-05-25 Alcon, Inc. Use of proteasome inhibitors to treat dry eye disorders
US7112588B2 (en) 2001-05-21 2006-09-26 Alcon, Inc. Use of proteasome inhibitors to treat dry eye disorders
WO2003084551A1 (fr) 2002-04-05 2003-10-16 Viromics Gmbh Agent de traitement d'infections par flaviviridae
US7759068B2 (en) 2002-04-15 2010-07-20 Proteosys Ag Use of substances for treating tumors
US9713607B2 (en) 2002-06-24 2017-07-25 The Regents Of The University Of California Salinosporamides and methods of use thereof
US9078881B2 (en) 2002-06-24 2015-07-14 The Regents Of The University Of California Salinosporamides and methods of use thereof
US10912764B2 (en) 2002-06-24 2021-02-09 The Regents Of The University Of California Salinosporamides and methods of use thereof
US8637565B2 (en) 2002-06-24 2014-01-28 The Regents Of The University Of California Salinosporamides and methods for use thereof
US10314818B2 (en) 2002-06-24 2019-06-11 The Regents Of The University Of California Salinosporamides and methods of use thereof
US8222289B2 (en) 2002-06-24 2012-07-17 The Regents Of The University Of California Salinosporamides and methods for use thereof
EP1565193A4 (fr) * 2002-11-06 2008-05-21 Dana Farber Cancer Inst Inc Procedes et compositions de traitement du cancer au moyen d'inhibiteurs de la proteasome
EP1565193A2 (fr) * 2002-11-06 2005-08-24 Dana-Farber Cancer Institute, Inc. Procedes et compositions de traitement du cancer au moyen d'inhibiteurs de la proteasome
US8026230B2 (en) 2002-11-06 2011-09-27 Dana-Farber Cancer Institute, Inc. Methods and compositions for treating cancer using proteasome inhibitors
US8685954B2 (en) 2002-11-06 2014-04-01 Dana-Farber Cancer Institute, Inc. Methods and compositions for treating cancer using proteasome inhibitors
US8168803B2 (en) 2003-06-20 2012-05-01 Nereus Pharmaceuticals, Inc. Methods of using [3.2.0] heterocyclic compounds and analogs thereof
US8217072B2 (en) * 2003-06-20 2012-07-10 The Regents Of The University Of California Salinosporamides and methods for use thereof
EP1720559A4 (fr) * 2004-02-06 2009-07-22 Univ Chicago Facteur anti-inflammatoire, cytoprotecteur pouvant etre derive d'un organisme probiotique
EP1720559A1 (fr) * 2004-02-06 2006-11-15 The University Of Chicago Facteur anti-inflammatoire, cytoprotecteur pouvant etre derive d'un organisme probiotique
US9403851B2 (en) 2005-05-27 2016-08-02 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US8282967B2 (en) 2005-05-27 2012-10-09 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US11691995B2 (en) 2005-05-27 2023-07-04 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US8956658B2 (en) 2005-05-27 2015-02-17 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US8962029B2 (en) 2005-05-27 2015-02-24 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US9403852B2 (en) 2005-05-27 2016-08-02 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
WO2007138116A3 (fr) * 2006-06-01 2008-05-08 Virologik Gmbh Composition pharmaceutique pour traiter des infections virales et/ou des maladies tumorales par inhibition du repliement et de la décomposition des protéines
EP2152252A4 (fr) * 2006-11-13 2010-06-02 Univ Columbia Inhibiteurs de protéasome sélectifs pour traiter le diabète
EP2088205A1 (fr) 2008-02-11 2009-08-12 Institut National De La Sante Et De La Recherche Medicale (Inserm) PSMB10 : marqueur de diagnostic et cible thérapeutique de rejet chronique
US8389564B2 (en) 2008-05-12 2013-03-05 Venkat Rami Reddy Macherla Proteasome inhibitors
AU2018247326B2 (en) * 2008-11-13 2020-04-09 Gilead Calistoga Llc Therapies for hematologic malignancies
US10376538B2 (en) 2009-08-21 2019-08-13 Novan, Inc. Topical gels and methods of using the same
US9737561B2 (en) 2009-08-21 2017-08-22 Novan, Inc. Topical gels and methods of using the same
US9919072B2 (en) 2009-08-21 2018-03-20 Novan, Inc. Wound dressings, methods of using the same and methods of forming the same
US11583608B2 (en) 2009-08-21 2023-02-21 Novan, Inc. Wound dressings, methods of using the same and methods of forming the same
US9526738B2 (en) 2009-08-21 2016-12-27 Novan, Inc. Topical gels and methods of using the same
DE102010039631A1 (de) * 2010-08-22 2012-02-23 Ulrich Schubert Pharmazeutische Zusammensetzungen mit immunmodulatorischen Eigenschaften
US8591876B2 (en) 2010-12-15 2013-11-26 Novan, Inc. Methods of decreasing sebum production in the skin
US9713652B2 (en) 2011-02-28 2017-07-25 The University Of North Carolina At Chapel Hill Nitric oxide-releasing S-nitrosothiol-modified silica particles and methods of making the same
US8981139B2 (en) 2011-02-28 2015-03-17 The University Of North Carolina At Chapel Hill Tertiary S-nitrosothiol-modified nitric—oxide-releasing xerogels and methods of using the same
WO2012164398A3 (fr) * 2011-06-01 2013-07-11 Netherlands Cancer Institute Modulation du système ubiquitine-protéasome (ups)
WO2024193529A1 (fr) * 2023-03-20 2024-09-26 杭州天玑济世生物科技有限公司 Utilisation d'un composé à petites molécules ayant une structure de naphtylamine

Also Published As

Publication number Publication date
EP0967976A1 (fr) 2000-01-05
AU9731898A (en) 1999-05-24
CA2219867A1 (fr) 1999-04-30
JP2001508465A (ja) 2001-06-26
AU770798B2 (en) 2004-03-04

Similar Documents

Publication Publication Date Title
AU770798B2 (en) The use of proteasome inhibitors for treating cancer, inflammation, autoimmune disease, graft rejection and septic shock
US20020049157A1 (en) Use of proteasome inhibitors for treating cancer, inflammation, autoimmune disease, graft rejection and septic shock
Stangl et al. Long‐term up‐regulation of eNOS and improvement of endothelial function by inhibition of the ubiquitin–proteasome pathway
Chandra et al. Proteasome inhibitors induce apoptosis in glucocorticoid-resistant chronic lymphocytic leukemic lymphocytes
Mandic et al. Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis
Marzo et al. Caspases disrupt mitochondrial membrane barrier function
Wang et al. Role of proteasomes in T cell activation and proliferation
Luo et al. A PROTEASOME INHIBITOR EFFECTIVELY PREVENTS MOUSE HEART ALLOGRAFT REJECTION1
Barnes New therapies for chronic obstructive pulmonary disease
Moon et al. PDE4 inhibitors activate a mitochondrial apoptotic pathway in chronic lymphocytic leukemia cells that is regulated by protein phosphatase 2A
Klisovic et al. Depsipeptide (FR901228) inhibits proliferation and induces apoptosis in primary and metastatic human uveal melanoma cell lines
Park et al. Monensin-mediated growth inhibition in NCI-H929 myeloma cells via cell cycle arrest and apoptosis
Schliep et al. Functional evaluation of the role of inhibitor of apoptosis proteins in chronic lymphocytic leukemia
Billard et al. Flavopiridol downregulates the expression of both the inducible NO synthase and p27kip1 in malignant cells from B-cell chronic lymphocytic leukemia
Petit et al. Role of CD95‐activated caspase‐1 processing of IL‐1β in TCR‐mediated proliferation of HIV‐infected CD4+ T cells
Wall et al. Bryostatin 1 induces ubiquitination and proteasome degradation of Bcl-2 in the human acute lymphoblastic leukemia cell line, Reh.
US7786170B2 (en) Histone deacetylase inhibitor enhancement of trail-induced apoptosis
US7364719B2 (en) Vasoregulating compounds and methods of their use
CA2276013A1 (fr) Utilisation d'inhibiteurs de pro-teasomes dans le traitement du cancer, de l'inflammation, de ma-ladies autoimmunes, du rejet du greffon et du choc septique
CA2372316A1 (fr) Utilisation d'inhibiteurs de proteasome pour le traitement du cancer, des inflammations, des maladies autoimmunes, des rejets de greffes, et du choc septique
Shav-Tal et al. Enhanced proteolysis of pre-mRNA splicing factors in myeloid cells
Leach et al. Influence of chloroquine on diet-induced pancreatitis
US9155774B2 (en) Scaffold-kinase interaction blockades and uses thereof in treating cancer
US7232570B2 (en) ERAAP modulators regulate immune responses
US5780513A (en) Method of inhibiting the release of bioactive IL-1

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 97318/98

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2276013

Country of ref document: CA

Kind code of ref document: A

Ref document number: 2276013

WWE Wipo information: entry into national phase

Ref document number: 1998951135

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 1999 525054

Country of ref document: JP

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 09341009

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1998951135

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWR Wipo information: refused in national office

Ref document number: 1998951135

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 97318/98

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1998951135

Country of ref document: EP