New antiherpetic nucleoside from a Basidiomycete

0 Acadbmie Pharmacology

des

/ Elsevier, Paris

sciences

/ Pharmacologic

New antiherpetic

nucleoside

from a Basidiomycete

Nouveuu nuclebside mbhevpe’tique isok b pmtir d’un Bmh?iomyc&e Didier

Saboulard*,

Alexandre

Gaspar,

Bernard

Roussel,

Jean Villard

Laboratoire de mycologic appliq&e aux biotechnologies industrielles, lnstitut des sciences pharmaceutiques de Lyon, universit& Claude-Bernard-Lyon-l, 8, avenue Rockefeller, 69373 Lyon cedex 08, France (Received 23 December

1997, accepted

et biologiques

after revision 2 May 1998)

Abstract - Antiviral

activity was characterized from the culture broth of the Basidiomycete Macrocystidia cucumis (Pers. ex Fr.) Heim. When the stationary phase was reached (21 d), the culture broth was shown through an ELISA assay to contain antiviral activity against herpes simplex virus type 1 (HSV-I), as assessed in baby hamster kidney cells (BHK-21). Once the presence of the anti-HSV-1 activity in the culture broth was demonstrated, we proceeded with the purification and isolation of the active principle using a semi-preparative HPLC technique. The activity was associated with a purine nucleoside designated McA. This compound displayed no cytotoxicity at antivirally effective concentrations and proved to be a novel nucleoside analogue. (0 AcadPmie des sciences / Elsevier, Paris.) Macrocystidia

cucumis

(Pers. ex Fr.) Heim / Basidiomycetes

/ herpesvirus

/ antiviral

/ nucleoside

analogue

R&m& - L’activitt antiherpttique du surnageant de culture du Basidiomycete Macroytidia cucumis (Pers. ex Fr.) Heim, a CtC mise en kidence par test Elisa lors de la phase stationnaire de croissance (21 j). Le milieu de culture prCsente alors une activitt antivirale in vitro vis-a-vis du virus herpes simplex de type 1 (HSV-1) sur cellules BHK-21. Apres dCtection de l’activit6 antiherpttique, une stratCgie a et6 mise au point afin de purifier et d’isoler le principe actif par chromatographie liquide haute performance (CLHP) en phase inverse. Un mitabolite antiherpetique nommC McA a CtCis016 11 s’agit d’un nouvel analogue de nuclCoside dont l’activit6 antivirale n’est associCe B aucune cytotoxicite dans les conditions exptrimentales retenues. (0 Academic des sciences I Elsevier, Paris.) basidiomycbtes

I virus

herpes simplex

/ antiviral

I analogue

nucl&sidique

I Mwwy~ridia

cwuml

(Pers.

ex FL)

Heim

Version

abrCg6e

A la suite d’un large programme de criblage realis& sur des souches de basidiomycetes (Laboratoire de mycologic, Lyon), Macrocystidia cucwnis a et& sClectionnC pour la production de substances antivirales dirigCes en particulier contre le virus herpes simplex de type 1 (HSV- 1). La stratCgie mise au point nous a permis de s&parer et purifier un mttabolite antiherpetique excr&C par le myctlium, g partir d’un extrait de culture liquide agitCe.

Note communicated * Correspondence Fax: (33)

by Alain and

Un test Elisa effect& sur des surnageants preleves apres 21 j de culture, a mis en kidence une activitt antivirale dirigee contre le virus herpts simplex de type 1 (HSV- 1). Aucune activite antiherpttique n’a ttC d&ectCe apr&s 7 et 14 j de culture. De plus, cette activite a et6 recherchke sur des &hantillons pr&lev& au 21e jour de culture, ayant subi une extraction liquide/solide (colonnes Sep-Pak@, C,,). Le surnageant a present6 une t&s nette activitC antiherpetique, ainsi que les extraits de culture obtenus sur colonnes Sep-Pak’.

Boudet

reprints

04

78 77 72

12

C. R. Acad. 1998.321,585-591

Sci. Paris,

Sciences

de

la vie

/ Life

Sciences

585

D. Saboulard et al. Un metabolite antiherpetique nommt McA a et& isole. La purification a et& rCalisCe par chromatographie liquide haute performance (CLHP) semi-preparative [colonne Waters Assoc. PBondapak C,,; 10 pm, 7,8 x 250 mm]. L’analyse par CLHP de l’extrait de culture a demontre la presence de nombreux produits absorbant a 254 nm. Le test Elisa a mis en evidence une diminution des antigenes de HSV-1 en presence du pit A (McA) dont le temps de retention est de 10 min. L’activite du compose antiherpttique a ete testee de facon quantitative sur des cellules BHK-2 1. Le test Elisa demontre que cette activite varie de 0 a 94,86 % pour des concentrations allant de 0,3 1 a 10 ug/mL. La concentration minimale d’inhibition (CMI) determinee selon la methode de dilutions binaires successives est de 0,72 pg/mL. Cette activite antiherpetique est associee a un effet cytotoxique negligeable sur les lignees cellulaires Vero et MRC-5. Des concentrations du metabolite antiherpetique comprises entre 1,5 et 50 yg/mL engendrent sur les cellules BHK-21 un pourcentage de cytotoxicite variant de 0 a 2 %. D’autre part, une etude prtliminaire de toxicitt a et& realisee chez la souris par administration de McA per OS et 1.P a la dose de 90 mg/ kg. Aucune difference significative due au traitement n’est relevte, tant en ce qui concerne l’kolution pond&ale que la consommation d’eau et d’aliment. L’activite antiherpetique de cette molecule, associee a sa nature nucleosidique, conferent toute son originalite g cette etude. 11 s’agit dun analogue de nucleoside de formule

1. Introduction Herpesvirus infections in immunocompromised patients often have severe consequences with significant morbidity and mortality stemming from them. Major accomplishments have been made in herpes antiviral therapy. These include various nucleoside analogues with selective antiviral activity, such as vidarabine, bromovinyldeoxyuridine and especially acyclovir. However, there are limitations to currently available drugs [l-4]. Some drugs have been associatedwithsignificantconcomitanttoxicitybecauseoftheir lack of selectivity, whereas references to drug resistant virus strains have been noted 15, 61. As a consequence, the need for new substances with potent antiviral activity will be increasing in the future. And screening programme reports involvingcompoundsof natural sourcescan be found more and more frequently [7-l 31. Secondary metabolites from fungi display a wide range of biological activities, making the Basidiomycetes an alternative target for antiviral research. In our screening programme on biologically active principles of mushrooms, we examined mycelial culture of Macrocystidia cucumis (Pers. ex Fr.) Heim. When the stationary phase was reached (21 d), culture broth exhibited potent anti-

586

C1*H21N505 (PM: 339). La structure atypique de cette molecule ne correspond a aucun modele existant synthetique ou naturel, ceci &ant confirmt par une etude bibliographique approfondie, McA souleve de nombreuses questions quant a son mecanisme d’action. Ce compose pourrait agir a l’image d’analogues de nucleosides antiherpetiques tels que la vidarabine (A&, ou l’acyclovir (ACV). La majorite des medicaments disponibles a l’heure actuelle et diriges contre le virus HSV-1, inhibe l’ttape de replication du cycle viral. L’etude in vitro du spectre d’activite antiviral de McA sur des HerpCtoviridCs tels que les virus VZV, CMV, et HSV-1 deficients en thymidine kinase (resistants a 1’ACV) constitue une perpective tres interessante. Cette liste n’est pas exhaustive et comprend des retrovirus dont le VIH (Virus d’Immunodeficience Humaine). Neanmoins, une activite antivirale et un effet cytotoxique sont susceptibles de varier d’une lignee cellulaire a l’autre. De plus, l’eficacite et/au la selectivite des composes antiherpttiques en culture cellulaire ne predit pas necessairement leur effrcacite sur le modele animal ou en situation clinique. De ce fait, la necessite dune experimentation de McA sur un modtle animal approprie est done obligatoire, une prudence extreme devant etre prise lorsque l’on veut extrapoler des resultats experimentaux in vitro Q un eventuel effet chez l’homme. Et seules des etudes in vivo pourraient reveler si le nucleoside McA peut etre considere comme un candidat adequat au traitement clinique des infections herpetiques.

viral activity against herpes simplex type 1 (HSV-1) in baby hamster kidney cells (BHK-21). In this paper, we wish to present the purification, isolation and biological characterization of this antiherpetic substance. A specially developed strategy was set in order to characterize the fungal metabolites and led us to isolate a new compound, hereby named McA. This compound has not yet been described from natural or synthetic sources. Its molecular formula is C,,H,,N,O, (MW 339).

2. Materials 2.1.

Macrocystidia

and methods cucumis

(Pers.

ex Fr.)

Heim

Mycelial cultures of Macrocystidia cucumis were obtained from spore prints of fruiting bodies collected near Lyon, France. The producing strain was deposited in the culture collection of the Mycology Laboratory, University of Lyon. A voucher specimen was preserved at the Pasteur Institute Culture Collection (Paris, France) under the accession number l-l 813. 2.2.

Cell

lines

Baby hamster kidney fibroblast cells (BHK-21), green monkey kidney (Vera) cells and Human C. R. Acad.

Sci. Paris, Sciences

African embryo

de la vie / Life Sciences 1998.321.585-591

New antiherpetic lung fibroblasts Whittaker-France. in Glasgow medium serum

(MRC-5) BHK-21 modification

(MEM; Gibco) (FCS) and 10

%

cells were purchased from Bioand MRC-5 cells were grown of Eagle’s minimal essential

supplemented tryptose

with phosphate

cells were grown in the same medium non-essential amino acids (Gibco). maintained at 37 “C in a humidified bator) containing 5 % CO,. 2.3.

Virus

10 % fetal calf broth. Vero

supplemented The cultures atmosphere

with were (incu-

stock

Herpes simplex virus type 1 (HSV-1; strain F) was supplied by Dr Roizman (University of Chicago, USA). The virus was propagated in BHK-21 cells. The concentration of virus in the stock suspension was determined by the plaque reduction method [141, and adjusted mL PFU (plaque-forming units). The stock sion was preserved in liquid nitrogen. 2.4.

Fungal

growth

The maintenance medium g/L), glucose: 16.5, malt

for M. cucumis extract: 3.5,

MgS0,.7H,O 0.25, (NH&C,H,06 tartrate): 0.35, casein hydrolysate: 0.5 in 1 L deionized water. Agar solid adjusted For

cultures.

The

to 6.2 liquid

to 2 x IO-‘/ virus suspen-

with

culture

pH

of the

(in 0.5,

(di-Ammonium L(+)0.5, L(+) asparagine: (10.5 g/L) was added for

maintenance

1 M HCI an

contained KH,PO,

before

inoculum

medium

was

sterilization.

was

prepared:

samples

fractions

were

pooled,

nucleoside methanol

from

was

a Basidiomycete

evaporated

to dry-

ness in vacua and the residue was dissolved with 5 % acetonitrile (Merck, Darmstadt, Germany). Final purification was achieved by semi-preparative high performance liquid chromatography C,,-silica gel column perature. The HPLC Model 510 solvent

(HPLC) on Waters Assoc. pBondapak (IO pm, 7.8 x 250 mm) at room temsystem consisted of a Waters Asssoc. delivery system, equipped with a

Waters Model 480 254 nm. The mobile

variable wavelength detector set at phase consisted of acetonitrile-water

(5:95; v/v). It was filtered through a 0.45~pm HPLC-certified membrane filter and degassed under vacuum and sonicated. Elution was monitored at a flow rate of 3.5 mL per minute. on individual compound

Antiviral activity was assessed fractions. The retention time named McA was 10 min.

The purity of McA was asserted (Merck), and by analytical HPLC pBondapak C,,-silica gel column (5 flow rate: 1 mL /min) with diode array positive ion fast atom bombardment trum was cerol was on Perkin

by ELISA test of the antiviral

by tic on silica gel on a Waters Assoc. pm, 4.6 x 250 mm; detection (DAD). A (FABMS) mass spec-

recorded on a Nermag spectrometer. used as a matrix. ToF-SIMS data were Elmer Physical Electronics Static

Model PHl720. Nuclear magnetic resonance 13C NMR) spectra were determined in DMSO-d, with a Bruker AM 400 NMR spectrometer and tions are given in parts per million downfield

Thyoglyrecorded ToF-SIMS (‘H

NMR, solution peak posifrom tetra-

of surface growth (3 mg) from a 2 1 -day solid agar medium were transferred to a 100 mL Erlenmeyer flask containing 25 mL of broth medium. Flasks were incubated at 24 “C in darkness for 21 days with agitation (150 rpm) on a Gyratory shaker (Model G25, New Brunswick Scientific

methylsilane as internal standard (signals s, singlet; d, doublet; t, triplet; q, quadruplet; br-s, broad singlet).

Co., New Brunswick, NJ, USA). The mycelium separated from the culture broth by filtration carded. The filtrate was stored at -20 “C. The

The medium was removed from 24-h-old BHK-21 cultures in 96-well culture plates. The HSV-1 stock was diluted 1 :I0 in medium containing 2 % FCS. A 40-pL virus dilution was distributed in all wells except rows 1

of the antiherpes compound was followed linked immunosorbent assay (ELISA). 2.5.

Isolation

and

purification

was then and disproduction

by the enzyme-

of bioactive

2.6.

Bioassay

are described m, multiplet;

as

procedure

and 2. In these rows 40 pL of the growth medium were added. Following virus adsorption for 1 h at 37 “C, the medium was discarded by aspiration.

metabolites

The following procedure was used to isolate the active agent from the M. cucumis culture broth. First, the production of the antivirally active metabolite was followed by the bioassay (ELISA) procedure. At the end of a 21 -day

In rows 1 to 4, 100 pL medium were added. Rows 1 and 2 served trol, rows 3 and 4 served as the other rows dilutions of acyclovir

cultivation period, the mycelium containing compounds was separated from the culture tration through a 0.45~pm nylon membrane,

come Research Laboratories), culture broth or fungus extracts were distributed in serial two-fold dilutions in a range from 2 to 64. To each dilution, 100 pL per well were

carded. This filtrate was at 4 000 g for 10 min. C,,

An aliquot Sep-Pak

first

(4 mL) of the classic cartridge

clarified

supernatant (Waters

no active broth by filand dis-

by a centrifugation was Assoc.,

loaded Milford,

onto a MA,

containing 5 % FCS as the uninfected coninfected control. In the (ACV, Zovirax, Well-

added. The assays were run in quadruplicate. After 24 h incubation (37 “C, 5 % CO,), the plates were washed with phosphate-buffered saline (PBS; pH 7.2) containing 1 % bovine albumin. Counter ion of the buffer was added (NaH,PO,).

USA), preactivated with 2 mL methanol (Merck, Darmstadt, Germany) and 4 mL deionized distilled water. The antiviral product was adsorbed into the cartridge while most inactive impurities remained in the supernatant.

The plates were fixed with 50 pL 0.05 % glutaraldehyde (Sigma, France) in PBS and incubated for 1 h at 4 “C. The wells were washed three times with a solution containing

The cartridge antiviral product

0.05 % Tween 20 in PBS (PBS-Tw). anti-HSV-1 antibody (Virus Stat.,

was washed with 4 mL distilled water. was eluted with 2 mL methanol.

C. R. Acad. Sci. Paris, Sciences 1998.321,X5-591

de la vie / Life Sciences

The The

Then 100 pL of a goat Maine, USA; diluted

507

D. Saboulard

et al

1:4 000 in PBS-Tw) was added, and the plates were further incubated for 2 h at 30 “C. The plates were washed three times with PBS-Tw and 200 pL of peroxidase-conjugated rabbit anti-goat immunoglobulin G (IgC) (Sigma, France; diluted 1:25 000 in PBS) were added in each well. The plates were incubated again for 1 h at 37 “C. The wells were washedthree times with PBS-Tw and 200 pL of o-phenylenediamine dihydrochloride (SIGMA, France) 0.4 mg/mL in 0.05 M phosphate-citrate buffer (pH 5) containing 0.03 % sodium perborate (Sigma, France). The reaction was stopped after 20 min with 50 FL of 2 M H,SO, and the optical density (O.D.) was read at 492 nm with a microplate reader (Thermomax Molecular Devices, CA, USA). 2.7.

Cytotoxicity

1

-

01

0

1=

8

I

I

I

I

I

I

I

I

2

4

6

8

10

12

14

16

18

Culture

K I

IO

21

time (days)

Figure 1. Time course cultivation diagram of Macrocystidia W pH value; 0 mycelial dry weight (g/L).

cucumis.

assay

In-vitro cellular cytotoxicity assays were performed with MRC-5, Vero and BHK-21 cells with a tetrazolium salt (MTT) assay [151. The medium was removed by aspiration from 96-well culture plates containing 24-h-old culture cells (20 000 cells per well). In rows 1 and 2, 100 pL of medium containing 5 % FCS were added in each well (0 % cytotoxicity control). In rows 3 and 4, 100 FL of the same medium containing 20 Mm iodacetamid were added (100 % cytotoxicity control). In the other wells we added 100 FL of serial two-fold dilutions of culture broth or McA in a range from 2 to 64. The plates were incubated at 37 “C in a humidified atmosphere containing 5 % CO,. After a 24-h incubation, 10 pL MTT (50 pg per mL medium) [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazoliumbromide, Sigma Chemical Company, France] were added to all wells. The plates were incubated for 4 h at 37 “C. After incubation, 10 % sodium dodecyl sulfate (SDS) in 0.01 M HCI was added to all wells. The optical density (O.D.) at 570 nm was measured in a microplate reader (Thermomax Molecular Devices, California, USA). The average O.D. of each culture broth or McA dilution was plotted against the logarithm of culture broth or McA concentration. The linear portion of the curve was used to calculate the concentration corresponding to 50 % cytotoxicity (CC,,).

3. Results and discussion During the stationary phase of growth, M. cucumis released intheextracellularmediumacompoundwithantiviral activity against herpes simplex virus type 1 (HSV-1). This result is an additional illustration of the ability of fungi to produce secondary metabolites depending on developmental stage (data not shown). The time course for the culture of M. cucumis is shown in figure 1. The pH, which was not controlled, decreased from 6.2 to 3.5 while the mycelial dry weight increased from 0.8 to 3.5 g/L. This biological activity was assessed in an ELISA test system using a series of culture broth dilutions. The ELISA assay proved to be a useful and reproducible method to

580

-4

follow the production of anti-HSV-1 activity of the culture fluid. Using the ELISA assay, we were able to demonstrate that the culture broth produced a significant decrease in the HSV-1 antigen expression when the stationary phase was reached (21 d). Antiviral activity was found neither on the 7th day nor on the 14th day of cultivation period. Moreover, the antiviral activity was still present in culture broth (21 d) dilutions as high as 64-fold. Little or no cytotoxic effect could be detected in culture broth dilutions producing a marked inhibition of HSV-1 replication (cytotoxicity less than 4 % at dilutions up to 64-fold). Once the synthesis of secondary metabolites with antiviral activity by M. cucumis was proved, we proceeded with the purification and isolation of the active agent. In order to perform a preliminary purification of the culture broth, an extraction and cleaning step using Sep-Pak C,, reverse-phase columns was carried out. The isolation procedure is outlined in figure 2. An HPLC system was developed to analyse the extracts from the culture broth of M. cucumis as indicated in the Materials and methods. A representative semi-preparative HPLC chromatogram is shown in figure 3. Fractionation of the methanolic extract resulted in different samples that were tested for their inhibitory effect on the replication of HSV-1 in BHK-21 cells. Only the fraction containing the McA compound elicited marked anti-HSV-1 activity. In general, secondary metabolites are produced in small quantities; therefore, only a few milligrams of McA could be isolated from the filtrate. Fractions were collected from the HPLC and pooled in an ice bath. Lyophilization of McA fractions resulted in a white amorphous powder, the yield being 30 mg of antiviral compound per litre culture broth. The minimum inhibitory concentration (MIC) was determined by a serial two-fold dilution method, and with acyclovir as the positive control (100 % inhibition at an acyclovir concentration of 100 pg/mL). McA was shown to exhibit a marked inhibitory effect on HSV-1 replication (table 0; the minimum inhibitory concentration was calculated as 0.72 pg/mL. C. R. Acad.

Sci. Paris, Sciences

de la vie / Life Sciences 1998. 32lI585-591

New

antiherpetic

nucleoside

from

a Basidiomycete

filter (0.45 pm)

Filtrate

I

Mycelium 1. Centrifuge

I SepPak (‘A) c=bidw

10 min., 4000 x g I discard

1. 2. 3. 4.

A&ate mtrige (2ml MeOH, 4 ml H20) Add sample (4 ml) Wash with 4 ml Hz0 Elute with 2 ml MeOH

1. Evaporate 2. Dissolve

to dryness in vacua in 5% acetonitrile

semi-preparative HPLC

I

1. HPLC fracbnati~~ 2. ELBA monitoring

Figure 2. Isolation fluid of Macrocystidia

(BHK-21

procedure of the McA cucumis.

cells. HSV-1)

compound

from

i

the culture 0

Every time that the antiviral activity was tested, the cytotoxicity was determined in parallel. Thus, the chance of false interpretation of cytotoxicity as an antiviral effect was annihilated. McA displayed no cytotoxicity to Vero and MRC-5 cells and very little cytotixicity to BHK-21 cells at the antiviral effective concentrations (0 to 2.05 % cytotoxicity for McA concentrations in the range 1.5 to 50 pg/mL).

This compound has not been described previously [16, 171. Its molecular formula is C,,H,,NsO, (MW 339). The positive fabms (thioglycerol) of McA displayed a significant ion at m/z 340 corresponding to M+ (C,,H,,N,O,) + H. Examination of the ‘H NMR, 13C NMR and COSY Table I. Susceptibility of herpes simplex virus type inhibitory effects of McA compound in baby hamster tures. Sample

and

assay

were

as presented

in the

1 (HSV-1) to the kidney cell cul-

Materials

and

methods

section. All values shown assays. The percentage of termined by the formula: ODx: optical density of

in the table are the means of quadruplicate antiviral activity of McA on HSV-1 was de100 x [l -(ODx - ODuc)/(ODic - ODuc)]. infected cells in the presence of antiviral

compound (McA). ODic: optical density of uninfected

optical density control. McA

Antiviral

0.31

0.62

0

0

of infected

control.

ODuc:

2.5

5

10

77.12

90.86

94.86

concentration 1 .25 44.22

[pg/mL]

activity WI

C. R. Acad. 1998.321,585-591

Sci.

Paris,

Sciences

de

la vie

/ Life Sciences

L

ki Retention

1’0

15

time (minutes)

Figure 3. Semi-preparative HPLC profile of the 21st day culture fluid extract from Macrocystidia cucumis. Column: p6ondapak Cl 8-silica gel (10 pm, 7.8 x 250 mm). Mobile phase: acetonitrile-water (5:95; v/v), 3.5 mL /min. Detection: UV 254 nm. Fractions from peak A (elution time: 10 min) were found to contain anti-HSV-1 activity and were pooled.

spectra in DMSO-d6 revealed the structure. ‘H NMR: 6, 5.86 (1 H, d, 6 Hz, 1 ‘-H); 6, 4.52 (1 H, ddd, 6 Hz, 5.8 Hz, 5.5 Hz, 2,-H); 6, 4.12 (lH, ddd, 4.6 Hz, 4.6 Hz, 3.6 Hz, 3*-H); 6,3.94 (1 H, ddd, 3.6 Hz, 3.6 Hz, 3.6 Hz, 4,-H); 6, 3.64 (lH, ddd, 12.2 Hz, 3.6 Hz, 3.6 Hz, 5,-H); 6, 3.55 (1 H, ddd, 12.2 Hz, 7.6 Hz, 2.7 Hz, 5,-H); 6, 5.38 (1 H, d, 6.2 Hz, 2’-OH); 6, 5.34 (1 H, d, 4.6 Hz, 3’-OH); 6, 5.00 (1 H, dd, 6.0 Hz, 3.9 Hz, 5’-OH); S, 7.24 (2H, br-s, NH,); 6, 8.11 (1 H, s, 2-H); 6,8.31 (1 H, s, 8-H); 6, 8.51 (1 H, s, 4-H); 6, 1.26 (3H, t, 6.5 Hz, CH,-CH,); 6, 1.97 (2H, s, CHICO) ppm. 13C NMR: 6c87.7 (C-l ‘); 6c73.4 (C-2’); 6, 70.0 (C-3’); SC 84.7 (C-4’); 6, 61 .O (C-5’); tic 152.1 (C-2); 6, 140.6 (C-8); 6, 173.0 (C-4); 6, 163.0 (C-5); 8, 118.0 (C-6); SC 40.89 (C-l”); 6,189.O (C-2”); 6,29.45 (C-3”), 6c 14.0 (C-4”) ppm. The ToF-SIMS showed the expected fragmentation pattern: 362 (M+Na) and 378 (M+K), 71 (R: CH,-CO-CH2-CH,), 268 (M-R), 205 (Base+R), 135 (Base+H), 206 (Base+R+H). Figure 4 shows a structural formula of McA. From these characteristics, McA could be differentiated from any antiviral agent already reported and thus, McA should be considered as a new nucleoside analogue with potent anti-HSV-1 activity.

569

D. Saboulard

et al.

leukemia patients or tissue or organ transplant recipients). As Griffiths correctly underlined “there is evidence that the prevalence and impact of some herpesvirus infections are likely to grow is an urgent need agents. Although

over the next decade” [24]. Thus, there for a continuing search for new antiviral a number of new antiherpetic com-

pounds have been chemically exhibited unacceptable toxicity result, only a few have proceeded trials 125-291. An alternative approach agents consists in the metabolites from fungal

OH Figure

4. Structure

of McA. 1” 2”

R = CH,COCH,CH,

More

research

3”

work

OH

viral

is needed

to

answer

a

number of questions concerning its biosynthesis, metabolism and mechanism of action against HSV-1. Since McA was found to be a nucleoside analogue, its mechanism of action and biological behaviour could be comparable to those of acyclovir or other antiviral (i.e. inhibition of the HSV-1 DNA In addition, McA against the last years that has

This

is related

compromised

nucleoside polymerase)

analogues [I 8-201.

it is important to determine the activity of ACV-resistant HSV-1 strains. Indeed, during the risk of encountering herpesvirus strains

are resistant to acyclovir increased 121-231. to

the

(ACV)

or ganciclovir

growing

individuals

effective

Moreover, intraperitoneally

McA

number

(i.e.

in the search for new antiviral isolation of natural secondary origin [30-331. Thus, we were

able to isolate a new nucleoside (McA) from the Basidiomycete M. cucumis, that was found to possess potent antiHSV-1 activity in BHK-21 cells (MIC 0.72 ug/mL). Interestingly, McA proved to be free of any cytotoxicity at anti-

4”

on

synthesized, many have in vitro or in vivo. As a to evaluation in clinical

of

HIV-infected

(GCV) immunopatients,

concentrations. a preliminary toxicology administered McA

study at 90

of orally and mg/kg doses

was conducted in female and male mice. examined body weight evolution, water and sumption. No difference was observed between treated blished

group and results).

It could

the

non-treated

be interesting

control

to extend

these

This food the

group

previous

study conMcA(unpu-

studies

to a larger number of viral strains. Further in vitro studies are planned to demonstrate the potential activity against herpesviruses such as cytomegalovirus (CMV) or varicella-zoster virus (VZV), and against retroviruses (HIV). It now seems important to examine the efficiency and safety of McA in HSV-l-infected addition, the metabolism should be studied. These confirm the potentiality didate

drug

for

appropriate animal model. In and pharmacology of McA in vitro and in vivo studies could qualities of McA as a good can-

antiherpesvirus

therapy.

Acknowledgments:

This investigation was supported by a financial grant from Etudes & expertises industrielles (Mulhouse, France) to which we are deeply indebted. We gratefully acknowledge Pr Dr E. De Clercq and Dr L. Naesens (Rega Institute for Medical Research, Leuven, Belgium], Pr E. Bahraoui and Pr A. Boudet (Universite Paul-Sabatier, Toulouse, France) for helpful critical reading of the manuscript, We are grateful to T. Saboulard for his valuable advice and help. We wish to express our sincere appreciation to H. Astbury for her dedicated editorial assistance.

[6] Field A.K., Biron K.K., ‘The End of Innocence’ herpesviruses to antiviral drugs, Clin. Microbial.

4. References [l] Darby G., A history other. 5 (1994) 3-9. [2] De Clercq J. Antimicrob.

of antiherpes

research,

the treatment 121-132.

131 De Clercq E., Descamps J., Verhelst Torrence P. F., Shugar D:, Comparative against different strains of herpes simplex

ment,

D., Barton S., Johnson Antiviral. Chem. Chemother.

[5] Chatis PA., Crumpacker drugs, Antimicrob. Agents

590

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