WO2003038032A2 - Produit inhibiteur de virus lents - Google Patents

Produit inhibiteur de virus lents Download PDF

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Publication number
WO2003038032A2
WO2003038032A2 PCT/DE2002/004052 DE0204052W WO03038032A2 WO 2003038032 A2 WO2003038032 A2 WO 2003038032A2 DE 0204052 W DE0204052 W DE 0204052W WO 03038032 A2 WO03038032 A2 WO 03038032A2
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vpr
hiv
cypa
inhibitors
cyclophilin
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PCT/DE2002/004052
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German (de)
English (en)
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WO2003038032A3 (fr
WO2003038032A8 (fr
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Uwe Tessmer
Karsten Bruns
Ulrich Schubert
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Viromics Gmbh
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Priority to AU2002347102A priority Critical patent/AU2002347102A1/en
Priority to DE20220744U priority patent/DE20220744U1/de
Priority to EP02782752A priority patent/EP1439853A2/fr
Publication of WO2003038032A2 publication Critical patent/WO2003038032A2/fr
Publication of WO2003038032A8 publication Critical patent/WO2003038032A8/fr
Publication of WO2003038032A3 publication Critical patent/WO2003038032A3/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
    • A61K38/12—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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the invention relates to agents for inhibiting lentiviruses and in particular to the use of PPIase inhibitors as agents for inhibiting the lentivirus protein R of primates (virus protein R - Vpr), especially the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2 ) and the monkey (Simian) immunodeficiency virus (SIV).
  • PPIase inhibitors as agents for inhibiting the lentivirus protein R of primates (virus protein R - Vpr), especially the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2 ) and the monkey (Simian) immunodeficiency virus (SIV).
  • Vpr interacts with the enzyme cyclophilin A (CyPA) and that this interaction can be inhibited by the agent cyclosporin (CsA).
  • CsA cyclophilin A
  • This Vpr-CyPA interaction is essential for the protein stability, folding and function of Vpr.
  • CsA as well as other PPIase inhibitors, the function of Vpr can be blocked due to ⁇ 5 of this newly recognized connection.
  • the invention describes the new finding that after treatment of an HIV-infected cell with the inhibitor CsA, the Vpr becomes unstable and therefore can no longer function in the replication cycle of HIV. Due to the fact that Vpr is an important pathogenicity factor in the clinical picture of an HIV infection, CsA and other PPIase-
  • inhibitors for the treatment of AIDS can be used as a novel anti-retroviral therapy.
  • PPIases 25 peptidylprolyl isomerases are a group of cytosolic enzymes that were originally characterized by their ability to catalyze the cis-trans isomerization of c / s-peptidylprolyl bonds within a protein. It was later discovered that these PPIases are the direct target of certain immunosuppressive drugs, including cyclosporin A (CsA), FK506 and rapamycin. Newer me-
  • CsA immunosuppressive are the substances "SDZ NIM811" and “SanglipherinA” developed by Novartis. These PPIases are also called immunophilins. Immunophilins are widespread and highly expressed in higher eukaryotes. It it can therefore be assumed that PPIases play an important role in cell physiology. This role is not yet understood, essential and according to the nature of PPIases, these enzymes have an important function for the correct folding and expression of proteins, including multiprotein complexes such as Ca (2+) ion channels, steroid-receptor complexes and receptors for the thyrosine kinase.
  • multiprotein complexes such as Ca (2+) ion channels, steroid-receptor complexes and receptors for the thyrosine kinase.
  • the PPIase inhibitors CsA and FK506 are natural products from microorganisms and potent inhibitors because they form a specific complex with PPIases.
  • Serine / threonine protein phosphatase calcineurin (CN) is a well-characterized target for the effects of CsA.
  • the HIV replication cycle begins when the virus binds to various cell receptors, among which the CD4 glycoprotein acts as the primary receptor. Virus entry occurs through pH-independent membrane fusion, followed by the "uncoating" of the virus particles in the cytosol.
  • the viral RNA genome is transcribed by means of enzymatic activities of reverse transcriptase (RT), RNase H and polymerase into double-stranded DNA, which is then transported in association with the pre-integration complex into the cell nucleus and is inserted as a provirus genome in chromosomal DNA using viral integrase.
  • RT reverse transcriptase
  • RNase H reverse transcriptase
  • polymerase reverse transcriptase
  • Gag / Gag-Pol polyproteins and envelope proteins are transported to the cell membrane, where virions are assembled.
  • the virus particles mature through proteolytic processing of the Gag / Gag-Pol polyproteins (for review see Martin, 1993).
  • HIV-1 encodes other genes: tat and rev are essential for virus replication; Tat is the main transactivator of the virus promoter and Rev performs post-transcriptional functions by regulating the transport of unspliced gaglpol and simply spliced vif, vpr and vpu / env mRNAs from the cell nucleus into the cytoplasm (for review see Trono, 1995).
  • nef vif vpr and vpu are not absolutely necessary for virus replication in cell cultures and are therefore usually referred to as "accessory" genes.
  • the gene vpr was first described in 1987 as a small open reading frame in the genome of the human immunodeficiency virus type 1 (HIV-1) and was designated with the letter "R".
  • the vpr gene is conserved among all known isolates of HIV-1 and HIV-2 as well as the monkey immunodeficiency virus (SIV) and is therefore also called the lentivirus protein Vpr.
  • Vpr has various biological activities such as: oligomerization, localization in and transport of the viral pre-integration complex to the cell nucleus, transcriptional activation of HIV-1 and heterologous virus promoters, induction of cell differentiation and cell cycle arrest, binding and interaction with various cellular proteins, especially the glucocorticoid receptor (GR) and enhancement of the effect of steroid hormones (for review see Emerman, 1996).
  • oligomerization localization in and transport of the viral pre-integration complex to the cell nucleus
  • transcriptional activation of HIV-1 and heterologous virus promoters induction of cell differentiation and cell cycle arrest
  • binding and interaction with various cellular proteins especially the glucocorticoid receptor (GR) and enhancement of the effect of steroid hormones (for review see Emerman, 1996).
  • GR glucocorticoid receptor
  • Vpr is necessary for the development of AIDS-like symptoms in SlV-infected rhesus monkeys, or that point mutations in the vpr gene revert to functional Vpr in vivo as the infection spreads (Lang et al., 1993 ). Furthermore, the double mutations in vpx and vpr of SIV- mac 239 prevent AIDS progression (Gibbs et al, 1995). Vpr is the only accessory protein that is incorporated into virus particles in significant amounts and through a specific mechanism.
  • Vpr occurs early in the HIV replication cycle, immediately after membrane fusion and "uncoating" of the invading virus particle.
  • anti-Vpr drugs showed that vpr-specific antisense constructs can suppress HIV-1 virus replication in macrophages (Balotta et al, 1993). Vpr supports virus replication in differentiated, no longer dividing primary human monocytes / macrophages (Heinzinger et al, 1994). This fact is justified by the fact that Vpr is no longer part of the import of the viral pre-integration complex into the cell nucleus. lent cells is necessary and HIV can infect differentiated cells regardless of cell mitosis.
  • Vpr induces cell differentiation and cell cycle arrest and causes transport and various functions in the cell nucleus. Since Vpr, as part of the pre-integration complex, influences elementary processes of the cell in the interest of virus replication, it is understandable that Vpr enters into specific connections with cellular factors. For example, an interaction of Vpr with an approximately 200 kDa protein and with a 41 kDa cellular protein as a complex with the human glucocorticoid receptor type II (GR-II) was observed (Refaeli et al., 1995).
  • GR-II human glucocorticoid receptor type II
  • Vpr also suppresses T cell receptor-mediated apoptosis and T cell activation (activation of lymphokine expression IL-2, IL-10, IL-12, TNF-alpha and IL-4) by influencing NF- K B activity (Ayyavoo et al ., 1997). Vpr is also said to enter into a specific interaction with the second UBA (ubiquitin associated) domain of the human DNA repair protein HHR23A, which forms a three-helix bundle structure (Diekmann et al., 1998).
  • UBA ubiquitin associated
  • Vpr also interacts directly with the glucocorticoid receptor and various transcription factors and can thus, as a co-activator, increase the effect of glucocorticoids in different cell lines (Kino et al., 1999).
  • the effect of Vpr as a glucocorticoid coactivator is believed to contribute to increased glucocorticoid sensitivity and thereby to the pathogenesis of AIDS.
  • CyPA and Cyclophilin B interact with HIV-1 Gag proteins and are therefore incorporated in significant amounts into HIV-1 virions during virus assembly (Luban et al., 1999; Franke et al., 1994) , CyPA binds to a proline-rich sequence in HIV-1 gag, but a cis / trans isoproline phenomenon is not known for gag. It is therefore assumed that CyPA only binds to Gag, but does not have any enzymatic function for folding Gag.
  • the incorporation of CyPA in HIV-1 virions is regulated by an exposed proline-rich loop sequence in the N-terminal region of the HIV-1 capsid (CA), p24 CA , within the Pr55 Gag polyprotein.
  • CyPA - / - cell line is also used in the present description of the invention, but only to test the effect of CyPA on the expression and function of Vpr.
  • the HIV-1 Vpr is conserved in all lentiviruses, including the immunodeficiency-inducing viruses HIV-1 and HIV-2, the causative agents of AIDS in humans, and SIV, the causative agents of monkey AIDS and in lower and higher primates. Vpr is therefore also referred to as the lentivirus protein R. Vpr plays a crucial role in the pathogenesis of AIDS. Blocking Vpr therefore has decisive potential for novel anti-viral strategies.
  • Vpr For the biological function of Vpr, it is necessary that this protein is expressed in sufficient quantities in the infected cell in order to also be incorporated into the maturing virus particle in the process of assembly, budding, and the release of progeny viruses . It is essential that Vpr is the only regulatory HIV protein that is incorporated into progeny viruses in significant quantities. As a result, Vpr is part of the invading virus in the process of infection and can therefore influence important processes in the early phase of HIV infection before the new synthesis of viral proteins is initiated. Inhibiting the incorporation of Vpr in progeny viruses is therefore a relevant approach for an anti-viral strategy.
  • the CyPA-Gag interaction is limited to HIV-1 only.
  • the HIV-2 and SIV gag proteins do not contain CypA binding motifs, while the CyPA binding motif is contained in all of the HIV-1, HIV-2 and SIV Vpr proteins.
  • the invention has for its object to provide means for inhibiting the virus protein R (Vpr).
  • Vpr virus protein R
  • the problem was solved by using inhibitors of peptidylprolyl isomerases (PPIases) - the peptidylprolyl isomerase inhibitors (PPIase inhibitors).
  • PPIases peptidylprolyl isomerases
  • PPIase inhibitors peptidylprolyl isomerase inhibitors
  • all compounds are suitable which block the enzymatic function of PPIases.
  • the inhibitors according to the invention are administered in pharmaceutical preparations. These active substances are compounds which contain inhibitors of the cellular chaperones of the class of the PPIases or immunophilins. In particular, these inhibitors are said to inhibit the PPIases CyPA and CyPB.
  • These agents, the PPIase inhibitors include the drugs CyclosporinA (CsA), FK506, Rapamycin, SDZ NIM811, and Sanglipherin
  • CyPA an 18 kDa protein in the cytosol, human genome name PPIA;
  • CyPB an 18-20 kDa protein in the endoplasmatic see reticulum, human genome name PPIB;
  • CyPC an 18 kDa protein in the secretory pathway, human genome name PPIC;
  • CyPF or Cyp3 an 18-22 kDa protein in the inner membrane of the mitochondrion, human genome name PP1F;
  • CyPM or PPIL1 an 18 kDa protein in the cytosol, human genome name PPIL1; USA-CyP or Cyp20: a 20 kDa protein in the cell nucleus, human genome name USA-CYP;
  • CyPE or Cyp33A a 40 kDa protein in the cell nucleus, human genome name PPIE;
  • Cyp40 a 40 kDa protein in the cytosol and cell nucleus, human genome name PP1D;
  • Cyp60 a 60 kDa protein in the cell nucleus, human genome name PPIL2;
  • Hal539-CyP a 60kDa protein, human genome name CYP; - NK-TRCyP: an 89 kDa protein in the cell nucleus, human genome name NKTR;
  • NUP-358 a 358 kDa protein in the cell nucleus, human genome name RAN-BP1 (Braaten and Luban, 2001).
  • An essential characteristic of these inhibitors is that they block the enzymatic function of PPIases. As a result, the cis / trans isomerization of prolyl-peptide bonds in proteins can no longer take place. As a major consequence of the use of these agents, the high proportion of prolyl residues in the cis conformation within the N-terminus of Vpr is preserved. However, this cis conformation is disadvantageous for the expression and the stability function of Vpr.
  • Vpr no functional Vpr molecules can be expressed in an HIV-infected cell.
  • Vpr is inactivated in the course of HIV infection. Since Vpr is an important pathogenicity factor in the course of an HIV infection, these agents can be used for the treatment of AIDS and for the treatment and prevention of infection with the lentiviruses HIV-1, HIV-2 and SIV.
  • Vpr interacts with certain cellular factors. In the HIV host cell, these factors are chaperones, enzymes which regulate the folding of proteins and are generally assigned to the class of cis / trans prolylpeptidyl isomerases (PPIases).
  • the class of PPIases also includes enzymes with the names immunophilins or cyclophilins.
  • the enzymes CyclophilinA and B (CyPA and CyPB) belong to the immunophilins.
  • Essential to the invention is our finding that conserved prolines occur in the N-terminus of Vpr in an unusually high proportion in the cis conformation.
  • a recognition sequence for PPIases is contained in the N-terminus of Vpr.
  • the PPIase CyPA binds to Vpr. This binding is released by the PPIase inhibitor CsA. After inactivation of CyPA by CsA or after inactivation of CyPA, Vpr becomes unstable.
  • Vpr from HIV-1 binds to CyPA.
  • Vpr has the ability to interact with PPIases and whether this interaction is important for the protein stability of Vpr.
  • a sequence comparison of over 150 known Vpr sequences from a wide variety of HIV-1 isolates clearly shows that all four proline residues that contribute to the above-mentioned s / tr ⁇ ns phenomenon (proline residues in positions 4, 10, 14 and 35) are contained in all previously known Vpr sequences; A biological function of these proline residues was therefore further investigated according to the invention (FIG. 4).
  • CyPA inhibitors reduce the protein stability and expression of HIV-1 Vpr and thereby block the biological function of Vpr in an HIV-infected cell.
  • the inhibitors of Cypa reduce the expression, the stability and thus the amount of Vpr in an HIV-infected cell and consequently the amount of Vpr that can be incorporated into a maturing progeny virus. drastically reduced to such an extent that after application of CyPA inhibitors, no biological activity of Vpr is detectable. Since a specific interaction between CyPA and Vpr was determined and this influences the folding and the stability of Vpr, it was investigated - derived from the molecular, biochemical and structural findings according to the invention (as stated above) - whether inhibitors of CyPA (these are known and have been used in the treatment of certain diseases as immunosuppressants for years), i.e.
  • Vpr-transmitted increase in virus production in HIV-infected monocytes / macrophages as well - Vpr-mediated co-activation of the glycocorticoid receptor. It was also found
  • CsA prevents the interaction of Vpr with other cellular factors, such as with the glycocorticoid receptor, and
  • CsA prevents the transport of Vpr in the cell, especially to the cell nucleus.
  • CsA reduces the amount of Vpr in an HIV-infected cell (FIG. 9). It was further observed that after the addition of CsA in a dose-dependent mechanism, the amount of Vpr in the cell (FIG. 10) as well as in the released virions is reduced. It is now observed for the first time that the binding of CyPA to Vpr is essential for its stability. It was further recognized that after inactivation of CyPA by the administration of CsA, the Vpr after inhibition of the main protease and after application of proteasome inhibitors, the extremely rapid loss of Vpr cannot be restored.
  • Vpr-containing virions are detected that are released by HIV-1 infected CyPA - / - cells. It is also found that the Vpr-induced G2 cell cycle arrest is undetectable in CyPA - / -.
  • the solution according to the invention thus provides evidence that the function of Vpr is lost without the action of the folding enzyme CyPA and / or other PPIases (either after inhibition by PPIase inhibitors, such as, for example, the CyPA inhibitor CsA, or after genetic inactivation) ,
  • PPIase inhibitors such as CsA
  • CsA PPIase inhibitors
  • CsA and other PPIase inhibitors switch off the function of the essential regulatory protein Vpr by the agents according to the invention and thereby prevent, switch off, block, negatively regulate or influence the HIV replication cycle.
  • CyPA only interacts with p24 A of HIV-1, but not with the gag proteins of other lentiviruses, -
  • the anti-viral effect of CsA on HIV-1 does not include the function of Vpr.
  • a cell already infected with HIV-1 is no longer affected by CsA; the inhibitory effect of CsA on p24 CA is only active in the infection process.
  • CyPA co-translationally regulates the folding, expression and stability of a viral protein. No such mechanism has been demonstrated for any viral or cellular protein.
  • the essence of the invention lies in the use of known agents for a new purpose and in a combination of known elements - the inhibitors of peptidylprolyl isomerases (PPIase inhibitors) - and a new effect - their use in influencing the primates lentiviruses HIV-1, HIV -2 and SIV - which, in their new overall effect, result in an advantage and the desired success, which lies in the fact that means are now available for inhibiting the virus protein R necessary for virus replication.
  • the invention further relates to the use of PPIase inhibitors for the production of agents for the inhibition of primates lentiviruses.
  • HIV-induced dementia HIV-induced disorders in lipid metabolism, especially HLS syndrome (HIV-associated lipodystrophy syndrome).
  • HIV-induced disorders in kidney function especially the HIV AN syndrome (HIV associated nephropathy).
  • the invention relates in summary to the use of PPIase inhibitors as agents for the inhibition of primates lentiviruses, human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and Simian immunodeficiency viruses (SIV).
  • Vpr HIV-1 virus protein R
  • PPIases cellular cis / trans peptidylprolyl isomerases
  • the anti-viral effect of PPIase inhibitors, especially CyclosporinA is based on a specific interaction of PPIases, especially CyclophilinA, with chocolates in the N-terminus of Vpr.
  • Areas of application are anti-retroviral therapy and prevention of infections with lentiviruses that cause immunodeficiency in animals and humans, in particular AIDS or HIV-induced pathological symptoms, also in combination with other anti-retroviral drugs.
  • Example 1 Detection of the Vpr-CyPA interaction using BIAcore spectroscopy
  • Flow cell 1 was used as a reference without CyPA coupling Peptide Vpr 1'40 was injected into a flow buffer (10 mM Hepes, 150 mM NaCl, 50 ⁇ M EDTA, 0.005% Tween 20, pH 7.4) over all flow cells in a concentration range from 1 to 250 ⁇ M, at a flow rate of 5 ⁇ l / min. All data were measured at 2.5 Hz during a 120 sec association and dissociation phase. In general, the CyPA-Vpr 1 "40 interaction reached equilibrium within 30 sec.
  • Vpr 1 "40 was used in a concentration range of 1, 2.5, 5, 10, 25, 50, 100 and 250 ⁇ M, CyPA was bound to the chip matrix with a constant concentration. All data were corrected on the RU data of the control cell (without CyPA). The measurements were repeated at least three times with different CyPA and Vpr preparations and were reproducible at all times.
  • the following mutants were also tested, which contain Pro-Asn amino acid changes in the positions critical for the CyPA interaction: Mutant Vpr 1 "40 , P 5,10 '14 -N (Pro-Asn exchange in positions 5, 10 and 14, Fig.
  • Example 3 The expression of Vpr in HIV-1 infected cells CyPA - / - knock out cells is reduced
  • Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host_cells. Proc.Natl.Acad.Sci.U.S.A. 91: 7311-7315.
  • the HIV-1 virion-associated protein Vpr is a coactivator of the human glucocorticoid receptor.
  • Figure 1 Evidence of structural instability of Vpr N-terminus using CD spectroscopy.
  • FIG. 2 High-resolution structure of Vpr N-terminus by means of 1H NMR at Vpr 1 "40 .
  • Figure 3 Detection of cis / trans isomerism in proline residues of Vpr by means of ⁇ NMR spectroscopy.
  • FIG. 4 A CyPA binding motif is conserved by the Vpr in the N-terminus.
  • the amino acid sequence is shown of Vpr of the isolate HIV-I N L ".
  • the amino acid positions conserved in all previously known HIV-1 isolates are marked in bold. It can be seen that the amino acid positions, Pro-4, Pro-10, Pro-14 and Pro-35, which are found in the context of the invention and which are important for the interaction between Vpr and CypA, have a high degree of conservation.
  • the secondary structural elements in the N-terminus of Vpr are shown.
  • Figure 5 Detection of Vpr-CyPA interaction using Far-Western blot.
  • 6 to 8 binding studies on Vpr and CypA using BlAcore or plasmon resonance spectroscopy.
  • HeLa cells were transfected with the proviral pNL4-3, radioactively labeled with [ 35 S] -methionine for 25 min and then subjected to an 8-hour chase in the absence of radioactivity.
  • Vpr was immunoprecipitated with polyclonal antiserum and Gag proteins with anti-p24 CA antibodies. The relatively small amount of Vpr as well as the percentage stability are shown.
  • FIG. 10 CsA causes a very rapid loss of Vpr.
  • HeLa cells were transfected with the Vpr expression vector CMV-VprFlag, pulse-labeled for 5 min with [ 35 S] -met and subjected to a 60 min chase. After cell lysis, the cell lysates were separated by centrifugation into nuclear and cytosol fractions, and Vpr was immunoprecipitated using anti-flag monoclonal antibodies. As a result, it is found that in the presence of CspA, less Vpr is contained in both the cell nucleus and the cytosol fraction.
  • FIG. 12 The expression on Vpr in CypA knock out cells is reduced.
  • Jurkat cells, human CD4 + T cells, either as wild type or as a genetically modified variant in which both CypA alleles are mutated by genetically knocking out (CypA - / -), were infected with HIV-1NL4-3 and for 7 days cultured.
  • CypA - / - genetically knocking out
  • both cultures were subjected to a pulse chase experiment (5 min pulse, 8 hr chase).
  • Gag and Vpr proteins were immunoprecipitated using anti-Gag and anti-Vpr antibodies, separated in 10-16% SDS-PAGE and visualized by fluorography.
  • Vpr is significantly reduced relative to the amount of p24CA in the knock out culture CypA - / -. This demonstrates according to the invention that CypA is necessary for the expression and stability of Vpr.

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Abstract

L'invention concerne un produit destiné à l'inhibition de virus lents primates et notamment l'utilisation d'inhibiteurs de PPIase comme produit d'inhibition de virus de l'immunodéficience humaine de type 1 et 2 (HIV-1 et HIV-2) et de virus de l'immunodéficience simienne (SIV). A l'appui de la protéine virale R (Vpr), on démontre que les inhibiteurs de cis/trans peptidylprolyl-isomérases (PPIases) cellulaires inhibent l'expression, le pliage, la stabilité protéique tout comme l'efficacité biologique de Vpr. L'action anti-virale d'inhibiteurs de PPIase, plus particulièrement de Cyclosporine A, se fonde sur une interaction spécifique de PPIases, spécialement de cyclophiline A, avec des prolines sur le N-terminal de Vpr. Les domaines d'application sont la thérapie antirétrovirale et la prévention en cas d'infections par des virus lents provoqués par l'immunodéficience chez l'homme et les animaux, notamment de phénomènes pathologiques induits par AIDS ou HIV, également en combinaison avec d'autres médicaments anti-rétroviraux.
PCT/DE2002/004052 2001-10-25 2002-10-25 Produit inhibiteur de virus lents WO2003038032A2 (fr)

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AU2002347102A AU2002347102A1 (en) 2001-10-25 2002-10-25 Means for inhibition of the synthesis of viral proteins
DE20220744U DE20220744U1 (de) 2001-10-25 2002-10-25 Mittel zur Hemmung von Lentiviren
EP02782752A EP1439853A2 (fr) 2001-10-25 2002-10-25 Produit inhibiteur de virus lents

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005063281A2 (fr) * 2003-12-31 2005-07-14 Viromics Gmbh Agents pour inhiber la reproduction de virus par regulation du plissement de proteines

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0484281A2 (fr) * 1990-11-02 1992-05-06 Sandoz Ltd. Cyclosporines
WO1997033604A1 (fr) * 1996-03-12 1997-09-18 Picower Institute For Medical Research Traitement de l'infection a vih par action sur le recepteur de la cyclophiline de cellules hotes
WO2003038056A2 (fr) * 2001-11-02 2003-05-08 The Regents Of The University Of California Methodes permettant d'inhiber la replication d'un lentivirus

Patent Citations (3)

* Cited by examiner, † Cited by third party
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WO2003038032A8 (fr) 2003-08-14

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