Phosphorylation and anti-leishmanial activity of formycin B

Vol. 100, No. 3,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1377-1383

June 16, 1981

PHOSPHORYLATION AND ANTI-LEISHMANIAL

ACTIVITY

OF FORMYCIN B

DENNIS A. CARSON KWANG-PO0 CHANG Department of Clinical Research Scripps Clinic and Research Foundation 10666 North Torrey Pines Road La Jolla, California 92037 Laboratory of Parasitology The Rockefeller University New York, New York 10021

Received

May 6,

1981 SUMMARY

The inosine analog formycin B (l-10 uM) inhibited the in vitro growth of Leishmania promastigotes and amastigotes. When administered to Syrian hamsters infected with Leishmania donovani, formycin B (10 mg qd X 5) decreased by greater than 90% the number of liver amastigotes, with a concomitant reduction in hepatosplenomeqaly. Both extracts and intact cells of Leishmania, unlike mammalian cells, effectively phosphorylated formycin B. The resultinq form.ycin B monophosphate inhibited dose-dependently the conversion-of IMP to adenylosuccinate in parasite extracts. This effect may be related to the potent anti-leishmanial activity of formycin B. The inosine

analog

allopurinol

ribonucleoside,

ribofuranosylpyrazolo(3,4-d)pyrimidine) vitro

growth

of the

and intracellular -al

also

hosts, that

showed

that

contained converted

(3).

the

of several of Leishmania

Leishmania

an unusual allopurinol

Subsequent

was found

promastigotes amastigotes

with

resulting

reported infection

inhibition

whether

or not

unlike

to the

that of growth

allopurinol

in vitro

Nelson

their

mammalian

ribonucleoside

nucleotide

incorporated It is

et -

(EC 2.7.1.77)

5'-monophosphate

(l-4).

(l),

(2).

yielded

were

the -in species

phosphotransferase

and phosphorylation

of 4-aminopyrazolo(3,4-d)pyrimidine

inhibit

leishmanial

parasites,

nucleoside

to

donovani --

ribonucleoside

amination

(4-hydroxy-l-B-D-

derivative derivatives

into

RNA,

has not

been

effective

in Leishmania

--in vivo.

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0006-291X/81/111377-07$01.00/0 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. lOO,No.3,1981

BIOCHEMICAL

Formycin

analog

by mammalian

inhibits

inosine

of

in mammalian

is rather

low.

In mice,

when

injected

present

--in vitro

growth

converted

the

was a potent ligase

Growth

of

COMMUNICATIONS

nucleoside

Leishmania

formycin

the

in vivo

6 competitively diphosphoribose

toxicity

of the

compound

B was estimated

dose,

nor

and greater

than

to be 200 mg/kg

(8).

to

we show that promastigotes

formycin

in parasite

therapeutically

formycin

synthetase extracts.

active

B inhibits

and amastigotes.

B monophosphate.

of adenylosuccinate

EC 6.3.4.4)

donovani

phosphorylated

and polyadenosine

as a single

Leishmania

B was also

Leishmania

RESEARCH

neither

LD50 of formycin

experiments,

inhibitor

(GDP),

formycin

of

(6)

the

qd X 10 days

is

Although

cells,

when injected

In the

that

(5,6).

phosphorolysis

(7)

mg/kg

inosine

cells

synthesis

1,000

BIOPHYSICAL

B (7-hydroxy-3-8-D-ribofuranosylpyrazolo(4,3-d)pyrimidine)

is a C-nucleoside cleaved

AND

The parasites

The latter (IMP:

with

compound,

L-aspartate

In a preliminary

in hamsters

the

trial,

established

infection.

MATERIALS AND METHODS Promastigotes and Amastigotes

Promastigotes 04 Leishmania donovani and Leishmania mexicana were obtained and grown at 27 C in a semi-defined medium defined medium (10) with-modifications (11). Parasites in late610g phase were resuspended in fresh medium at an average density of 10 ml, either supplemented or not with formycin B. After three days, the number of viable parasites was determined under a microscope in a hemocytometer. Amastigotes of Leishmania mexicana were grown 'in the J77468-mouse macrophage-cell line in RPM1 1640 medium SUDDlemented with 20% heat inactivated fetal bovine serum, exactly as described previously (12). The number of cells and parasites per cell, and the percent infected cells were determined as described after three days incubation with formycin B at various concentrations (12). Treatment

of

Hamsters

Infected

with

Leishmania

donovani

Six 85 gram, month old, male Syrian golden hamsters each received lo8 Leishmania donovani amastigotes by intracardiac inoculation. Treatment began two weeks later. Formycin B was dissolved in distilled water at a concentration of 10 mg/ml and sterilized by ultrafiltration. Three mice received 1 ml formycin B in water administered ip qd X 5; control mice received water only. One day after stopping treatment, all mice were sacrificed and the spleens gnd livers were removed and weighed. The number of amastigotes per 10 liver cells was determined by microscopic counts of Giemsa stained impression smears.

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Enzyme Assays Washed pellets (10' cells) of Leishmania mexicana or Leishmania donovani promastigotes were suspended in 1 ml of distilled water and lysed by sonication. After clarification of the extracts by centrifugation (20,000 g, 1 hour), low molecular weight materials were removed by Sephadex G-25 chromatography in water. Nucleoside phosphotransferase activity in the extracts was determined as described by Nelson -et -3 al using 100 mM p-nitrophenyl phosphate as the phosphate donor, 2.5 mM formycin B or inosine as the acceptor, and 10 ug Leishmania extract in a total volume of 50 ul of 100 mM sodium acetate phm The products were isolated by high performance liquid chromatography (HPLC) on a Partisil 10 SAX column (Whatman, Maidstone, Kent) eluted with 7 mM potassium phosphate pH 3.6 at a flow rate of 1.5 ml/minute. Formycin B monophosphate and IMP eluted at 20 and 18.5 minutes respectively, and were well separated from other ultraviolet absorbing peaks. The nucleotides were identified by comparison of their retention volumes with authentic standards, and by the ratio of absorbance at 280 nm/254 nm. Viable Leishmania mexicana promastigotes were also treated with 100 pM formycin B for 24 hours. After washing and extraction, formycin B monophosphate levels were determined by HPLC. Adenylosuccinate synthetase activity in Leishmania extracts was determined by the method of Lieberman, using 100 mM glycine pH 8 contaiydng 10 mM magnesium, 200 fan IMP or formycin B monophosphate, 20 l.nn IUCl-L-aspartate (specific activity 233 mCi/mmole, New England Nuclear, Boston, MA.), and 140 ug protein either with or without 100 p GTP in a total volume of 100 pl (13). After 15 and 30 minutes at 37 C, the incubations were terminated in a boiling water bath, and radioactive reactants and products were separated by HPLC in 0.125M K&, 0.068M potassium phosphate, pH 3.5 at a flow rate of 1.5 ml/minute; [ Cl-adenylosuccinate eluted at 20 minutes. Product formation averaged 75 pmols/hr/mg protein, and was linear with time up to 30 minutes, and with protein concentration. Reagents Formycin B was obtained from Calbiochem (La Jolla, CA). monophosphate was prepared from formycin A monophosphate by the action of adenylate deaminase (Sigma, St. Louis) by HPLC.

Formycin B (Calbiochem) and was purified

RESULTS Effects

of

Formycin

B on In Vitro

Growth

of

Leishmania

One m formycin B inhibited the proliferation of Leishmania donovani and Leishmania mexicana promastigotes by 85%, and Leishmania mexicana amastigotes in macrophages by 52% (Table 1). The drug at this concentration was primarily leishmanistatic , since the non-growing parasites remained viable. One um formycin 8 had no effect on the growth of murine 577468 macrophages. Ten ldn formycin B showed leishmanicidal activity against intracellular amastigotes, with a 30% decrease in the growth rate of the host macrophages. Effects

of Formycin

B on In Vivo

Infection

Formycin B, 10 mg ip qd X 5, markedly decreased the number of amastigotes in the livers of Leishmania donovani infected hamsters, with an accompanying reduction in hepatosplenomegaly, when compared to control animals (Table 2 and Figure 1).

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BIOCHEMICAL

TABLE 1: A.

EFFECT

AND

OF FORMYCIN

BIOPHYSICAL

RESEARCH

B UPON THE GROWTH

OF LEISHMANIA

COMMUNICATIONS

IN VITRO

Promastigotes Treatment

Percent

Control

Parasites

L. donovani

8.

L. mexicana

100(14.8x106/ml)

None

at 72 hours

lOO( 14.8x106/ml)

Formycin

B, 1~

14 + la

16 + 2

Formycin

B, 1Ofl

13 + 1

11 + 1

Amastigotes

of L. mexicana

in 577468 Mouse Macrophaqes Percent Infected

C”:lloy

Treatment None

Parasites Infected

per Cell

Parasits No. x 10

5.0 -+ 1.3a

35 2 5

5.9 + 0.8

1.12 + 0.1

Fortnycin

B, lug

5.0 + 0.6

18 + 6

5.5 + 1.4

0.48 + 0.17

Formycin

B, 1Ofl

3.5 + 0.4

11 + 2

2.2 + 0.5

0.09 + 0.03

aS.E,M.,

Formation

n=3

of Formycin

B Monophosphate

Leishmania promastigotes incubated with formycin B (100 ~ni) for 24 hours accumulated 2.76 Fmoles/mg protein formycin B monophosphate, as determined by HPLC. No formycin A monophosphate was detected. Similarly, in the presence of p-nitrophenyl phosphate, but not in its absence, Leishmania extracts effectively phosphorylated formycin B (0.75 mmoles/hr/mg protein). The glycosidic bond of formycin B was not cleaved detectably by Leishmania extracts. Inhibition

of Adenylosuccinate

Synthetase

In the presence ofl@TP, and [14C]-L-aspartate, converted IMP to [ Cl-adenylosuccinate.

TABLE 2:

by Formycin Product

B Monophosphate

extracts of formation

Leishmania was entirely

EFFECT OF FORMYCIN B ON LEISHMANIA DONOVAN1 INFECTION IN HAMSTERS

Hamster

Treatment

Liver weight (gm)

Spleen weight (gm)

+astigotes per, 10 liver nuclei

:

Control

5.54 7.24

0.78 0.47

1,710 2,394

8.28

0.80

1,316

3.46

0.14

264

3.81 3.34

0.18 0.28

220 0

3 4 z 'Giemsa

Formycin ip, stained

B, 10 mg qd x 5 impression

smears (microscopic

1380

counts).

Vol. 100, No. 3,198l

Figure

1:

BIOCHEMICAL

AND

Photograph of spleens treated with formycin (right side).

BIOPHYSICAL

RESEARCH

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removed from C. donovani infected hamsters 6 (left side) as compared to controls

GTP dependent, consistent with the described properties of an adenylosuccinate synthetase (13). Since radiolabelled formycin B was not available, we could not determine unequivocally the metabolism of formycin B monophosphate. Nevertheless, if formycin B monophosphate was substituted for IMP in no new radioactive peaks were the adenylosuccinate synthetase reaction, detected by HPLC. Instead, the addition of formycin B monophosphate to IMP containing reaction mixtures dose dependently inhibited adenylosuccinate formation (Figure 2). At an IMP concentration of 200 W, 2Ol.1771formycin B monophosphate inhibited product formation by 50%. In other experiments, formycin B monophosphate inhibited similarly the adenylosuccinate synthetase activity in extracts of Bacillus subtilis. DISCUSSION Formycin B inhibited the in vitro growth of Leishmania promastigotes and amastigotes at concentrations that were non-toxic to mammalian cells. The drug also was effective in treating established Leishmania donovani infections in hamsters. Formycin B was phosphorylated by intact parasites, in contrast to mammalian cells. The probable enzyme mediating the phosphorylation was the recently described nucleoside phosphotransferase of Leishmania, since p-nitrophenyl phosphate served as an efficient phosphate donor in the reaction (3).

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Vol. 100, No. 3,19Bl

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100 5 2 0 :E 36 E s4 E E2 a. Pl 0

Figure

2:

2

20 200 FormycinB Monophosphate,PM

Inhibition of adenylosuccinate synthetase in a C. mexicana et&t-act by formycin B monophosphate. Percent controlactivity= 1 Cl-L-aspartate incorporatpd into adenylosuccinate with formycin B monophosphate 4 1 Cl-L-aspartate incorporated without formycin B monophosphate x 100. The IMP concentration was 200 pM. The brackets show the range of two experiments.

Formycin B monophosphate inhibited the conversion of IMP to adenylosuccinate in Leishmania extracts, but was not itself a detectable substrate for the Moreover, the inhibition of adenylosuccinate synthetase parasite enzyme. activity by formycin B monophosphate was not unique to the Leishmania, but was also observed in extracts of Bacillus subtilis. The results suggest that the selective toxicity of formycin B toward Leishmania results from the phosphorylation and trapping of the nucleoside by the parasite, but not the host cells. This route of metabolism of formycin B is shared in common with allopurinol ribonucleoside. However, the mechanism of action of formycin B monophosphate differs markedly Thus, formycin B monophosphate is from allopurinol ribonucleotide. a potent inhibitor of Leishmania adenylosuccinate synthetase activity; in contrast, allopurinol ribonucleotide is a substrate, and indeed requires amination to exert its biological activity. Konigk (14) reported inhibition of inosine nucleosidase and adenosine phosphorylase of C. tropica by formycin B, but the inhibitory effect was marginal at very high drug concentrations. It is unlikely that this effect contributes to the anti-leishmanial activity of the drug. In summary, the in viva anti-leishmanial with its distinctive effects on nucleotide detailed investigation of its therapeutic action.

activity of formycin B, combined metabolism, warrants further potential and mechanism of

Acknowledgements: Supported by NIH grants GM 23200, AI 15183, U.N. Development Programme, World Bank-World Health Organization Program for Research and Training in Tropical Diseases Contract and by an Irma T. Hirsch1 Career Scientist Award (To KPC).

by the Special 780040,

REFERENCES

1. 2.

Berens, R.L., Marr, J.J., Nelson, D.J., and LaFon, S.W. (1980) Biochem. Pharm. 29,2397-2398. R.L. (1977) J. Infect. Dis. 136,724-730. Marr, J.J., and Berens,

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3. 4. 5. 6. 7. 8. 9. if:

::: 14.

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Nelson, D.J., LaFon, S.W., Tuttle, J.V., Miller, W.H., Miller, R.L., Krenitsky, T.A., Elion, G.B., Berens, R.L., and Marr, J.J. (1979) J. Biol. Chem. 254,11544-11549. Nelson, D. J., Bugge, C.J.L., Elion, G.B., Berens, R.L., and Marr, J.J. (1979) J. Biol. Chem. 254,3959-3964. Umezawa, H. Sawa, T., Fukagawa, Y., Hommo, I., Ishizuka, M. and Takeuchi, T. (1967) J. Antibiotics (Tokyo) 20,308-316. Sheen, M.R., Kim, B.K., and Parks, R.E. (1968) Mol. Pharmacol. 4,293299. Muller, M., Zahn, R.K., and Umezawa, H. (1975) Cant. Res. 35,36733681. Ishizuka, M., Sawa, T., Hori, S., Takazama, H., Takeuchi, T., and Umezawa, H. (1968) J. Antibiotics (Tokyo) 21,5-12. Chang, K.P. (1979) Exp. Parasitol. 48,175-189. Steiger, R.F. and Steiger, E. (1977) J. Parasitol. 62,1010-1011. Chang, K.P. and Fish, W.R. (1981) In --In Vitro cultivation of protozoa pathogenic to man and domestic animals, Ed. J.B. Jensen, CRC Press, Florida (In Press). Chang, K.P. (1980) Science 209,1240-1242. Lieberman, I. (1956) J. Biol. Chem. 223,327-339. Konigk, E. (1978) Tropenmed. Parasit. 29,435-438.

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