key: cord-0808077-sr4ap98j
authors: de Clercq, Erik; Murase, Junichi; Marquez, Victor E.
title: Broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at CTP synthetase
date: 1991-06-15
journal: Biochemical Pharmacology
DOI: 10.1016/0006-2952(91)90120-t
sha: fccbe2d1dab29119bf24e065b589725df5ae2c7a
doc_id: 808077
cord_uid: sr4ap98j

Abstract Cyclopentenylcytosine (Ce-Cyd) is a broad-spectrum antiviral agent active against DNA viruses [herpes (cytomegalo), pox (vaccinia)], (+)RNA viruses [picorna (polio, Coxsackie, rhino), toga (Sindbis, Semliki forest), corona], (−)RNA viruses [orthomyxo (influenza), paramyxo (parainfluenza, measles), arena (Junin, Tacaribe), rhabdo (vesicular stomatitis)] and (±)RNA viruses (reo). Ce-Cyd is a more potent antiviral agent than its saturated counterpart, cyclopentylcytosine (carbodine, C-Cyd). Ce-Cyd also has potent cytocidal activity against a number of tumor cell lines. The putative target enzyme for both the antiviral and antitumor action of Ce-Cyd is assumed to be the CTP synthetase that converts UTP to CTP. In keeping with this hypothesis was the finding that the antiviral and cytocidal effects of Ce-Cyd are readily reversed by Cyd and, to a lesser extent, Urd, but not by other nucleosides such as dThd or dCyd. In contrast, pyrazofurin and 6-azauridine, two nucleoside analogues that are assumed to interfere with OMP decarboxylase, another enzyme involved in the biosynthesis of pyrimidine ribonucleotides, potentiate the cytocidal activity of Ce-Cyd. Ce-Cyd should be further pursued, as such and in combination with OMP decarboxylase inhibitors, for its therapeutic potential in the treatment of both viral and neoplastic diseases.

such as dThd or dCyd. In contrast, pyrazofurin and 6-azauridine, two nucleoside analogues that are assumed to interfere with OMP decarboxylase, another enzyme involved in the biosynthesis of pyrimidine ribonucleotides, potentiate the cytocidal activity of Ce-Cyd. Ce-Cyd should be further pursued, as such and in combination with OMP decarboxylase inhibitors, for its therapeutic potential in the treatment of both viral and neoplastic diseases.

Various pyrimidine nucleoside analogues, i.e. 5fluoro-2'-deoxyuridine

[ 11, 5-iodo-2'-deoxyuridine [2] , (E)-5-(2-bromovinyl)-2'-deoxyuridine [3] , Sethyl-2'-deoxyuridine [4] , 3'-amino-2',3'-dideoxythymidine [S], 5-iodo-2'-deoxycytidine [6] and 5iodo -1 -(2 -deoxy -2 -fluoro -/3 -Darabinofurano-NH, NH2

N' 3 N'

N syl)cytosine [7] , are known as potent antiviral or antitumor agents. To make these nucleoside analogues resistant to degradation by nucleoside HO phosphorylases that cleave the N-glycosidic bond, carbocyclic derivatives of these nucleosides have been synthesized, whereby the sugar part is replaced by a cycloalkyl ring [l-7] . As a rule, these carbocyclic HOdI (-,' HO derivatives retained only part of the antitumor or OH HO OH antiviral activity of the parent compounds. Also, carbocyclic analogs, both cyclopentyl and cyclo-Ce-Cyd C-Cyd pentenyl derivatives, of the normal. nucleoside Fig. 1 . Formulae of cyclopentenylcytosine (Ce-Cyd) cytidine have been prepared, and these derivatives, and cyclopentylcytosine (C-Cyd, carbocyclic cytidine, termed C-Cyd (carbodine) and Ce-Cyd respectively carbodine). Ce-Cyd has shown antitumor activity in several human tumor xenografts in athymic mice; in viho it has proved active against herpesviruses (herpes Both C-Cyd and Ce-Cyd are assumed to interact simplex, cytomegalo and varicella-zoster), vaccinia, with CTP synthetase, the enzyme that converts UTP influenza A, vesicular stomatitis virus, Japanese to CTP, after they have been phospho~lated encephalitis virus and Punta Toro virus 1111.

intra~eliuIarly to the 5'-triphosphate [9, 12, 131 . Direct measurements with CTP synthetase (from t To whom all correspondence should be addressed at the murine leukemia L1210 cells) have indicated that Rega Institute for Medical Research, Minderbroedersstraat the 5'-triphosphate of Ce-Cyd is indeed inhibitory 10, B-3000 Leuven, Belgium.

to the enzyme [14] . The depletion of CTP pools that I Present address: Toyo Jozo Co., 4-5-13 Shibaura, results from such an inhibitory effect has been held Minato-ku, Tokyo 108, Japan.

responsible for the cytocidal action of Ce-Cyd in vitro [12] and its antitumor activity in uiuo (131. It has not been ascertained, however, whether the antiviral action of Ce-Cyd also results from an inhibitory effect on CTP synthetase. The present studies were undertaken to (it delineate the antiviral activity spectrum of Ce-Cyd (in comparison with the activity spectrum of C-Cyd [15]), and to (ii) assess the influence of exogenously added cytidine and other nucleosides on both the antiviral and cytocidal action of Ce-Cyd. In agreement with previous studies 1111, Ce-Cyd was found to be a highly potent inhibitor of both virus replication and tumor cell proliferation. It proved active against a remarkably broad range of RNA and DNA viruses. Both the antiviral and cytocidal action of Ce-Cyd could be reversed by Cyd, but not dThd or dCyd. Combination of Ce-Cyd with pyrazofurin or &azauridine, two compounds which are known to inhibit OMP decarboxylase, resulted in an enhancement of its cytocidal activity [16-191. [28] , and influenza types A, B. C.

Cells. The cell lines used in the antiviral activity assays were: PRK (primary rabbit kidney), HEL (human embryonic lung fibroblast). HeLa (a human epithelial cell line derived from a cervix carcinoma), Vero (a simian fibroblast cell line derived from African green monkey kidney), WI-38 (human embryonic lung fibroblast), MDCK (Madin-Darby canine kidney), EhSM (human embryonic skinmuscle fibroblast), RK13 (a rabbit kidney cell line). BSC-1A (a simian epithelial cell line derived from African green monkey kidney), GBK (Georgia bovine kidney), CV-1 (a simian fibroblast cell line derived from African green monkey kidney) and L-929 (a murine fibroblast cell line originally derived from an adult C3H mouse and mutagenized with methylcholanthrene).

The cell lines used in the cytocidal activity assays were, besides Vero and HeLa cells, murine leukemia (L1210). murine mammary carcinoma (FM3A). human B-lymphoblast (Raji) and human T lymphoblast (Molt/ 4F) cells. The cells were grown in Eagle's mjnimum essential medium supplemented with 10% fetal calf serum.

Antiviral activity. Inhibition of virus-induced cytopathogenicity was measured following wellestablished procedures [20.24] . In all viral cytopathogenicity assay,s the virus inoculum was 100 CClD~"permicrotiterwel~ (1 ~CID~~~corresponding to the virus stock dilution that proved infective for 50% of the cell cultures). In the cytomegalovirus assays, plaque formation was measured instead of viral cytopathogenicity.

In these assays the virus inoculum was 20 PFU (plaque forming units).

Anti~zeta6olic actiuiry. Inhibition of host cell macromolecule (DNA, RNA and protein synthesis) was monitored by incorporation of [nredzgl-"HjdThd, ~ytocidal activity. Inhibition of the proliferation of Vero, HeLa, L1210. FM3A. Raji and Molt/JF cells was assessed during their exponential growth phase and monitored by counting the number of viable cells (following staining with trypan blue). The procedure was similar to that initially described for L1210 ceils [30] .

[15] and two other broad-spectrum antiviral agents, neplanocin A 1241 and ribavirin [31] , against a wide variety of RNA and DNA viruses (Table 1) . Of the four compounds, Ce-Cyd showed the most pronounced antiviral activity. Against some viruses (i.e. TK-herpes simplex, vaccinia. polio. reo, Junin, Tacaribe, SSPE). Ce-Cyd proved effective at a 50% inhibitory concentration of ~0.1 pg/mL. whereas normal cell morphology was not altered (at concentrations up to 4OO~g/mL). Ce-Cyd was active against all viruses that are sensitive to C-Cyd, but. in addition, Ce-Cyd proved also active against picornaviruses (polio, Coxsackie. rhino) which are not sensitive to C-Cyd. Thus, the activity spectrum of Ce-Cyd includes all major virus groups [herpes (TK-herpes simplex, cytomegalo). pox (vaccinia), picorna ( polio, /I Data taken from Hosoya et al. [28] .

1 Data taken from Shigeta et al. [29] . ND, not determined.

Coxsackie, rhino), toga (Sindbis, Semliki forest), corona, orthomyxo (influenza), paramyxo (parainfluenza, measles), arena (Junin, Tacaribe), rhabdo (vesicular stomatitis) and reo] ( Table 1) . Ce-Cyd and C-Cyd were also examined for their inhibitory effect on the replication of human immunodeficiency virus type 1 (HIV-l) in (exponentiallygrowing) MT-4cells (a humanT-lymphocyte cell line). Ce-Cyd and C-Cyd were inhibitory to the growth of MT-4 cells at an lcso of 1.2 and 50 ng/mL, respectively. They were not inhibitory to HIV-l replication at concentrations below their ICKY for host cell growth (data not shown).

The antiviral potency of antiviral agents may differ considerably depending on the choice of the cells, as has been previously noted with C-Cyd and ribavirin when evaluated for their activity against vesicular stomatitis virus in different cell lines [15] .

In contrast with C-Cyd and ribavirin, however, Ce-Cyd inhibited the cytopathogenicity of vesicular stomatitis virus in all cell lines in which it was examined ( Table 2) . Differences between C-Cyd and Ce-Cyd were particularly striking in RK13 and BSC-lA, two cell lines in which Ce-Cyd exhibited marked antiviral activity whereas C-Cyd did not. The concentrations at which Ce-Cyd inhibited vesicular stomatitis virus in the different cell lines ranged from 0.1 to 2 pg/mL, whereas, again, no alteration of normal cell morphology was observed at C-Cyd concentrations up to 400 pg/mL. Ce-Cyd was not toxic to resting (confluent) cell monolayers, as used in the antiviral assays (Tables  1 and 2) , at concentrations up to 4OOpg/mL. However, Ce-Cyd proved inhibitory to (Vero) cell DNA and RNA synthesis (as monitored by the incorporation of [methyl-3H]dThd and [S3H]Urd, respectively), at a concentration of 1.4 and 0.7~g/ mL, respectively (Table 3) . These concentrations coincide with, or are only lo-to loo-fold higher than, the concentrations at which Ce-Cyd exhibit antiviral activity in Vero cells (Tables 1 and 2 ). Yet, it should be pointed out that during macromolecule synthesis the cells were in their exponential growth phase, whereas they were resting (confluent) in the antiviral activity assays.

Ce-Cyd proved inhibitory to the growth of HEL any of the viruses (listed in Table 1 ) that were found susceptible to Ce-Cyd.

To obtain further insight into the mechanism of action of Ce-Cyd at the cellular level, the reversing effects of exogenously added nucleosides (dThd, Urd, dCyd and Cyd) on the antiviral and cytocidal activity of Ce-Cyd was examined (Tables 5 and 6 ). The antiviral assays were carried out with four viruses, representative of the DNA virus (vaccinia). (-)RNA virus (vesicular stomatitis), (+-)RNA virus (reo) and (+)RNA virus (Semliki forest) groups. The deoxyribonucleosides dThd and dCyd did not affect the antiviral potency of Ce-Cyd. even if added at a concentration of lOOpg/mL (Table 5 ). In marked contrast, the ribonucleosides Cyd and, to a lesser extent, Urd reversed the activity of Ce-Cyd against all viruses tested: if added at 100 pg/mL, Cyd completely abolished the antiviral activity of Ce-Cyd. whereas Urd caused a reduction in antiviral potency (increase in ICKY) of about 25 to 50-fold. Even if added to a concentration of 10 pg/mL, Cyd annihilated the activity of Ce-Cyd against vaccinia virus and Semliki forest virus, whereas Urd reduced it by 3-to .5-fold.

Similar results were obtained with regard to the reversing effects of the nucleosides on the cytocidal activity of Ce-Cyd (Table 6 ). Thus, dThd and dCyd did not markedly influence the inhibitory effect of , DNA polymerase (cytarabine)] were examined in conjunction with Ce-Cyd. Whereas 5-fluorouracil, neplanocin A and cytarabine did not influence the inhibitory effect of Ce-Cyd on L1210 cell growth, pyrazofurin and Gazauridine potentiated the cytotidal activity of Ce-Cyd. All antimetabolites were Values are mean f SD. * Required to reduce L1210 cell proliferation (during the exponential growth phase) by 50%. The nucleoside analogues effected a 50% reduction in the growth of L1210 cells at the following concentrations: pyrazofurin (0.025 pg/mL), 6-azauridine (0.034 pg/ mL). 5-fluorouracil(O.32 pg/mL), neplanocin A (0.40 pg/mL) and cytarabine (0.016 ng/ mL). used at concentrations below and above their lcso for L1210 cell growth (Table 7) . Under these conditions, pyrazofurin and 6-azauridine, when added at a concentration of 0.1 ,ug/mL (which is above their lcso for L1210 cell growth), brought about a 6-to lo-fold decrease in the lcso of Ce-Cyd.

Various enzymes of both viral or cellular origin have been shown to interact as either activators or targets for antiviral agents. The activity spectrum of these antiviral agents is determined by the enzymes with which they interact. For example, acyclovir [9-(2-hydroxyethoxymethyl)guanine] needs to be phosphorylated by the virus-encoded thymidine kinase (TK) before it will interact, as a chain terminator, with the viral DNA polymerase [33] . Hence, acyclovir is only active against those viruses, in particular herpes simplex virus type 1 (HSV-1) and 2 (HSV-2). that encode a viral TK recognizing acyclovir as substrate. The phosphonylmethoxyalkyl derivatives, i.e. HPMPA [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine]

and HPMPC [(S)-l-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], are not dependent on the viral TK for their phosphorylation, and, hence, these compounds are effective against a much broader spectrum of viruses, including TK-herpesviruses and various other DNA viruses (i.e. adeno-, pox-, irido-and hepadnaviruses) ]341.

Most compounds that have been recognized as selective antiviral agents interact with a specific viral protein:

i.e. influenza A virus matrix (M,) protein (amantadine) [35] . HSV-1 DNA polymerase (acyclovir triphosphate) [36] , HIV-l reverse transcriptase (azidothymidine triphosphate) [37] . Yet, the target protein should not necessarily be of viral origin. Under certain conditions, host cell enzymes may serve as targets for antiviral agents. Thus. while not being a target for anti-HSV compounds in TK+ HSV-l-infected cells. thymidylate synthase may be an important target in TK-HSV-l-infected cells [21] . Also, S-adenosylhomocysteine (SAH) hydrolase, another host cell enzyme, has been identified as a target for the broad-spectrum antiviral action of several acyclic and carbocyclic adenosine analogues [38], including neplanocin A [24], 3-deazaneplanocin A and their 5'-nor derivatives [39] . Through their inhibitory effect on SAH hydrolase these compounds enhance the intracellular levels of SAH and thus inhibit transmethylation reactions starting from Sadenosylmethionine (SAM) as the methyl donor. This includes a number of transmethylation reactions catalysed by virus-associated methyltransferases that are required for the maturation of viral mRNAs.

Like SAH hydrolase, CTP synthetase is an example of a host cell enzyme that may be envisaged as a target enzyme for antiviral agents. CTP synthetase catalyses the last step in the biosynthesis of pyrimidine ribonucleoside 5'-triphosphates, that is the conversion of UTP to CTP (Fig. 2) . There is circumstantial evidence for the role of CTPsynthetase as target for the cytocidal activity of Ce-Cyd. As has been demonstrated in several cell lines, i.e. murine is closely correlated with the cytocidal activity of the compound. The depletion of CTP pools is most likely due to the inhibition of CTP synthetase, and, in fact, the triphosphates of both C-Cyd [9] and Ce-Cyd [14] have been shown to inhibit CTP synthetase. CTP pools were not measured in the host cells (i.e. PRK, HEL, Vero) used for the antiviral assays. However, we surmise that, since in all cell systems examined thus far [12] [13] [14] 40, 41] CTP pools are depleted as a consequence of Ce-Cyd treatment, this may also be the case for the cells used in the present study. At the concentrations at which Ce-Cyd was found effective in inhibiting virus-induced cytopathogenicity [for vaccinia virus, a concentration as low as 0.08 PM (Table l) ], it causes a significant reduction in CTP pools [12-14.40,41] .

Also, the fact that exogenous addition of Cyd or Urd, but not dThd or dCyd, reverses the antiviral activity of C-Cyd [15] and Ce-Cyd (Table 5 ) supports the notion that the antiviral action of these cytidine analogues may be mediated by the inhibition of CTP synthetase (Fig. 2) .

There are only a few compounds known to exert an antiviral effect through interference with the biosynthesis of pyrimidine ribonucleotides. Two antimetabolites that are supposed to do so are pyrazofurin [16,17] and 6-azauridine [18] . These compounds are targeted at OMP decarboxylase (Fig.  2) . Whether targeted at CTP synthetase (C-Cyd, Ce-Cyd) or OMP decarboxylase (pyrazofurin, 6azauridine), all four compounds have antitumor, in addition to, antiviral properties.

In fact, the antitumor potential of pyrazofurin [17] and Ce-Cyd [ll] has been considered the more attractive for initial development of these drugs.

Why may some compounds, such as Ce-Cyd, have potential as both antitumor and antiviral agents? If viruses or tumor cells impose similar requirements on some essential metabolites for their growth, one may expect that a shortage in the supply of these metabolites leads to suppression of both virus growth and tumor cell growth. Through its inhibitory effect on CTP synthetase, Ce-Cyd abrogates the CTP supply that is needed for both virus replication and tumor cell growth. Consequently, both the antiviral and cytocidal effects of the compound are reversed if the CTP pools are restored by the exogenous addition of cytidine.

Whether Ce-Cyd has any practical utility for the treatment of virus infections in humans remains to be established. It could be argued that the antitumor and antiviral properties of Ce-Cyd may be mutually exclusive. This is certainly not the case if the antiviral and antitumor (or cytocidal) assays are conducted under different conditions, that is with resting confluent cells for the antiviral assays and exponentially growing cells for the cytocidal assays. Under these conditions, Ce-Cyd exhibits antiviral activity at concentrations which are by several orders of magnitude lower than the concentrations that are toxic to the host cells. If, however, rapidly growing cells have to be used to monitor antiviral activity, as is the case for HIV, the toxicity of the compound for the host (MT-4) cells precludes any specific action against the virus. If these considerations are extrapolated to the in vivo situation, it follows that, if Ce-Cyd were to be used as an antiviral drug, proper conditions have to be conceived so as to prevent the toxic effect of the compound on rapidly growing cells (i.e. bone marrow, small intestine). This goal may well be achieved by site-specific delivery whereby the drug is directly delivered to the site of virus infection, i.e. if the compound is administered as an aerosol in the treatment of virus infections of the respiratory tract.

Similar recommendations may be made for pyrazofurin, which also exhibits broad-spectrum antiviral activity in vitro [42] , and, in addition, has proved inhibitory to the replication of respiratory syncytial virus (RSV) in vivo, in cotton rats given daily doses of 10 mg/kg [43] . Pyrazofurin may be too toxic for systemic administration, but, as an aerosol, it may prove particularly useful, not only in the treatment of RSV infection, but also in the treatment of other respiratory tract virus infections. The high potency and wide-spectrum activity of pyrazofurin [26-281 and Ce-Cyd (Table 1) against 10. ortho-and paramyxoviruses in vitro make these compounds attractive candidates in the pursuit of an effective chemotherapy of respiratory virus 11. infections.

As Ce-Cyd and pyrazofurin interact with different steps of the biosynthesis of pyrimidine ribonucleotides (Fig. 2) , they may act synergistically if combined. Such combinations should be pursued 12. from both an antiviral and antitumor viewpoint. The combination of pyrazofurin with Ce-Cyd has already been examined in one tumor cell system (i.e. L1210) ( Table 7) , and in this system pyrazofurin potentiated J3, the cytocidal activity of Ce-Cyd. 

biochemical effects of pyrazofurin in humans

Carbocyclic analogues of 5-fluorouracil nucleosides

Carbocyclic analogues of 5-substituted uracil nucleosides: synthesis and antiviral activity

Synthesis and antiviral activity of the carbocyclic analogues of (E)-5-(2-halovinyl)-2'-deoxyuridines and (E)-5-(2-halovinyl-2'-deoxycytidines

Synthesis and antiviral activity of the carbocyclic analogues of 5-ethyl-2'-deoxyuridine and of 5-ethynyl-2'-deoxvuridine

Carbocyclic analogues of 5'-amino-5'-deoxy-and 3'-amino-3'-deoxvthvmidines

Carbocyclic analogues of 5-halocytosine nucleosides

Fluoro carbocyclic nucleosides: synthesis and antiviral activity of 2'-and 6'-fluoro carbocyclic pyrimidine nucleosides including carbocyclic l-(2-deoxy-2-fluoro-B-D-arabinofuranosyl)-5-methyluracil and carbocyclic l

Carbocyclic analogs of cytosine nucleosides

Evaluation of carbodine, the carbocyclic analog of cytidine, and related carbocyclic analogs of pyrimidine nucleosides on respiratory syncytial virus replication in uitro

Inhibitory 'effegt of selected antiviral compounds' on measles (SSPE) virus replication in uitro

Comparative activities of several nucleoside analogs against influenza A, B, and C viruses in vitro

Thymidylate' synthetase as target enzyme for the inhibitory activity of 5-substituted 2'-deoxyuridines on mouse leukemia LI210 cell growth

Broad-spectrum antiviral activity of virazole: l-/I-D-ribofuranosyl-I,2,4-triazole-3-carboxamide

Homocysteine potentiates the antiviral and cytostatic activity of those nucleoside analogues that are targeted at 9 adenosylhomocysteine hydrolase

Biochemic~ aspects of the selective antiherpes activity of nucleoside analogues

Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrmidines

The molecular basis of the specific anti-influenza action of amantadine

Herpes simplex virus type 1 DNA polymerase

Azido: 3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase

Adenosylhomocysteine hydrolase inhibitors as broad-spectrum antiviral agents

Broad-spectrum antiviral activities of neplanocin A, 3-deazaneplanocin A, and their S-nor derivatives

Induction of differentiation in the human promyelocytic leukemia cell line HL-60 by the cyclopentenyl analogue of cytidine

Enhancement of the toxicity and DNA incorporation of arabinosyl-5-~acytosine and 1-~~-arabino~ranosylcytosine by cyclopentenyl cytosine

Broad-spectrum antiviral activity of pyrazofurin (pyrazomycin)

Comparison of the anti-respiratory syncytial virus activity and toxicity of papaverine hydrochloride and pyrazofurin in vitro and in vivo