WO2010020676A1 - The use of topoisomerase type i inhibitors to treat viral infections - Google Patents

Abstract

The present invention relates to the use of compounds, inducing isgylation to treat viral infections. More specifically, the invention relates to topoisomerase type I inhibitors, such as camptothecin and camptothecin derivatives to treat viral infections, preferably infections by RNA viruses. The invention relates further to the measurement of the level of ISG15 and or the level of isgylation during or after treatment as a method to measure the efficacy of the antiviral treatment.

Description

The use of topoisomerase type I inhibitors to treat viral infections

The present invention relates to the use of compounds, inducing isgylation to treat viral infections. More specifically, the invention relates to topoisomerase type I inhibitors, such as camptothecin and camptothecin derivatives to treat viral infections, preferably infections by RNA viruses. The invention relates further to the measurement of the level of ISG15 and/or the level of isgylation during or after treatment as a method to measure the efficacy of the antiviral treatment. Interferon (IFN) Stimulated Gene 15 (ISG15) formerly known as UCRP (Ubiquitin-Cross Reacting Protein) or G1 P2, is an Ubiquitin (Ub)-Like molecule (UbL), highly induced upon type I IFN treatment. It is expressed as a 17kDa protein, containing 2 Ubiquitin domains and a C- terminal octapeptide (in humans) or hexapeptide (in mice). Splicing of the C-terminal peptide gives a 15kDa protein, which can be conjugated via an isopeptide binding on the ε-amino group of a Lysine residue in the substrate (Loeb and Haas, 1992). Alternatively, the processed 15kDa molecule can be secreted and exerts immunoregulatory functions on peripheral blood lymphocytes (D'Cunha et al., 1996).

Conjugation of Ub and UbL requires the cooperative activity of at least 3 enzymes. First, a Ubiquitin-Activating Enzyme (termed Uba or E1 ) adenylates the C-terminus of Ub or UbL, forming a thioester bond between the C-terminal Glycine of Ub or UbL, and a catalytic Cysteine residue in E1. In humans, Ub molecules are activated by UbE1 (also known as A9S1 ) (Haas et al., 1982), ISG15 by UbE1 L (Yuan and Krug, 2001 ) SUMO by AOS-Uba1 and Nedd8 by APPBP-Uba3.

After this ATP-dependent activation step, Ub or UbL molecules are transferred to a Ubiquitin- Conjugating Enzyme (termed Ubc or E2), also by a thioester linkage on a Cysteine residue. UbcH8 has been identified as the major E2 involved in ISGylation (Kim et al., 2004; Zhao et al., 2004). Recently, also UbcH6 has been shown to be able of forming a thioester intermediate with ISG15 (Takeuchi et al., 2005). Around 400 proteins are recognized as E3 or Ubiquitin Ligases. Roughly, they can be discerned as RING (Really Interesting New Gene)- finger proteins, acting as a molecular scaffold, and HECT (Homologous to E6-AP C-Terminus)- domain proteins, which also exert a catalytic contribution. E3 ligases confer specificity, and place the Ub or UbL molecule in close proximity to the Lysine residue of the substrate. The formation of mono- and/or polyubiquitin chains is a process mediated by E2 together with E3. Recently, the IFN-induced HERC5 has been identified as an ISG15 E3 ligase in human cells (Dastur et al., 2006; Wong et al., 2006). The Estrogen-response Finger Protein (EFP), also an IFN-induced protein, functions as an E3 ligase for ISGylation of 14-3-3δ (Zou and Zhang, 2006). Definitely, these recent discovered ISG15 ligases are only the onset of a more extensive list.

In some cases, a Ubiquitin Chain Elongation Factor Enzyme (termed E4) is required for the poly-Ubiquitination process. It is not clear whether poly-ISG15 chain formation occurs (Giannakopoulos et al., 2005).

As Ubiquitin, most UbLs are synthesized as inactive precursors, being processed by De- UBiquitinating Enzymes (DUBs) exposing the mature protein with a C-terminal Glycine residue. DUBs not only exert their function by protein processing, they also have a function in removal of the Ub(L) from their substrate. lsopeptidase T(USP5) has been identified as a protease with a dual specificity for Ub an ISG15 (Hemelaar et al., 2004). UBP43 (USP18) is identified as a specific DUB for ISG15 (Malakhov et al., 2002). Moreover, UBP43 interferes at another level in ISG15 attenuation as it competes with Jak1 binding to the Type I IFN receptor IFNaR2 (Malakhova et al., 2006). There are indications that ISG15 is involved in the antiviral defense against several RNA viruses. ISG15 is markedly upregulated upon viral infection, but its precise mechanism of action remains unclear. Several viruses have developed mechanisms to counteract ISG15 function. For example, the influenza B Non-Structural NS1 B protein binds ISG15, thereby preventing its association to UbEI L (Yuan and Krug, 2001 ). Also the papain-like protease of the Severe Acute Respiratory Sydrome (SARS) Coronavirus, counteracts ISG15 functioning by removing it from its substrate (Lindner et al., 2005). The Hepatitis C virus NS3/4A protease cleaves IFN Promoter-Stimulator-1 (IPS-1 ), causing a subcellular distribution of IPS-1 , which is in vivo linked with a deficiency of ISG15 expression and conjugation (Loo et al., 2006). In a more direct way, ectopic expression of ISG15 in cells lacking a functional IFN response reduces Newcastle Disease Virus and Influenza Virus replication (Bazzigher et al., 1992), and also overcomes fatal intracerebral infection in IFNaR-/- mice by Sindbis Virus (Lenschow et al., 2005). An inverse dose-response correlation of exogenously expressed ISG15 and release of Human Immunodeficiency Virus (HIV) virions is also found and suppression of ISG15 expression by siRNA counteracts IFN-mediated inhibition of HIV virion release (Okumura et al., 2006). Notwithstanding this support for a role of isgylation in the innate immunity, there is a lack of ISG15 based antiviral treatments. WO2006004907 describes a method of inhibiting the viral replication comprising contacting a cell infected with a virus with a compound that modulates the activity or expression of a polypeptide involved in the ubiquitination pathway used by the virus during viral replication, thereby inhibiting the replication of the virus. Hereby, the application indicates that this can be realized by upregulation of UbEI L or by downregulation of the ISG15 deconjugation enzyme UBP43. However, the application doesn't give any indication that increased ISGylation can be obtained by treatment of the cells with chemical compounds, and it is neither disclosing the fact that mutant or variant forms of ISG15 may show an increased ISGylation activity and/or a broader ISGylation pattern. Surprisingly we found that compounds, inducing ISG15 and/or consequent ISGylation, such as topoisomerase type I inhibitors or ISG15 mutants can be used to inhibit or slow down viral replication of RNA viruses. The use of topoisomerase inhibitors to treat infection by RNA viruses is specially unexpected, as no DNA intermediate, that could function as a target for topoisomerase inhibitors, is present in the replication cycle.

One aspect of the invention is the use of a compound inducing ISG 15 and/or ISGylation to treat infections by RNA viruses. Preferably, said compound inducing ISG15 and/or ISGylation is selected from the group consisting of topoisomerase type I inhibitors, and ISG15 mutants. In one preferred embodiment, said compound is a topoisomerase type I inhibitor. Topoisomerase type I inhibitors are known to the person skilled in the art and comprise, but are not limited to camptothecin, camptothecin derivatives, and lamellarin D or further derivatives of those structures. Camptothecin derivatives, as used here include but are not limited homocamptothecin, irinotecan, topotecan, DB67, BNP1350, exatecan, lurtototecan, ST1481 and CKD602. Derivatives as used here are compounds that share a same core structure, but differ in side chains and/or groups placed on that core structure. Preferably, said inhibitor is topotecan. In another preferred embodiment, said compound is a mutant ISG15 characterized by high ISGylation activity, comprising an amino acid at position 89 which is not N. Preferably, said amino acid is selected from the group consisting of A, R, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and Z. Even more preferably, said amino acid is selected from the group consisting of D, E, H, K and R. Even more preferably said mutant or variant has a D at position 89. Even more preferably, said mutant or variant comprises SEQ ID N°1 (human sequence, with N89D), most preferably said mutant or variant is consisting of SEQ ID N°1. Position 89 refers to the position in the human sequence (Genbank AAP35961 ). The numbering of the position may differ in other species, but is determined in that case by a protein-protein BLAST (Altschul et al., 1997; Schaffer et al., 2001 ) with said human sequence and refers then to the position that coincides with the position 89 of the human sequence. Mutant or variant as used here means that either the endogenous sequence of ISG15 is mutated into a form according to the invention (mutant) or that a heterologous ISG15 sequence, or a mutant thereof, according to the invention is expressed in a host, where it is boosting the ISGylation activity (variant). In the latter case, the heterologous ISG15 variant is either replacing the endogenous activity, or additional to the endogenous activity. High ISGylation activity, as used here, means that the D89 form is ISGylating a broader range of proteins and/or a bigger fraction of one or more specified proteins, when compared with the N89 form of the same protein (in case of a mutant), or when compared with the endogenous ISG15 sequence, in case of a variant, tested under identical experimental conditions. RNA viruses as used here do include double stranded (ds) RNA viruses, positive sense single stranded (ss) RNA viruses and negative sense single stranded RNA viruses, but do not include the retroviruses where DNA intermediates are used for replication. Preferably said RNA virus is a positive sense ssRNA virus, more preferably it belongs to the family of the Flaviviridae, even more preferably it is a Hepatitis C virus (HCV).

Still another aspect of the invention is the use of the measurement of the level of ISG15 expression and/or the level of ISGylation during or after treatment as a method to measure the efficacy of the antiviral treatment. The measurement of the level of ISG15 or ISGylation during or after the treatment is then compared with the basal levels of ISG 15 or ISGylation before the treatment. Indeed, the induction of ISG15 and/or the induction of ISGylation by the treatment can be used as a marker for the antiviral effect.

The level of ISGylation, as used here, is preferably be measured by measuring the level of ISGylated proteins. Alternatively, the level of ISGylation can be measured indirectly, by measuring the level of ISG15, either at protein level or at expression level.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Alignment of the ISG15 sequence of different species

Figure 2: Requirement for ectopic expression of either UbE1 or UbEI L and UbcH8 for ISGylation by Hu or AGMk ISG15. HekT cells were transfected with flag-TAGged mock, Hu or AGMk ISG15, and the indicated Ubiquitinating enzymes. Western blot using anti-flag antibody revealing the transfected ISG15.

Figure 3: ISGylation of different E2s by Hu and AGMk ISG15. Mock, Hu ISG15 or AGMk ISG15 was co-transfected with the indicated E2 fused to a V5-TAG. SDS-PAGE was performed and the different E2 enzymes were revealed by their V5-TAG. The ISGylated form of the E2 enzymes is shown by a purple asterisk.

Figure 4: Sequence alignment of AGMk and Hu ISG15. Amino acids of hulSG15 mutated in AGMk residues are shown in dark. If not further specified, the human allelic variant with N at position 83 and S at position 94 was used. Allelic variants N83S and S94N were also tested.

Figure 5: ISGylation of E2s by Hu ISG15 and mutants. Hek293T cells were co-transfected with the indicated V5-TAGged E2, and the different ISG15 mutants. 48h after transfection, cells were lysed and SDS-PAGE was performed on the whole lysates. The E2 was revealed by its V5 tag. The unconjugated form of the E2 and ISGylated form of E2 is indicated by arrows. Figure 6: Western blot analysis of ISG15 induction in Jurkat cells. Samples were taken after 2, 5, 10, 24 and 48 hours. It should be noted that, at 48 hrs, all camptothecin treated cells have died, β-actin is used as control.

Figure 7: Western blot analysis of ISG15 induction in Huh7.5 cells. The left panel shows the ISG15 induction, the right panel the β-actin control. ATRA: all trans retoinic acid.

Figure 8: Evolution of number of HCV colonies in function of the topotecan in pretreatment. Compound: concentration of topotecan, present during the pretreatment. The concentration is diluted upon addition of the virus.

EXAMPLES

Example 1 : comparison of ISG15 sequences Unlike Ubiquitin, ISG15 is not well conserved among different species. ISG15 is not found in lower eukaryotes, such as yeast, insects, nematodes or plants, and is less than 50% conserved between mammalians. In Figure 1 , various orthologues of ISG15 are aligned.

Example 2: differences in activity between AGMk and Hu ISG15 Tandem Array Purification (TAP) experiments were performed to identify ISGylated substrates. N-terminal of the Human (Hu) ISG15 or African Green Monkey {Cercopithecus aetiops) (AGMk) ISG15, a tandem proteinA binding domain and Flag-TAG, separated by a Tobacco Etch Virus cleavage domain, was cloned. This allowed tandem purifications of ISGylated proteins in different cell-lines and under different conditions (+/- IFNβ or the proteasome inhibitor MG132). Table 1 gives the result of gel-free LC-MS/MS identifications.

Table 1 Identifications by LC-MS/MS of TAP analysis experiments with either Hu or AGMk ISG15 as bait. The amount of peptides found of the corresponding protein is indicated. The TAP experiments identified substantially more substrates being ISGylated by AGMk ISG15 compared to Hu ISG15 in human cell-lines. As nor the Ubiquitin activating enzyme nor the Ubiquitin conjugating enzymes were picked up as conjugation partners for Hu ISG15, a qualitative difference between AGMk and Hu ISG15 is hinted. It is of note that UbcH I O, UbcH16, UbcH17 and Proliferating Cell Nuclear Antigen (PCNA) and some of the Heat Shock Proteins (HSPs) are not-yet published substrates of ISGylation.

Figure 2 exposes a clear difference between ISGylation by Hu and AGMk ISG15. Hu ISG15 requires co-transfection of its E1 and E2 enzymes for conjugation. AGMk ISG15 is not so dependent on for high expression levels of the human E1 L and UbcH8. Even a redundancy for the activating enzyme of Ubiquitin, UbE1 and of ISG15, UbEI L is apparent. Note that although both UbEI L and UbcH8 are IFN-induced genes, endogenous UbEI L and UbcH8 levels suffice in case of AGMk ISG15. UbcH8 could be readily amplified by rt-PCR in unstimulated HekT cells. We next isolated various Ubiquitin-Conjugating enzymes previously identified by TAP analysis by rt-PCR from 2fTGH cells treated with IFNβ and coupled them C-terminal with a V5- TAG. These constructs were transfected in HekT cells together with a mock construct, Hu ISG15 or AGMk ISG15. In Figure 4, ISGylation of UbcHI O, H13 and H17 can be observed by AGMk ISG15 but not with Hu ISG15. ISGylation of UbcH13 and UbcH6 was previously described, ISGylation of UbcHI O and UbcH17 not.

Example 3: a Hu ISG15 variant shows enhanced ISGylation capacity

In order to map the residues of AGMk ISG15 involved in its efficient conjugating to its substrates, we mutated residues in Hu ISG15 to the corresponding residues in the AGMk orthologue.

Five mutants were generated based on the predicted interaction interface between ISG15 and its activating enzyme, UbE1 L (Narasimhan et al., 2005).

One single amino acid replacement, N89D, causes a dramatic change in ISGylation efficiency of the Hu ISG15. As with AGMk ISG15, no co-transfection of any E1 or E2 in HekT cells is needed for this conjugation. By homology modeling (Narasimhan et al., 2005 and Dr. F. Peelman) the critical position of residue 89 could be confirmed. This residue is located in close neighborhood of the C-terminal Glycine, the residue conjugating the Lysine of the substrate.

The Aspargine (N) residue at position 89 is shared by humans, chimpanzees and dogs. As can be seen in Figure 2, Old World AGMks share this Aspartic Acid (D) residue with sheep and cow. In rodents, this residue is evolved to a Glutamic Acid (E) residue. Example 4: Camptothecin is inducing ISG15

ISG15 induction by camptothecin was tested in Jurkat cells, 21TGH cells, and Huh7.5 cells. At different time intervals, cell lysates were prepared and ISG15 was detected in a Western blot using mouse anti-human-ISG15 (Dr. Borden). Beta actin was used as control, using rabbit anti- human-actin for the blot. IFNα, IFNβ, poly I/C and all-trans retoinic acid were used as control. All cell lines showed ISG15 induction by camptothecin, but the response was dependent upon the cell line used. Results are summarized in figure 6 and 7. A similar effect was seen with the camptothecin derivative topotecan.

Example 5: Camptothecin derivative topotecan inhibits HCV replication

Huh7.5 cells were seeded in a 96 well, PDL coated plate, at a concentration of 10⁴ cells/well. 10Oμl medium was used per well, without or with 30, 100 or 30OnM topotecan. 4 wells were used for every concentration . After 24 h rs i ncu bation 1 00 μ I H CV virus solution (H77/JFH1 mtAD) was added. Plates were further incubated for 40 hours, and then the virus count was determined. The results are shown in Figure 8. Camptothecin treated cells showed a clear and significant inhibition of the viral growth, already at lowest concentration tested (3OnM in the pretreatment).

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Claims

1. The use of a compound inducing isgylation to treat infections caused by an RNA virus.

2. The use of a compound according to claim 1 , whereby said compound is a topoisomerase type I inhibitor.

3. The use of a compound according to claim 2, whereby said topoisomerase I inhibitor is camptothecin or a camptothecin derivative.

4. The use of a compound according to claim 1 , whereby said compound is a mutant or variant ISG15.

5. The use of a compound according to claim 4, whereby said mutant or variant ISG15 has D at position 89.

6. The use of a compound according to any of the preceding claims, whereby said RNA virus belongs to the family of the Flaviviridae.

7. The use of a compound according to claim 6, whereby said RNA virus is Hepatitis C virus.