WO2003038032A2 - Means for inhibition of the synthesis of viral proteins - Google Patents

Abstract

The invention relates to means for inhibition of primate lentiviruses, in particular the use of PPIase inhibitors as means for inhibiting human immune deficiency viruses type 1 and 2 (HIV-1 and HIV-2) and simian immune deficiency viruses (SIV). It can be shown using viral protein R (Vpr), that inhibitors of cellular peptidyl-prolyl cis/trans isomerases (PPIases) inhibit the expression, folding protein stability and the biological efficacy of Vpr. The anti-viral effect of PPIase inhibitors, in particular, cyclosporin A, is based on a specific interaction of PPIases, in particular of cyclophilin A, with prolines on the N-terminus of Vpr. Applications are in anti-retroviral therapy and prevention of infections with lentiviruses which cause immune deficiency in humans and animals, in particular of AIDS or HIV-induced pathological conditions, also in combination with other anti-retroviral medicaments.

Description

 Lentivirus inhibitor

description

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). These agents are particularly suitable for the interaction of Vpr with a class of cellular chaperones,

IO to prevent the cis / trans peptidylprolyl isomerases (PPIases). In particular, it is found that Vpr interacts with the enzyme cyclophilin A (CyPA) and that this interaction can be inhibited by the agent cyclosporin (CsA). This Vpr-CyPA interaction is essential for the protein stability, folding and function of Vpr. By means of CsA, as well as other PPIase inhibitors, the function of Vpr can be blocked due to ι _{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-

20 inhibitors for the treatment of AIDS can be used as a novel anti-retroviral therapy.

State of knowledge

1. Immunophilins (PPIases) as cellular chaperones

25 peptidylprolyl isomerases (PPIases) 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-

30 dicaments which do not like

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. 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. For a review of this section, see Ivery, 1999, 2000.

2. HIV and PPIases

2.1. HIV replication cycle

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. After transcription and translation, Gag / Gag-Pol polyproteins and envelope proteins are transported to the cell membrane, where virions are assembled. After budding and constriction, the virus particles mature through proteolytic processing of the Gag / Gag-Pol polyproteins (for review see Martin, 1993).

2.2. Importance of accessory viral gene products in the replication cycle of HIV-1

In addition to the prototype retrovirus genes gag, pol and env, 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). In contrast, nef vif vpr and vpu are not absolutely necessary for virus replication in cell cultures and are therefore usually referred to as "accessory" genes. However, there are increasing indications in the Lite- rature, according to which these proteins essentially determine the spread of infection and the disease progression of individual viruses in vivo. Furthermore, experiments in vitro have shown that individual of these accessory proteins in certain cell cultures, in particular in cultivated primary human monocytes / macrophages, have an essential function for virus replication.

2.3. The accessory regulatory protein Vpr

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).

2.4. Biological functions of Vpr studies in animal models showed that 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. This fact led to the presumption that the effect of Vpr occurs early in the HIV replication cycle, immediately after membrane fusion and "uncoating" of the invading virus particle. Studies of a possible use of "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.

It is also known that 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). 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). 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.

2.5. HIV gag and CyPA

An interaction of Vpr with the cellular chaperone CyPA or other cyclophilins, as is shown for the first time in the present description of the invention, has so far been reported neither in the technical nor in the patent literature. So far it has only been reported that CyPA is necessary for the maximum infectivity of HIV-1, but not of HIV-2 or other lentiviruses (for review see Luban, 1996). It is known that CyPA and Cyclophilin B (CypB) 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. This interaction is said to be essential for the folding of Gag after virus assembly (Dietrich et al., 2001). Since HIV-1 gag binds to a class of peptidyl prolyl isomerases, which also include CyPA and CypB, it was shown that after genetic inactivation by homologous recombination (knock out) of both CyPA genes in a human CD4 ⁺ T cell line (CyPA - / -) in fact, in the absence of CyPA, the infectivity of HIV-1 is reduced and, of course, the CyPA inhibitor CsA no longer had an additional inhibitory influence on the infectivity of HIV-1 (Braaten and Luban, 2001) , This finding was the essential evidence that CyPA regulates the infectivity of HIV-1. The same 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 novel interaction of CyPA and Vpr, which is presented for the first time in the present description of the invention, was not recognized, mentioned and / or described in all of the above-mentioned publications on CyPA and HIV-1.

Furthermore, it has recently been reported that an extracellular receptor, CD 147, interacts with virus-associated CyPA and that this Vpr-CyPA interaction is necessary for the early steps of an HIV-1 infection (Pushkarsky et al, 2001). A compound of CyPA and Vpr, as it is mentioned for the first time in the present description of the invention, was also not described in this publication.

U.S. Patent 4,814,323 by Andrieu et al. In 1989 it was reported for the first time that the treatment of AIDS patients with the immunosuppressive drug CyclosporinA (CsA) leads to an improvement in the symptoms of the disease, especially to an increase in the CD4 number. In this description, it is assumed that CsA brings about a general improvement in the immune status of HIV-infected persons, and direct inhibition of HIV-1 was not recognized and was also not protected. The class of PPIase inhibitors is also not protected, only the drug CsA. The subject of the present description of the invention, the interaction of CyPA and Vpr, is not affected by this patent. invention description

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.

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.

It is known that various cellular proteins are contained in HIV virions, but only CyPA is incorporated in significant amounts. The prevention of the incorporation of CyPA, either in CyPA - / - knock out or by treatment with CsA, has a significant influence on the infectivity of HIV-1. So far, however, this relationship has only been examined for the interaction between CyPA and HIV-1 gag. This already known CyPA-Gag interaction is only necessary to regulate the incorporation of CyPA into virions. However, the inhibitory effect of CsA on HIV replication cannot be explained by the CyPA-Gag interaction alone. It is therefore demonstrated for the first time in the present description of the invention that the enzymatic activity of CyPA is necessary for the folding, protein stability and the function of Vpr and therefore the inhibitor CsA has a novel effect which is completely independent of the CyPA-Gag interaction on the replication of HIV-1 affects. This novel inhibitory effect is not limited to HIV-1 alone, but affects all lentiviruses, HIV-1, HIV-2 and SIV. A major advantage compared to the effects of CsA on HIV replication described so far is the Vpr-CyPA interaction mentioned for the first time in the present description of the invention is the fact that all previously known AIDS pathogens are affected by this interaction. In contrast - and to the main disadvantage for the previously described the CyPA-Gag interaction - 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). The problem was solved by using inhibitors of peptidylprolyl isomerases (PPIases) - the peptidylprolyl isomerase inhibitors (PPIase inhibitors). In principle, 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 SanglipherinA.

All cyclophilins that can interact with Vpr can be used as targets for these agents. This includes:

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;

- KIAA0073 or Hal539-CyP: a 60kDa protein, human genome name CYP; - NK-TRCyP: an 89 kDa protein in the cell nucleus, human genome name NKTR;

- RAΝ-BP2 or 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. As a consequence of the use of these agents, no functional Vpr molecules can be expressed in an HIV-infected cell. As a result of using these agents, 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. One of the findings of the invention is that 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. Furthermore, a recognition sequence for PPIases is contained in the N-terminus of Vpr. In particular, it was shown that 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. The inactivation of Vpr by CsA is a very quick effect and presumably takes place even in the course of protein synthesis, i.e. co-translational. As a result of the inactivation of Vpr by CsA, there is an almost complete loss of Vpr function in an HIV-1 infected cell. As a consequence of the invention and essentially new is the fact that after inactivation of CyPA the Vpr mediated biological activities such as Example cell cycle arrest, apoptosis and the incorporation of Vpr in progeny viruses can be completely blocked. This results in a novel anti-retroviral strategy based on PPIase inhibitors. In the N-terminus of Vpr, a high proportion of proline peptide bonds are contained in the cis conformation.

Using methods of protein structure elucidation (Η NMR, CD and infrared spectroscopy) it was found that a special cs / trαrts phenomenon occurs in the N-terminus of Vpr. This means that an unusually large proportion of the proline residues in positions 4, 10, 14 and 35 are in the cz ^' s conformation in the absence of CyPA. As a result, Vpr tends to extreme instability, which can be regulated by the folding enzyme CyPA. In particular, it is observed as part of the invention that without the structure-stabilizing effect of CyPA, the N-terminus of Vpr is not stable and cannot assume a defined secondary structure. This is determined by CD measurements (Fig.l), NMR structure calculation (Fig. 2). The proportion of proline residues in the cw conformation is determined by high-resolution TOCSY (1H NMR) spectra (FIG. 3).

Vpr from HIV-1 binds to CyPA.

Based on this completely unexpected and novel finding of a cis / trans phenomenon in the N-terminus of Vpr, it was investigated whether 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 / trαns 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). First, the suspected interaction between Vpr and CyPA was demonstrated using Far-Western blot, a specific form of Western blot on nitrocellulose. It has been found that purified recombinant CyPA binds to synthetic Vpr (FIG. 5). Furthermore, the Vpr-CyPA interaction was detected by means of plasmon resonance (BIAcore) spectroscopy (FIGS. 6 to 7). It was specifically found that Vpr only binds to the N-terminus, and only to the first 40 amino acid positions. Vpr does not bind to the C-terminus. Furthermore, it has been found that these prolines are important for binding to CyPA, in particular the rest of proline-35 is essential for the interaction with CyPA. Mutations in these positions prevent Vpr from binding to CyPA. As an essential core of the invention, it was shown that the mutation of proline-35 after asparagine (a conserved amino acid exchange) on the one hand inhibits the binding of Vpr to CyPA in the BIAcore studies and later it was shown in biological studies that this mutation completely suppresses the stability and thus the function of Vpr in HIV-1 infected cells.

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.

It is also among the findings of the invention that 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. whether inhibitors of CyPA, especially the inhibitor CsA, regulate, possibly block or even block the expression of Vpr. It was surprisingly found that CsA prevents the interaction of Vpr with CyPA. Against all expectations, it was recognized that the inhibitor CsA dramatically reduced the stability and thus the expression of Vpr. Surprisingly, it was also found that inhibitors of CyPA, for example the drug CyclosporinA (CsA), can prevent the function of Vpr in an HIV-1-infected cell, in particular Vpr-induced apoptosis and cell cycle arrest. At first it was found that after the addition of CsA Vpr no longer binds to CyPA. As a result, it was shown that all essential functions of Vpr can be blocked by CsA. These include - the Vpr-induced cell cycle arrest in the G2 phase (G2 arrest),

- Vpr-induced apoptosis,

- The 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

- that CsA prevents the incorporation of Vpr into virions,

- that CsA prevents the interaction of Vpr with other cellular factors, such as with the glycocorticoid receptor, and

- that CsA prevents the transport of Vpr in the cell, especially to the cell nucleus.

As an essential mechanism in these observations, it was surprisingly recognized that 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. From this it can be deduced that the interaction of CyPA with Vpr is necessary for the de novo synthesis of Vpr, that is to say for expression, that is to say for the co-translational folding of Vpr. This is the first time that a function of CyPA for the co-translational folding of a protein, in particular a viral, in particular an HIV protein, has been established. The mechanism that CyPA co-translationally regulates the folding of Vpr is relatively new. Although there are preliminary indications that newly synthesized proteins interact with folding enzymes immediately after synthesis in order to ensure optimal protein folding (for review see Frydman, 2001), there are no indications that CyPA is involved in these co-translational processes is involved.

The mechanism of observation is seen in the fact that CyPA has to bind to the N-terminus of Vpr in the process of protein synthesis in order to ensure the further stability and folding and thus the full biological effectiveness of the protein. In a further consequence of the invention it is stated that, in the complete absence of CyPA, significantly less Vpr is expressed in an HIV-infected cell. For this purpose, a genetically manipulated human CD4 ⁺ T cell line was used, which is mutated in both alleles of the CyPA gene and therefore cannot express cellular CyPA. In this CyPA knock out cell line, called CyPA - / -, the amount of viral Vpr is very strong intracellularly reduced or almost undetectable (Fig. 12). Also, no 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) ,

The inhibitory effect of PPIase inhibitors, such as CsA, on the Vpr stability can be demonstrated in various expression systems for Vpr. Which includes:

Infection with HIV-1, HIV-2, SIV or chimeric viruses thereof,

Transfection with the DNA of expression vectors encoding Vpr,

- infection with recombinant and Vpr-coding retroviruses, - infection with recombinant and Vpr-coding adenovirus,

- infection with recombinant and Vpr-encoding vaccinia viruses,

- Infection with recombinant and Vpr-encoding bacculoviruses.

The novel anti-viral action of CsA and other PPIase inhibitors is that they 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.

Since Vpr is an important pathogenicity factor of an HIV infection and the associated clinical pictures - such as AIDS - the use of CsA and other PPIase inhibitors results in a completely new anti-retroviral strategy, in particular in that a large part of the previous known PPIase inhibitors have already been used as medicines and are now used as anti-retroviral agents. The discovery that CyPA interacts with the HIV-1 structural protein p24 ^CA and the inhibitor CsA thereby reduces the infectivity of HIV has two major disadvantages compared to the inventive solution:

- 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.

The main novelty value of the invention in comparison with data previously published in connection with HIV and CyPA can be summarized as follows:

- There is a new target for the anti-viral effect of CyPA - the lentivirus protein Vpr.

- A new mechanism exists, that 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.

- This mechanism involves not only the mere binding (as in the case of HIV-1 p24 ^CA ), but also the enzymatic activity of CyPA, which leads to the conformational conversion of the proline-peptidyl bonds in the N-terminal region of Vpr from a ice into a more stable trarcs conformation. It is also new and advantageous that the new target for PPIase inhibitors is contained in all hitherto known lentiviruses (HIV-1, HIV-2 and SIV) and is therefore essentially different from the inhibitory effect of CsA on HIV-1 described so far p24 ^{CA is} not limited to HIV-1 alone. 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. This includes their use in the manufacture of medicaments for the treatment and prophylaxis of HIV infections, AIDS and related pathological symptoms, such as HIV-induced dementia, HIV-induced disorders in lipid metabolism, especially HLS syndrome (HIV-associated lipodystrophy syndrome). as well as 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). Using the example of the HIV-1 virus protein R (Vpr), it is shown that inhibitors of cellular cis / trans peptidylprolyl isomerases (PPIases) inhibit both the expression, folding, protein stability and the biological activity of Vpr. 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.

The invention is to be explained in more detail on the basis of exemplary embodiments, without being restricted to these examples.

embodiments

Example 1: Detection of the Vpr-CyPA interaction using BIAcore spectroscopy

Surface plasmon resonance biosensor analysis (BIAcore spectroscopy) was used to investigate the novel interaction between CyPA and Vpr. For this purpose, the synthetically produced peptide Vpr ^{1 "40} and purified recombinant CypA (Sigma) were used. Surface plasmon resonance measurements were carried out at 25 ° C. with a BIACORE 2000 (Biacore AB, Uppsala Sweden) spectrometer, which uses a CM5 research grade sensor chip CypA was immobilized in an amount of 4200 to 1200 RU (resonance units) (flow cells 2, 3 and 4) using standard amino coupling reactions. 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 μM EDTA, 0.005% Tween 20, pH 7.4) over all flow cells in a concentration range from 1 to 250 μM, at a flow rate of 5 μl / 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 μM, 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. In addition to the wild-type sequence of HIV-IN _L4-3 Vpr (FIG. 6, Panel A, Vpr ^{1 "40} ), 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. 6 Panel B); Mutant Vpr ^{1 "40} , P ³⁵ -N (Pro-Asn exchange in position 35, Fig. 6 Panel C); Mutant Vpr ^{1" 40} , P ^{5>10>14> 35} _N (Pro-Asn exchange in positions 5, 10, 14 and 35 Fig. 6 Panel D). Similar BlAcore results were obtained using the peptide Vpr ^{21 "40} and its mutant Vpr ^{21" 40} , P ³⁵ -N (Fig. 7). This fragment of Vpr has specific affinity for CyPA, which can be canceled by mutation of Pro-35. In contrast to this, no binding to CyPA could be detected for the N-terminal fragment Vpr ^" (FIG. 8). As a result of these BlAcore studies, the novel CyPA-Vpr interaction effect was convincingly demonstrated. On the basis of this data it can be established that CyPA binds to the N-terminus of Vpr in the form of the peptide Vpr ^{1 "4} and that proline in position 35 is essential for this binding.

Example 2: Detection of the Vpr-CyPA interaction using Far-Western blot

The Far-Western blot technique was used to investigate the interaction between Vpr and Cyp in the context of total Vpr. The interaction of total Vpr with CyPA could not be investigated directly using BlAcore, since total Vpr shows a strong tendency towards self-association and can therefore result in non-specific binding in resonance measurements.

For the Far-Western blot technique, recombinant CyPA was separated in a 15% SDS PAGE at a concentration of approximately -150 ng CyPA per lane in SDS-PAGE. The protein was then electrotransferred to nitrocellulose and individual strips of this western blot were blocked for 4 hr with 3% BSA in TBS. The CyPA-Western blot strips obtained in this way were then for 12 hr at 4 ° C. with synthetic total Vpr, the peptide Vpr ^{1 "96} , at a concentration of 1 μg / ml either without detergents (FIG. 5, lane 1 ) or in TBS / T with 0.5% Triton XI 00 (Fig. 5, lane 2). The strips were then washed 3 times in TBS / T for 10 min, then with anti-Vpr ^{1 "96} (polyclonal antiserum from rabbits) in a dilution of 1: 2000 in 3% BSA TBS / T, incubated for 4 hr at room temperature. After washing, the bound anti-Vpr antibodies were visualized by anti-rabbit IgG-peroxidase conjugate and enhanced chemiluminescence (ECL) reaction. A 5 min exposure to Kodak X-ray film is shown in Figure 5. These studies show for the first time that both proteins, Vpr and CyPA, interact specifically, that this interaction affects not only the N-terminus of Vpr, but also the entire protein, and that the high affinity of CyPA for Vpr itself interacts enabled with CyPA, which was bound to nitrocellulose and renatured to nitrocellulose after SDS-PAGE.

Example 3: The expression of Vpr in HIV-1 infected cells 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 gene alleles are mutated by a genetically knock out (CypA - / -) (Braaten and Luban, 12001), were included HIV-IN _L4-3 infected and cultured for 7 days. At the time of maximum virus expression, 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 (FIG. 12). It can be seen that the amount of 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.

literature

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Kino, T .; Gragerov, A .; Kopp, JB; Stauber, RH; Pavlakis, GN; Chrousos, GP (1999) The HIV-1 virion-associated protein Vpr is a coactivator of the human glucocorticoid receptor. J. Exp.Med. 1: 51-61. Lang, SM; Weeger, M .; Stahl-Hennig, C; Coulibaly, C; Hunsmann, G .; Müller, J .; Müller-Hermelink, H .; Fuchs, D .; Wächter, H .; Daniel, MM; Desrosiers, RC; Fleckenstein, B. (1993) Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus. JNirol. 67: 902-912. Luban, J. (1996) Absconding with the chaperone: essential cyclophilin-gag interaction in

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192nd

Legends for the figures

Figure 1: Evidence of structural instability of Vpr N-terminus using CD spectroscopy.

Figure 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 Η NMR spectroscopy.

Figure 4: A CyPA binding motif is conserved by the Vpr in the N-terminus.

In the figure, 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.

In the context of the invention it is found that the N-terminus in the form of the peptide Vpr ^{1 "40} specifically binds to CypA,

- especially the fragment Vpr21-40 binds to CypA,

- the mutant Vpr21-40 with Pro-Asn exchange does not bind to CypA,

- The C-terminal fragment Vpr47-96 does not bind to CypA.

Figure 9: The CyPA inhibitor CsA causes instability of Vpr in HIV-1 infected cells.

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. In the context of the invention, it is shown that after the addition of CsA (50 microg / ml), the amount of Vpr is reduced approximately 10-fold immediately after the [S] -met pulse marking and the half-life of Vpr is significantly reduced , As a control, treatment with CspA has no influence on the expression and proteolytic processing of HIV Gag structural protein. Although it is known that CypA also binds to HIV-1 Gag proteins, only the Vpr-CypA interaction, which was found to be novel in the context of the invention, brings about a dramatic reduction in the expression and stability of Vpr.

Figure 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. Since this loss occurs very quickly - already after the short pulse time of 5 min - it is determined according to the invention that the inactivation of CypA by Csp causes the instability and therefore the loss of Vpr expression. Figure 11: Proteasome inhibitors do not restore the stability of Vpr after CsA treatment.

Figure 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. At the time of maximum virus expression, 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. In the context of the invention it can be seen that the amount of 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.

List of abbreviations

AIDS Aquired Immunodeficiency Syndrome

BSA Bovine serum albumin

CD cluster of differentiation

CD4 glycoprotein

CsA cyclosporin, cyclosporin A

CyPA cyclophilin A

CyPB cyclophilin B etc.

DNA deoxyribonucleic acid (deoxyribonucleic acid)

EDTA ethylenediaminetetraacetic acid

HIV Human Immunodeficiency Viruses

'H NMR Proton NMR Spectroscopy hr hours

IL Interleukin kDa Kilodalton

NMR Nuclear Magnetic Resonance

PPIases peptidylprolyl isomerases

RT reverse transcriptase RNA ribonucleic acid

SDS- sodium dodecyl sulfate (sodium dodecyl sulfate)

SDS-PAGE SDS polyacrylamide gel electrophoresis

SIV monkey immunodeficiency viruses

TBS Tris Borat Saline

TBS / T Tris Borat Saline / T ween

TNF tumor necrosis factor

TOCSY total correlation spectroscopy

Vpr lentivirus protein R

Claims

claims 1. Means for inhibiting the primate lentivirus protein R (Vpr) of the immunodeficiency viruses HIV-1, HIV-2 and SIV, characterized in that they contain, as effective components, inhibitors of cellular chaperones which are necessary for the correct folding, stability and thus the biological function of Vpr are necessary. 2. Agents for inhibiting the lentivirus protein R (Vpr), characterized in that they contain, as active components, inhibitors of peptidylprolyl isomerases - PPIases- in pharmaceutical preparations. 3. Composition according to claims 1 and 2, characterized in that they contain, as active components, compounds which block the enzymatic function of PPIases. 4. Composition according to claims 1 and 2, characterized in that the inhibitors inhibit PPIases which belong to the immunophilins or cyclophilins. 5. Composition according to claims 1, 2 and 4, characterized in that the inhibitors bind to cis-trans peptidylprolyl isomerases (PPIases) or otherwise with these in Interact and thereby influence, regulate or inhibit their enzymatic activity. 6. Composition according to claims 1 and 2, characterized in that Vpr interacts with cyclophilines, that this interaction is important for the expression, protein stability and biological functions of Vpr, and that this cyclophilin-Vpr interaction by the PPIase inhibitors 6.1. Cyclosporin A (CsA) and / or 6.2. FK506 and / or 6.3. Rapamycin is inhibited. 7. Composition according to claims 1 and 2, characterized in that Vpr interacts with cyclophilines and that this interaction is caused by the non-immunosuppressive PPIase inhibitors 7.1 SDZ NIM811 and / or 7.2 SanglipherinA is inhibited. 8. Composition according to claims 1, 2, 6 and 7, characterized in that Vpr with one or more of the enzymes 8.1 Cyclophilin A (CyPA) 8.2 Cyclophilin B (CyPB) 8.3 Cyclophilin C (CyPC) 8.4 Cyclophilin F (CyPF) / 8.5 Cyclophilin 3 (CyP3) 8.6 Cyclophilin M (CyPM) 8.7 PPIL1 8.8 USA cyclophilin (USA CyP) 8.9 Cyclophilin 20 (CyP20) 8.10 Cyclophilin E (CyPE) 8.1 1 Cyclophilin 33 A (CyP33A) 8.12 Cyclophilin 40 (CyP40) 8.13 Cyclophilin 60 (CyP60) 8.14 KIAA0073 8.15 Hal539-Cyclophilin (Hal539-CyP) 8.16 NK-TR-Cyclophilin (NK-TRCyP) 8.17 RAN-BP2 8.18 NUP-358 interacts and that these interactions are inhibited by PPIase inhibitors according to claims 6 and 7. Agent according to claims 1, 2, 6, 7 and 8, characterized in that Vpr binds primarily to the cellular PPIase CyPA, this interaction regulates the trans conformation of proline-peptidyl bonds in the N-terminus of Vpr, - and that this c / s-to-trαrcs rearrangement can be inhibited by PPIase inhibitors. 10. Composition according to claim 9, characterized in that the CyPA-Vpr interaction includes the proline residues in positions 5, 10, 14 and 35 of Vpr and that the cis-after-tra «s rearrangement in this proline -Positions can be inhibited by PPIase inhibitors. 11. A composition according to claim 10, characterized in that the PPIase inhibitors block the CyPA-Vpr interaction by neutralizing one, several or all of the four prolines in positions 5, 10, 14 and 35 of Vpr. 12. Composition according to claims 1, 2 and 6, characterized by a mechanism by which CyPA co-translationally regulates the expression, folding and stability of Vpr. 13. Composition according to claim 12, characterized in that the mechanism of the stabilizing effect of CyPA or other PPIases on the expression and folding of Vpr in a cell infected with HIV-1, HIV-2, SIV, or chimeric viruses thereof, one Cell transfected with the DNA of expression vectors encoding Vpr, a cell infected with recombinant and Vpr-encoding retroviruses, a cell infected with recombinant and Vpr-encoding adenoviruses, - a cell infected with recombinant and Vpr-encoding vaccine viruses, a cell infected with recombinant and Vprovirus encoding , takes effect. 14. Composition according to claims 1, 2 and 6, and claims 9-13, characterized in that genetic or other methods influence the expression and / or the enzymatic activities of CyPA or other PPIases and thereby the biological functions of Vpr - in HIV-1 host cells, in CD4 ⁺ T cells and in monocytes or macrophages - influence, regulate or block. 15. Composition according to claim 14, characterized in that these methods include the expression and / or transfection of anti-sense DNA or RNA which specifically inhibit the translation of CyPA and / or other PPIases. 16. Agent according to claims 1, 2 and 6 and claims 9 to 15, characterized in that all the essential biological functions of Vpr are the Vpr-induced cell cycle arrest in the G2 phase (G2 armrest), the Vpr-induced apoptosis, the Vpr-transmitted increase in virus production in HIV-infected monocytes / macrophages and the Vpr-mediated co-activation of the glycocorticoid receptor are blocked by the application of PPIase inhibitors and genetic anti-PPIase methods. 17. Composition according to claims 1, 2 and 6 and claims 9 to 16, characterized in that PPIase inhibitors and genetic anti-PPIase methods prevent the incorporation of Vpr into virions, the interaction of Vpr with other cellular factors, such as Glycocorticoid receptor, prevent and - prevent the transport of Vpr in the cell and to the cell nucleus. 18. Use of the agent according to claims 1 to 16, characterized in that by using lentiviruses a) human immunodeficiency virus type 1 (HIV-1) or b) human immunodeficiency virus type 2 (HIV-2) or c) monkey immunodeficiency virus (SIV) be inhibited. 19. Use according to claim 17, characterized in that the active components and methods used in the compositions contain the lentivirus protein R (Vpr) influence, regulate or block. 20. Use according to claims 17 and 18 for combating / treating diseases / pathological phenomena which were caused by infections with lentiviruses. 21. Use according to claims 17 to 19 for combating / treating AIDS. 22. Use according to claims 17 to 20, characterized in that PPIase inhibitors are used as a novel anti-retroviral therapy for the treatment of AIDS. 23. Use according to claims 17 and 18 for blocking the essential functions of Vpr in vivo, in the infected organism, the Vpr-induced cell cycle arrest in the G2 phase (G2 arrest), the Vpr-induced apoptosis, - the Vpr-transmitted Increased virus production in HIV infected monocytes / Macrophages and the Vpr-mediated co-activation of the glycocorticoid receptor by PPIase inhibitors. 24. Use according to claims 17 and 18, characterized in that PPIase inhibitors in vivo, in the infected organism, - the Vpr-induced cell cycle arrest in the G2 phase (G2 arrest) - Vpr-induced apoptosis - the incorporation of Vpr in virions and / or - the transport of Vpr in the cell and to the cell nucleus - prevent and - prevent the interaction of Vpr with other cellular factors, such as the glycocorticoid receptor. 25. Use of PPIase inhibitors according to claims 6 and 7 for the preparation of agents for inhibiting the lentivirus protein R (Vpr). 26. Use of PPIase inhibitors according to claims 6 and 7 for the manufacture of medicaments for the treatment and prevention of the outbreak of AIDS in HIV-infected people. 27. Use of PPIase inhibitors according to claims 20 and 21 for the manufacture of medicaments for the treatment and prophylaxis of an HIV infection. 28. Use according to claims 20 to 27, in combination with 28.1 other anti-retro viral drugs 28.2 blockers of reverse transcriptase and / or the viral protease 28.3 anti-retro viral therapies based on gene therapy interventions 28.4 intracellular immunization 28.5 the introduction of anti-HIV-1 / HIV-2 effective genes into stem cells and / or peripheral CD4 ⁺ lymphocytes. 29. Use according to claim 28 for combating / treating AIDS in the advanced phase of the disease. 30. Use according to claim 20 to 29 for preventing the onset of the disease and for reducing the spread of infection in the organism (reduction of "viral load") of symptom-free HIV-1 / HIV-2 seropositive and HIV-1 / HIV-2 infected persons. 31. Use according to claim 20 to 30 for the treatment / control / prevention of HIV-induced dementia, in particular for the prevention of HIV infection of neurons, glia, and endothelial cells in capillaries of the brain. 32. Use of PPIase inhibitors according to claims 20 to 31 for the manufacture of medicaments for the treatment and prophylaxis of HIV-induced disorders in the lipid metabolism, especially the HLS syndrome (HIV-associated lipodystrophy syndrome). 33. Use of PPIase inhibitors according to claim 20-32 for the manufacture of medicaments for the treatment and prophylaxis of HIV-induced disorders in kidney function, especially the HIV AN syndrome (HIV associated nephropathy). 34. Use according to claims 20 to 33 to prevent the establishment of a systemic HIV-1 / HIV-2 infection immediately after contact with infectious viruses, such as needle-prick injuries with HIV-contaminated blood or blood products.