The effects of cordycepin on malaria parasites

514 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.

Vol. 65.

No. 4.

1971.

THE E F F E C T S OF C O R D Y C E P I N ON M A L A R I A P A R A S I T E S P. I. T R I G G , W. E. G U T T E R I D G E

AND J. W I L L I A M S O N

Division of Parasitology, National Institute for Medical Research, Mill Hill, London, N.W.7 Cordycepin is a purine nucleoside antibiotic which has been isolated from cultures o f the mould, Cordyceps militaris (Linn.) Link (CuNNINGHAM, HUTCHINSON, MASON and SPRING, 1951). It has been shown to be the 3'-deoxy-analogue of adenosine (KACZKA, TRENNER, ARISON, WALKER and FOLKERS, 1964). T h e potential of cordycepin as an antibiotic and an anti-tumour agent has been known for some time. CUNNINGHAM et al. (1951) showed that cordycepin inhibited the growth of both Bacillus subtilis and an avian-tubercle bacillus and JAGGER, KEDRICH and GUARINO (1961) demonstrated that the administration of cordycepin prolonged the survival time of mice inoculated with Ehrlich's ascites tumour cells. Little is known of the effect of cordycepin on protozoa, although WILLIAMSON (1966, 1969) has shown that it inhibits the development of infections by African trypanosomes, particularly Trypanosoma congolense in laboratory animals. This observation p r o m p t e d us to determine whether cordycepin has any antimalarial activity. Initially only a few mg. o f the drug were available and so we studied the effects of cordycepin on cultures of the erythrocytic stages of Plasmodium knowlesi incubated in vitro. Later when more cordycepin was available, in vivo tests against P. berghei berghei were performed. Materials and methods

In vitro incubation The Nuri strain of P. knowlesi was maintained in male and female rhesus monkeys by weekly intravenous subinoculation of infected blood. When the parasitaemia reached 1-5 parasites per 100 red ceils, the monkeys were anaesthetised by intramuscular injections of 5 mg./kg, phenycyclidine HCI (Sernylan, Parke-Davis) and bled by cardiac puncture. One part of 1 : 1000 heparin/Ringer previously warmed to 37'5°C. was added to 10 parts whole blood to prevent coagulation. The parasitized blood was incubated in siliconized 50 ml. conical flasks as described by TRIGG (1968). Tissue culture medium '199' (Glaxo Laboratories), supplemented with extra glucose (to 3 g./1.) and extra N a H C O a (to give a p H of 7-45 at 37.5 °C. in an atmosphere of 5°o v/v CO.o/95°o air) was used as the growth medium (TRIGG, 1969a). 4"0 ml. of sterile medium was allowed to equilibrate to 37.5 C . with a moist stream of 5 o{; v/v CO 2/9500 air. 0-4 ml. of parasitized blood suspension was then added to each flask to give a red cell density of 4 ~ 108/ml. and a parasite density of 4 × 106-2 :, 107-2 × 107 ml. Care was taken to ensure that the time between bleeding the monkeys and the onset of the incubation in vitro was reduced to a minimum. Samples were taken from the cultures at times during the incubation period. Red cell, parasite and differential parasite counts were made as described by TRIGG (1969b). All cultures were tested for sterility by inoculation on to nutrient and blood agar plates. We thank Dr. J. Melling of the Microbiological Research Establishment, Porton and Dr. H. Klenow for supplying purified samples of cordycepin, Dr. F. Hawking for helpful discussions, Mr. B. Cover, Mr. T. J. Scort-Finnigan and Miss Jane Dunnett for technical assistance. One of us (P.I.T.) has received financial assistance from the World Health Organization.

P. I. TRIGG~ W. E. GUTTFRIDGE AND J. WILLIAMSON

515

Biockemica/ stndie.~ The effect of cordycepin on protein and nucleic acid metabolism was examined. Cultures of ring stage parasites of P. knowlesi were incubated in the absence or presence of cordycepin (10 1--10 9 M ) . 100 Ixc of I~C algal protein hydrolysate (52 m C m Atom carbon) and 100 :xc of '~H-adenosine (2340 m C / m M in the presence of 10 tzg/ml, nonradioactive adenine) or 100 iJ-c of :~H-orotic acid (22-5 C/raM) were added to each culture. Th e cultures were incubated for 16 hours at 37'5 C. during which time the parasites in the control flasks grew from the ring to the late trophozoite stage. The incorporation of radioactive tracer into parasite protein and parasite nucleic acid was then measured. Cultures were centrifuged at 1300 g at 0 for 5 minutes and the packed cells washed twice in cold Krebs Ringer solution and precipitated by the addition of 5 ml. of cold 5",, w v trichloracetic acid. T h e precipitated material was extracted twice with the cold trichloracetic acid to yield the cold acid soluble fraction (~ pool). Th e residual material was then suspended in phosphate buffer, split into two parts and incubated with DN'ase or RN'ase to yield D N A and R N A fractions as described by GUTTERIDGE and TRIOG (1970). The residual material after R N A extraction was suspended in 5 ml. 5",, w/v trichloracetic acid and heated at 9 0 C . for 30 minutes. After cooling, 1 ml. portions were collected on to Millipore filters, washed thoroughly with 5 %, w v trichloracetic acid and then dried. Liquid fractions were mixed with Bray's fluid (BRAY, 1960) and then estimated for radioactivity in a liquid scintillation counter (efficiency ~ 30°. for 3H and ~ 60'!. for ~C.). Dried filters were covered with a toluene-based scintillation fluid before estimation of radioactivity. Glucose and lactate estimations were made on centrifugal supernatant fractions of cultures precipitated by the addition of 0.6M perchloric acid. Glucose was estimated spectrophotometrically using hexokinase and glucose-6-phosphate dehydrogenase (Boehringer test kit, T C - X - 1 ) ; lactate was estimated with tactic dehydrogenase (Boehringer test kit TC-B).

P. berghei berghei (strain N, clone 3) was maintained by twice weekly passage of infected blood into male, Parkes strain mice. 15-20 g. mice were used throughout this study. Infected donor mice were bled when 1-5 % of the erythrocytes were infected and heparinized infected blood was diluted with ice-cold 50°i, (v,v) calf serum in Ringers solution. T h e effect of cordycepin on P. b. berghei in vivo was tested using two techniques. Firstly, mice were infected intravenously with approximately 10 ~ parasites and 3 hours after infection 5 groups of 5 mice were treated with 15, 25, 50, 100 and 150 mg./kg, respectively of cordycepin dissolved in distilled water. A control group of mice received distilled water. Smears were taken from all mice at intervals of 24 hours after infection and the number of parasitized cells in 500 erythrocytes was counted. T h e prepatent period ( . pre 2", period) of infection in each mouse was determined by the technique of WARHURSTand FOLWELL(1968). Secondly groups of mice were also treated with three successive daily doses of 50 mg./kg, of cordycepin given intraperitoneally when the initial parasitaemia reached 1-5%. Control groups of mice received distilled water intraperitoneally on the same days. Cordycepm All our in vitro studies were carried out with a sample of cordycepin provided by Dr. H. Klenow. Cordycepin used in the m vivo tests was produced for us at the Microbiological Research Establishment, Porton, Wilts, from culture filtrates of Cordyceps militaris. T h e infra-red spectrum of this material was identical to that of the Klenow's sample of cordycepin, it melted sharply at 231 C . and was chromatographically pure. Results

M o r p h o l o g i c a l s t u d i e s o n P. knowlesi in vitro T h e r e was a marked effect on the growth of P. knowlesi when ring stage parasites were incubated in vitro for 22 hours in the presence o f cordycepin. At concentrations of 10 :'M and 1 0 - ~ M , all the parasites were killed. At 10 ~M some thickening of the cytoplasm occurred during the first 4 hours but there was little further growth and by 22 hours the vast majority of the parasites were abnormal and no nuclear division occurred. In the presence of 10- ~M cordycepin the growth cycle was delayed and a number of abnormal

516

C O R D Y C E P I N A N D /vlALARIA PARASITES

parasites occurred. Lower concentrations had no observable effect on the parasite (Table I), so that 1 0 - 6 M can be regarded as the m i n i m u m growth inhibitory concentration of cordycepin in vitro. TABLE I. Effect of cordycepin on the growth in vitro of P. knowlesi. I

0 hours I

e ~

22 hours

J

m

0

Differential parasite count

J

O v

Differential parasite count

o/

(%)

+.a

(,o)

m ,

R

1"34 × 107 96

T -

II

1'34 × 107 96

10 -4

1.30 × 107 96 - !

1"28 >,~ 107

_ 4 1

10 -3

10- 5

S !GAb

--

4

1"24 × 107

--I 4

1-27 x 107

--i

1'30 × 107 96

R

T

S

G'tA--b-

1

18

77

4 ~--

100

96

!100

73

i

1 ' 2 3 × 10 v - -

25

4

--

71

29

1'25 ,'~ 107 - -

51

19

4

26

0

i1"33 × 10 v - -

13

85

2

--

0

4

I

10 - 6

10-7 10- s

!1.30

× 10 v 96

--

- - ~ 41

1

!1"35 × 107 96

: 4

i 1"34 × 107 96

--,

1"32 × 107

4

2

18 75

4 I

1

0

Key to Table: R = ring; T = trophozoite; S = schizont; G = gametocyte; Ab = abnormal parasite• T h e effect of 1 0 - + M cordycepin could be partially prevented by the addition to the cultures of adenosine b u t 2'-deoxyadenosine, hypoxanthine and 3', 5' cyclic acid A M P had no effect on the inhibition of growth (Table I I). TABLE II. Attempts to reverse inhibition by cordycepin of growth of P. knowlesi in vitro. % Abnormal ~arasites --

Control Cordycepin (10- 5M)

Biochemical

Adenosine

- -

~

- -

T

74

studies

!

9

Hypoxanthine

2'-deoxyadenosine

3'-5'-AMP

10-4M

10-~M

10 4M

10-4M

2

4

2

5

26

71

79

83

o n P. knowlesi m vitro

T h e structural similarity of cordycepin to adenosine and 2'-deoxyadenosine suggested the possibility that this drug might be affecting pathways in the parasite in which these

P. 1. T R I G G , W. E. G U T T E R I D G E A N D I . W I L L I A M S O N

517

metabolites are involved. W e therefore studied the effects on the drug on the catabolism of glucose to lactate, on the uptake of all-adenosine and aH-orotic acid into the pool, D N A and R N A and on the incorporation of t~C-amino acids into protein over a 16 hour period (Table III). T h e results indicated that protein and nucleic acid synthesis were sensitive to inhibition by the drug at the minimum growth inhibitory concentration, whereas lactate production and the uptake of the radioactive precursor into the pool were markedly less sensitive to inhibition. It was clear, therefore, that the main effect o f c o r d y cepin was on macromolecular biosynthesis. TABLE III. The effect of cordycepin on the metabolism of P. knowlesi in vitro. % inhibition of synthesis Cordycepin conc. (xM)

Protein

DNA

RNA

Pool

Lactate

10

84

I00

100

88

68

10 _r,

68

100

100

16

39

38

49

0

0

0

0

0

0

J

10--"

26

10--;

0

;

A detailed study of the time course of effect of cordycepin using all-adenosine to label the nucleic acids is shown in the Figure. This showed that protein synthesis was less sensitive to inhibition by the drug than D N A or R N A synthesis and it also revealed that there was a 21 hour longer lag period before inhibition occurred than in the case of D N A or R N A synthesis. A similar result was obtained when :'H-orotic acid was used to label the nucleic acids. T h e primary effect on the malaria parasite is therefore on nucleic acid synthesis.

5 ? 0 74

PROTEIN

25-

RNA

/

121

20-

/

DNA

1°

Z

~3

gz

uD t-Z

/o~O

~2

0 (J

"

1510 -

Z /~;""

0

I

4

I

8

8"

0

1'2 1'6 0

i

4

o-o-o- -°

.... -o. ..... 2-

/

i

i

8 12 16 T]ME(hr)

C)

0

i

4

t

~

i

1

12 16

FIGURE. Time course of effect of 10 ~M cordycepin on the incorporation of all-adenosine into D N A and R N A and l~C-algal hydrolysate into protein of P. knowlesi in vitro. Cordycepin was without effect on the glucose metabolism of normal red cells, as measured by lactate production, and in addition, there was no obvious change in osmotic fragility of the red cells after incubation in the presence of cordycepin.

518

CORDYCEPINAND MALARIAPARASITES

In vivo t e s t s The prepatent period ( .... pre 2°/o period) of P. b. berghei infections in mice was significantly increased by single doses of cordycepin of 50 mg./kg, and above. The time of death from the infection of these mice was also delayed (Table IV). TABLE IV. Effect of cordycepin on P. berghei berghei in mice. Cordycepin (mg./kg.)

Pre-2'!i, period (Days)

Significance

Day of Death

Significance

11 - 2

2-46 - 0-15

None

15

2.77

~ 0'10

None

12 - 2

25

3"04

: 0.11

None

12 :j: 2

i

None

5O

3.41 - 0"20

P <0.05

16 ~:1

I

P ~ O.Ol

100

3"89 -- 0"08

P -: 0.001

17 := 1

[

P ~: 0.001

19±1

I

150

5.84

- 0'08

P < 0-001

I

i P ~ 0"001

Three daily doses of cordycepin at 50 mg./kg., given to mice after the initial parasitaemia reached approximately 1°/'o,reduced the parasitaemia for 2-4 days but the infection recurred and the mice subsequently died. There was no significant difference between the time of death in the control and treated groups. On one occasion it was possible to test the drug on a P. knowlesi infection in a rhesus monkey and this gave a similar picture to that of P. b. berghei. The monkey, showing an initial parasitaemia of 42%, was given 3 daily doses of 50 mg./kg, cordycepin intraperitoneally. This reduced the parasitaemia for 4 days and the monkey developed a chronic infection. Toxicity

JAGGER, KREDICH and GUARINO (1961) reported that cordycepin could be administered to mice at doses up to 900 mg./kg, without visible effects. We found that when mice infected with P. b. berghei were given 3 daily doses of 150 mg./kg., 4 of 5 mice died between 4-7 days after the end of drug treatment and in the absence of a detectable parasitaemia. Subsequently, it was found that the L D 5 0 in mice (15-20 g.) of our samples of cordycepin was about 400 mg./kg, when the drug was given as a single dose intraperitoneally. Discussion

Cordycepin (10-4M) had no observable effect on the metabolism of mature monkey red cells in vitro. One hundredth the concentration ( 1 0 - a M ) however, markedly affected the development of erythrocytic stages of P. knowlesi, indicating that the drug has a direct selective effect on the metabolism of the malaria parasite. The earliest effect of cordycepin that we could detect in vitro was an inhibition of nucleic acid synthesis. This effect could be due to an inhibition of purine biosynthesis de novo similar to that described by ROTTMAN and GUARINO (1964), OVERGAARD-HANSEN (1964) and TYRSTED and SARTORELLI(1968) but as P. knowlesi appears to be unable to synthesize the purine ring (TRIGG and GUTTERIDGE, 1971) this is unlikely. Alternatively the inhibition could be the result of an effect on an enzyme such as adenosine kinase,

P. I. TRIGG, W. E, GUTTERIDGE AND .~, WILLIAM~ON

51c~

which prevents the replenishment of the purine nucleotide pool bv the phosphorylation of exogenous purines. We cannot rule out this possibility but it seems more likely that inhibition is the result of a direct effect on the biosynthesis of nucleic acids since similar effects of cordycepin on nucleic acid synthesis have been described by KLENOW (1963a) with Ehrlich's ascites cells in vitro and on RNA synthesis by SIEV, WEINBERGand PENMAN (1969) with HeLa cells in vitro. In these cases the effects on RNA synthesis have been shown to be due to an accumulation within the cells of cordycepin triphosphate (KLENOW, 1963b) which subsequently affects the DNA-primed RNA-polymerase (KLENOW and FREDERICKSEN,1964) by being incorporated into the nascent RNA chain and thus terminating the synthesis due to its lack of a 3'-OH group (SHIGEURAand BOXER,1964). A similar effect on the DNA-primed-DNA-polymerase could explain the effects on DNA synthesis in Ehrlich's ascites cells. These mechanisms can also explain the effects of cordycepin on malarial DNA and RNA synthesis. Although cordycepin is therapeutically active against blood induced infections of P. b. berghei at doses above 50 mg./kg., a curative dose was not obtained. WILLIAMSON (1966) however, found the drug to be therapeutically active at 6,25 mg./kg, and curative at 25 mg./kg, against Trypanosoma congolense in vivo and, more recently, WILLIAMSON and COVER (unpublished results) have obtained activity in vivo against T. cruzi. It is of interest that cordycepin has both trypanocidal and anti-malarial activity whereas its close analogue, the aminonucleoside of puromycin, is only trypanocidal. The aminonucleoside of puromycin is in fact, more active against the intracellular than against the extracellular forms of T. cruzi and it seems possible that the tissue cells in which the intracellular forms are grown metabolise the amino nucleoside to a compound which is more active against the trypanosome (GUTTERIDGE,KNOWLER and COOMBES, 1969). In this context it would be interesting to see whether cordycepin is more active against sporozoite than blood-induced infections of malaria. The clinical use ofpurine analogues has usually been prevented by their high toxicity. The report by JAGGERet al. (1961) which showed that cordycepin could be given to mice at doses up to 900 mg./kg, without visible toxic effects, suggested that a non-toxic purine analogue had been found. Under our experimental conditions however, our samples of cordycepin were lethal to some mice at doses as low as 200 rag. kg. and renal toxicity was observed at 400 mg./kg. Cordycepin itself is not likely therefore to be a clinically useful antimalarial drug. Nevertheless, our investigations show that malarial parasites are sensitive to inhibition by some purine analogues so it may be possible to synthesise a structural analogue of cordycepin which is more active against the malaria parasite than the parent compound and less toxic to the host. Such an analogue could prove a particularly useful drug since its mode of action is likely to be so different from any of the currently used antimalaria drugs that problems of cross-resistance should not arise. Summary 1. The effects ofcordycepin (3'-deoxyadenosine) both on cultures of the erythrocytic stages of Plasmodium knowlesi incubated in vitro and Plasmodium berghei berghei in vivo have been studied. 2. Concentrations of drug as low as 106M affected the growth in vitro of the parasite after a lag period of about 4 hours. These effects could be partially prevented by adenosine but not by other purines tested. 3. Of the various anabolic and catabolic processes studied in the malaria parasite in vitro, the drug affected nucleic acid synthesis first and to the greatest extent. 4.

It was without observable effect on the host red cell.

520

CORDYCEPINAND MALARIAPARASITES

5. Cordycepin was therapeutically active at doses of 50 mg./kg, and above against blood-induced P. b. berghei infections. Complete cure was not obtained even at the highest doses and toxic effects were observed at doses of 200 mg./kg, and above. REFERENCES BRAY, G. A. (1960). Anal. Biochem., 1, 279. CUNNINGHAM,K. G., HUTCHINSON,S. A., MASON,W. & SPRING,F. S. (1951). J. Chem. Soc., 2, 2299. GUTTERIDGE,~r. E., KNOWLER,J. & COOMBES,J. D. (1969). J. Protozool., 16, 521. - & TRIGG, P. I. (1970). Ibid., 17, 89. JAGGER, D. V., KREDICH, N. M. & GUARINO,A. J. (1961). Cancer Res., 21, 216. KACZKA,E. A., TRENNER,N. R., ARISON,B., WALKER,R. W. & FOLKERS,K. (1964). Biochem. Biophys. Res. Commun., 14, 456. KLENOW, H. (1963a). Biochim. Biophys. Acta., 76, 354. - (1963b). Ibid., 76, 347. & FREDERICKSEN,S. (1964). Ibid., 87, 495. OVERGAaRD-HANSEN,K. (1964). Ibid., 80, 504. ROTTMAN, F. & GUARINO,A. J. (1964). Ibid., 89, 465. SHIGEtrRA, H. T. & BOXER, G. E. (1964). Biochem. Biophys. Res. Commun., 17, 758. SIEV, M., WEINBERG,R. & PENMAN, S. (1969). J. Cell Biol., 41, 510. TRIGG, P. I. (1968). Trans. R. Soc. trop. Med. Hyg., 62, 371. (1969a). Parasitology, 59, 925. ---(1969b). Ibid., 59, 915. ---& GUTTERIDGE,W. E. (1971) ibid., 62, 113. TYRSTED, G. & SARTORELLI,A. C. (1968). Biochim. Biophys. Acta, 155, 619. WaRHURST, D. C. & FOLWELL,R. O. (1968). Ann. trop. Med. Parasit., 62, 349. WILLIAMSON,J. (1966). Trans. R. Soc. trop. Med. Hyg., 60, 8. (1969). Parasitology, 59, 9P. -

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