Cyclin-dependent kinases as cellular targets for antiviral drugs

Luis M. Schang*

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


    Abstract
 Top
 Abstract
 Introduction
 Pharmacological cyclin-dependent...
 In vitro antiviral activity...
 PCIs appear to have...
 Future directions
 Conclusion
 References
 
Cyclin-dependent kinases (cdks) are required for replication of viruses that replicate only in dividing cells, such as adeno- and papillomaviruses. Recently, cdks have been shown to be required also for replication of viruses that can replicate in non-dividing cells, such as HIV-1 and herpes simplex virus types 1 and 2 (HSV-1 and -2). In these experiments, pharmacological cdk inhibitors (PCIs) were shown to have potent antiviral activity in vitro against HIV-1, HSV-1 and -2, human cytomegalovirus, varicella–zoster virus, and to inhibit specific functions of other viruses. Since two PCIs, flavopiridol and roscovitine, are proving to be non-toxic in human clinical trials against cancer, PCIs may be useful as antivirals. As significant advantages, PCIs are active in vitro against many viruses, including drug-resistant strains of HIV-1 and HSV-1, and mutant strains of HIV-1 or HSV-1 resistant to PCIs have not been identified in spite of intense efforts. Furthermore, the antiviral effects of a PCI and a conventional antiviral drug are additive. The aetiopathogenesis of several diseases, such as Kaposi’s sarcoma, HPV-induced cervical carcinoma and HIV-associated nephropathy (HIVAN), among others, includes replication or expression of proteins by viruses that require cdks. Thus, PCIs could target both the aetiological agent (the virus) and the pathogenic mechanisms (cell replication). Two important questions regarding the antiviral activities of PCIs are the focus of current research efforts, (i) the identity of the specific cdks that mediate the antiviral activities of PCIs, and (ii) whether PCIs have antiviral activity in vivo at non-toxic doses.


    Introduction
 Top
 Abstract
 Introduction
 Pharmacological cyclin-dependent...
 In vitro antiviral activity...
 PCIs appear to have...
 Future directions
 Conclusion
 References
 
In order to avoid toxicity, antiviral drugs are designed to target viral proteins. Many antivirals have been (and are being) developed successfully following this approach, and novel viral targets are being discovered continuously (for example, see1,2). Yet, antivirals that target viral proteins tend to have a relatively narrow spectrum of action and often need to be replaced with novel drugs because viruses develop resistance against the available ones. Moreover, two novel drugs must be developed for a combination therapy against a single virus, and small viruses encode only a limited number of proteins that can be targeted by drugs. In sum, as stated by Phelps and colleagues, ‘ . . . drug discovery leaders in the industry will have to champion new drug targets . . .’.3

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).


    Pharmacological cyclin-dependent kinase inhibitors
 Top
 Abstract
 Introduction
 Pharmacological cyclin-dependent...
 In vitro antiviral activity...
 PCIs appear to have...
 Future directions
 Conclusion
 References
 
Among several other cellular proteins, cyclin-dependent kinases (cdks) are required for replication of many clinically important viruses, such as papillomaviruses, human immunodeficiency virus type 1 (HIV-1), human cytomegalovirus (HCMV) and herpes simplex virus (HSV) types 1 and 2. Cdks are a family of serine/threonine kinases involved in regulation of cell division (cdks1, 2, 3, 4, 6 and 7), transcription (cdks7, 8 and 9) or maintenance of the structure of the cytoskeleton (cdk5) (Table 1). Because many cdks are critical regulators of cell division, pharmacological cdk inhibitors (PCIs) have been, and continue to be, developed as potential anticancer drugs.413 Surprisingly, PCIs are proving to have no major toxic effects for humans in clinical trials (as anticancer drugs). Several recent publications have proven that PCIs also have potent antiviral effects in vitro, as expected from the requirements for cdks in viral replication. If PCIs inhibited viral replication in vivo at non-toxic doses, they could be developed as clinical antiviral drugs in the near future.


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Table 1.  Known roles of cdks in cellular and viral functions
 
In comparison with protein cdk inhibitors, PCIs are low molecular weight compounds (of the order of 600 Da). PCIs contain cyclic carbon–nitrogen rings; otherwise their chemical structures have little in common (Figure 1). According to their specificities, PCIs can be loosely classified in three categories. ‘Non-specific PCIs’ inhibit cdks and a variety of other kinases, ‘broad-spectrum PCIs’ inhibit cdks indiscriminately and ‘narrow-spectrum’ (specific) PCIs inhibit preferentially only a subset of cdks (Table 2).



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Figure 1. Structures of selected PCIs. The structure of three ‘non-specific’ (a); three ‘wide-spectrum’ (b) and five ‘narrow-spectrum’ (c) PCIs are presented. Of the ‘wide-spectrum’ PCIs presented, flavopiridol is the least specific. Among the ‘narrow spectrum’ PCIs presented, the purine-derived PCIs preferentially inhibit cdks1, 2, 5 and 7; kenpaullone, cdks1, 2 and 5; and T276339, cdk1 and cdk9.

 

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Table 2.  Biochemical and cellular effects of selected PCIs
 
To date, the purine-derived PCIs (P-PCIs), such as olomoucine, roscovitine and purvalanols, are the most specific and most extensively studied PCIs.7,1416 P-PCIs are classified amongst the ‘specific’ PCIs, as they inhibit cdks1, 2, 5 and 7, but do not inhibit cdk 4, 6 or 8. P-PCIs inhibit cdk9 in vitro,17 but do not appear capable of binding to cdk9 in vivo.18 Extracellular receptor activated kinases (erks) are inhibited at concentrations ~50- to ~1000-fold above the cdk-inhibitory concentrations. Other enzymes studied (kinases, phosphatases, ATPases, topoisomerases, DNA polymerases, for a total of 38 enzymes) are not significantly inhibited by P-PCIs at concentrations >=100-fold above the cdk-inhibitory concentrations (Table 2). P-PCIs are competitive with ATP and bind to the ATP-binding pocket of the cdks, although they contact some amino acids that do not make contacts with ATP.15,19,20 These critical amino acids are not positionally conserved in other kinases and thus explain in part the specificity of PCIs. A recent study further indicates that the ‘cavities’ of the ATP-binding pockets of different cdks differ in size. As PCIs also differ in their molecular sizes, the fitting between the different PCIs and the ‘cavities’ of the ATP-binding pockets plays a major role in determining the specificity of at least some PCIs.21 All known cellular effects of P-PCIs appear to be accounted for by the inhibition of the target cdks.

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


    In vitro antiviral activity of PCIs
 Top
 Abstract
 Introduction
 Pharmacological cyclin-dependent...
 In vitro antiviral activity...
 PCIs appear to have...
 Future directions
 Conclusion
 References
 
In studies originally designed to identify cellular proteins required for viral replication, P-PCIs (olomoucine and roscovitine) were found to inhibit replication of HCMV.34 Since HCMV replicates in dividing cells and induces cdk2 activity, these findings were perhaps not too surprising. More surprisingly, several PCIs (olomoucine, roscovitine, purvalanol and flavopiridol) were also found to inhibit replication of HSV-1 and HIV-1,17,32,3539 two viruses that are capable of replicating in non-dividing cells. Moreover, several non-purine-derived PCIs (such as flavopiridol, DRB and T276339) were also found to inhibit replication of HIV-1.32,40,41

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 12–24 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.


    PCIs appear to have no major toxicity for humans
 Top
 Abstract
 Introduction
 Pharmacological cyclin-dependent...
 In vitro antiviral activity...
 PCIs appear to have...
 Future directions
 Conclusion
 References
 
A plethora of compounds commonly used as therapeutic human drugs target cellular proteins and have no toxic effects. For example, the statins, which are widely used to lower blood cholesterol levels (without major undesirable side-effects), target 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, a cellular enzyme that is required for essential post-translational protein modifications and cell-cycle progression. Nonetheless, the potential toxic effects that might result from inhibition of cellular cdks have constrained speculations that PCIs might serve as clinical antivirals. In the past few years, however, PCIs have been tested in animals and humans (as anticancer drugs) with surprisingly few and minor toxic effects. Plasma concentrations of PCIs above those that inhibit cell-cycle progression and viral replication in vitro have been shown to be non-toxic for mice, dogs and even humans.913,2225,6366 On first sight, this lack of toxicity of drugs that target proteins required for cell division may seem surprising. However, it must be considered that these drugs do not kill dividing cells (as other anticancer drugs do), but rather simply arrest cells in specific phases of the cell cycle. In fact, other drugs that inhibit cell-cycle progression are currently used in humans. The aforementioned statins, for example, are efficient inhibitors of the cell cycle,68,69 and have been used in humans for many years without major toxic effects.

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.


    Future directions
 Top
 Abstract
 Introduction
 Pharmacological cyclin-dependent...
 In vitro antiviral activity...
 PCIs appear to have...
 Future directions
 Conclusion
 References
 
Two questions that need to be addressed in the near future are the effectiveness of PCIs as antivirals in vivo, and the specific targets of these drugs that mediate their antiviral effects.

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, Kaposi’s 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.


    Conclusion
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 Abstract
 Introduction
 Pharmacological cyclin-dependent...
 In vitro antiviral activity...
 PCIs appear to have...
 Future directions
 Conclusion
 References
 
For some time, drugs that target cellular proteins have been proposed to possess the potential to become clinical antivirals. Shugar,75 for example, has reviewed studies on the antiviral properties of drugs that inhibit cellular (and viral) protein kinases. Hydroxyurea (HU), which inhibits cellular ribonucleotide reductase, has been tested repeatedly as a potential anti-HIV-1 drug (for recent discussion and examples, see76,77). Ribavirin, which has long been known to have antiviral effects, appears to target no viral proteins, and hence may be an antiviral that targets cellular proteins.79 Immunomodulatory drugs have been tested, and used, against several viruses. Yet no antiviral drug currently available has been designed to target cellular proteins that are required for viral replication. However, in the light of the recent results from several groups, perhaps the time has come to start evaluating whether cellular proteins should be considered valid targets for antiviral drugs.

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.


    Acknowledgements
 
I would like to acknowledge Dr Priscilla A. Schaffer for her support and collaboration from 1996 to 2000, and Drs Laurent Meijer, Peter Nelson, Fatah Kashanchi and Jennifer Moffat for extensive and productive discussions. I would also like to thank Drs Herbert E. M. Kaufman, Yung-Chi Cheng and Masanori Baba for their invitation to present our results to the antiviral community for the first time; and, finally, to extend my most sincere appreciation to the anonymous reviewers who provided excellent comments and suggestions. Our research is supported by operating grants MOP 49551 from the Canadian Institute for Health Research (CIHR) and 200100459 from the Alberta Heritage Foundation for Medical Research (AHFMR). L.M.S. is supported by the CIHR and the AHFMR, and wishes to acknowledge the support from the Department of Biochemistry, the Department of Medical Microbiology and Immunology and the Faculty of Medicine of the University of Alberta.


    Footnotes
 
* Tel: +1-780-492-6265; Fax: +1-780-492-3383; E-mail: luis.schang{at}ualberta.ca Back


    References
 Top
 Abstract
 Introduction
 Pharmacological cyclin-dependent...
 In vitro antiviral activity...
 PCIs appear to have...
 Future directions
 Conclusion
 References
 
1 . Crute, J. J., Grygon, C. A., Hargrave, K. D., Simoneau, B., Faucher, A. M., Bolger, G. et al. (2002). Herpes simplex virus helicase-primase inhibitors are active in animal models of human disease. Nature Medicine 8, 386–91.[ISI][Medline]

2 . Kleymann, G., Fischer, R., Betz, U. A. K., Hendrix, M., Bender, W., Schneider, U. et al. (2002). New helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease. Nature Medicine 8, 392–8.[ISI][Medline]

3 . Underwood, M. R., Shewchuk, L. M., Hassell, A. M. & Phelps, W. C. (2000). Searching for antiviral drugs for human papillomaviruses. Antiviral Therapy 5, 229–42.[ISI][Medline]

4 . Meijer, L. & Damiens, E. (2002). CDK inhibitors: small molecular weight compounds. In Tumor Suppressing Viruses, Genes and Drugs: Innovative Cancer Therapy Approaches (Maruta, H., Ed.), pp. 145–67. Academic Press, New York, NY, USA.

5 . Arris, C. E., Boyle, F. T., Calvert, A. H., Curtin, N. J., Endicott, J. A., Garman, E. F. et al. (2000). Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles. Journal of Medicinal Chemistry 43, 2797–804.[ISI][Medline]

6 . Kim, K. S., Sack, J. S., Tokarski, J. S., Qian, L., Chao, S. T., Leith, L. et al. (2000). Thio- and oxoflavopiridols, cyclin-dependent kinase 1-selective inhibitors: synthesis and biological effects. Journal of Medicinal Chemistry 43, 4126–34.[ISI][Medline]

7 . Meijer, L., Borgne, A., Mulner, O., Chong, J. P., Blow, J. J., Inagaki, N. et al. (1997). Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. European Journal of Biochemistry 243, 527–36.[Abstract]

8 . Murthi, K. K., Dubay, M., McClure, C., Brizuela, L., Boisclair, M. D., Worland, P. J. et al. (2000). Structure–activity relationship studies of flavopiridol analogues. Bioorganic and Medicinal Chemistry Letters 10, 1037–41.[Medline]

9 . Sausville, E. A., Johnson, J., Alley, M., Zaharevitz, D. & Senderowicz, A. M. (2000). Inhibition of CDKs as a therapeutic modality. Annals of the New York Academy of Sciences 910, 207–22.[Abstract/Free Full Text]

10 . Senderowicz, A. M., Headlee, D., Stinson, S. F., Lush, R. M., Kalil, N., Villalba, L. et al. (1998). Phase I trial of continuous infusion flavopiridol, a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasms. Journal of Clinical Oncology 16, 2986–99.[Abstract]

11 . Senderowicz, A. M. (1999). Flavopiridol: the first cyclin-dependent kinase inhibitor in human clinical trials. Investigational New Drugs 17, 313–20.[ISI][Medline]

12 . Senderowicz, A. M. & Sausville, E. A. (2000). Preclinical and clinical development of cyclin-dependent kinase modulators. Journal of the National Cancer Institute 92, 376–87.[Abstract/Free Full Text]

13 . Stadler, W. M., Vogelzang, N. J., Amato, R., Sosman, J., Taber, D., Liebowitz, D. et al. (2000). Flavopiridol, a novel cyclin-dependent kinase inhibitor, in metastatic renal cancer: a University of Chicago Phase II Consortium study. Journal of Clinical Oncology 18, 371–5.[Abstract/Free Full Text]

14 . Vesely, J., Havlicek, L., Strnad, M., Blow, J. J., Donella-Deana, A., Pinna, L. et al. (1994). Inhibition of cyclin-dependent kinases by purine analogues. European Journal of Biochemistry 224, 771–86.[Abstract]

15 . Gray, N. S., Wodicka, L., Thunnissen, A. M., Norman, T. C., Kwon, S., Espinoza, F. H. et al. (1998). Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors. Science 281, 533–8.[Abstract/Free Full Text]

16 . Legraverend, M., Ludwig, O., Bisagni, E., Leclerc, S., Meijer, L., Giocanti, N. et al. (1999). Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors. Bioorganic and Medicinal Chemistry 7, 1281–93.[ISI][Medline]

17 . Wang, D., de la Fuente, C., Deng, L., Wang, L., Zilberman, I., Eadie, C. et al. (2001). Inhibition of human immunodeficiency virus type 1 transcription by chemical cyclin-dependent kinase inhibitors. Journal of Virology 75, 7266–79. [Abstract/Free Full Text]

18 . Schang, L. M., Bantly, A., Knockaert, M., Shaheen, F., Meijer, L., Malim, M. H. et al. (2002). Pharmacological cyclin-dependent kinase inhibitors inhibit replication of wild type and drug-resistant strains of HSV and HIV-1 by targeting cellular, not viral proteins. Journal of Virology 76, 7874–82.[Abstract/Free Full Text]

19 . Schulze-Gahmen, U., Brandsen, J., Jones, H. D., Morgan, D. O., Meijer, L., Vesely, J. et al. (1995). Multiple modes of ligand recognition: crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors, olomoucine and isopentenyladenine. Proteins 22, 378–91.[ISI][Medline]

20 . De Azevedo, W. F., Leclerc, S., Meijer, L., Havlicek, L., Strnad, M. & Kim, S. H. (1997). Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine. European Journal of Biochemistry 243, 518–26.[Abstract]

21 . Ikuta, M., Kamata, K., Fukasawa, K., Honma, T., Machida, T., Hirai, H. et al. (2001). Crystallographic approach to identification of cyclin-dependent kinase 4 (CDK4)-specific inhibitors by using CDK4 mimic CDK2 protein. Journal of Biological Chemistry 276, 27548–54.[Abstract/Free Full Text]

22 . Sausville, E. A., Zaharevitz, D., Gussio, R., Meijer, L., Louarn-Leost, M., Kunick, C. et al. (1999). Cyclin-dependent kinases: initial approaches to exploit a novel therapeutic target. Pharmacology and Therapeutics 82, 285–92.[Medline]

23 . Innocenti, F., Stadler, W. M., Iyer, L., Ramirez, J., Vokes, E. E. & Ratain, M. J. (2000). Flavopiridol metabolism in cancer patients is associated with occurrence of diarrhea. Clinical Cancer Research 6, 3400–5.[Abstract/Free Full Text]

24 . Stinson, S. F., Hill, K., Siford, T. J., Phillips, L. R. & Daw, T. W. (1998). Determination of flavopiridol (L86 8275; NSC 649890) in human plasma by reversed-phase liquid chromatography with electrochemical detection. Cancer Chemotherapy and Pharmacology 42, 261–5.[ISI][Medline]

25 . Schwartz, G. K., Ilson, D., Saltz, L., O’Reilly, E., Tong, W., Maslak, P. et al. (2001). Phase II study of the cyclin-dependent kinase inhibitor flavopiridol administered to patients with advanced gastric carcinoma. Journal of Clinical Oncology 19, 1985–92.[Abstract/Free Full Text]

26 . Leclerc, S., Garnier, M., Hoessel, R., Marko, D., Bibb, J. A., Snyder, G. L. et al. (2000). Indirubins inhibit glycogen synthase kinase-3beta and CDK5/P25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer’s disease. A property common to most cyclin-dependent kinase inhibitors? Journal of Biological Chemistry 276, 251–60.[Abstract/Free Full Text]

27 . Oikonomakos, N. G., Schnier, J. B., Zographos, S. E., Skamnaki, V. T., Tsitsanou, K. E. & Johnson, L. N. (2000). Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site. Journal of Biological Chemistry 275, 34566–73.[Abstract/Free Full Text]

28 . Bible, K. C., Bible, R. H., Jr, Kottke, T. J., Svingen, P. A., Xu, K., Pang, Y. P. et al. (2000). Flavopiridol binds to duplex DNA. Cancer Research 60, 2419–28.[Abstract/Free Full Text]

29 . Hooijberg, J. H., Broxterman, H. J., Scheffer, G. L., Vrasdonk, C., Heijn, M., de Jong, M. C. et al. (1999). Potent interaction of flavopiridol with MRP1. British Journal of Cancer 81, 269–76.[ISI][Medline]

30 . Hooijberg, J. H., Broxterman, H. J., Heijn, M., Fles, D. L., Lankelma, J. & Pinedo, H. M. (1997). Modulation by (iso)flavonoids of the ATPase activity of the multidrug resistance protein. FEBS Letters 413, 344–8.[ISI][Medline]

31 . De Azevedo, W. F., Jr, Mueller-Dieckmann, H. J., Schulze-Gahmen, U., Worland, P. J., Sausville, E. & Kim, S. H. (1996). Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase. Proceedings of the National Academy of Sciences, USA 93, 2735–40.[Abstract/Free Full Text]

32 . Chao, S. H., Fujinaga, K., Marion, J. E., Taube, R., Sausville, E. A., Senderowicz, A. M. et al. (2000). Flavopiridol inhibits P-TEFb and blocks HIV-1 replication. Journal of Biological Chemistry 275, 28345–8.[Abstract/Free Full Text]

33 . Lam, L., Pickeral, O., Peng, A., Rosenwald, A., Hurt, E., Giltnane, J. et al. (2001). Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol. Genome Biology 2, 0041.1–0041.11.

34 . Bresnahan, W. A., Boldogh, I., Chi, P., Thompson, E. A. & Albrecht, T. (1997). Inhibition of cellular Cdk2 activity blocks human cytomegalovirus replication. Virology 231, 239–47.[ISI][Medline]

35 . Schang, L. M., Phillips, J. & Schaffer, P. A. (1998). Requirement for cellular cyclin-dependent kinases in herpes simplex virus replication and transcription. Journal of Virology 72, 5626–37.[Abstract/Free Full Text]

36 . Schang, L. M., Rosenberg, A. & Schaffer, P. A. (1999). Transcription of herpes simplex virus immediate-early and early genes is inhibited by roscovitine, an inhibitor specific for cellular cyclin-dependent kinases. Journal of Virology 73, 2161–72.[Abstract/Free Full Text]

37 . Schang, L. M., Rosenberg, A. & Schaffer, P. A. (2000). Roscovitine, a specific inhibitor of cellular cyclin-dependent kinases, inhibits herpes simplex virus DNA synthesis in the presence of viral early proteins. Journal of Virology 74, 2107–20.[Abstract/Free Full Text]

38 . Jordan, R., Schang, L. & Schaffer, P. A. (1999). Transactivation of herpes simplex virus type 1 immediate-early gene expression by virion-associated factors is blocked by an inhibitor of cyclin-dependent protein kinases. Journal of Virology 73, 8843–7.[Abstract/Free Full Text]

39 . Nelson, P. J., Gelman, I. H. & Klotman, P. E. (2001). Suppression of HIV-1 expression by inhibitors of cyclin-dependent kinases promotes differentiation of infected podocytes. Journal of the American Society of Nephrology 12, 2827–31.[Abstract/Free Full Text]

40 . Mancebo, H. S., Lee, G., Flygare, J., Tomassini, J., Luu, P., Zhu, Y. et al. (1997). P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes and Development 11, 2633–44.[Abstract/Free Full Text]

41 . Flores, O., Lee, G., Kessler, J., Miller, M., Schlief, W., Tomassini, J. et al. (1999). Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication. Proceedings of the National Academy of Sciences, USA 96, 7208–13.[Abstract/Free Full Text]

42 . Advani, S. J., Brandimarti, R., Weichselbaum, R. R. & Roizman, B. (2000). The disappearance of cyclins A and B and the increase in activity of the G(2)/M-phase cellular kinase cdc2 in herpes simplex virus 1-infected cells require expression of the alpha22/U(S)1.5 and U(L)13 viral genes. Journal of Virology 74, 8–15.[Abstract/Free Full Text]

43 . Advani, S. J., Weichselbaum, R. R. & Roizman, B. (2000). The role of cdc2 in the expression of herpes simplex virus genes. Proceedings of the National Academy of Sciences, USA 97, 10 996–1001.

44 . Yanagi, K., Talavera, A., Nishimoto, T. & Rush, M. G. (1978). Inhibition of herpes simplex virus type 1 replication in temperature-sensitive cell cycle mutants. Journal of Virology 25, 42–50.[ISI][Medline]

45 . Umene, K. & Nishimoto, T. (1996). Inhibition of generation of authentic genomic termini of herpes simplex virus type 1 DNA in temperature-sensitive mutant BHK-21 cells with a mutated CCG1/TAF(II)250 gene. Journal of Virology 70, 9008–12.[Abstract]

46 . Goto, H., Motomura, S., Wilson, A. C., Freiman, R. N., Nakabeppu, Y., Fukushima, K. et al. (1997). A single-point mutation in HCF causes temperature-sensitive cell-cycle arrest and disrupts VP16 function. Genes and Development 11, 726–37.[Abstract]

47 . Hilton, M. J., Mounghane, D., McLean, T., Contractor, N. V., O’Neil, J., Carpenter, K. et al. (1995). Induction by herpes simplex virus of free and heteromeric forms of E2F transcription factor. Virology 213, 624–38.[ISI][Medline]

48 . Hossain, A., Holt, T., Ciacci-Zanella, J. & Jones, C. (1997). Analysis of cyclin-dependent kinase activity after herpes simplex virus type 2 infection. Journal of General Virology 78, 3341–8.[Abstract]

49 . Kawaguchi, Y., Van Sant, C. & Roizman, B. (1997). Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3. Journal of Virology 71, 7328–36.[Abstract]

50 . Wilcock, D. & Lane, D. P. (1991). Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells. Nature 349, 429–31.[ISI][Medline]

51 . Schang, L. M., Bantly, A. & Schaffer, P. A. (2002). Explant-induced reactivation of herpes simplex virus occurs in neurons expressing nuclear cdks2 and 4. Journal of Virology 76, 7724–35.[Abstract/Free Full Text]

52 . Advani, S. J., Hagglund, R., Weichselbaum, R. R. & Roizman, B. (2001). Posttranslational processing of infected cell proteins 0 and 4 of herpes simplex virus 1 is sequential and reflects the subcellular compartment in which the proteins localize. Journal of Virology 75, 7904–12.[Abstract/Free Full Text]

53 . Davido, D. & Schaffer, P. (2002). The cyclin-dependent kinase inhibitor roscovitine inhibits the transactivating activity and alters the posttranslational modification of herpes simplex virus type 1 ICP0. Journal of Virology 76, 1077–88.[Abstract/Free Full Text]

54 . Ehmann, G. L., Burnett, H. A. & Bachenheimer, S. L. (2001). Pocket protein p130/Rb2 is required for efficient herpes simplex virus type 1 gene expression and viral replication. Journal of Virology 75, 7149–60.[Abstract/Free Full Text]

55 . Jenkins, H. L. & Spencer, C. A. (2001). RNA polymerase II holoenzyme modifications accompany transcription reprogramming in herpes simplex virus type 1-infected cells. Journal of Virology 75, 9872–917.[Abstract/Free Full Text]

56 . Hayes, T. E., Valtz, N. L. & McKay, R. D. (1991). Downregulation of CDC2 upon terminal differentiation of neurons. New Biologist 3, 259–69.[ISI][Medline]

57 . Zhu, Y., Pe’ery, T., Peng, J., Ramanathan, Y., Marshall, N., Marshall, T. et al. (1997). Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes and Development 11, 2622–32.[Abstract/Free Full Text]

58 . Taylor, S. L. & Moffat, J. F. (2001). V2V replication in vitro is prevented by Roscovitine, an inhibitor of the cell cycle. In Proceedings of the Fourth International Conference on V2V, Abstract A35. The V2V Research Foundation, La Jolla, CA, USA.

59 . Ye, M., Duus, K. M., Peng, J., Price, D. H. & Grose, C. (1999). Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase. Journal of Virology 73, 1320–30.[Abstract/Free Full Text]

60 . Okamoto, H., Cujec, T. P., Peterlin, B. M. & Okamoto, T. (2000). HIV-1 replication is inhibited by a pseudo-substrate peptide that blocks Tat transactivation. Virology 270, 337–44.[ISI][Medline]

61 . Pisell, T., Ho, O., Lee, G. & Butera, S. (2001). Spectrum of cdk-9 inhibitory activity against HIV-1 replication among various models of chronic and latent infection. Antiviral Chemistry and Chemotherapy 12, Suppl. 1, 33–41.[Medline]

62 . Rokyta, D., Badgett, M. R., Molineux, I. J. & Bull, J. J. (2002). Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus. Molecular Biology and Evolution 19, 230–8.[Abstract/Free Full Text]

63 . Arguello, F., Alexander, M., Sterry, J. A., Tudor, G., Smith, E. M., Kalavar, N. T. et al. (1998). Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against human leukemia and lymphoma xenografts. Blood 91, 2482–90.[Abstract/Free Full Text]

64 . Drees, M., Dengler, W. A., Roth, T., Labonte, H., Mayo, J., Malspeis, L. et al. (1997). Flavopiridol (L86–8275): selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. Clinical Cancer Research 3, 273–9.[Abstract]

65 . Patel, V., Senderowicz, A. M., Pinto, D., Jr, Igishi, T., Raffeld, M., Quintanilla-Martinez, L. et al. (1998). Flavopiridol, a novel cyclin-dependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis. Journal of Clinical Investigation 102, 1674–81.[Abstract/Free Full Text]

66 . Shenfeld, M., Senderowicz, A. M., Sausville, E. A. & Barrent, K. E. (1997). A novel chemotherapeutic agent, flavopiridol, modulates intestinal epithelial chloride secretion. Gastroenterology 112, A404.

67 . Keyomarsi, K., Sandoval, L., Band, V. & Pardee, A. B. (1991). Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Research 51, 3602–9.[Abstract]

68 . Jakobisiak, M., Bruno, S., Skierski, J. S. & Darzynkiewicz, Z. (1991). Cell cycle-specific effects of lovastatin. Proceedings of the National Academy of Sciences, USA 88, 3628–32.[Abstract]

69 . Pippin, J. W., Qu, Q., Meijer, L. & Shankland, S. J. (1997). Direct in vivo inhibition of the nuclear cell cycle cascade in experimental mesangial proliferative glomerulonephritis with Roscovitine, a novel cyclin-dependent kinase antagonist. Journal of Clinical Investigation 100, 2512–20.[Abstract/Free Full Text]

70 . Nutley, P. M., Goddard, L. R., Kelland, M., Valenti, L., Brunton, D., Eady, G. B. et al. (2000). Antitumour activity and oral bioavailability of the cyclin dependent kinase (CDK) inhibitor roscovitine. Clinical Cancer Research 6, Suppl., 317.[Free Full Text]

71 . Hoessel, R., Leclerc, S., Endicott, J. A., Nobel, M. E., Lawrie, A., Tunnah, P. et al. (1999). Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nature Cell Biology 1, 60–7.[ISI][Medline]

72 . Schultz, C., Link, A., Leost, M., Zaharevitz, D. W., Gussio, R., Sausville, E. A. et al. (1999). Paullones, a series of cyclin-dependent kinase inhibitors: synthesis, evaluation of CDK1/cyclin B inhibition, and in vitro antitumor activity. Journal of Medicinal Chemistry 42, 2909–19.[ISI][Medline]

73 . Zaharevitz, D. W., Gussio, R., Leost, M., Senderowicz, A. M., Lahusen, T., Kunick, C. et al. (1999). Discovery and initial characterization of the paullones, a novel class of small-molecule inhibitors of cyclin-dependent kinases. Cancer Research 59, 2566–9.[Abstract/Free Full Text]

74 . Kunick, C., Schultz, C., Lemcke, T., Zaharevitz, D. W., Gussio, R., Jalluri, R. K. et al. (2000). 2-Substituted paullones: CDK1/cyclin B-inhibiting property and in vitro antiproliferative activity. Bioorganic and Medicinal Chemistry Letters 10, 567–9.[Medline]

75 . Shugar, D. (1999). Viral and host-cell protein kinases: enticing antiviral targets and relevance of nucleoside, and viral thymidine, kinases. Pharmacology and Therapeutics 82, 315–35.[Medline]

76 . Zala, C., Rouleau, D. & Montaner, J. S. (2000). Role of hydroxyurea in treatment of disease due to human immunodeficiency virus infection. Clinical Infectious Diseases 30, S143–50.[ISI][Medline]

77 . Gibbs, M. A. & Sorensen, S. J. (2000). Hydroxyurea in the treatment of HIV-1. Annals of Pharmacotherapy 34, 89–93.[Abstract]

78 . Tam, R. C., Lau, J. Y. N. & Hong, Z. (2001). Mechanisms of action of ribavirin in antiviral therapies. Antiviral Chemistry and Chemotherapy 12, 261–72.[ISI][Medline]

79 . Pleschka, S., Wolff, T., Ehrhardt, C., Hobom, G., Planz, O., Rapp, U. R. et al. (2001). Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade. Nature Cell Biology 3, 301–5.[ISI][Medline]

80 . Karpas, A., Lowdell, M., Jacobson, S. K. & Hill, F. (1992). Inhibition of human immunodeficiency virus and growth of infected T cells by the immunosuppressive drugs cyclosporin A and FK 506. Proceedings of the National Academy of Sciences, USA 89, 8351–5.[Abstract]

81 . Rosenwirth, B., Billich, A., Datema, R., Donatsch, P., Hammerschmid, F., Harrison, R. et al. (1994). Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonimmunosuppressive cyclosporine analog. Antimicrobial Agents and Chemotherapy 38, 1763–72.[Abstract]

82 . Franke, E. K. & Luban, J. (1996). Inhibition of HIV-1 replication by cyclosporine A or related compounds correlates with the ability to disrupt the Gag–cyclophilin A interaction. Virology 222, 279–82.[ISI][Medline]

83 . Lori, F., Malykh, A. G., Foli, A., Maserati, R., De Antoni, A., Minoli, L. et al. (1997). Combination of a drug targeting the cell with a drug targeting the virus controls human immunodeficiency virus type 1 resistance. AIDS Research and Human Retroviruses 13, 1403–9.[ISI][Medline]

84 . Andrus, L., Szabo, P., Grady, R. W., Hanauske, A. R., Huima-Byron, T., Slowinska, B. et al. (1998). Antiretroviral effects of deoxyhypusyl hydroxylase inhibitors: a hypusine-dependent host cell mechanism for replication of human immunodeficiency virus type 1 (HIV-1). Biochemical Pharmacology 55, 1807–18.[ISI][Medline]

85 . Yura, Y., Kusaka, J., Tsujimoto, H., Yoshioka, Y., Yoshida, H. & Sato, M. (1997). Effects of protein tyrosine kinase inhibitors on the replication of herpes simplex virus and the phsophorylation of viral proteins. Intervirology 40, 7–14.[ISI][Medline]

86 . Everett, R. D., Orr, A. & Preston, C. M. (1998). A viral activator of gene expression functions via the ubiquitin–proteasome pathway. EMBO Journal 17, 7161–9.[Abstract/Free Full Text]

87 . Bonjean, K., De Pauw-Gillet, M. C., Defresne, M. P., Colson, P., Houssier, C., Dassonneville, L. et al. (1998). The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells. Biochemistry 37, 5136–46.[ISI][Medline]

88 . Kurokawa, M., Hozumi, T., Basnet, P., Nakano, M., Kadota, S., Namba, T. et al. (1998). Purification and characterization of eugeniin as an anti-herpesvirus compound from Geum japonicum and Syzygium aromaticum. Journal of Pharmacology and Experimental Therapeutics 284, 728–35.[Abstract/Free Full Text]

89 . Moreau, P., Anizon, F., Sancelme, M., Prudhomme, M., Bailly, C., Carrasco, C. et al. (1998). Syntheses and biological evaluation of indolocarbazoles, analogues of rebeccamycin, modified at the imide heterocycle. Journal of Medicinal Chemistry 41, 1631–40.[ISI][Medline]

90 . Briggs, C. J., Ott, D. E., Coren, L. V., Oroszlan, S. & Tozser, J. (1999). Comparison of the effect of FK506 and cyclosporin A on virus production in H9 cells chronically and newly infected by HIV-1. Archives of Virology 144, 2151–60.[ISI][Medline]

91 . Murata, T., Goshima, F., Koshizuka, T., Takakuwa, H. & Nishiyama, Y. (2001). A single amino acid substitution in the ICP27 protein of herpes simplex virus type 1 is responsible for its resistance to leptomycin B. Journal of Virology 75, 1039–43.[Abstract/Free Full Text]

92 . Fruh, K., Simmen, K., Luukkonen, B. G. M., Bell, Y. C. & Ghazal, P. (2001). Virogenomics: a novel approach to antiviral drug discovery. Drug Discovery Today 6, 621–7.[ISI][Medline]

93 . van den Heuvel, S. & Harlow, E. (1993). Distinct roles for cyclin-dependent kinases in cell cycle control. Science 262, 2050–4.[ISI][Medline]

94 . Nikolic, M., Dudek, H., Kwon, Y. T., Ramos, Y. F. & Tsai, L. H. (1996). The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes and Development 10, 816–25.[Abstract]

95 . Chae, T., Kwon, Y. T., Bronson, R., Dikkes, P., Li, E. & Tsai, L. H. (1997). Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality. Neuron 18, 29–42.[ISI][Medline]

96 . Gilmore, E. C., Ohshima, T., Goffinet, A. M., Kulkarni, A. B. & Herrup, K. (1998). Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex. Journal of Neuroscience 18, 6370–7.[Abstract/Free Full Text]

97 . Kwon, Y. T., Tsai, L. H. & Crandall, J. E. (1999). Callosal axon guidance defects in p35(–/–) mice. Journal of Comparative Neurology 415, 218–29.[ISI][Medline]

98 . Fesquet, D., Morin, N., Doree, M. & Devault, A. (1997). Is Cdk7/cyclin H/MAT1 the genuine cdk activating kinase in cycling Xenopus egg extracts? Oncogene 15, 1303–7.[ISI][Medline]

99 . Larochelle, S., Pandur, J., Fisher, R. P., Salz, H. K. & Suter, B. (1998). Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity. Genes and Development 12, 370–81.[Abstract/Free Full Text]

100 . Wu, L., Chen, P., Hwang, J. J., Barsky, L. W., Weinberg, K. I., Jong, A. et al. (1999). RNA antisense abrogation of MAT1 induces G1 phase arrest and triggers apoptosis in aortic smooth muscle cells. Journal of Biological Chemistry 274, 5564–72.[Abstract/Free Full Text]

101 . Pagano, M., Pepperkok, R., Lukas, J., Baldin, V., Ansorge, W., Bartek, J. et al. (1993). Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts. Journal of Cell Biology 121, 101–11.[Abstract]

102 . Pagano, M., Pepperkok, R., Verde, F., Ansorge, W. & Draetta, G. (1992). Cyclin A is required at two points in the human cell cycle. EMBO Journal 11, 961–71.[Abstract]

103 . . Schang, L. M. (2002). The cell cycle, cyclin-dependent kinases, and viral infections: new horizons and unexpected connections. In Progress in Cell Cycle Research, vol. 5 (Meijer, L., Jezequel, A. & Roberge, M., Eds). Plenum, London, UK (in press).

104 . Schang, L. M. (2001). Cellular proteins (cyclin dependent kinases) as potential targets for antiviral drugs. Antiviral Chemistry and Chemotherapy 12, 157–78.[Medline]

105 . Carlson, B. A., Dubay, M. M., Sausville, E. A., Brizuela, L. & Worland, P. J. (1996). Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cancer Research 56, 2973–8.[Abstract]

106 . Gray, N., Detivaud, L., Doerig, C. & Meijer, L. (1999). ATP-site directed inhibitors of cyclin-dependent kinases. Current Medicinal Chemistry 6, 859–75.[ISI][Medline]

107 . Alessi, F., Quarta, S., Savio, M., Riva, F., Rossi, L., Stivala, L. A. et al. (1998). The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity. Experimental Cell Research 245, 8–18.[ISI][Medline]

108 . Alevizopoulos, K., Catarin, B., Vlach, J. & Amati, B. (1998). A novel function of adenovirus E1A is required to overcome growth arrest by the CDK2 inhibitor p27(Kip1). EMBO Journal 17, 5987–97.[Abstract/Free Full Text]

109 . Binarova, P., Dolezel, J., Draber, P., Heberle-Bors, E., Strnad, M. & Bogre, L. (1998). Treatment of Vicia faba root tip cells with specific inhibitors to cyclin-dependent kinases leads to abnormal spindle formation. Plant Journal 16, 697–707.[ISI][Medline]

110 . Choi, K. S., Eom, Y. W., Kang, Y., Ha, M. J., Rhee, H., Yoon, J. W. et al. (1999). Cdc2 and Cdk2 kinase activated by transforming growth factor-beta1 trigger apoptosis through the phosphorylation of retinoblastoma protein in FaO hepatoma cells. Journal of Biological Chemistry 274, 31775–83.[Abstract/Free Full Text]

111 . David-Pfeuty, T. (1999). Potent inhibitors of cyclin-dependent kinase 2 induce nuclear accumulation of wild-type p53 and nucleolar fragmentation in human untransformed and tumor-derived cells. Oncogene 18, 7409–22.[ISI][Medline]

112 . Frade, J. M. (2000). Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones. Journal of Cell Science 113, 1139–48.[Abstract/Free Full Text]

113 . Hsu, S. L., Yin, S. C., Liu, M. C., Reichert, U. & Ho, W. L. (1999). Involvement of cyclin-dependent kinase activities in CD437-induced apoptosis. Experimental Cell Research 252, 332–41.[ISI][Medline]

114 . Iseki, H., Ko, T. C., Xue, X. Y., Seapan, A., Hellmich, M. R. & Townsend, C. M., Jr (1997). Cyclin-dependent kinase inhibitors block proliferation of human gastric cancer cells. Surgery 122, 187–94.[ISI][Medline]

115 . Iseki, H., Ko, T. C., Xue, X. Y., Seapan, A. & Townsend, C. M., Jr (1998). A novel strategy for inhibiting growth of human pancreatic cancer cells by blocking cyclin-dependent kinase activity. Journal of Gastrointestinal Surgery 2, 36–43.[Medline]

116 . Krucher, N. A., Meijer, L. & Roberts, M. H. (1997). The cyclin-dependent kinase (cdk) inhibitors, olomoucine and roscovitine, alter the expression of a molluscan circadian pacemaker. Cellular and Molecular Neurobiology 17, 495–507.[ISI][Medline]

117 . Krude, T. (2000). Initiation of human DNA replication in vitro using nuclei from cells arrested at an initiation-competent state. Journal of Biological Chemistry 275, 13699–707.[Abstract/Free Full Text]

118 . Lee, H. R., Chang, T. H., Tebalt, M. J., 3rd, Senderowicz, A. M. & Szabo, E. (1999). Induction of differentiation accompanies inhibition of Cdk2 in a non-small cell lung cancer cell line. International Journal of Oncology 15, 161–6.[ISI][Medline]

119 . Matsumoto, Y., Hayashi, K. & Nishida, E. (1999). Cyclin-dependent kinase 2 (Cdk2) is required for centrosome duplication in mammalian cells. Current Biology 9, 429–32.[ISI][Medline]

120 . Mermillod, P., Tomanek, M., Marchal, R. & Meijer, L. (2000). High developmental competence of cattle oocytes maintained at the germinal vesicle stage for 24 hours in culture by specific inhibition of MPF kinase activity. Molecular Reproduction and Development 55, 89–95.[ISI][Medline]

121 . Mgbonyebi, O. P., Russo, J. & Russo, I. H. (1998). Roscovitine inhibits the proliferative activity of immortal and neoplastic human breast epithelial cells. Anticancer Research 18, 751–5.[ISI][Medline]

122 . Padmanabhan, J., Park, D. S., Greene, L. A. & Shelanski, M. L. (1999). Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis. Journal of Neuroscience 19, 8747–56.[Abstract/Free Full Text]

123 . Patrick, G. N., Zhou, P., Kwon, Y. T., Howley, P. M. & Tsai, L. H. (1998). p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) is degraded by the ubiquitin–proteasome pathway. Journal of Biological Chemistry 273, 24057–64.[Abstract/Free Full Text]

124 . Planchais, S., Glab, N., Trehin, C., Perennes, C., Bureau, J. M., Meijer, L. et al. (1997). Roscovitine, a novel cyclin-dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY-2 cell suspension. Plant Journal 12, 191–202.[ISI][Medline]

125 . Rousseau, A. & Vilain, J. P. (2000). Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs. Zygote 8, 3–14.[ISI][Medline]

126 . Sankrithi, N. &Eskin, A. (1999). Effects of cyclin-dependent kinase inhibitors on transcription and ocular circadian rhythm of Aplysia. Journal of Neurochemistry 72, 605–13.[ISI][Medline]

127 . Schutte, B., Nieland, L., van Engeland, M., Henfling, M. E., Meijer, L. & Ramaekers, F. C. (1997). The effect of the cyclin-dependent kinase inhibitor olomoucine on cell cycle kinetics. Experimental Cell Research 236, 4–15.[ISI][Medline]

128 . Yakisich, J. S., Boethius, J., Lindblom, I. O., Wallstedt, L., Vargas, V. I., Siden, A. et al. (1999). Inhibition of DNA synthesis in human gliomas by roscovitine. Neuroreport 10, 2563–7.[ISI][Medline]

129 . Yakisich, J. S., Siden, A., Idoyaga Vargas, V., Eneroth, P. & Cruz, M. (1998). Early inhibition of DNA synthesis in the developing rat cerebral cortex by the purine analogues olomoucine and roscovitine. Biochemical and Biophysical Research Communications 243, 674–7.[ISI][Medline]

130 . Abraham, R. T., Acquarone, M., Andersen, A., Asensi, A., Belle, R., Berger, F. et al. (1995). Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases. Biology of the Cell 83, 105–20.[ISI][Medline]

131 . Buquet-Fagot, C., Lallemand, F., Montagne, M. N. & Mester, J. (1997). Effects of olomoucine, a selective inhibitor of cyclin-dependent kinases, on cell cycle progression in human cancer cell lines. Anti-Cancer Drugs 8, 623–31.[ISI][Medline]

132 . Corellou, F., Bisgrove, S. R., Kropf, D. L., Meijer, L., Kloareg, B. & Bouget, F. Y. (2000). A S/M DNA replication checkpoint prevents nuclear and cytoplasmic events of cell division including centrosomal axis alignment and inhibits activation of cyclin-dependent kinase-like proteins in fucoid zygotes. Development 127, 1651–60.[Abstract/Free Full Text]

133 . Glab, N., Labidi, B., Qin, L. X., Trehin, C., Bergounioux, C. & Meijer, L. (1994). Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions. FEBS Letters 353, 207–11.[ISI][Medline]

134 . Huang, D., Farkas, I. & Roach, P. J. (1996). Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae. Molecular and Cellular Biology 16, 4357–65.[Abstract]

135 . Kwon, Y. G., Lee, S. Y., Choi, Y., Greengard, P. & Nairn, A. C. (1997). Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase. Proceedings of the National Academy of Sciences, USA 94, 2168–73.[Abstract/Free Full Text]

136 . Malecz, N., Foisner, R., Stadler, C. & Wiche, G. (1996). Identification of plectin as a substrate of p34cdc2 kinase and mapping of a single phosphorylation site. Journal of Biological Chemistry 271, 8203–8.[Abstract/Free Full Text]

137 . Park, D. S., Farinelli, S. E. & Greene, L. A. (1996). Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC12 cells and sympathetic neurons. Journal of Biological Chemistry 271, 8161–9.[Abstract/Free Full Text]

138 . Park, D. S., Morris, E. J., Greene, L. A. & Geller, H. M. (1997). G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis. Journal of Neuroscience 17, 1256–70.[Abstract/Free Full Text]

139 . Paulson, J. R., Patzlaff, J. S. & Vallis, A. J. (1996). Evidence that the endogenous histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not protein phosphatase 2A. Journal of Cell Science 109, 1437–47.[Abstract/Free Full Text]

140 . Veeranna, Shetty, K. T., Amin, N., Grant, P., Albers, R. W. & Pant, H. C. (1996). Inhibition of neuronal cyclin-dependent kinase-5 by staurosporine and purine analogs is independent of activation by Munc-18. Neurochemical Research 21, 629–36.[ISI][Medline]

141 . Kaur, G., Stetler-Stevenson, M., Sebers, S., Worland, P., Sedlacek, H., Myers, C. et al. (1992). Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275. Journal of the National Cancer Institute 84, 1736–40.[Abstract]

142 . Losiewicz, M. D., Carlson, B. A., Kaur, G., Sausville, E. A. & Worland, P. J. (1994). Potent inhibition of CDC2 kinase activity by the flavonoid L86-8275. Biochemical and Biophysical Research Communications 201, 589–95.[ISI][Medline]

143 . Chang, Y. T., Gray, N. S., Rosania, G. R., Sutherlin, D. P., Kwon, S., Norman, T. C. et al. (1999). Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors. Chemistry and Biology 6, 361–75.[ISI][Medline]

144 . Schnier, J. B., Kaur, G., Kaiser, A., Stinson, S. F., Sausville, E. A., Gardner, J. et al. (1999). Identification of cytosolic aldehyde dehydrogenase 1 from non-small cell lung carcinomas as a flavopiridol-binding protein. FEBS Letters 454, 100–4.[ISI][Medline]

145 . Marko, D., Schatzle, S., Friedel, A., Genzlinger, A., Zankl, H., Meijer, L. et al. (2001). Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells. British Journal of Cancer 84, 283–9.[ISI][Medline]