Vol. 35, No. 11

Brugia malayi and Acanthocheilonema viteae: Antifilarial Activityof Transglutaminase Inhibitors In Vitro

U. R. RAO,'* K. MEHTA,2 D. SUBRAHMANYAM,3 AND A. C. VICKERY'

College of Public Health, University of South Florida, Tampa, Florida-336121; Department of MedicalOncology, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 770302;

and CIBA-GEIGY Ltd., and Swiss Tropical Institute, Basel, Switzerland2

Received 3 April 1991/Accepted 22 August 1991

The possible involvement of transglutaminase-catalyzed reactions in survival of adult worms, microfilariae(mf), and infective larvae of the Milarial parasite Brugia malayi was studied in vitro by using the specificpseudosubstrate monodansylcadaverine (MDC) and the active-site inhibitors cystamine or iodoacetamide.These inhibitors significantly inhibited parasite mobility in a dose-dependent manner. This inhibition was

associated with irreversible biochemical lesions followed by filarial death. A. structurally related, inactiveanalog ofMDC, dimethyldansylcadaverine, did not affect the mobility or survival of the parasites. Adult wormsfailed to release mf when they were incubated in the presence ofMDC or cystamine, and this inhibitory effecton mf release was concentration dependent. Similar embryostatic and macrofilaricidal effects of MDC were

observed in Acanthocheilonema viteae adult worms. These studies suggest that transglutaminase-catalyzedreactions may play an important role in the growth, development, and survival of filarial parasites.

Despite the severe toxic effects associated with its use,

diethylcarbamazine, a microfilaricide, has been a drug ofchoice in the treatment of human filariasis for several years

(19). Treatment of onchocerciasis with suramin as a macro-

filaricide was encouraging, but its use is also limited by itsrelatively high toxicity (13). Recently, ivermectin, a semi-synthetic analog of macrocyclic lactone, was found to havemicrofilaricidal activity against filarial parasites and limitedside effects; it is currently considered an effective alternativedrug to DEC (25). Several other macro- and microfilaricidalagents have been tested in vivo and in vitro, but with onlypartial success (22). Although in vitro culture and drugscreening are regarded as fundamental bioassays for filari-cidal studies, the limitations of these assays are consider-able, because the results are poorly predictive (24). Yet, it isgenerally recommended by the World Health Organizationthat a rational approach to the design of macrofilaricidaldrugs as chemotherapy be used. In particular, the designshould be based on still unidentified parasite-specific meta-bolic pathways or key regulatory enzymes so essential to theparasite that their interruption leads to slow or instantaneousparasite death. Recently, we described the presence of a

novel protein cross-linking enzyme, transglutaminase(TGase; EC 2.3.2.13), in adult worms of B. malayi (18).TGases are a family of enzymes that catalyze the Ca2+-

dependent acyl transfer reaction between peptide-boundglutamine residues and primary amines, including thee-amino group of lysine residues in the appropriate peptides(9, 11). TGase of filarial parasites (pTGase) is a 22-kDaprotein whose activity was shown to be essential to the inutero growth and differentiation of microfilariae (mf) (18).Our recent studies (18a) have revealed several unique fea-tures of pTGase. For example, pTGase is a much smallerenzyme than its counterparts in mammalian system (4, 10).Unlike mammalian TGases, pTGase does not require Ca2+ions for its catalytic activity. Finally, pTGase cannot utilizecasein as an acyl donor, unlike other known TGases. Re-

* Corresponding author.

cently, a bacterial TGase with a molecular size of 40 kDa hasbeen identified in the genus Streptoverticillium; this bacterialTGase resembles pTGase in terms of its activity to functionin the absence of Ca2" (1). These differences in host andparasite enzymes would be useful in the design and synthesisof enzyme inhibitors specific for pTGase. In this report, wedemonstrate that pTGase-catalyzed reactions may be es-

sential for the survival of adults, mf, and infective-stagelarvae (L3) of the filarial parasites Brugia malayi and Acan-thocheilonema viteae. Thus, inhibition of pTGase by variousenzyme-specific inhibitors led to the death of these para-sites, as determined by their ability to transform the tetra-zolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) into its blue formazan derivative.

MATERUILS AND METHODS

Parasites. Brugia malayi infections were routinely main-tained in our laboratory by using Meriones unguiculatusjirds and Aedes aegypti mosquitoes. Adult worms and mf ofB. malayi were recovered from the peritoneal cavities ofjirds 4 to 6 months after infection with L3. The filarialparasite A. viteae was maintained in the jird and the softtick, Ornithodorus moubata, as described earlier (26). Adultworms were obtained from the skin and underlying tis-sue surface of jirds. Worms were washed three times withHanks balanced salt solution and separated into male andfemale worms in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered RPMI 1640 containing 100p.g of streptomycin ml-, 100 U of penicillin ml-', and 10%heat-inactivated fetal calf serum (referred to from now on as

medium). The mf collected from the peritoneal cavity were

subjected to centrifugation on lymphocyte separation me-

dium, to eliminate peritoneal cell contamination in the mfsuspension (14). After centrifugation, mf were washed threetimes with the medium and adjusted to 0.15 x 105/100 ,ul. L3were recovered from A. aegypti mosquitoes, 12 days afterthey were fed on jirds with patent infections. L3 were

washed thoroughly with the medium containing high concen-

trations of antibiotics (200 ,ug of streptomycin ml-1, 200 U of

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TABLE 1. In vitro effect of MDC, cystamine, and iodoacetamideon B. malayi adult female worms

Mobility of parasites at theInhibitor following times (h) after

(concn [mM]) incubation:4 24

None (control) +++ +++

MDC0.1 +++ +++0.2 +++ +++0.3

DDC (0.3) +++

Cystamine0.1 +++ +++0.4 +++1.0

Iodoacetamide0.0025 +++ ++0.01 +++0.06 +

a Two adult worms were incubated per each inhibitor concentration at 370Cin an atmosphere of 5% CO2-95% air. At the indicated times, worm viabilitywas scored under an inverted microscope and designated as -, inactive ordead; +, less active; + +, moderately active; and + + +, highly active. Resultsare from two independent experiments performed in duplicate. At 1 h afterincubation, the viability was scored as + + +.

penicillin ml-1, and 0.25 jig of fungizone ml-') and werethen counted and resuspended in normal medium.

Inhibitors. The TGase inhibitors monodansylcadaverine(MDC), a competitive substrate inhibitor; dimethyldansylca-daverine (DDC), a structurally related inactive analog ofMDC; and cystamine and iodoacetamide, both active-siteinhibitors, were purchased from Sigma Chemical Company(St. Louis, Mo.). Different concentrations of these inhibitorswere prepared fresh before each experiment in medium, andthe pH of the working solution was adjusted t6 7.5.

Effect of enzyme inhibitors on parasite viability. Adult maleor female worms (n = 2), mf (n = 0.15 x 105), or L3 (n = 20)were incubated in 1 ml of medium alohe or medium contain-ing MDC, DDC, cystamine, or iodoacetamide at appropriateconcentrations in a 24-well tissue culture plate (Costar,Cambridge, Mass.). Plates containing parasites were incu-bated at 37°C in a 95% air-5% CO2 atmosphere. Frozenparasites Were thawed and used as negative controls. Exper-iments were carried out in duplicate, and the results wereexpressed as an average of two independent experimentalvalues.

Assessment of parasite viability. (i) Visual inspection. Par-asite viability and killing were assessed visually by using aLeitz inverted microscope, and the observations werescored as -, inactive or dead; +, less active; + +, moder-ately active; and + + +, highly active.

(ii) MTT reduction assay. Parasite viability was also as-sessed quantitatively by the MTT reduction assay. Theprocedure used was essentially as described by Comley et al.(6), with a slight modification. Briefly, after microscopicassessment of parasite viability, adult wormns were trans-ferred to 0.5 ml of phosphate-buffered saline (PBS) contain-ing 0.5 mg of MTT (Sigma) per ml. The mf or L3 weresuspended in 0.1 ml of PBS containing 0.5 mg of MTT ml-'.Adult worms, mf, and L3 were incubated for 2 h at 37°C in

the dark. The mf were pelleted and washed once with PBSby centrifugation. To the mf pellet, 100 ,ul of dimethylsulfoxide (DMSO) was added to dissolve the dark bluecrystals of formazan, and the mixture was then transferredto a 96-well microtiter plate (Costar). Adult worms incubatedin MTT were washed once with PBS and blotted, and eachadult worm was transferred into each well of a microtiterplate containing 200 P,u of DMSO. The parasite-DMSOmixture was incubated for 1 h at room temperature forcomplete solubilization of formazan from the parasites intothe solvent. Formazan quantification was performed with anautomatic microtiter plate reader (Flow Laboratories,McLean, Va.) at a 595-nm wavelength by using DMSO as ablank and frozen worms as negative controls. Greater than50% inhibition in MTT reduction was considered significant.These absorbance values relative to those for the controlswere compared for significance (Student's t statistic) byusing Statworks software of the Macintosh Plus computer.The filaricidal activity of TGase inhibitors against L3 wasdetermined by microscopic examination.

Effect of enzyme inhibitors on mf release by adult worms invitro. Female B. malayi worms (n = 5) were incubated in 5ml of medium containing MDC or cystamine, and appropri-ate controls were incubated in medium alone. After 72 h ofincubation, the adult worms were removed and the culturefluid was centrifuged at 1,000 x g for 10 min to collect the mfreleased into the spent medium. The supematant was aspi-rated completely, and 100 ,ul of MTT (0.5 mg/ml) was addedto the pellet. This mixture was incubated for 2 h at 37°C. Thepellet was washed once with PBS, centrifuged, and resus-pended in 100 ,ul ofDMSO. The parasite-DMSO nmixture wasthen transferred to a microtiter plate and was incubated for1 h at room temperature, and the solubilized formazan wasquantitated by reading the A595. Statistical comparisonswere made by the t test.

TABLE 2. Effect of MDC, cystamine, and iodoacetamideon B. malayi L3 viability

% Viable larvae at the followingInhibitor times (h) after incubationa:

(concn [mM])4 24

None (control) 100 100

MDC0.1 100 1000.2 100 490.3 72 00.5 0 0

DDC (0.3) 100 100

Cystamine0.4 100 1001.0 100 05.0 100 0

lodoacetamide0.0025 100 1000.01 100 00.06 0 0a Twenty L3 parasites were incubated per each inhibitor concentration at

370C. At the indicated times, cultures were examined under a microscope forparasite viability. At 1 h after incubation, the viability was 100%. Results arefrom two experiments performed in duplicate.

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RESULTS

In an initial experiment to determine whether pTGase-catalyzed reactions have any role in the survival of filarialparasites, we tested the effect of various known TGaseinhibitors on the viability of B. malayi adult worms, L3, andmf. MDC, a potent competitive amine substrate inhibitor(16), and cystamine or iodoacetamide, the active-site inhib-itors of TGase (9, 23), inhibited adult worm motility in a

time- and dose-dependent manner (Table 1). A significantinhibition of adult female worm motility was evident after 4h of incubation in the presence of 0.3 mM MDC. However,at equimolar concentrations, DDC, which lacks the primaryamine group necessary for TGase activity, showed no mac-

rofilaricidal effect, even after 72 h of incubation. Adult wormmobility and viability were also reduced by cystamine andiodoacetamide at the higher concentrations. Interestingly,the viability of adult male worms was also affected by theenzyme inhibitors (data not shown).A similar effect of enzyme inhibition on the viability of B.

malayi L3 was observed (Table 2). For example, 28% of L3became immobile and died after 4 h of incubation in thepresence of 0.3 mM MDC. However, DDC, the inactiveanalog of MDC, showed no larvicidal effect, and all the L3remained highly motile and active, even after 24 h ofincubation in the presence of 0.3 mM DDC. L3 mobility wasinhibited to 100% in 4 h when L3 were incubated with 0.5mM MDC. A relatively high concentration of cystamine (1mM) was needed to accomplish a similar larvicidal effect;iodoacetamide was more active. At a 0.1 mM concentrationof iodoacetamide, all the L3 became immobile after 24 h ofincubation (Table 2).

The mf obtained from the peritoneal cavity of infectedjirds were also susceptible to TGase inhibitors (Table 3). Allthree inhibitors tested, but not the inactive analog (DDC),caused a dose- and time-dependent inhibition of mf mobility,which eventually led to the death of the parasites. Theinhibitor-induced effect on the mobility of the parasites wasirreversible; they failed to recover even 24 h after theremoval of inhibitors.The visual microscopic observations on the effect of

enzyme inhibitors on viability were confirmed in a colori-metric assay by determining the ability of the parasites toreduce the tetrazolium salt MTT to its blue formazan deriv-ative. As reported earlier by Comley et al. (6), viable B.malayi parasites are able to reduce MTT to formazan. Theeffects of enzyme inhibitors on B. malayi mf and femaleworms are given in Tables 3 and 4. MDC, cystamine, andiodoacetamide significantly (>50%) reduced formazan for-mation by the parasites in a time- and dose-dependentmanner. Conversely, incubation of mf (Table 3) or adultworms (Table 4) in the presence of DDC did not significantlyaffect their ability to reduce MTT. Parasites killed by freeze-thawing were unable to reduce MTT to formazan and thuswere used as negative controls (data not shown).Although the adult worms incubated in the presence of 0.1

to 0.3 mM MDC were found to be active and alive bymicroscopic examination at 24 h (Table 1), their ability totransform MTT into formazan was greatly reduced (Table 4).About 65% inhibition in formazan formation by adult wormswas observed within 1 h when they were incubated in thepresence of 0.3 mM MDC (Table 4).The effect ofMDC and cystamine on mf release from adult

worms was studied by the MTT reduction assay (Table 5).

TABLE 3. In vitro effect of pTGase inhibition on B. malayi mf survival determined by the MTT reduction test

Inhibitor mf mobility ata: A595 atb:(concn [mM) l h 24 h l h 24 h

None (control) +++ +++ 1.640 ± 0.11 1.345 ± 0.06

MDC0.1 +++ +++ 1.366 ± 0.03 (16.7) 0.827 ± 0.04 (38.5)0.2 +++ ++ 1.321 ± 0.14 (19.4) 0.434 ± 0.20 (67.7)c0.3 +++ 1.245 ± 0.03 (24.0) 0.266 ± 0.01 (80.2)d0.5 ++ 0.824 ± 0.02 (49.7) 0.097 ± 0.10 (92.7)d

DDC0.2 +++ +++ 1.324 ± 0.12 (19.2) 1.140 ± 0.08 (15.2)0.3 +++ +++ 1.340 ± 0.06 (18.2) 1.230 ± 0.13 (8.5)

Cystamine0.1 +++ +++ 1.442 ± 0.01 (12.0) 0.746 ± 0.04 (44.5)0.4 +++ +++ 1.407 ± 0.15 (12.2) 0.683 ± 0.06 (49.2)1.0 +++ 1.307 ± 0.04 (20.3) 0.044 ± 0.11 (96.7)d5.0 ++ 0.998 ± 0.12 (39.1) 0.065 ± 0.09 (95.1)d

lodoacetamide0.00015 +++ +++ 1.539 ± 0.01 (6.1) 0.813 ± 0.02 (39.5)0.0012 +++ +++ 1.606 ± 0.04 (2.0) 0.703 ± 0.04 (47.7)0.01 +++ 1.555 ± 0.01 (5.1) 0.661 ± 0.01 (50.8)c0.06 +++ 1.052 ± 0.02 (35.8) 0.087 ± 0.12 (93.5)da A total of 0.15 x 105 mf were incubated at 37°C per each inhibitor concentration, and at the indicated times, culture supernatants were examined under a

microscope for parasite motility. Motility was scored as indicated in footnote a of Table 1.b A total of 0.15 x 101 mf were incubated at each inhibitor concentration. mf mobility at 1 and 24 h was determined by the MTT test as described in the text.

The mobility was scored as described in footnote a of Table 1. Values are means + standard deviations of two experiments performed in duplicate. Values inparentheses are percent inhibition of formazan formation.

c P < 0.05.d p < 0.01.

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TABLE 4. Effect of pTGase inhibitors on B. malayi adult worm viability measured by the MTT reduction test

Inhibitor A595 at the following times (h) after incubationa:(concn [mM]) 1 4 24

None (control) 1.388 ± 0.05 1.274 ± 0.14 1.463 ± 0.10

MDC0.1 1.134 ± 0.02 (18.2) 0.836 ± 0.14 (34.3) 0.666 ± 0.14 (54.4)b0.2 1.007 ± 0.12 (27.4) 0.674 ± 0.14 (47.0) 0.486 ± 0.06 (66.7)c0.3 0.508 ± 0.07 (63.4)c 0.500 ± 0.07 (60.7)b 0.263 ± 0.12 (82.0)c

DDC0.2 0.989 ± 0.12 (28.7) 1.040 ± 0.01 (18.3) 0.992 ± 0.01 (32.1)0.3 0.994 ± 0.21 (28.3) 0.990 ± 0.12 (22.2) 0.986 ± 0.12 (32.6)

Cystamine0.1 1.285 ± 0.03 (7.4) 0.768 ± 0.22 (39.7) 1.015 ± 0.15 (30.6)0.4 1.045 ± 0.06 (24.7) 0.694 ± 0.27 (45.5) 0.461 ± 0.11 (68.4)b1.0 1.036 ± 0.08 (25.3) 0.347 ± 0.12 (72.7)b 0.087 ± 0.04 (94.0)c

lodoacetamide0.0025 1.356 ± 0.19 (2.3) 1.419 ± 0.29 (11.3) 1.313 ± 0.15 (10.2)0.01 1.240 ± 0.18 (10.6) 1.443 ± 0.16 (13.2) 0.334 ± 0.01 (77.2)c0.06 1.188 ± 0.08 (14.4) 0.668 ± 0.23 (47.5) 0.326 ± 0.01 (77.7)Ca Two adult female worms were incubated per each inhibitor concentration, and at the indicated times, MTT reduction was carried out separately on individual

worms as described in the text. Values are means ± standard deviations of two experiments performed in duplicate. Values in parentheses are percent inhibitionof formazan formation compared with that in untreated controls.

b P < 0.05.c P < 0.01.

At lower concentrations of MDC (0.2 mM) and cystamine(0.1 mM), the observed inhibitory effect on mf productionand release was reversible; the female worms started releas-ing mf into the culture medium after removal of the inhibitorsand culture in fresh medium (data not shown). However, thefemale worms pretreated with MDC or cystamine at rela-tively higher concentrations (>0.3 mM) were unable torecover from such an inhibitory effect. Formazan formationwas greatly reduced in samples containing mf released fromadult worms incubated in the presence of MDC (0.1 and 0.2mM) or cystamine (0.1 and 0.4 mM) when it was comparedwith that in the controls, suggesting the inhibitory effect ofMDC and cystamine on mf release by female worms and/oron their viability (Table 5). A similar inhibitory effect ofMDC on mf production by female worms and on adult wormsurvival was observed in A. viteae. Incubation of A. viteae

TABLE 5. Effect of MDC and cystamine on mf releaseby B. malayi female worms

Inhibitor aSgiiac(concni[mM]) A595' % inhibition (p)b

None (control) 0.130 ± 0.020

MDC0.1 0.012 ± 0.008 90 <0.050.2 0.010 ± 0.014 92 <0.05

Cystamine0.1 0.070 ± 0.016 46 <0.10.4 0.012 + 0.004 90 <0.05a Five female worms were incubated per each inhibitor concentration. The

spent media were centrifuged to pellet the released mf, which were thensubjected to the MTT reduction test. Values are means ± standard deviationsfrom two experiments performed in duplicate.

b Levels of significance were determined by the t test.

adult female worms in the presence ofMDC (0.05 to 0.2 mM)led to a complete inhibition of mf production and releaseafter 4 h of incubation. The adult worms died by 84 h ofincubation with MDC, while controls (in medium alone)were active and alive (data not shown).

DISCUSSION

Candidate filaricides may be developed by in vitro cultureof the different stages of parasites with the compounds.Ginger (12) suggested that enzyme inhibitors of knownspecificity can provide the basis of drug development andshould be used for the selection of target enzymes ormetabolic pathways that would be lethal to parasites. Re-cently, we have reported the presence of a novel TGaseenzyme (pTGase) in adult worms of B. malayi (18). In orderto determine the significance of pTGase-catalyzed reactionsin the survival of these parasites, we tested the effects ofseveral TGase inhibitors on the viability of B. malayi adults,L3, and mf by the colorimetric method. The method, de-scribed by Comley et al. (6), provides a useful in vitromethod for determining filarial viability (7). It is based on thecapacity of mitochondrial enzymes of viable parasites totransform the tetrazolium salt MTT into its blue formazanderivative.A concentration-dependent effect of TGase inhibitors

against B. malayi adults, mf, and L3 was observed in thepresent study. MDC, a highly potent competitive amineinhibitor of TGase (16, 17), and cystamine, an active-siteinhibitor, had a parasiticidal effect against B. malayi in vitroat various stages. MDC also significantly inhibited mf pro-duction and release by female worms. Similar inhibitoryeffects were caused by MDC against A. viteae in vitro.Furthermore, a parallel between viability and MTT incorpo-ration was observed in inhibitor-treated parasites. Thepresent results further corroborate evidence presented in

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previous reports (6, 7) that the MTT reduction assay couldbe an effective tool for evaluating the filaricidal activity oftherapeutic agents.

Several studies with Onchocerca volvulus, Litomosoidescarinii, and A. viteae suggested that ivermectin inhibitsembryogenesis and mf release from adult worms (2, 21, 27).In the present study, TGase inhibitors showed a similarinhibitory effect on the production and release of mf in vitro,indicating the possible involvement of TGase in embryogen-esis and mf release by the adult female worms. It is possiblethat the filaricidal or embryostatic effect of ivermectin maybe associated in some way with the intervention of TGase-catalyzed reactions in these parasites. Further studies toelucidate this hypothesis and to determine the precise bio-logical and biochemical roles of pTGase in parasites arebeing conducted.TGases have been implicated to participate in a variety of

disease states, including viral infections (3, 8, 15, 20), and aneed for a specific and active TGase inhibitor has beensuggested. Until such inhibitors are available, one mustcontinue using alternative approaches with caution to an-swer pertinent questions regarding the significance ofTGase-catalyzed reactions. We have used MDC, one of themost potent substrate inhibitors of TGase (17), to study therole of pTGase-catalyzed reactions in the growth and sur-vival of filarial parasites. The MDC-induced effects on theseparasites seemed quite specific in view of the fact that itsstructurally related analog (DDC) failed to induce either anembryostatic or a parasiticidal effect. Furthermore, structur-ally unrelated compounds that inhibit TGase by binding to itsactive site exerted similar effects on parasites.

Recently, synthesis of several compounds that specificallyinactivate the mammalian TGase by binding to the active sitehave been reported by Syntex (USA) Inc., Palo Alto, Calif.(5). One such inhibitor was N-benzyloxycarbonyl-D,L-P-(3-bromo-4,5-dihydroisoxazol-5-yl)-alanine benzylamide. ThisTGase inactivator was tested against helminthic infectionsand was found to be active in vitro against Nippostrongylusbraziliensis. Incubation of N. braziliensis in the presence ofthis inhibitor at micromolar concentrations markedly re-duced viability, mobility, and molting in vitro. The sameinhibitor was also effective in vivo against Nematosporoidesdubius and Hymenolepis nana in Swiss-Webster mice, sig-nificantly reducing parasite numbers. The results of thepresent study envisage a potential target for the chemother-apy of filarial infections based on the newly identified keyregulatory enzyme pTGase. However, identification of theregulatory biochemical pathways of the pTGase enzyme isimperative in order to develop efficacious and less toxicinhibitors. The results also suggest a method for blockingproduction of mf in adult worms and thereby interruptingtransmission of infection and ultimately preventing the adultworm- or mf-induced pathogenesis.

In conclusion, results of this study strongly support thenotion that pTGase may act as a key enzyme in the growth,development, and survival of the filarial parasites and thatpTGase could serve as a potential target for new therapeuticagents for treating filariasis.

ACKNOWLEDGMENTSWe thank J. K. Nayar, Florida Medical Entomology Laboratory,

University of Florida, Vero Beach, for providing A. aegypti mos-quitoes infected with B. malayi for L3 recovery. The materials usedin this study were also provided by NIAID supply contract AI02642, U.S.-Japan Cooperative Medical Science Program. U.R.R.was supported by the filarial component of UNDP/World Bank/

WHO special program for Research and Training in Tropical Dis-eases. We thank Colin D. Ginger, WHO/OCP/OCT, Geneva, foruseful comments on this project and for proposing additional exper-iments with Onchocerca species.

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