Departments of Biochemistry and Medical Microbiology and Immunology, Signal Transduction Research Group, University of Alberta, 315C Heritage Medical Research Center, Edmonton, Alberta T6G 2S2, Canada
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Abstract |
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Introduction |
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As an alternative to the traditional approach, some of the novel targets for antiviral drugs could be cellular, not viral, proteins. Antiviral drugs that targeted cellular proteins could be active against several unrelated viruses, including small-genome viruses, because many cellular proteins are required for replication of many viruses. Antiviral drugs of this sort might not select for viral mutants resistant to them because mutations in viral genes would have no effect on the proteins targeted by these drugs. Furthermore, antiviral drugs that target cellular proteins should be active even against viral mutants that are already resistant to conventional antiviral drugs. These drugs could be active against many viruses, including small ones, because often replication of several unrelated viruses requires the same cellular proteins, and replication of small viruses requires a large number of cellular proteins. Finally, the same drug could be used as a component of combination therapies against all viruses that require the cellular proteins targeted by the drug. In sum, antiviral drugs that target cellular proteins would not be constrained by the same limitations as current antivirals. On the negative side, targeting cellular proteins can certainly result in cytotoxic or other undesirable side-effects, notwithstanding the many clinical drugs that target cellular proteins without major negative side-effects, such as statins or non-steroidal anti-inflammatory drugs (NSAIDs).
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Pharmacological cyclin-dependent kinase inhibitors |
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The PCI that has been studied most extensively in human clinical trials is the non-purine-derived PCI, flavopiridol, a flavonoid.913,2225 Flavopiridol is classified as a wide-spectrum PCI, as it inhibits cdks1, 2, 4, 9 and possibly 7. The effects of flavopiridol on cdks5, 6 and 8 have not been reported. Flavopiridol inhibits several other non-cdk kinases, such as glycogen synthase kinase-3ß (GSK-3ß) and glycogen phosphorylase a and b.26,27 Flavopiridol also stimulates the ATPase activity of multidrug-resistance protein 1 (MRP1) and binds to duplex DNA and to cytosolic aldehyde dehydrogenase.2830 Like the P-PCIs, flavopiridol is competitive with ATP for cdks1 and 4 and binds to the ATP-binding pocket of cdk2.31 However, flavopiridol inhibits cdk9 non-competitively.32 The cellular effects of flavopiridol appear to be mediated by inhibition of cdks other than cdk1, 2 or 4. For example, flavopiridol inhibits global cellular transcription in vitro and in vivo at concentrations that are too low to inhibit cdk1 or 2 in the presence of physiological concentrations of ATP.32,33 In fact, inhibition of cellular transcription by flavopiridol is most likely a direct consequence of inhibition of cdk9.32
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In vitro antiviral activity of PCIs |
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Paradoxically, drugs that target cellular proteins activated during cell-cycle progression inhibit viruses that replicate in non-dividing cells. However, this paradox is more apparent than real. Cellular proteins that are normally activated during progression into the cell cycle may be active in non-cycling cells infected with HIV-1 or HSV-1. Furthermore, replication of HIV and HSV may require cdks (or other kinases) that are sensitive to PCIs but whose activity is not regulated during progression into the cell cycle. There is substantial experimental evidence for both models. Replication of HSV-1 and other viruses that replicate in non-dividing cells requires cellular proteins that are normally activated during progression into the cell cycle.4250 For example, HSV-1 replicates in neurons only under conditions in which these cells express cdk2, and HSV-1 reactivates from latency only in cdk2-expressing neurons (ex vivo).51 Conversely, PCIs inhibit several cdks whose activity is not regulated during progression into the cell cycle. P-PCIs inhibit cdk5 as efficiently as they inhibit cdks1 and 2, and flavopiridol inhibits cdk9 more potently than it inhibits cdk1 or 2.32 Roscovitine inhibits cdk9 in vitro,17 but it does not appear likely to do so in vivo.18 Other PCIs, such as DRB, inhibit cdk7 (which is required for cell-cycle progression and for transcription) or cdk9 (which is required for transcription but not for cell-cycle progression) preferentially over cdk1 (which is required for cell-cycle progression but not for transcription).41 The activity of these latter drugs on cdk2, cdk4 and other cdks has not been addressed. Finally, PCIs may inhibit other, as yet unidentified, proteins that are required for replication of PCI-sensitive viruses.
P-PCIs inhibit replication of HSV-1 and HSV-2 in a variety of cell lines.35 The concentrations of P-PCIs that inhibit HSV replication correlate directly with the concentrations that inhibit cell-cycle progression, a known consequence of cdk inhibition. These concentrations depend on cell type, and not on viral strain or multiplicity of infection.35 These findings are consistent with the hypothesis that P-PCIs inhibit viral replication as a consequence of inhibition of cellular proteins such as cdks, and not as a consequence of inhibition of viral proteins. Further supporting this hypothesis, we have recently found that P-PCIs do not bind to novel targets in HSV-infected cells in comparison to mock-infected cells18 (since P-PCIs are competitive inhibitors, they must bind to a protein in order to inhibit its activity). In contrast to traditional antivirals, P-PCIs inhibit several HSV functions, including transcription of genes of all kinetic classes, viral DNA replication and reactivation from latency.36,37 Remarkably, P-PCIs inhibit transcription of HSV immediate-early genes, a function that is not inhibited by any other drug or treatment, in as little as 2 h.38 In contrast, P-PCIs inhibit cell-cycle progression (in non-infected cells) only after 1224 h.35,36,38 Thus, inhibition of HSV transcription (and hence of HSV replication) by P-PCIs is not secondary to their effects on cell-cycle progression.
Roscovitine has been shown recently to inhibit phosphorylation and other as yet uncharacterized post-translational modifications of two HSV regulatory proteins, ICP0 and ICP4.52,53 Roscovitine was further found to inhibit the transcriptional regulatory activity of one of these proteins, ICP0.53 However, roscovitine also inhibits HSV transcription independently of its effects on ICP0,36,38 and HSV DNA replication in the presence of viral DNA replication proteins37 (viral DNA replication per se does not require direct participation of ICP0 or ICP4). Thus, the effects of roscovitine on ICP0 and ICP4 do not account for all of the multiple effects of roscovitine on viral functions. It is likely that roscovitine inhibits phosphorylation of cellular proteins whose activities are required for HSV replication. The specific mechanisms whereby roscovitine (and other PCIs) inhibit HSV functions are currently under active investigation.
In contrast to viral transcription and DNA replication, viral protein synthesis (from pre-made transcripts) is not inhibited by P-PCIs.36,37 In this regard, cdks are not required for translation of cellular transcripts either. As expected from the multiple viral functions that are inhibited by P-PCIs, P-PCIs inhibit HSV replication even if the treatment is started at relatively late times after infection.36,37
It has been hypothesized that inhibition of HSV transcription by P-PCIs might result from inhibition of the cdks that both participate in cellular transcription and are susceptible to PCIs, for instance cdk7 and perhaps cdk9.54 Remarkably, however, P-PCIs inhibit HSV-1 transcription but not cellular transcription (which requires cdk7 and cdk9).33,35,36,39 Moreover, the complexes engaged in transcription of HSV genomes are depleted in both cdk7 and one of its activators (TFIIE), indicating that cdk7 likely plays no critical role in HSV transcription.55 Finally, P-PCIs inhibit HSV DNA replication, whereas cdk7 or 9 is not involved in DNA replication. Thus cdk7 or 9 does not appear to mediate all the multiple inhibitory effects of P-PCIs on HSV replication. We and others have postulated that cdk1 or 2 is the P-PCI-sensitive cdk that is required for HSV-1 replication.36,43,52 In support of this hypothesis, HSV establishes latent, but not productive, infections in resting neurons, cells that express no cdk1 or 251 but express cdk757 and cdk9 (unpublished observations). Furthermore, we have recently observed that HSV reactivates from latency specifically in neurons that express cdk2 and its cyclin partners (but not cdk1), and that roscovitine inhibited HSV reactivation from latency.51 It has been shown recently that HSV replicates more efficiently if (wild-type) cells are infected when they express highest levels of cdk2 activity.54 This correlation, however, was lost in p130Rb2 /, in which HSV could not replicate efficiently whereas cdk2 was highly active.53 Although the authors interpreted these results as indicating that cdk2 inhibits HSV replication, an alternative explanation is that p130Rb2 phosphorylated by cdk2 plays a major role in HSV replication. Further experiments are necessary to clarify the role of the differentially phosphorylated forms of p130Rb2 in HSV replication.
PCIs also inhibit replication of varicella-zoster virus (VZV), HCMV and HIV-1, and in all three cases inhibition of viral replication appears to be mediated by inhibition of cellular cdks, and not by direct inhibition of viral proteins.32,34,40,41,57,58 However, inhibition of which specific cdks accounts for the effects of PCIs against these viruses has yet to be determined. For VZV, it has been demonstrated that cdk1 is required for phosphorylation of a structural viral protein (gI), and that this phosphorylation is inhibited by roscovitine.59 Whether this effect of roscovitine fully accounts for its anti-VZV effect has not been addressed, but recent evidence indicates that cdk2 is required for transcription and replication of the VZV genome.58 Thus, roscovitine may inhibit VZV replication as a result of inhibition of cdk2 as well as cdk1. For HCMV, it has been shown that cdk2 is activated in infected cells, and that non-pharmacological inhibition of cdk2 resulted in inhibition of viral DNA replication.34 However, it should be noted that cdk1 activity was most likely inhibited in these experiments secondarily to the inhibition of cdk2.
Regarding HIV-1, several reports postulate that cdk9, or perhaps cdk7, is the cellular target of the anti-HIV activity of some PCIs. Thus, flavopiridol was found to inhibit HIV-1 transcription at concentrations that are below the cdk1/cdk2-inhibitory concentrations.32 Moreover, flavopiridol specifically inhibited elongation of HIV-1 transcripts, the HIV-1 function that requires cdk9.32 Flavopiridol also inhibits cdk7, which appears to be required for HIV-1 transcription as well, and a substrate-like inhibitor of cdk7 was found to inhibit HIV-1 transcription and replication as efficiently as flavopiridol.60 However, when Flores and co-workers40,41 conducted a large-scale screening (more than 100 000 compounds), all the drugs that inhibited HIV-1 transcription and replication were found (or previously known) to be cdk9 inhibitors. Many of these drugs were not very potent inhibitors of cdk1 or 7, whereas their activities against other cdks, such as cdk2 or cdk4, were not evaluated. In a follow-up study, non-pharmacological inhibition of cdk9 was also found to inhibit HIV-1 replication. Finally, flavopiridol has been shown recently to inhibit cellular transcription,33 which requires cdk9 (and cdk7) activity. Thus, flavopiridol most likely inhibits HIV-1 transcription because of its inhibitory activity on cdk9.
Whether inhibition of HIV-1 replication by P-PCIs is also a consequence of inhibition of cdk7 or 9 has not been established. In contrast to flavopiridol, P-PCIs inhibit HIV-1 replication only at concentrations that are inhibitory for cdks1 and 2, and for cell-cycle progression.17,39 Further contrasting with flavopiridol, roscovitine does not inhibit cellular transcription at these concentrations,33 suggesting that cdks7 and 9 are not inhibited efficiently in vivo by antiviral concentrations of the drug. Furthermore, roscovitine inhibits both Tat-dependent and independent HIV-1 transcription,17,61 whereas cdk9 is required for Tat-dependent transcription only. Thus, the limited and circumstantial evidence available to date suggests that inhibition of HIV-1 replication by P-PCIs may not be a consequence of inhibition of cdk7 or 9 exclusively. Clearly, the identification of the targets of P-PCIs that mediate their anti-HIV effects is an area that merits further study.
Interestingly, and as discussed above, P-PCIs may inhibit different functions for different viruses. PCIs inhibit HSV-1 transcription and DNA replication, while having been shown to inhibit only transcription of HIV-1 and only DNA replication of HCMV.32,34,40,41,57 These differences may indicate that different cdks are required for the different functions of different viruses. However, the effects of PCIs on HIV-1 functions other than transcription, or on HCMV functions other than DNA replication, have not been evaluated exhaustively. Moreover, the effects of PCIs on other viral functions, such as reactivation from latency, are just starting to be evaluated.17,51,61 It is thus possible that PCIs inhibit more viral functions than the ones currently recognized. PCIs have also been reported to induce apoptosis of HCMV- and HIV-1-infected cells (but not of control uninfected cells), without inducing release of infectious viral progeny.17,34 These pro-apoptotic effects of PCIs could result from unmasking of the pro-apoptotic effects of HCMV and HIV-1 themselves, viruses which both promote and inhibit apoptosis. The viral anti-apoptotic proteins would not be able to counteract the viral pro-apoptotic effects if PCIs inhibited the former but not the latter. Regardless of the specifics of the mechanisms, selective killing of infected cells could contribute greatly to the antiviral activities of these compounds. The contribution of apoptosis to the antiviral effects of these drugs should be evaluated carefully.
Our attempts to isolate PCI-resistant strains of HSV-1 have been unsuccessful to date35 and (unpublished observations), which is consistent with the fact that several HSV-1 functions are inhibited by PCIs. No PCI-resistant strain of HCMV or HIV-1 has been reported either, although an exhaustive effort to isolate such an HIV-1 mutant was undertaken recently.17 The number of viral functions that require the cellular protein(s) inhibited by a drug plays a major role in determining how easily viruses may develop resistance to this drug. Viruses should be able to develop resistance easily if the protein targeted by the drug is required to activate just one viral protein, but not if it is required to activate many viral functions. In the former scenario, a mutation in a single viral gene may result in a mutant viral protein that needs no activation by the cellular protein inhibited by the drug. In the latter case, multiple mutations would be required to generate a viral mutant that can replicate in the presence of the drug. In sum, the activity of a single drug that targets a cellular protein required for several viral functions is equivalent to the conjoint activities of several drugs where each targets a different viral function. Recently, Rokyta et al.62 have shown that deletion of a cellular protein that is required for a single viral function was only partially compensated by five co-compensatory mutations acting together with several other (non-compensatory) mutations. It can be inferred from these results that the antiviral activity of a drug that inhibits cellular proteins required for multiple viral functions will not be easily overcome by mutations in the viral genome. This hypothesis, however, will only be tested if, and after, PCIs are used as clinical antivirals extensively.
As expected for drugs that target cellular, not viral, proteins, P-PCIs are active against mutants of HSV-1 and HIV-1 that are resistant to multiple conventional antiviral drugs (which all target viral proteins).18 Thus, roscovitine and purvalanol are both active against HSV-1 mutants that are resistant to aciclovir and phosphonoacetic acid (a drug structurally related to foscarnet), and roscovitine is active against HIV-1 mutants that are resistant to multiple nucleoside or non-nucleoside reverse transcriptase and protease inhibitors (NRTIs, NNRTIs and PIs, respectively).18 Furthermore, the antiviral effects of P-PCIs against wild-type and drug-resistant strains of HSV-1 were additive to the antiviral effects of aciclovir.18 These results open the possibility of evaluating the antiviral effects of P-PCIs in clinical trials as components of a combination therapy. This approach would allow testing for the antiviral effects of PCIs in humans without suspending a (partially) effective antiviral treatment.
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PCIs appear to have no major toxicity for humans |
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In Phase I and II clinical trials as an anticancer drug, flavopiridol is proving to have no major toxicities at plasma concentrations above those required to inhibit cell-cycle progression and viral replication in vitro. Toxicity involved secretory diarrhoea (which responded to standard treatments), asthenia and fatigue, but, perhaps surprisingly, no anaemia or immunosuppression. In two recent Phase II clinical trials, flavopiridol treatment resulted in major fatigue in several patients.13,25 A relatively high incidence of thrombosis was also observed, but this effect appears to have been a consequence of the route of application (continuous infusion through an intravenous catheter), rather than of the drug itself.13,25 The toxicity of flavopiridol for humans is being evaluated further in several ongoing clinical trials against cancers.
Roscovitine is the second-best-studied PCI in vivo (after flavopiridol). It has proven non-toxic in several animal models (for examples, see69,70). In our own unpublished analyses, roscovitine had no toxic effects for mice, including no negative effect on weight gain during long-term treatments. The purified R-enantiomer of roscovitine has entered human clinical trials. In Phase I clinical trials, (R)-roscovitine has proven to be orally bioavailable and to have no acute toxicity. The first Phase I/II clinical trial to study the chronic (i.e. 1 year) toxicity for humans and the efficacy (against cancer) of (R)-roscovitine has just been completed. Thus, more detailed and larger-scale information about its toxicity for humans should soon be available.
Indirubins and paullones are other PCIs currently being tested as potential anticancer drugs. Indirubins have been used in humans for centuries as a component of Chinese and herbal medicines, with no major toxicities (for a discussion, see71 and its supplementary information). In contrast, the anticancer and cdk inhibitory activities of the paullones were discovered only recently.7274 Yet, this new family of PCIs is moving rapidly towards clinical trials as anticancer drugs. The antiviral properties of these two families of compounds, if any, have not been reported yet.
Two words of caution regarding the lack of toxicity of PCIs for humans are required. First, the number of human clinical trials is still rather small, and consequently relatively few patients have been treated with these drugs. More severe toxicities may become apparent in clinical trials involving larger numbers of patients. Secondly, the effects of these drugs on individuals possessing different genetic backgrounds have not been analysed. Thus, PCIs might be more toxic for individuals possessing certain alleles of genes whose products are involved in the pathways targeted by PCIs, or in their metabolism.
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Future directions |
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To address the effectiveness against viruses and toxicity of PCIs in vivo, clinical trials will ultimately be necessary. The toxicity of PCIs for humans will most likely be addressed during clinical trials against cancer. Regarding their effectiveness as antivirals, two approaches to test these drugs can be envisioned. The first approach would take advantage of the fact that the aetiopathogenesis of several proliferative diseases includes viral replication and/or viral gene expression. Thus, Kaposis sarcoma, HPV-induced cervical carcinoma, and HIV-associated nephropathy (HIVAN), among others, all involve both cellular proliferation as well as expression of viral genes and/or viral replication. In these cases, a single drug (a PCI) could target both replication of the aetiological agent (the virus) and the pathogenic mechanism (cell replication). Thus, the antiviral properties of PCIs could be tested in vivo first against diseases where the pathogenicity involves uncontrolled cell replication. From their known antiproliferative effects, it can be expected that PCIs would at least ameliorate the pathogenesis of these diseases. Meanwhile, the antiviral effects of PCIs could be analysed. Promisingly, flavopiridol was recently shown to ameliorate the pathogenesis of HIVAN, and to minimize HIV gene expression in a transgenic mouse model of this disease (P. Nelson, Mount Sinai School of Medicine, New York, USA, personal communication). No evidence of an antiproliferative effect of flavopiridol on kidneys was observed through an indirect analysis, even though the histopathological analyses demonstrated that the cell proliferation associated with this disease was ameliorated in the treated animals. Independently of these studies, (R)-roscovitine has just entered clinical trials against glomerulonephritis, a (non-virally induced) proliferative kidney disease. The possible therapeutic value of PCIs against HIVAN should be tested as soon as their (lack of) toxicity for humans is well established.
As discussed, all PCIs with proven antiviral activity target several cdks and even some other enzymes. The identities of the specific kinases, the inhibition of which accounts for the antiviral effects of PCIs, have yet to be established. Although it is now clear that the antiviral effect of PCIs results from inhibition of cellular, and not viral, proteins,17,18,32,34,51 it is as yet unknown whether inhibition of a single kinase accounts for all antiviral activities of PCIs. Alternatively, inhibition of replication of different viruses (or even inhibition of different functions of a single virus) may be a consequence of inhibition of different cellular targets of these drugs. Studies to address these outstanding and technically challenging issues are currently under way.
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Conclusion |
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This review has focused on the recent studies on the antiviral properties of PCIs. However, cellular proteins other than cdks are known to be required for viral replication and may be good targets for antiviral drugs.7591 Fruh, Gazhal and colleagues92 have recently proposed that those cellular proteins showing upregulation of expression during viral infection should be analysed as potential targets for antiviral drugs. This approach has been called virogenomics because the targets of antiviral drugs would be identified by a genomics approach.92
In conclusion, considering cellular proteins as potential targets for antiviral drugs may open the path to novel antivirals that could overcome some of the limitations of the ones available. Furthermore, this new approach would expand the pool of potential new antivirals significantly. Careful and extensive analyses of toxicity must be paramount in the evaluation of drugs that target cellular proteins as potential antivirals. However, in many instances the toxicity studies have been performed, or will be performed, during the development of these drugs for uses other than as antivirals. Several laboratories are currently studying the antiviral properties and in vivo safety of PCIs. We can expect that the full potential of PCIs as antiviral drugs will be thoroughly evaluated in the coming years.
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Acknowledgements |
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Footnotes |
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References |
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