- Email: [email protected]
Antiviral activity of cyclopentenone prostanoids
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8 Southwick,F.S. and Purich, D.L. (1996) New Engl. J. Med. 334, 770-776 9 Cossart, P. (1995) Curr. Opin. Cell Biol. 7, 94-101 10 Pollard, T.D. (1995) Curr. Biol. 5, 837-840 11 Cossart, P. and Kocks, C. (1994) Mol. Microbiol. 13, 395-402 12 Lasa, I. and Cossart, P. (1996) Trends Cell Biol. 6, 109-114 13 Pistor, S. etal. (1994) EMBOJ. 13, 758-763 14 Die&rich, E. et al. (1995) EMBO ]. 14, 2731-2744 15 Smith, G.A., Pormoy, D.A. and Theriot, J.A. (1995) Mol. Microbiol. 17, 945-951 16 Kocks,C. et al. (1995) Mol. Microbiol. 18,413-423 17 Armstrong, D. (1990) in Principles and Practice of Infectious Disease (Douglas, G.L. et al., eds), pp. 1587-1593, Churchill Livingston 18 Theriot, J.A. et al. (1994) Cell 76, 505-517 19 Mar&and, J.B. et al. (1995)]. Cell Biol. 130, 331-343 20 Niebuhr, K. et al. (1993) Infect. Immun. 61, 2793-2802 21 Kocks,C. et al. (1993)]. Cell Sci. 105, 699-710 22 Smith, G.A., Theriot, J.A. and Portnoy,D.A. (1996)]. Cell Biol. 135,647-660 23 Safer,D., Elzinga, M. and Nachmias, V.T. (1990)J. Biol. Chem. 266, 4029-4032 24 Pistor, S. et al. {1995)Curr. Biol. 5, 517-525
25 Lasa, I. et al. (1995) Mol. Microbiol. 18, 425-436 26 Kuhn, M. et al. (1990) Infect. Imrnun. 58, 3477-3486 27 Zhukarev,V. et al. (1995) Cell Motil. Cytoskeleton 30, 229-246 28 Zhukarev,V. et al. (1995) Mol. Biol. Cell 6, 139 29 Pring, M., Weber, A. and Bubb, M.R. (1992) Biochemistry 31, 1827-1836 30 Pantaloni, D. and Carlier, M-F. (1993) Cell 75, 1007-1014 31 Weber, A. et al. (1992) Biochemistry 31, 6179-6185 32 Theriot, J.A. et al. (1992) Nature 357, 257-260 33 Chakraborty,T. etal. (1995) EMBOJ. 14, 1314-1321 34 Reinhard, M. et aL (1995) EMBO J. 14, 1%27 35 Gertler, F.B. et al. (1996) Cell 87, 227-239 36 Pollard, T.D. (1986)]. Cell BioL 103, 2747-2754 37 Southwick,F.S. and Purich, Di. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5168-5172 38 Southwick,F.S. and Purich, D.L. (1995) Infect. Irnmun. 63, 182-190 39 Tilney, L.G., Connelly,P.S. and Pormoy, D.A. (1990)J. Cell Biol. 111, 2979-2988 40 Tilney,L.G. et al. (1992)]. Cell Biol. 118, 83-93 41 Machesky,L.M. et al. (1994)J. Cell Biol. 127, 107-115 42 Welch, M.D., Iwamatsu, A. and Mitchison, T.J. (1997) Nature 385,265-269
Antiviral activity of
cyclopentenone prostanoids M. Gabriella Santoro 'n an age when so many antiCyclopentenone prostanoids inhibit virus levels while triggering the synbiotics are available to comreplication by turning on an intracellular thesis of cytoprotective proteins .bat almost any bacterial indefence response that involves the by the host cell. They appear to fection, advances in the control induction of cytoprotective heat-shock act differently from any other and cure of viral diseases are proteins, the modification of viral known antiviral agents and offer still limited. Toxicity and lack glycoprotein maturation and the control the prospect of novel strategies of therapeutic efficacy in longOf NF-~B activation. These molecules to combat viral infections. term treatments are the major represent an interesting model for disadvantages of the antiviral the development of novel antiviral Prostaglandins control virus drugs now in use. The failure to drugs that can affect different targets replication develop nontoxic antiviral drugs during the virus life cycle. Prostaglandins are a class of has traditionally been blamed naturally occurring cyclic C20 M.G. Santoro is in the Institute of on the intracellular parasitic nafatty acids with potent biologiExperimental Medicine, CNR, 00137 Rome, Italy, ture of viruses. Consequently, cal properties. In eukaryotic and also in the Dept of Experimental Medicine, most of the drugs that affect cells, they are synthesized from University of L'Aquila, 67100 L'Aquila, Italy. virus replication also interfere arachidonic acid and other polye-mail: [email protected] with important metabolic prounsaturated fatty acid precurcesses in uninfected cells. Although more selective antisors derived from the phospholipid pool of the cell viral compounds that inhibit the function of specific membrane, and they function as intracellular signal meviral proteins are being developed, the high mutation diators in the regulation of physiological and pathorate seen in several viruses represents a further obstacle logical processes, including inflammation and the febrile for long-term effective treatment. One successful apresponse, the immune response, cell proliferation and proach in combating viral diseases appears to be the differentiation, and cytoprotection 1. simultaneous use of two or more drugs that affect difIn the early 1980s, the use of virus models to invesferent targets during the virus life cycle. tigate the molecular events that follow the exposure of A group of prostaglandins (PGs) and PG derivatives mammalian cells to prostaglandins led to the serendipare able to interfere with virus replication at multiple itous discovery that specific arachidonic acid derivatives
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are potent inhibitors of virus replication 2. Table 1. Viruses sensitive to the antiviral activity Based on the early observation that A-type of prostaglandins a,b prostaglandins (PGAs) inhibit Sendal virus (SV) replication2, the antiviral activity of Family Virus Host cell lines Refs natural and synthetic cyclopentenone PGs (cy-PG) has been described in a wide variety RNA viruses of DNA and RNA virus experimental models, Paramyxoviruses Sendal 37RC (monkey) 2,21 both in vitro and in vivo (Table 1). It soon Sendal HEp-2 (human) 34 became evident that the antiviral activity had Sendal VERO (monkey) 34 a wide spectrum of action, affecting both Orthomyxoviruses Influenza A PR8 Mouse (in vivo) 35 naked and enveloped DNA and RNA viruses; EMC L cells (mouse) 10 Picornaviruses however, only the cyclopentenone arachiPoliovirus HeLa (human) 11 donic acid derivatives (Fig. 1) were effective in inhibiting virus replication. The unique Rhabdoviruses VSV L cells (mouse) 7 characteristic of cy-PG is the presence of an VSV M A I 0 4 (monkey) 8 VSV HeLa (human) 11 (x,~-unsaturated carbonyl group in the cyclopentane ring, which allows this portion of Togaviruses Sindbis VERO (monkey) 36 the molecule to form Michael adducts with Retroviruses HTLV-I CBL (human) 37 cellular nucleophilics and to bind covalently HIV-1 C8166 (human) 38 to specific proteins 3. This molecular strucHIV-1 VB-8 (human) 39 ture is essential for antiviral activity4,L It was HIV-1 CEM-SS (human) 24 also obvious from the beginning that the DNA viruses antiviral activity is obtained at micromolar Poxviruses Vaccinia L cells (mouse) 40 concentrations of PGs that do not inhibit macromolecular synthesis in the host celF. Herpesviruses HSV-1 VERO (monkey) 39 HSV-1 Mouse (in vivo) 41 Moreover, cy-PG do not affect early events in HSV-2 HEF (human) 12 virus replication (i.e. adsorption, penetration HSV-2 HFF (human) 39 or uncoating) and, in contrast to interferon, treatment with cy-PG can be started in relaaCyclopentenoneprostaglandins inhibit the replication of a variety of RNA and DNAviruses tively late stages of the virus replication cycle in different types of mammalian cells. The antiviral activity of a long-acting synthetic and still be effective (reviewed in Refs 5,6). analogue of prostaglandin A2 (PGA2)(16,16 dimethyI-PGA2-methylester) and of prostaglandin D2 (PGD2),the precursor of A12-PGJ2,has also been shown in vivo in mice infected While fuelling a rapidly growing interest with influenza A virus 35and herpesvirus4:, respectively. in this new class of antiviral compounds, bAbbreviations:CBL, human cord blood mononuclear cells; EMC, encephalomyocarditis these initial observations left scientists with •virus; HEF, human embryonic fibroblasts; HFF, human foreskin fibroblasts; HSV, herpes the puzzling question of how these molsimplex virus; HTLV-I, human T-cell leukaemia/lymphoma virus type I; VSV, vesicular stomatitis virus. ecules could inhibit the replication of viruses that have as diverse replicative strategies as herpesviruses, paramyxoviruses, retroviruses and picornaviruses. What is the common target? impair virus morphogenesis and invasion of new cells (Fig. 2). Inhibition of viral glycoprotein maturation Even though this hypothesis is appealing, reports One of the first observations on the mechanism of in- from different laboratories indicate that PGAs can inhibition of virus replication came from studies on two hibit the replication of non-enveloped viruses, such as negative-stranded viruses: a rhabdovirus [vesicular encephalomyocarditis virus 1°or poliovirus 11. Moreover, stomatitis virus (VSV)] and a paramyxovirus (SV). A7-PGA1 has been shown to inhibit herpesvirus RNA Prostaglandin A~ (PGA0 was shown to inhibit the transcription in human cells 12,and PGA1 treatment preglycosylation of the VSV glycoprotein G in murine vents the synthesis of three late vaccinia virus proteinslL L fibroblasts 7,8, altering its electrophoretic mobility Cy-PG also selectively inhibit VSV and SV protein synand decreasing its molecular weight of -4 kDa. PGA1, thesis in monkey and human cells but not in murine cells and subsequently A12-prostaglandin JR (a12-PGJ2),was (reviewed in Ref. 5). In conjunction, these results indishown to selectively inhibit the glycosylation, matu- cate that cy-PG affect different events during virus repliration and intracellular translocation of the SV glyco- cation and that the effects on the virus are also dependproteins HN (haemagglutinin-neuroaminidase) and ent on the host cell species. Does a common molecular F (fusion) 9. The mechanism by which cy-PG alter mechanism underlie these remarkable differences? virus protein maturation is still unknown. In the case of VSV, it has been hypothesized that PGs can bind Induction of heat-shock proteins directly to specific sequences of the newly synthesized Heat-shock proteins (HSPs) are induced in mammalian proteins, competing for acylation with the natural cells in a variety of pathophysiological states, including fatty acid precursor and causing structural modifi- fever, inflammation, oxidant injury and virus infeccations of the G protein that are incompatible with tion 14,is. Induction requires the activation, translocation its further processing and intracellular translocation. to the nucleus and phosphorylation of a transregulatory Inhibition of virus protein glycosylation could then protein, the heat-shock transcription factor (HSF),
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tions are expressed during normal growth conditions and can be induced by biologically active molecules (i.e. haemin and PGs), whereas others are expressed upon stressactivated regulation of transcriptional and translational switches during exposure to adverse environmental conditions. HSP induction is not only a signal for the detection of physiological stress but is also used by the cells in the repair process following different types of injury to prevent damage resulting from the accumulation of nonnative proteins. HSPs are widely recognized to have a cytoprotective role in a variety of human diseases, including ischaemia, inflammation and infection (reviewed in Ref. 15). Following the observation that P G A 1 induces the synthesis of a 70-kDa HSP (hsp70) in K562 cells16, it became clear that one major characteristic common to prostaglandins with antiviral activity is the ability to function as a signal for the induction of HSP synthesis via cycloheximide-sensitive activation of HSF (Refs 16,17) (Fig. 2b). Hsp70 expression can be selectively induced by cy-PG in non-stressing circumstances and is associated with the cytoprotection of human cells by PGA1 during thermal injury TM. Cy-PG can trigger hsp70 synthesis in a variety of monkey, canine, porcine and human cell lines, human peripheral blood lymphocytes, macrophages and primary cells derived from cord blood (reviewed in Ref. 6). Interestingly, PGA1 is not able to induce hsp70 synthesis in murine cells 19.
Do HSPs interfere with virus replication? The possibility that HSPs could be involved in the control of virus replication was sugFig. 1. Structure of cyclopentenone prostanoids (cy-PG). Prostaglandins A (PGA) and J (PGJ) are the dehydration products of prostaglandins E (PGE) and D (PGD), respectively3. gested by the facts that only PGs with Clavulones were initially isolated from the stolonifer Clavularia viridis 32. PGA2 was also antiviral activity are able to induce hsp70 originally isolated from the gorgonian Plexaura homomalla 33. Cy-PG all possess a reactive synthesis and that inhibition of virus replic(,~unsaturated carbonyl group in the cyclopentane ring of the molecule. cation is always associated with hsp70 induction s,z0. which binds to multiple arrays of heat-shock elements Treatment with cy-PG in an early phase of the rep(HSEs) located in the promoters of heat-shock genes TM. lication cycle of SV in primate cells causes a selective In mammalian cells, several HSPs with chaperonin func- block of viral protein synthesis that persists for as long
OAc
Fig, 2. (Faci~i~ Page) Multiple targets for cyclopentenone prostaglandin (cy-PG) activity. Cy-PG are rapidly and actively transported into the cytoplasm and nucleus of mammalian cells, where they bind to specific proteins 3. (a) Selective block of viral protein synthesis (1) and inhibition of virus glycoprotein maturation and intracelluiar translocation 7,9(2). Cy-PG do not affect virus adsorption, penetration and uncoating but do inhibit virus protein synthesis 5,21.This effect appears to be dependent on the expression of heat-shock proteins (HSPs) by the host cell. A role for the 70-kDa HSP (hsp70) in the control of viral protein synthesis has been hypothesized2°.21. (b) Synthesis of cytoprotective HSPs is induced by cy-PG via the activation of heat-shock transcription factor (HSF). HSF converts from a monomeric non-DNA-binding form to an oligomeric form that translocates to the nucleus and binds to specific promoter elements (HSEs) located upstream of heat-shock genes (e.g. HSP70). (¢) Inhibition of nuclear factor KB (NF-KB) activation and NF-KB-Controlled transcription by cy-PG. NF-~B normally exists in an inactive cytoplasmic complex, of which the predominant form is a heterodimer composed of p50 and p65 subunits bound to the inhibitory protein IKB-c~ (Ref. 25). Activation by a variety of stimuli, including inflammatory cytokines and viruses, triggers the phosphorylation and degradation of IEB-~, resulting in NF-KB translocation to the nucleus, where it binds to DNA at specific KB sites and induces genes encoding signalling proteins2~2T. Cy-PG act by inhibiting IKB-~ phosphorylation and degradation2~. Abbreviations: ER, endoplasmic reticulum; vRNA, genomic RNA; mRNA, messenger RNA.
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azetidine and heat shock, selectively inhibit SV protein synthesis for as long as HSP synthesis is occurring in infected cells21 P~_ (Fig. 3a,b). In contrast, PGA 1 treatment has HNno effect on SV protein synthesis in murine Friend erythroleukaemic cells, which lack Fothe ability to synthesize hsp70 (Ref. 21). Similar results have been reported in monkey and human cells infected with rhabdoviruses. Induction of hsp70 synthesis by molecules as diverse as PGAs, PGJs, sodium (-9 -r< arsenite, azetidine or 2-cyclopenten-l-one, or heat shock itself, is always associated with a dramatic decrease of VSV protein syn(d) (c) thesis and protection of the host cell from the virus-induced shut-off of cellular protein i ii iiiill ¸ synthesis4,s,8 (Fig. 3c,d). However, in VSVinfected murine fibroblasts, in the absence -G G_ of hsp70 induction, PGA1 has no effect on kDa68_43_ viral protein synthesis but does inhibit glycoprotein G maturation 7. Finally, during in~ _N/NS N/NSfection of human cells with poliovirus, which prevents hsp70 induction by cy-PG (Ref. 7i!!i¸ ~'!i,,~ 11), PGA1 does not inhibit virus protein synthesis. The picture emerging from the data now ~i!iiiiii¸ ~!i!!i!i!¸¸¸¸ !il!!!~i!i!!~¸ ii!~i~iliiiiiii~il available suggests that high hsp70 levels M antagonize virus protein synthesis in the early phase of acute infection by an unknown 0 2 4 6 8 0 2 4 6 8 mechanism. It is possible that hsp70 could interact directly with the nascent viral polyT i m e (h) T i m e (h) peptides, causing a translational block. Fig. 3. Selective inhibition of viral protein synthesis by cyclopentenone prostaglandins Schlesinger et al. have shown that during and different heat-shock protein inducers. (a) Uninfected or (b) Sendai virus (SV)in vitro translation of Sindbis virus mRNA, infected cells were treated soon after infection with either sodium arsenite (As02), cadmium chloride (Cd), prostaglandin J2 (PGJ2) or control diluent (C), or subjected to heat hsp70 interferes with normal polypeptide shock (HS) starting 3 h postinfection. Samples were labelled with [3sS]methionine 9 h synthesis 22. Our studies on paramyxovirus postinfection. SV proteins P, HN, Fo and NP are indicated. All treatments resulted in ininfection suggest that hsp70 could interfere duction of heat-shock proteins (indicated by asterisks) and in the suppression of SV with viral mRNA translation only during protein synthesis 21. Hsp70 is indicated by arrowheads. MAI04 cells infected with vesicits synthesis by the host cell21. One possiular stomatitis virus (VSV) were treated with (¢) control diluent or (d) A12-pGJ2 soon after infection and labelled with [3sS]methionine at different time intervals postinfection. bility is that HSP and virus messages, both Virus proteins G, N, NS and M are indicated, and hsp70 is indicated by an arrowhead. of which can be translated in conditions A~2-pGJ2 induced hsp70 expression and inhibited VSV protein synthesis, protecting host where cellular protein synthesis is impaired cells by the virus-induced shut-off of protein synthesis 8. (i.e. under conditions of elevated cytoplasmic ionic concentrations and in the as hsp70 is synthesized by the host cell21. The block absence of functional eukaryotic initiation factor 4F), occurs at the translational level and is cell-mediated21. could possess similar mechanisms for preferential A role for hsp70 as the cellular mediator interfering with translation and might compete with each other s,2°. A better understanding of the functional role of HSPs SV protein synthesis has been suggested, as different hsp70 inducers, including sodium arsenite, cadmium, in virus replication is necessary to establish whether they act as an intracellular defence against pathogens and whether we can manipulate the host cell response Questionsfor future research for virus therapy.
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• Do heat-shock proteins function as an intracellular defence against invading viruses? • Is fever beneficial in viral infections? • Could nuclear factor KB (NF-KB) be the target for prostaglandin antiviral activity against viruses other than retroviruses? • Is there a link between heat-shock transcription factor activation and NF-~B inhibition by prostaglandins? Do these factors share a common switch on/off signal? • Is the antiviral activity of cyclopentenone prostaglandins only a pharmacological effect?
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Role of nuclearfactor ~cB In addition to inhibiting virus protein synthesis and maturation, cy-PG may also affect viral RNA transcription lz,e3, raising more questions about their molecular targets. In the case of HIV-1, both PGA1 and PGJ2 were found to inhibit viral RNA transcription in human cells24. A dramatic reduction in HIV-1 mRNA levels was detected 48-72 h after infection following a single PG treatment. This observation has
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led to the discovery of a novel target for cy-PG in human cells. Nuclear factor KB (NF-Fd3), an inducible eukaryotic transcription factor of the rel family, normally exists in an inactive cytoplasmic complex, of which the predominant form is a heterodimer composed of p50 and p65 subunits bound to inhibitory proteins of the I~:B family 2s. NF-Fd3 is activated in response to a variety of stimuli, including inflammatory cytokines, bacteria and viruses 26. Stimulation triggers the release of NF-F,B from IcB, resulting in NF-~B translocation to the nucleus where it binds to DNA at specific ~B sites and rapidly induces a variety of genes that encode signalling proteins. NF-~:B is considered to be an immediate-early mediator of immune and inflammatory responses and is involved in many pathological events, including the progression to AIDS, because of its ability to enhance HIV-1 RNA transcription 27. We have recently shown that cy-PG are potent inhibitors of NF-~B activation and of NF-~B-dependent HIV-1 transcription in human cells 28. Cy-PG act by inhibiting phosphorylation and preventing degradation of the NF-K:B inhibitor IKB-ix (Fig. 2c). This effect is associated with HSF activation 2s, suggesting that a common molecular mechanism could underlie these different events. Conclusions
Even though major advances have taken place in identifying the molecular targets of cy-PG, many questions still need to be answered to unravel the complex mechanisms of their antiviral effect. The information now available indicates that cy-PG inhibit virus replication differently from any other known antiviral agent, by acting on multiple cellular and viral targets. This raises the question of the potential use of these molecules in the treatment of viral diseases. PGs are used clinically in the treatment of several diseases, including gastric ulcers and congenital heart disease, and to facilitate labour, and they are generally effective and well-tolerated 29. Administration of the PGA 1 precursor, PGEI, has also been shown to be beneficial in patients with fulminant viral hepatitis 3°. In studies on volunteers with hypertension, infusion with PGAt has beneficial effects on blood pressure, and has no deleterious effects on kidney function or other significant side effects 31. Cy-PG could be made readily available, as they can be synthesized chemically, and a large variety of PGA analogues can be obtained from natural sources 32,33 (Fig. 1). However, the fact that different types of PGs are synthesized in different tissues throughout the body and have multiple effects on blood pressure, muscle contraction and inflammatory and immune responses poses several questions concerning the possible use of natural PGs as antiviral compounds. The recent finding that one component of the PGA molecule, 2-cyclopenten-l-one, is able to activate HSF and exerts antiviral activity 4 indicates that a new class of molecules that are devoid of the pleiotropic effects of natural PGs could be designed, opening new avenues in the search for novel antiviral and cytoprotective drugs.
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Acknowledgements
This work was supportedby a grant from the Italian Ministryof Public Health, 1997AIDSresearchprojectand CNR ProgettoStrategico'Stress Proteins'. References
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8 Southwick,F.S. and Purich, D.L. (1996) New Engl. J. Med. 334, 770-776 9 Cossart, P. (1995) Curr. Opin. Cell Biol. 7, 94-101 10 Pollard, T.D. (1995) Curr. Biol. 5, 837-840 11 Cossart, P. and Kocks, C. (1994) Mol. Microbiol. 13, 395-402 12 Lasa, I. and Cossart, P. (1996) Trends Cell Biol. 6, 109-114 13 Pistor, S. etal. (1994) EMBOJ. 13, 758-763 14 Die&rich, E. et al. (1995) EMBO ]. 14, 2731-2744 15 Smith, G.A., Pormoy, D.A. and Theriot, J.A. (1995) Mol. Microbiol. 17, 945-951 16 Kocks,C. et al. (1995) Mol. Microbiol. 18,413-423 17 Armstrong, D. (1990) in Principles and Practice of Infectious Disease (Douglas, G.L. et al., eds), pp. 1587-1593, Churchill Livingston 18 Theriot, J.A. et al. (1994) Cell 76, 505-517 19 Mar&and, J.B. et al. (1995)]. Cell Biol. 130, 331-343 20 Niebuhr, K. et al. (1993) Infect. Immun. 61, 2793-2802 21 Kocks,C. et al. (1993)]. Cell Sci. 105, 699-710 22 Smith, G.A., Theriot, J.A. and Portnoy,D.A. (1996)]. Cell Biol. 135,647-660 23 Safer,D., Elzinga, M. and Nachmias, V.T. (1990)J. Biol. Chem. 266, 4029-4032 24 Pistor, S. et al. {1995)Curr. Biol. 5, 517-525
25 Lasa, I. et al. (1995) Mol. Microbiol. 18, 425-436 26 Kuhn, M. et al. (1990) Infect. Imrnun. 58, 3477-3486 27 Zhukarev,V. et al. (1995) Cell Motil. Cytoskeleton 30, 229-246 28 Zhukarev,V. et al. (1995) Mol. Biol. Cell 6, 139 29 Pring, M., Weber, A. and Bubb, M.R. (1992) Biochemistry 31, 1827-1836 30 Pantaloni, D. and Carlier, M-F. (1993) Cell 75, 1007-1014 31 Weber, A. et al. (1992) Biochemistry 31, 6179-6185 32 Theriot, J.A. et al. (1992) Nature 357, 257-260 33 Chakraborty,T. etal. (1995) EMBOJ. 14, 1314-1321 34 Reinhard, M. et aL (1995) EMBO J. 14, 1%27 35 Gertler, F.B. et al. (1996) Cell 87, 227-239 36 Pollard, T.D. (1986)]. Cell BioL 103, 2747-2754 37 Southwick,F.S. and Purich, Di. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5168-5172 38 Southwick,F.S. and Purich, D.L. (1995) Infect. Irnmun. 63, 182-190 39 Tilney, L.G., Connelly,P.S. and Pormoy, D.A. (1990)J. Cell Biol. 111, 2979-2988 40 Tilney,L.G. et al. (1992)]. Cell Biol. 118, 83-93 41 Machesky,L.M. et al. (1994)J. Cell Biol. 127, 107-115 42 Welch, M.D., Iwamatsu, A. and Mitchison, T.J. (1997) Nature 385,265-269
Antiviral activity of
cyclopentenone prostanoids M. Gabriella Santoro 'n an age when so many antiCyclopentenone prostanoids inhibit virus levels while triggering the synbiotics are available to comreplication by turning on an intracellular thesis of cytoprotective proteins .bat almost any bacterial indefence response that involves the by the host cell. They appear to fection, advances in the control induction of cytoprotective heat-shock act differently from any other and cure of viral diseases are proteins, the modification of viral known antiviral agents and offer still limited. Toxicity and lack glycoprotein maturation and the control the prospect of novel strategies of therapeutic efficacy in longOf NF-~B activation. These molecules to combat viral infections. term treatments are the major represent an interesting model for disadvantages of the antiviral the development of novel antiviral Prostaglandins control virus drugs now in use. The failure to drugs that can affect different targets replication develop nontoxic antiviral drugs during the virus life cycle. Prostaglandins are a class of has traditionally been blamed naturally occurring cyclic C20 M.G. Santoro is in the Institute of on the intracellular parasitic nafatty acids with potent biologiExperimental Medicine, CNR, 00137 Rome, Italy, ture of viruses. Consequently, cal properties. In eukaryotic and also in the Dept of Experimental Medicine, most of the drugs that affect cells, they are synthesized from University of L'Aquila, 67100 L'Aquila, Italy. virus replication also interfere arachidonic acid and other polye-mail: [email protected] with important metabolic prounsaturated fatty acid precurcesses in uninfected cells. Although more selective antisors derived from the phospholipid pool of the cell viral compounds that inhibit the function of specific membrane, and they function as intracellular signal meviral proteins are being developed, the high mutation diators in the regulation of physiological and pathorate seen in several viruses represents a further obstacle logical processes, including inflammation and the febrile for long-term effective treatment. One successful apresponse, the immune response, cell proliferation and proach in combating viral diseases appears to be the differentiation, and cytoprotection 1. simultaneous use of two or more drugs that affect difIn the early 1980s, the use of virus models to invesferent targets during the virus life cycle. tigate the molecular events that follow the exposure of A group of prostaglandins (PGs) and PG derivatives mammalian cells to prostaglandins led to the serendipare able to interfere with virus replication at multiple itous discovery that specific arachidonic acid derivatives
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are potent inhibitors of virus replication 2. Table 1. Viruses sensitive to the antiviral activity Based on the early observation that A-type of prostaglandins a,b prostaglandins (PGAs) inhibit Sendal virus (SV) replication2, the antiviral activity of Family Virus Host cell lines Refs natural and synthetic cyclopentenone PGs (cy-PG) has been described in a wide variety RNA viruses of DNA and RNA virus experimental models, Paramyxoviruses Sendal 37RC (monkey) 2,21 both in vitro and in vivo (Table 1). It soon Sendal HEp-2 (human) 34 became evident that the antiviral activity had Sendal VERO (monkey) 34 a wide spectrum of action, affecting both Orthomyxoviruses Influenza A PR8 Mouse (in vivo) 35 naked and enveloped DNA and RNA viruses; EMC L cells (mouse) 10 Picornaviruses however, only the cyclopentenone arachiPoliovirus HeLa (human) 11 donic acid derivatives (Fig. 1) were effective in inhibiting virus replication. The unique Rhabdoviruses VSV L cells (mouse) 7 characteristic of cy-PG is the presence of an VSV M A I 0 4 (monkey) 8 VSV HeLa (human) 11 (x,~-unsaturated carbonyl group in the cyclopentane ring, which allows this portion of Togaviruses Sindbis VERO (monkey) 36 the molecule to form Michael adducts with Retroviruses HTLV-I CBL (human) 37 cellular nucleophilics and to bind covalently HIV-1 C8166 (human) 38 to specific proteins 3. This molecular strucHIV-1 VB-8 (human) 39 ture is essential for antiviral activity4,L It was HIV-1 CEM-SS (human) 24 also obvious from the beginning that the DNA viruses antiviral activity is obtained at micromolar Poxviruses Vaccinia L cells (mouse) 40 concentrations of PGs that do not inhibit macromolecular synthesis in the host celF. Herpesviruses HSV-1 VERO (monkey) 39 HSV-1 Mouse (in vivo) 41 Moreover, cy-PG do not affect early events in HSV-2 HEF (human) 12 virus replication (i.e. adsorption, penetration HSV-2 HFF (human) 39 or uncoating) and, in contrast to interferon, treatment with cy-PG can be started in relaaCyclopentenoneprostaglandins inhibit the replication of a variety of RNA and DNAviruses tively late stages of the virus replication cycle in different types of mammalian cells. The antiviral activity of a long-acting synthetic and still be effective (reviewed in Refs 5,6). analogue of prostaglandin A2 (PGA2)(16,16 dimethyI-PGA2-methylester) and of prostaglandin D2 (PGD2),the precursor of A12-PGJ2,has also been shown in vivo in mice infected While fuelling a rapidly growing interest with influenza A virus 35and herpesvirus4:, respectively. in this new class of antiviral compounds, bAbbreviations:CBL, human cord blood mononuclear cells; EMC, encephalomyocarditis these initial observations left scientists with •virus; HEF, human embryonic fibroblasts; HFF, human foreskin fibroblasts; HSV, herpes the puzzling question of how these molsimplex virus; HTLV-I, human T-cell leukaemia/lymphoma virus type I; VSV, vesicular stomatitis virus. ecules could inhibit the replication of viruses that have as diverse replicative strategies as herpesviruses, paramyxoviruses, retroviruses and picornaviruses. What is the common target? impair virus morphogenesis and invasion of new cells (Fig. 2). Inhibition of viral glycoprotein maturation Even though this hypothesis is appealing, reports One of the first observations on the mechanism of in- from different laboratories indicate that PGAs can inhibition of virus replication came from studies on two hibit the replication of non-enveloped viruses, such as negative-stranded viruses: a rhabdovirus [vesicular encephalomyocarditis virus 1°or poliovirus 11. Moreover, stomatitis virus (VSV)] and a paramyxovirus (SV). A7-PGA1 has been shown to inhibit herpesvirus RNA Prostaglandin A~ (PGA0 was shown to inhibit the transcription in human cells 12,and PGA1 treatment preglycosylation of the VSV glycoprotein G in murine vents the synthesis of three late vaccinia virus proteinslL L fibroblasts 7,8, altering its electrophoretic mobility Cy-PG also selectively inhibit VSV and SV protein synand decreasing its molecular weight of -4 kDa. PGA1, thesis in monkey and human cells but not in murine cells and subsequently A12-prostaglandin JR (a12-PGJ2),was (reviewed in Ref. 5). In conjunction, these results indishown to selectively inhibit the glycosylation, matu- cate that cy-PG affect different events during virus repliration and intracellular translocation of the SV glyco- cation and that the effects on the virus are also dependproteins HN (haemagglutinin-neuroaminidase) and ent on the host cell species. Does a common molecular F (fusion) 9. The mechanism by which cy-PG alter mechanism underlie these remarkable differences? virus protein maturation is still unknown. In the case of VSV, it has been hypothesized that PGs can bind Induction of heat-shock proteins directly to specific sequences of the newly synthesized Heat-shock proteins (HSPs) are induced in mammalian proteins, competing for acylation with the natural cells in a variety of pathophysiological states, including fatty acid precursor and causing structural modifi- fever, inflammation, oxidant injury and virus infeccations of the G protein that are incompatible with tion 14,is. Induction requires the activation, translocation its further processing and intracellular translocation. to the nucleus and phosphorylation of a transregulatory Inhibition of virus protein glycosylation could then protein, the heat-shock transcription factor (HSF),
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tions are expressed during normal growth conditions and can be induced by biologically active molecules (i.e. haemin and PGs), whereas others are expressed upon stressactivated regulation of transcriptional and translational switches during exposure to adverse environmental conditions. HSP induction is not only a signal for the detection of physiological stress but is also used by the cells in the repair process following different types of injury to prevent damage resulting from the accumulation of nonnative proteins. HSPs are widely recognized to have a cytoprotective role in a variety of human diseases, including ischaemia, inflammation and infection (reviewed in Ref. 15). Following the observation that P G A 1 induces the synthesis of a 70-kDa HSP (hsp70) in K562 cells16, it became clear that one major characteristic common to prostaglandins with antiviral activity is the ability to function as a signal for the induction of HSP synthesis via cycloheximide-sensitive activation of HSF (Refs 16,17) (Fig. 2b). Hsp70 expression can be selectively induced by cy-PG in non-stressing circumstances and is associated with the cytoprotection of human cells by PGA1 during thermal injury TM. Cy-PG can trigger hsp70 synthesis in a variety of monkey, canine, porcine and human cell lines, human peripheral blood lymphocytes, macrophages and primary cells derived from cord blood (reviewed in Ref. 6). Interestingly, PGA1 is not able to induce hsp70 synthesis in murine cells 19.
Do HSPs interfere with virus replication? The possibility that HSPs could be involved in the control of virus replication was sugFig. 1. Structure of cyclopentenone prostanoids (cy-PG). Prostaglandins A (PGA) and J (PGJ) are the dehydration products of prostaglandins E (PGE) and D (PGD), respectively3. gested by the facts that only PGs with Clavulones were initially isolated from the stolonifer Clavularia viridis 32. PGA2 was also antiviral activity are able to induce hsp70 originally isolated from the gorgonian Plexaura homomalla 33. Cy-PG all possess a reactive synthesis and that inhibition of virus replic(,~unsaturated carbonyl group in the cyclopentane ring of the molecule. cation is always associated with hsp70 induction s,z0. which binds to multiple arrays of heat-shock elements Treatment with cy-PG in an early phase of the rep(HSEs) located in the promoters of heat-shock genes TM. lication cycle of SV in primate cells causes a selective In mammalian cells, several HSPs with chaperonin func- block of viral protein synthesis that persists for as long
OAc
Fig, 2. (Faci~i~ Page) Multiple targets for cyclopentenone prostaglandin (cy-PG) activity. Cy-PG are rapidly and actively transported into the cytoplasm and nucleus of mammalian cells, where they bind to specific proteins 3. (a) Selective block of viral protein synthesis (1) and inhibition of virus glycoprotein maturation and intracelluiar translocation 7,9(2). Cy-PG do not affect virus adsorption, penetration and uncoating but do inhibit virus protein synthesis 5,21.This effect appears to be dependent on the expression of heat-shock proteins (HSPs) by the host cell. A role for the 70-kDa HSP (hsp70) in the control of viral protein synthesis has been hypothesized2°.21. (b) Synthesis of cytoprotective HSPs is induced by cy-PG via the activation of heat-shock transcription factor (HSF). HSF converts from a monomeric non-DNA-binding form to an oligomeric form that translocates to the nucleus and binds to specific promoter elements (HSEs) located upstream of heat-shock genes (e.g. HSP70). (¢) Inhibition of nuclear factor KB (NF-KB) activation and NF-KB-Controlled transcription by cy-PG. NF-~B normally exists in an inactive cytoplasmic complex, of which the predominant form is a heterodimer composed of p50 and p65 subunits bound to the inhibitory protein IKB-c~ (Ref. 25). Activation by a variety of stimuli, including inflammatory cytokines and viruses, triggers the phosphorylation and degradation of IEB-~, resulting in NF-KB translocation to the nucleus, where it binds to DNA at specific KB sites and induces genes encoding signalling proteins2~2T. Cy-PG act by inhibiting IKB-~ phosphorylation and degradation2~. Abbreviations: ER, endoplasmic reticulum; vRNA, genomic RNA; mRNA, messenger RNA.
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azetidine and heat shock, selectively inhibit SV protein synthesis for as long as HSP synthesis is occurring in infected cells21 P~_ (Fig. 3a,b). In contrast, PGA 1 treatment has HNno effect on SV protein synthesis in murine Friend erythroleukaemic cells, which lack Fothe ability to synthesize hsp70 (Ref. 21). Similar results have been reported in monkey and human cells infected with rhabdoviruses. Induction of hsp70 synthesis by molecules as diverse as PGAs, PGJs, sodium (-9 -r< arsenite, azetidine or 2-cyclopenten-l-one, or heat shock itself, is always associated with a dramatic decrease of VSV protein syn(d) (c) thesis and protection of the host cell from the virus-induced shut-off of cellular protein i ii iiiill ¸ synthesis4,s,8 (Fig. 3c,d). However, in VSVinfected murine fibroblasts, in the absence -G G_ of hsp70 induction, PGA1 has no effect on kDa68_43_ viral protein synthesis but does inhibit glycoprotein G maturation 7. Finally, during in~ _N/NS N/NSfection of human cells with poliovirus, which prevents hsp70 induction by cy-PG (Ref. 7i!!i¸ ~'!i,,~ 11), PGA1 does not inhibit virus protein synthesis. The picture emerging from the data now ~i!iiiiii¸ ~!i!!i!i!¸¸¸¸ !il!!!~i!i!!~¸ ii!~i~iliiiiiii~il available suggests that high hsp70 levels M antagonize virus protein synthesis in the early phase of acute infection by an unknown 0 2 4 6 8 0 2 4 6 8 mechanism. It is possible that hsp70 could interact directly with the nascent viral polyT i m e (h) T i m e (h) peptides, causing a translational block. Fig. 3. Selective inhibition of viral protein synthesis by cyclopentenone prostaglandins Schlesinger et al. have shown that during and different heat-shock protein inducers. (a) Uninfected or (b) Sendai virus (SV)in vitro translation of Sindbis virus mRNA, infected cells were treated soon after infection with either sodium arsenite (As02), cadmium chloride (Cd), prostaglandin J2 (PGJ2) or control diluent (C), or subjected to heat hsp70 interferes with normal polypeptide shock (HS) starting 3 h postinfection. Samples were labelled with [3sS]methionine 9 h synthesis 22. Our studies on paramyxovirus postinfection. SV proteins P, HN, Fo and NP are indicated. All treatments resulted in ininfection suggest that hsp70 could interfere duction of heat-shock proteins (indicated by asterisks) and in the suppression of SV with viral mRNA translation only during protein synthesis 21. Hsp70 is indicated by arrowheads. MAI04 cells infected with vesicits synthesis by the host cell21. One possiular stomatitis virus (VSV) were treated with (¢) control diluent or (d) A12-pGJ2 soon after infection and labelled with [3sS]methionine at different time intervals postinfection. bility is that HSP and virus messages, both Virus proteins G, N, NS and M are indicated, and hsp70 is indicated by an arrowhead. of which can be translated in conditions A~2-pGJ2 induced hsp70 expression and inhibited VSV protein synthesis, protecting host where cellular protein synthesis is impaired cells by the virus-induced shut-off of protein synthesis 8. (i.e. under conditions of elevated cytoplasmic ionic concentrations and in the as hsp70 is synthesized by the host cell21. The block absence of functional eukaryotic initiation factor 4F), occurs at the translational level and is cell-mediated21. could possess similar mechanisms for preferential A role for hsp70 as the cellular mediator interfering with translation and might compete with each other s,2°. A better understanding of the functional role of HSPs SV protein synthesis has been suggested, as different hsp70 inducers, including sodium arsenite, cadmium, in virus replication is necessary to establish whether they act as an intracellular defence against pathogens and whether we can manipulate the host cell response Questionsfor future research for virus therapy.
(a)
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• Do heat-shock proteins function as an intracellular defence against invading viruses? • Is fever beneficial in viral infections? • Could nuclear factor KB (NF-KB) be the target for prostaglandin antiviral activity against viruses other than retroviruses? • Is there a link between heat-shock transcription factor activation and NF-~B inhibition by prostaglandins? Do these factors share a common switch on/off signal? • Is the antiviral activity of cyclopentenone prostaglandins only a pharmacological effect?
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Role of nuclearfactor ~cB In addition to inhibiting virus protein synthesis and maturation, cy-PG may also affect viral RNA transcription lz,e3, raising more questions about their molecular targets. In the case of HIV-1, both PGA1 and PGJ2 were found to inhibit viral RNA transcription in human cells24. A dramatic reduction in HIV-1 mRNA levels was detected 48-72 h after infection following a single PG treatment. This observation has
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led to the discovery of a novel target for cy-PG in human cells. Nuclear factor KB (NF-Fd3), an inducible eukaryotic transcription factor of the rel family, normally exists in an inactive cytoplasmic complex, of which the predominant form is a heterodimer composed of p50 and p65 subunits bound to inhibitory proteins of the I~:B family 2s. NF-Fd3 is activated in response to a variety of stimuli, including inflammatory cytokines, bacteria and viruses 26. Stimulation triggers the release of NF-F,B from IcB, resulting in NF-~B translocation to the nucleus where it binds to DNA at specific ~B sites and rapidly induces a variety of genes that encode signalling proteins. NF-~:B is considered to be an immediate-early mediator of immune and inflammatory responses and is involved in many pathological events, including the progression to AIDS, because of its ability to enhance HIV-1 RNA transcription 27. We have recently shown that cy-PG are potent inhibitors of NF-~B activation and of NF-~B-dependent HIV-1 transcription in human cells 28. Cy-PG act by inhibiting phosphorylation and preventing degradation of the NF-K:B inhibitor IKB-ix (Fig. 2c). This effect is associated with HSF activation 2s, suggesting that a common molecular mechanism could underlie these different events. Conclusions
Even though major advances have taken place in identifying the molecular targets of cy-PG, many questions still need to be answered to unravel the complex mechanisms of their antiviral effect. The information now available indicates that cy-PG inhibit virus replication differently from any other known antiviral agent, by acting on multiple cellular and viral targets. This raises the question of the potential use of these molecules in the treatment of viral diseases. PGs are used clinically in the treatment of several diseases, including gastric ulcers and congenital heart disease, and to facilitate labour, and they are generally effective and well-tolerated 29. Administration of the PGA 1 precursor, PGEI, has also been shown to be beneficial in patients with fulminant viral hepatitis 3°. In studies on volunteers with hypertension, infusion with PGAt has beneficial effects on blood pressure, and has no deleterious effects on kidney function or other significant side effects 31. Cy-PG could be made readily available, as they can be synthesized chemically, and a large variety of PGA analogues can be obtained from natural sources 32,33 (Fig. 1). However, the fact that different types of PGs are synthesized in different tissues throughout the body and have multiple effects on blood pressure, muscle contraction and inflammatory and immune responses poses several questions concerning the possible use of natural PGs as antiviral compounds. The recent finding that one component of the PGA molecule, 2-cyclopenten-l-one, is able to activate HSF and exerts antiviral activity 4 indicates that a new class of molecules that are devoid of the pleiotropic effects of natural PGs could be designed, opening new avenues in the search for novel antiviral and cytoprotective drugs.
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This work was supportedby a grant from the Italian Ministryof Public Health, 1997AIDSresearchprojectand CNR ProgettoStrategico'Stress Proteins'. References
1 Serhan, C.N., Haeggstrom,J.Z. and Leslie,C.C. (1996) FASEB J. 10, 1147-1158 2 Santoro, M.G. et al. (1980) Science 209, 1032-1034 3 Fukushima, M. (1990) Eicosanoids 3, 189-199 4 Rossi,A., Elia, G. and Santoro, M.G. (1996)J. Biol. Chem. 271, 32192-32196 5 Santoro, M.G. (1994) Experientia 50, 1039-1047 6 Santoro, M.G., Garaci, E. and Amici, C. (1990) in Stress Proteins: Induction and Function (Schlesinger,M.J. et al., eds), pp. 27-44, Springer-Verlag 7 Santoro, M.G., Jaffe, B.M. and Esteban, M. (1983)J. Gen. Virol. 64, 2797-2801 8 Pica, F. et al. (1993)Antiviral Res. 20, 193-208 9 Santoro, M.G. et al. (1989)J. Gen. Virol. 70, 789-800 10 Ankel, H., Mittnacht, S. and Jacobsen, H. (1985)J. Gen. Virol. 66, 2355-2364 11 Comi, C. et at. (1996)Antimicrob. Agents Chemother. 40, 367-377 12 Yamamoto, N. et al. (1987) Biochem. Biophys. Res. Commun. 146, 1425-1431 13 Benavente,J. et al. (1984)J. Gen. Virol. 65,599-608 14 Morimoto, R.I., Sarge, K.D. and Abravaya, K. (1992)J. Biol. Chem. 267, 21987-21990 15 Feige,U. et al. (1996) Stress-Inducible Cellular Responses, Birkhiiuser-Verlag 16 Santoro, M.G., Garaci, E. and Amici, C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8407-8411 17 Amici, C. et al. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6227-6231 18 Amici, C., Palamara, A.T. and Santoro, M.G. (1993) Exp. Cell Res. 207, 230-234 19 Rossi,A. and Santoro, M.G. (1995) Biochem. ]. 308,455-463 20 Santoro, M.G. (1996) in Stress-Inducible Cellular Responses (Feige,U. et al., eds), pp. 337-357, Birkhiiuser-Verlag 21 Amici, C. et al. (1994)]. Virol. 68, 6890-6899 22 Schlesinger,M.J. et al. (1991) in Heat Shock Proteins (Maresca, B. and Lindquist, S., eds), pp. 111-117, Springer-Verlag 23 Bader,T. and Ankel, H. (1990)J. Gen. Virol. 71, 2823-2832 24 Rozera, C. et al. (1996)J. Clin. Invest. 97, 1795-1803 25 Baeuerle,P. and Baltimore, D. (1988) Science 242, 540-546 26 Thanos, D. and Maniatis, T. (1995) Cell 80, 529-532 27 Lenardo,M.J. and Baltimore, D. (1989) Cell 58, 227-229 28 Rossi,A., Elia, G. and Santoro, M.G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 746-750 29 Shield, M.J. (1995) Pharmacol. Ther. 65, 125-147 30 Sinclair, S.B.et al. (1989)]. Clin. Invest. 84, 1063-1069 31 Lee,J. et al. (1971) Ann. New York Acad. Sci. 180, 218-240 32 Kikuchi, H. et al. (1982) Tetrahedron Lett. 23, 5171-5174 33 Weinheimer,A.J. and Spraggins, R.L. (1969) Tetrahedron Lett. 7, 5185-5188 34 Santoro, M.G. et al. (1983) Prostaglandins 25, 353-364 35 Santoro, M.G. etal. (1988) Arch. Virol. 99, 89-100 36 Mastromarino, P. et al. (1993) Antiviral Res. 20, 209-222 37 D'Onofrio, C. et al. (1990) Br. J. Cancer 61,207-214 38 Ankel, H., Turriziani, O. and Antonelli, G. (1991)]. Gen. Virol. 72, 2797-2800 39 Hughes-Fulford,M. et al. (1992) Antimicrob. Agents Chemother. 36, 2253-2258 40 Santoro, M.G. et aI. (1982)]. Gen. Virol. 63, 435-440 41 Tanaka, A. et al. (1986) Prostaglandins Leukot. Med. 25, 131-138
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