Proteinase inhibitors as antileishmanial agents

660

TRANSACTIONS OFTHBROYALSOCIETY OFTROPICAL MEDICINE ANDHYGIENE, VOL:76, No. 5, 1982

Proteinase

inhibitors

as antileishmanial

agents

G. H. COOMBS,D. T. HART* AND J. CAPALDO Dept of Zoology,

Leishmania

University

of Glasgow, Glasgow, G12 8QQ, Scotland

Summary mexicana mexicana amastigote pro-

teinase activity was largely inhibited by low concentrations of leupeptin, antipain and two epoxysuccinates, compounds known to affect cysteine proteinases. Of these inhibitors, only two had leishmanicidal activity. trans-Dicyclohexylepoxysuccinate at 10 pg/ ml inhibited the in vitro transformation of L. m. mexicana amastigotesto promastigotesby ireater than 50%. Antipain was a potent antileishmanial agent, which inhibited promastigote growth over seven days bv 50% at 0.5 wg/ml. The number of amastigotesthat transformed iA vitro to promastigotes was reduced 78% bv antinain at 0.1 &ml. Each of the three diamidnes kvestigated ‘(Gentamidine isothionate, amicarbilide and M and B 4596) exhibited marked antileishmanial activity, but only M and B 4596 had any significant effect (36% inhibition at 33 pg/ml) on L. m. mexicana amastigote proteinase activity.

studies, parasites were washed twice in growth medium. For the enzyme assays,they were washed in 0.25M sucrose and homogenized by suspension in 0*25M sucrose containing 0.1% (v/v) Triton X-100. Testing the sensitivity of L. m. mexicana to drags The effects of uroteinase inhibitors unon L. m. mexicana were assissed using two syste&. 1. Transformation in vitro of amastigotes to promastigotes: The methods described previously (HART et al.,

1981b) were used and progress of the parasites through transformation was monitored from samples taken-after 8, 12, 24, 36 and 48 hours incubation. 2. Promastigote mowth: The effects of comuounds upon L. m. kexic&a promastigotes were stud&d both in short-term (24 hours) and long-term (seven days) incubations. At the start of each experiment, promastigotes were suspended in growth medium, with drug at the appro@ate concentration, at densities of anm-oximatelv 10 /ml and lO’/ml in the short term and long-term experiments, respec&ly. At the termination of the experiments the parasites in the control cultures (no drug) were in late log phase of growth. Static cultures in flat-bottomed microtitre plates (Flow Laboratories, Irvine, Scotland) were incubated at 25°C in air. At the end of experiments parasites were counted with a Leitz inverted microscope.This assessmentwas only semi-quantitative (on a 7-point scale), but consistency was checked, both within and between experiments, by counting parasites in some experiments with an improved Neubauer haemocytometer. Parasite motility and morphology were assessed(on 6- and 5-point scales, respectively) by phase contrast microscopy of living promastigotes and by observations of methanol-fixed preparations stained with Giemsa’s stain. Doubling dilutions of drugs weie used and the sensitivities to the compounds were expressed as the LDsO (the lowest drug concentration used that reduced the number ofpromastigotes present by 50%) and the MLC (the lowest drug concentration used in which no proma&igotes were tiesent at the end of the experiment) . -1

Introduction Leishmania mexicana amastigoteshave been shown

to contain high proteinase activity compared not only with L. mexicana promastigotes but also with other flagellate protozoa and mammalian cells (NORTH & COOMBS, 1981; COOMBS, 1982). Over 90% of’the amastigote proteinase activity was due to cysteine proteinases, most of which were soluble and possibly associated with large organelles, named megasomes (ALEXANDER& VICKERMAN,1975),thatare characteristically abundant in Leishmania amastigotes (COOMBS,1982). The relatively high proteinase activity of the amastigotes could make the mammalian stage of the parasite susceptible to inhibitors of cysteine proteinases. We have studied a range of cbmpounhs reported to be proteinase inhibitors and measured their effects on the viabilitv of L. m. mexicana, both of amastigotestransform&g in vitro to promastigotes and of the latter form growing in vitro, as well as on proteinases in cell-free extracts of the parasite. Materials and Methods Parasites Leishmania m. mexicana amastigotes were isolated

from lesions in female NIH mice (Hacking and Churchill, Huntingdon, Cambridge, England) as described elsewhere (HART et aE., 1981a). Promastieotes were derived from amastieotes as described breviously (NORTH & COOMBS,19il) and maintained at 25°C in a modified Eagle’s minimal essential medium (designated HOGEk medium) (BERENSet al., 1976) with 10% (v/v) heat inactivated foetal calf serum (FCS). Air was used as the gas phase throughout. Only promastigotes passagedless than 11 times were used in this study. Before use in the growth

Proteinase assays The effects of the compounds upon the proteinase activities in homogenates of both L. m. mexicana amastigotes and promastigotes were estimated as

described previously (COOMBS,1982).

Materials Pentamidine isethionate, amicarbilide and M and B

4596 were obtained from May and Baker Ltd.,

*Present address: I.C.P. Unit for Research in Tropical Disease, Avenue Hippocrate 74, Bl200 Brussels, Belgium

H.

G.

Table I-Effects of drugs upon mexicana amastigote proteiaases

Leishmania

ES1

8f

33 3.3 0.33 33 20 2 0.2 20 2 0.2

ES2

Leupeptin Antipain Pentamidine isethionate Amicarbilide

cysteine proteinase inhibitors (peptide analogues and epoxysuccinates) at very low concentrations, whereas the diamidines, reported to inhibit other proteinases (ASGHAR, 1977), had little effect even at relatively high concentrations. The effects of the compounds upon growth of L. m. mexicana promastigotes are given in Table II. The three diamidines inhibited growth at relatively low concentrations, whereas the only cysteine proteinase inhibitor that had any detectable activity was antipain, which was effective at low concentration in the long-term test. The transformation in vitro of L. m. mexicana

l(4)

9(2) 8 + 3(3) 10 + l(4)

1w 19(2) 11 + l(4) 23(2)

amastigotes was inhibited markedly by diamidines (Table II and HART et al., 1981b), only slightly by

W)

leupeptin and ESl, but significantly by ES2 at 10 pg/ml although hardly at all at 1.0 pg/ml. Antipain, however, even at O-1 pg/ml causeda decreasein total parasite numbers and relatively few (22% of control) promastigotes developed.

95 + 13(S) 100 f 6(3) 64 2 6(3)

amean+ standard deviation from the number of experiments given in parentheses.

Discussion Pentamidine isethionate, amicarbilide and M and B 4596 have been shown to be potent inhibitors of some blood proteinases (ASGHAR, 1977) and also trypanotidal (BERGet al., 1961; NATHAN et al., 1979). Each of the compounds was leishmanicidal but none was a potent inhibitor of the parasite’s proteinases, indicating that the drugs probably act upon other targets. Leishmania polyamine and nucleic acid metabolism

Dagenham, Essex. Antipain and leupeptin were supplied by the Protein Research Institute, Osaka,

Japan. Potassium tranS-monobenzylepoxysuccinate (ESI) and trans-dicyclohexylepoxysuccinate (ES2) were synthesized as described previously (HANADA et al., 1978; INABA et aE., 1979). ES1 was prepared as a

white solid (M.pt 177°C) consisting of 85% active

have been suggested (GUTTERIDGE, 1969; BACHRACH et al., 1979). Antimonial compounds are important

component, and ES2 as a 100% pure, colourless oil.

ES2 was &ssolved in 10% (v/v) ethanol in water, all other compounds were dissolved in water. At the concentrations used the ethanol did not affect parasite viability.

drugs in the treatment of cutaneous leishmaniasis. Such compounds are thought to interact with thiol groups, and glycolytic kinases, especially phosphofructokmase, have been suggestedas the primary drug target in the parasite (GUTTERIDGE 81 COOMBS, 1977). The possibility that antimonials affect leishmanial amastigote cysteine proteinases was investigated with both trivalent and pentavalent compounds, but

Results The effects of the inhibitors upon L. m. mexicana amastigote proteinases are given in Table I. The Table II-The

effects

of drugs upon the growth

661

enzyme activities were greatly reduced by established

W

600 33 33

M & B 4596

mexicana

% control activitya

Concentration @W)

Compound

et al.

COOMJB

of Leishmania mexicana mexicana promastigotes*

Incubation Time

L&ob

Pentamidine isethionate Amicarbilide

MLC’

24 hours

7 days

Drug at LDsod

Drug at LDsod

Morphology’

Motility’ 3 3 4 5 5 5 4

2

25

5

100

>lOO

5

ES1 ES2

50 >lOO >lOO

Leupeptin Antipain

>I00 >lOO

>lOO >lOO >lOO >lOO

5 5 5 5 2

M & B 4596

“The figures bThe lowest cThe lowest d,Or highest

>lOO

LDmb

MLC’

0.5 1 5 25 0.02 25 >lOO >lOO >lOO >lOO >lOO >I00 0.5 >lOO

Morphology’

Motility’

5 5 5 5 5 5 2

4 4 4 5 5 5 2

given are the median results of 3 to 12 experiments. drug concentration @g/ml) that reduced the number of promastigotes present by 50%. concentration @g/ml) in which no promastigotes were present at the end of the experiment. concentration tested.

e On scale 5 (normal)-1 r On scale 5 (normal)-0

(entirely round). (immotile).

662

PROTEINASE INHIBITORS

AND

Leishmania

in vitro of Leishmania mexicanamexicana amastigotes

Table III-The effects of drugs upon the transformation to promastigotes

Cell type after 48h (%) Compound Control M & B 4596

ES1

ES2

Leupeptin

ug/ml 07 1.0 1:: 0.1 1.0 10 100 0.1 1.0 1;: 0.1 1.0

1:: Antipain

0.1 1-O 10 100

A ll?r 13k 14t 12t 12 k 12-t 15+ 13-t 17+ 15k 10-t 20* 16+ 15+ 18+ 14+ 21+ 11t 10f 3Ok 25+

TIl 5 3 6 6 10 3 5 6 8 6 6 7 6 5 6 2 7 3 5 9 5

T12 2 4 5

2+ 6+ 8+ lo+ 0

3

E

0

2+ 6+ lo+ 0 0 lo+

iti* 4c 3+ 8 0 3-t 6t 65 20 + 0

2 4 3 4

5 0 2

2 2 2 10

x 3+ 5+ 3f 4+ 2+ 0 125 15+ 5k 0

2f 8f 5+

1 4 2 4 1 3 5 5

1 3 5

8 0 : 2+

8

ii

P

T13

i 8k 6+ 1+

2k 2+ 0 17+ 2Ok i

2 2 4 1 2 2 8 6

TP

201 f 155 f

9 9

22Ok 8 181 + 11

1;; ; 0 210 + 206 k 191 * 190 -t 170 + 1;; ;

; 8 9 8 6 6 ;

150 62 + 2~ 11 12 k 10 220 + 6 218 t 7 220 + 11 217 t 10 189 t 9 161 60 rt 10 9

20 f 190 t 190 t 1522 145 i 45 k 18L

10 11 7 9 6 10 6

56 f 10 212 It 10 200 k 10 17Okll 160 4 10 93 t 11 6Ok 8

:

60f 25rtr 11 5

s % of original number of amastigotespresent. Mean + standard deviation from four experiments. Symbols: A, amastigotes;TIl, 2, 3, transformation intermediates 1,2 and 3; P, promastigotes; TP, total number of parasites.

in preliminary experiments at pH 6 little enzyme inhibition was detected (Coombs, unpublished). Leupeptin and ES1 were potent inhibitors of L. m. mexicana proteinases (Table I), but neither exhibited great leishmanicidal activity even at concentrations 100 times greater than those necessary for maximal inhibition of enzyme activity. The insensitivity of amastigotes and promastigotes (Table II and III) indicates that the drugs may not reach the enzymes within the cell. ES2 also inhibited L. m. mexicana proteinases, and it was leishmanicidal with respect to amastigote transformation; the drug at 10 ug/ml reducing promastigote numbers to only 12% of the control. Although a higher concentration of antipain than of leupeptin or the epoxysuccinates was required to give maximal inhibition of amastigote proteinases, it was by far the most effective antileishmanial agent of the four compounds. The greater drug-sensitivity of amastigote transformation than promastigote growth correlates with the differences in proteinase activities of the two forms (COOMBS, 1982). This peptide analogue may have other effects than inhibition of cysteine proteinases, although none have been reported. The hypothesis that the high cysteine proteinase activity of L. m. mexicana amastigoteswould make them particularly susceptible to proteinase inhibitors has not been proved by the results presented, but the findings with antipain are the best evidence available to support it. On a weight basis, antipain is as effective an inhibitor of amastigote transformation as is pentamidine isethionate. Whether the same is true with respect to amastigote

growth, both in vitro and in vivo, remains to be seen. The evidence presented here, however, suggeststhat some inhibitors of Leishmanzuamastigote proteinases may have potential as antileishmanial agents. Acknowledgements We thank the Fundacion Gran Mariscal de Ayacucho for sponsoring one of us (J.C.). This investigation received financial support from the LJNDPWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases and from the UK Medical Research Council.

References Alexander, J. & Vickerman, K. (1975). Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana-infected macrophages. Journal of Protozoology, 22, 502-508. Asghar, S. S. (1977). Diphenyldiamidines-a theoretical evolution their possible therapeutic uses. Journal of Molecular Medicine, 2, l-24. Bachrach, U., Brem, S., Wertman, B., Schnur, L. F. & Greenblatt, C. L. (1979). Leishmania spp.: cellular levels and synthesis of polyamines during growth cycles. Experimental Parasitology, 48, 457-463. Berens, R. L., Brun, R. & Krassner, S. M. (1976). A simple monophasic medium for axenic culture of hemoflagellates. Journal of Parasitology, 62, 360-365. Berg, S. S., Brown, K. N., Hill, J. & Wragg, W. R. (1961). A new prophylactic trypanocidal drug,2, 7-di-(m-amidi~bphe~~Idiazoamino)-1O-ethyl-9-phenylphenanthridinium chloridine dihydrochloride (M & B 4596). Nature, 192, 367-368. Coombs, G. H. (1982). Proteinases of Leishmania mexicana and other flagellate protozoa. Parasitology, 84, 149-155.

G. ?I. COOMBS

Gutteridge, W. E. (1969). Some effects of pentamidine di-isethionate on Crithidia fasciculata. 3ournal of Protozoology, 16, 306-311. Gutteridge, W. E. & Coombs, G. H. (1977). Biochemisry of Parasrttc Protozoa. London: Macmillan Press Ltd. Hanada, K., Tami, M., Ohmura, S., Sawada,J., Seki, T. & Tanaka, I. (1978). Structure andsynthesis of E-64, a new F;;!5;;hibitor. Agricultural Btological Chemtstry, 42, Hart, D. Ti, Vickerman, K. & Coombs, G. H. (1981a). A quick, simplemethodfor purifying Leishmania mexicana amastigotesin largenumbers.Parasitology, 82,345-355. Hart, D. T., Vickerman, K. & Coombs, G. H. (1981b). Transformation in Gtro of Leishmania mexicana amastigotes to promastigotes: nutritional requirements and the effect of drugs. Purasitoloygr, 83, 529-541.

et

663

d.

Inaba, T., Hirayama, Y. & Fujinaga, N. (1979). Inhibition of cathepsin B 1 by E-64 a thiol proteinase inhibitor and 2;5-$5va&s.

Agricultural

Biological

Chemistry, 43,

Nathan, H.‘C., Soto, K. V..M., Moreira, R., Chunosoff, L., Hutner, S. H. 8t Bacchi, C. J. (1979). Curative effects of the anupiroplasms amicarbilide and imidocarb on Trypanosoma bruca’ infection in mice. Journal of Protozoology, 26, 657-660. North, M. J. & Coombs, G. H. (1981). Proteinases of Leishmania mexicana amastigotes and promastigotes: analysis by gel electrophoresis.Molecular and Biochemical Parasitology, 3, 293-300. Accepted for publication 11th March, 1982.

Book Review Medical Laboratory

Manual

far

Tropical Countries.

Volume 1. Monica Cheesbrough, 1981.519 pp., illus. ISBN 095-074-3410. Obtainable from: M. Cheesbrough, 14 Bevills Close, Doddington, March, Cambridge&ire PE15 OTT, England. Price g4.70 + U.25 postage(developing countries); &7.60 + El.75 postage (elsewhere). This book of some 500 pages, labelled Volume I, is composed of four sections, (1) Introduction to the Laboratory, (2) Anatomy and Physiology, (3) Medical Parasitology, (4) Clinical Chemistry. Each of these sections is broken down into chapters, and there are three appendices. Finally, there are loose leaf charts for wall display. The index is adequate and cross reference is accurate. The idea of this manual is to be applauded, but its production is difficult to understand. There are numerous incongruous suggestions which hardly fit the concept of a manual for laboratory workers in developing countries. For example, the author recommends disposable plastic Pasteur pipettes and the use of sophisticated automatic pipettes but gives no description of how to make the time honoured universally acceptedglasscounterpart. The “comparison” plates and loose leaf charts of helminths and protozoa really cannot be accepted as

anywhere near descriptive. Paragonimus westermanni and Schistosoma haematobium, two important parasites, are missing from the African chart, whilst the protozoa are completely incomprehensible. During the discussion of the intestinal amoebaethe descriptive word “trophozoite” for some unknown reason is not used once! Instead the author substitutes the word amoeba, this is unexcusable. The use of such expressions as “left shift of the neutrophils” means nothing without explanation. There is no precise instruction given for the preparation of a reference solution. But, we are given a precise seating plan for collection of blood under field conditions! Measured against these criticisms, which are only a few of several that could be cited, are the very good chapters on “The Leishmanial Parasites” and “Insects and Arachnids of Medical Importance”. In summary this manual attempts to cover too much ground, ascending into areas best left to me&lists. and is entirelv out of balance. It would have been better to describe techniques in precise detail so that workers in peripheral laboratories could fabricate their own materials with the few resources that they have available. P. SARGEAUNT