acivicin: a highly active potential chemotherapeutic agent against visceral leishmaniasis

Vol. 170, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1990 Pages 426-432

ACIVLCIN : A HIGHLY ACTIVE POTENTIAL CHEMOTHERAPEUTIC AGENT

AGAINST VISCERAL LEISHMANIASIS

Tanmoy MUKHERJEE, Krishnendu ROY and Amar BHADURI*

Leishmania Group, Enzyme Division Indian Institute of Chemical Biology

4, Raja S.C. Mullick Road Calcutta 700032, India

Received .Iune 7, 1990

SUMMARY : Acivicin, a chlorinated amino acid antibiotic, is found to be remarkably effective in killing both the vector and the host form of the parasitic protozoa, Leishmania donovani, the causative agent for visceral leishmaniasis or Kala-azar. The ED50 (50 nM1 for the pathogenic amastigote form in invitro screening system is significantly lower than the reported values for other drugs under trial. The drug irreversibly inactivates both in vitro and in vivo carbamyl phosphate synthetase II, the first enzyme of the pyrimidine biosynthetic pathway. The irreversible inactivation of this sensitive target enzyme and lack of effective reversal by glutamine makes acivicin a preferred candidate for potential chemotherapy against increasing number of Kala-azar cases that are reported to be unresponsive to pentavalent antimonials. 01990 Academic Press. Inc.

Visceral leishmaniasis, caused by Leishmania donovani and commonly

known as Kala-azar is a fatal disease. The disease is endemic in many

parts of the tropical world and threatens to become a major health hazard

in coming decades (1). This protozoa1 pathogen has a digenic life cycle;

an extracellular, flagellated, non-pathogenic form in the insect vector

and an af lagellated , oval, pathogenic form in humans that reside and

proliferate exclusively in the host macrophages (2). The cultural form

corresponds to the vector form. Drugs currently available for treatment

of Kala-azar are few in number and have severe limitations (3.4). Penta-

valent antimonials, introduced in the early days of chemotherapy, are

still the drugs of choice. These are toxic, have inconvenient route of

administration and worst of all, give rise to frequent cases (lo-15%)

of clinical resistance (3.41. Pentamidine. a biguanidine and amphotericin

B. a polyene antibiotic, are used as a secondary line of defence. In

cases of total treatment failure, even splenectomy is reported (5.61.

Obviously, an urgent need exists for development of new lines of drugs

for combating the disease.

* To whom correspondence should be addressed.

0006-291XHl $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 426


Pyrimidine biosynthetic pathway, along with purine pathway consti-

tutes the gateway to nucleic acid synthesis and many chemotherapeutic

agents against different diseases have been successfully developed on

the basis of subtle biochemical differences of these pathways between

the host and the pathogen. These pathways in Leishmania have already

shown some striking differences from the host (7-91. For example, we

had shown that in Leishmania. the first three enzymes of the pyrimidine

pathway exist as discrete proteins unlike in mammalian systems where

the three enzymatic functions resides in a single protein (9). Based on

such differences, allopurinol riboside, an antimetabolite for the leishmanial

pur ine salvage pathway has been developed and is undergoing clinical

trial with moderate success (4,10,11). We have also shown that N-phospho-

nacetyl-L-aspartic acid (PALA), a transition state analogue inhibitor of

aspartate transcarbamylase, has significant leishmanicidal activity (9).

Encouraged by these observations, a more systemic search for clinically

significant leishmanicidal activity amongst anti-metabolites of these path-

ways that are already on clinical trial, was initiated, We now report

that acivicin (a-[% ,S-5Sl-a-amino-3-chloro-4,5-dihydroisoxazoloacetic acid),

under clinical trial as an anticancer drug, is remarkably effective against

both forms of the pathogen. This fungal antibiotic is a chlorinated subs-

trate affinity analogue of glutamine (12). Our preliminary studies clearly

identify the parasite carbamyl phosphate synthetase II ( CPSase II) as

a major target enzyme that is irreversibly inactivated at very low concen-

trations by acivicin both in vitro and in vivo conditions.

METHODS

A recent clinical isolate of Indian strain ((JR-61 of Leishmania donovani (MHOMIIN/1978/URbl was used for this work. The maintenance of promastigotes and growth inhibition studies on this form of organism were done as described recently (9). Inhibition studies with acivlcin on the multiplication of the pathogenic form of the organism i.e., amastigotes werls also done. The peritoneal exudates of BALB/C mice were with- drawn and washed twice with media containing RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine. 25 mM

Hepes, 100 ug/ml streptomycin and 100 u/ml penicillin. After spinning down at 200 g for 610 mins., macrophages were suspended in the above

media and about 10 cells were distributed and allowed to adhere for 4 hrs. on each coverslip (18 mm’). Non-adherent cells were removed and further incubated for 16 hrs. at 37°C in the same above media. The macrophagee culture was then inoculated with L. donovani promastigote suspension at a parasite to macrophage ratio 1O:ly After 2 hrs. incubation at 37°C the non-phagocytized parasites were removed and transformed

amastigotes were suspended in media. After 8 hrs. fresh medium was added alc’ng with acivicin. The medium including drug was replenished every alterndte day. The number of amastigotes per infected macrophages were then determined every 24 hrs. by examining at least 200 giemsa

stained infected macrophages which are made in triplicate assays.

427


Enzyme extract was prepared and carbamyl phosphate synthetase II was assayed by the method described by Mukherjee et. al. (9).

For in vivo effect of acivicin on the carbamyl phosphate synthetase II activity, the whole cells of L. donovani promastigotes were washed twice and resuspended in phosphate buffered saline, pH 7.4. The cell suspensions were then incubated separately at 25°C with different concentrations of acivicin and L-glutamine. After different time intervals, cell suspensions were immediately centrifuged and again washed twice with

phosphate buffered saline. The isolated cells were homogenized and crude extracts were assayed for CPSase II activity. Suitable controls were run in parallel whenever required.

RESULTS AND DISCUSSION

The growth of L. donovani promastigotes in semi-defined liquid -

media with an initial cell-density of 1 x lo6 cells/ml was assessed every

24 hrs. in presence and absence of acivicin (Fig. 1). Acivicin at a concen-

tration of 0.1 pg/ml (0.55 pM1 showed a profound growth inhibitory effect

virtually prevent:ng all cell-divisions. Under identical growth conditions

5 ug/ml pentamidine, 1 ug/ml of amphotericin B and 40 pg/ml of PALA

(N-phosphonacetyl-L-aspartic acid) were needed to arrest the growth

completely (Fig. 1). Even at a ten-fold reducing dose (0.01 pg/ml), acivicin

was more than 60% inhibitory towards the growth of L. donovani promasti- -

gotes.

Effect of acivicin was also checked on the multiplication of the

pathogenic form of the organism in mouse peritoneal macrophages in vitro.

It is clear that 0.05 pg/ml of acivicin effectively stops all cell divisions

within a day (Fig.2A). In another experiment, the promastigotes were

previously incubated with 0.2 pg/ml of acivicin for 30 mins. at 25°C

and then thoroughly washed cells were inoculated to the macrophages

with proper control. The macrophages eliminated all the parasite cells

within 48 hours whereas control showed a normal growth rate (Fig.ZB).

The concentration (0.05 pg/mll at which acivicin had shown total growth

arrest is substantially lower than that of any other known drugs (except

pentamidine, ED 5. 0.02 pg/ml) or potential drugs which are under clinical

trial or other experimental chemotherapeutic agents (10). Acivicin at

a dose of 0.5 pg/ml causes no alteration of the macrophages as assessed

by lactate dehydrogenase assay and trypan blue exclusion.

The highly lethal effect of acivicin is probably due to the irrever-

sible inactivation of critical glutamineamido transferases of the parasite.

This is evident by its effect on carbamyl phosphate synthetase II, the

first enzyme of the pyrimidine biosynthetic pathway of the organism that

utilizes glutamine for the synthesis of carbamyl phosphate. When viable,

motile, promastigote cells were exposed to the drug a rapid in vivo

428


I I

20 40 60 80 WI

TIME IN HOUR5

I (A)

6

4

t:_,

2

O0kk-Zk-F

TIME IN HOURS TIME IN HOURS

Pig.1 . Inhibition of growth of Leishmania donovani promastigotes in presence of acivicin and other antileishmanial drugs. The control growth curve in absence of acivicin or any drug is represented byU.O--Q,M, D------O, - , indicate growth pattern in presence of 0.01 pg/ml, 0.05 pglml, 0.10 ug/ml, 1.0 pg/ml of acivicin respectively. M , x-x (

*-* represent the same for 1 uglml amphotericin B, 5 Fe/ml pentamidine and 20 pg/ml PALA respectively.

Fig.2 . Effect of acivicin on Leishmania donovani amastigotes in mice peritoneal macrophages in vitro. (A) Multiplication of L. donovani amastigotes cultivated for 3 days in a media with different concentrations of acivicin. W , U , U -9 represent control (without acivicinl, 0.01 ug/ml. 0.0; pglml and 0.1 pg/ml of acivicin respectively. (Bl Here, L. donovani promastigotes were pretreated with 0.2 ug/ml of acTvicin for 30 mins. and cells free of acivicin were infected with macrophages as described in ‘methods’. The transformed amastigotes in macrophages were cultivated in medium alone. C\ and U indicate control f without treatment 1 and 0.1 pg/ml acivicin pretreated cell respectively.

inactivation of this enzyme could be subsequently demonstrated in cell

free extracts. About 90% inactivation was achieved within 5 mins with

1 pg/ml of acivicin or within 30 mins with 0.2 pg/ml of acivicin (Table I).

Under identical conditions no activity was lost for subsequent enzymes

like aspartate transcarbamylase and dihydroorotase of de novo pyrimidine

pathway (data not shown). Reasonable protection of CPSase II activity

with glutamine could be achieved only at extremely high molar ratio

of glutamine to the drug in the medium. Thus 10 mM glutamine was needed

to afford significant protection (only 26% inhibition) in presence of 0.2 pgl

ml (I. 1 uM) acivicin (Table I). This rapid in vivo inactivation with

acivicin and only partial protection with high concentrations of glutamine

429


TABLE I

In viva inactivation of CPSase II activity by acivicin in whole cells of L. donovani -

Preincubation Acivicin L-Glutamine CPSase II Inhibition Protection time concentration concentration activity ($1 (%I

(min) @g/ml) (mM) (nmoles/min/mgl

30 0.435

5 1.0 0.043 90

15 1.0 0.021 95

30 1.0 0.017 96

30 0.1 0.174 60

30 0.2 0.066 85

30 0.5 0.039 91

30 0.2 5.0 0.208 48

30 0.2 10.0 0.330 76

30 0.2 20.0 0.340 70

was further confirmed by the growth experiment with the promastigotes.

In a typical growth experiment, inhibition of promastigotes with 0.1 ug/ml

(0.55 PM) acivicin could only be partially protected (50%) by 10 mM

glutamine and no significant protection was achieved with glutamine less

than 5 mM. Aminoacids like tryptophan, histidine or several nucleobases

and nucleosides failed to provide any protection under these conditions

(data not shown).

The irreversible nature of CPSase II inactivation was further confir-

med in invitro experiment with partially purified enzyme that was eluted

through sephacryl S-200 column (91. Preincubation of leishmanial CPSase II

in vitro with 10 pM acivicin resulted in time-dependent rapid inactivation

of the enzyme. When excess acivicin was physically removed at various

time intervals by passing the incubation mixture through sephadex G-

50 columns, essentially the same inactivation pattern for the enzyme was

observed (Fig.31.

It is evident from our study that in model assay systems, acivicin

acts as an extremely potent leishmanicidal agent and compares very favo-

urabl y with established and potential drugs for Kala-azar. Leishmanral

CPSase II appears to be its site of action though other amidotransferases

may also be involved. Irreversible nature of inhibition of the target

enzyme by the drug (Fig.31 and lack of any effective reversal by gluta-

mine, unless it is added in higher concentrations (10 mM) than is normally

available in mammalian tissues (131 t makes acivicin a preferred candidate

430

Vol. 170, Nlo. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 8 16 2L TIME IN MINUTES

Fig.3 . In vitro inactlvation of CPSase II with acivicin. The L. donovani promastigote extract was passed through sephacryl a ST200 column. The extract was then incubated at 25°C with 10 PM acivicin. After indicated time intervals one portion each was assayed dir- ectly for CPSase II activity. The other equivalent portion was passed through a small Sephadex G-50 column to remove acivicin from the incubated extract and CPSase II activity was assayed .

U indicate no acivicin treated sephadex G-50 passed enzyme (control ) ; H indicate time-course of CPSase II inactivation with 10 pM acivicin and w represent the same time course with sephadex column passed enzyme.

for further exploration as a potential chemotherapeutic agent for Kala-

azar. Moreover, regulatory and inhibition characteristics of parasite

CPSase II are significantly different from that reported for mammalian

systems [ Ki for acivicin; 0.2 pM parasite enzyme and 7.0 ~,IM for mamma-

lian enzyme (13). Manuscript in preparation] to suggest some fundamental

differences in structure and organisation of the enzymes of pyrimidine

biosynthetic pathway between the protozoa1 parasite and higher eucaryotes.

This was already indicated by our earlier studies of parasite aspartate

transcarbamylase (9).

Like all other potential anti-tumor drugs, we cannot exclude the

problems of toxicrty in this case also. However, in the context of increa-

sing reports of clinical resistance to pentavalent antimonials in the treat-

ment of Kala-azar leading to splenectomy (3-61, drugs like acivicin can

be seriously considered as second line of defence. Combination therapy

with allo:purinol riboside blocking simultaneously de novo pyrimidine and

pur ine salvage pathway can be explored. Further, in experimental models

of leishmaniasis encapsulation in liposomes leading to passive targeting

to liver macrophages (3.4) or active direct targeting via ‘Scavenger’

receptor of the macrophages (14) or via neoglycoconjugate (151 have been

shown to reduce the toxicity drastically. In short, the chemotherapeutic

potential of acivicin for visceral leishmaniasis and the biochemical basis

of its profound leishmanicidal effect should be further explored.

431


ACKNOWLEDGMENTS

We are grateful to Dr. D.A. Conney of NIH, USA for sending acivicin

as a gift. This work was partially supported by a project funded by

UNDP. T.M. is a Post-doctoral Fellow of CSIR, India.

1.

2. 3. 4. 5. 6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

REFERENCES

'The Leishmaniases' (1987) Eighth programme report of UNDP/World Bank/WHO for research and training in tropical diseases. WHO, Geneva. Chang, K.P. (1983) Int. Rev. Cytol. Suppl. 14, 267-302. Berman, J.D. (1988) Rev. Infec. Dis. 10, 560-586. Croft, S.L. (1988) Trends in Pharmacol. Sci. 9, 376-381. Basu, D. and Mallik, K.K. (1988) Cal. Med. Jour. 85, 18-21. Locksley, R.M. and Plorde, J.J. (1987) In 'Harrison's principles of Internal Medicine 1' (11th ed.) pp 785-787, Mcgraw-Hill Book Company, New York. Glew, R.H., Saha, A.K., Das, S. and Remaley, A.T. (1988) M:cro- biol. Rev. 52, 412-432. Dutta, A.K., Bhaumik, D. and Chatterjee, R. (1987) 3. Biol. Chem. 262, 5515-5521. Mukherjee, T., Ray, M. and Bhaduri, A. (1988) J. Biol. Chem. 263, 708-713. Neal, R.A. (1987) In 'The Leishmaniases in Biology and Medicine' Vol.11 (Peters, W. and Killick-Kendrick, R. eds.) PP. 793-845, Academic Press, London. Berman, J.D. (1985) In 'Leishmaniasis' (Chang, K.P. and Bray R.S. eds.) Elsevier Science Publishers B.V. (Biochemical division) Amsterdam, pp. 112-138. Kensler. T.W. and Conney, D.A. (1981) Adv. in Pharmacol. and chemother. 18, 273-351. Aoki, T., Sebolt. J. and Weber, G. (1982) Biochem. Pharmacol. 31, 927-932. Mukhopadhyay, A., Chaudhuri. G.. Arora, S.K., Seghal, S., Basu, S.K. (1989) Science 244, 705-707. Chakraborty, P.. Bhaduri, A.N. and Das, P.K. (1990) Biochem. Biophys. Res. Commun. 166, 404-410.

432

acivicin: a highly active potential chemotherapeutic agent against visceral leishmaniasis

Documents