THE KINETICS

OF MEBENDAZOLE CONTORTUS

BINDING TUBULIN

JENNIFERH.GILL*~~~ C.S.I.R.O.

Division

of Animal

Health.

McMaster

ERNEST

Laboratory, Private Australia

TO HAEMONCHUS

LACEY

Bag No.

I, P.O. Glebe,

New South

Wales 2037,

(Received9 March 1992: accepted 8 June 1992) Abstract- GILL J. H. and LACEY E. 1992. The kinetics of mebendazole binding to Haemonchus cantortu~ tubulin. international Journalfor Parasitology 22:939-946. The kinetics of the binding of mebendazole (MBZ) to tubulin from the third-stage (L3) larvae of the parasitic nematode. Haemonchus contortus, have been characterized. In partially purified preparations, the association of[‘H]MBZ to nematode tubulin was rapid, k, = (2.6 f 0.3) x IO’ M 'min ‘, but dissociation was slow, k , = (1.58 & 0.02) x 10 ’ min ‘_The affinity constant (K,) for the interaction, determined by the ratio k,/k ,,was(l.6 f 0.2) X 10”~ ‘. Similar results were obtained with crude cytosolic fractions. In equilibrium studies, performed with partially purified nematode tubulin under similar conditions, a K, of (5.3 f I .6) x 10” M ’ was obtained. The best estimate for the K, of the MBZ-nematode tubulin interaction is considered to be the ‘kinetic’ value determined from the ratio of rate constants. The slow dissociation of MBZ from nematode tubulin. which contrasts with the rapid dissociation of MBZ from mammalian tubulin, supports the hypothesis that the selective toxicity of the benzimidazole anthelmintics results from a difference between the affinities of mammalian and nematode tubulins for these drugs. INDEX

KEY WORDS:

Haemonchus ccm~orfus: mebendazole;

INTRODUCTION benzimidazoles

* To whom all correspondence

tubulin.

has been shown that there is one high affinity binding site for colchicine on the tubulin heterodimer (Dustin, 1984; Bryan, 1972), although its exact location is, as yet, unresolved. In populations of the important sheep parasites,

(BZs) have been used widely as effective broad spectrum anthelmintics for the control of nematode parasites in livestock (Prichard, 1978). The development of resistance to this class of drugs within parasite populations has, however, limited their continued usefulness in control programmes (Wailer, 1986). The BZs act as microtubule poisons, binding to the ubiquitous cytoskeletal protein, tubulin, and hence disrupting the dynamic state of microtubule assembly and disassembly within eukaryotic cells. This action leads to a cascade of other biochemical and physiological effects which results in the displacement of the parasite from its predeliction site and its subsequent expulsion from the host (Lacey, 1988). Tubulin usually exists in solution as a heterodimer of CI- and @subunits (Dustin, 1984). Competitive binding experiments with tubulin from mammalian, invertebrate and fungal sources suggest that the BZs bind within the colchicine binding domain on tubulin (reviewed by Lacey, 1988). For mammalian tubulin it SUBSTITUTED

nematode;

Haemonchus

contortus,

Trichostrongylus

coluhr~formis

and Ostertagia circumcincta, the extent of charcoalstable BZ-tubulin binding has been demonstrated to be inversely proportional to the degree of BZ resistance (Lacey & Snowdon, 1988). The decrease in binding in resistant isolates results from a reduction in the amount of drug bound to tubulin in a charcoalstable manner, with no significant change in the association constant of the charcoal-stable BZ-tubulin complex (Lacey, Snowdon, Eagleson & Smith, 1987). It has been suggested that the species specificity of BZ action results from differences in the affinities of tubulins from different sources for these drugs (Friedman & Platzer, 1980a,b). A better understanding of the kinetics of BZ-parasite tubulin complex formation should allow further insights into the selective toxicity of the BZ anthelmintics and the mechanism of BZ resistance in parasitic nematodes. In this paper we have characterized the charcoalstable binding of mebendazole (MBZ) to tubulin from

should be addressed. 939

J. H.

940 H. contortus

third-stage

(L3)

larvae

and

report

GILL

and E. LA(‘EY

rate

for the formation and dissociation of the MBZ-parasite tubulin complex in both crude cytosolic fractions and partially purified preparations. The binding of MBZ to parasite tubulin was analysed according to the mechanism illustrated in Fig. 1. The figure depicts an equilibrium between free MBZ and tubulin and the MBZ-tubulin complex, and irreversible degradation/denaturation reactions leading to formation of inactive tubulin (tubulin*). The affinity constant (K,) calculated for this interaction from the kinetic rate constants obtained in this study indicates a much tighter binding complex than is usually suggested by K,, values derived from equilibrium binding studies. constants

kl MBZ

+

tubulin

MBZ-tubulin

e k--l

I

kd’

kd

tubulin* FIN;. I. A scheme for the binding

tubulin*

+

MBZ

of MBZ to tubulin

MATERIALS AND METHODS Chemical.~. Mebendazole (MBZ) was a gift from Smith Kline, Australia. Colchicine, /%lumicolchicine. podophyllotoxin. vinblastine. poly-L-lysine-linked agarose (0.5-I .O mg polylysine per ml sedimented gel), 4.morpholineethane sulphonic acid (MES), ethylene glycol hi.c-(paminoethyl ether)-N,N.N’,N’-tetraacetic acid (EGTA), Norit A-activated charcoal and bovine serum albumin (BSA, fraction V) were obtained from the Sigma Chemical Company (St Louis, MO, U.S.A.). Calmodulin (bovine brain) was purchased from Calbiochem (CA. U.S.A.). Bio-Rad Protein Assay Dye Concentrate was purchased from Bio-Rad (Sydney, Australia). Dimethyl sulphoxide (DMSO, analytical grade), sodium dodecyl sulphate (SDS) and 2-mercaptoethanol were obtained from BDH Chemicals (Sydney. Australia). All other reagents used were of the highest grade commercially available. [‘HI-Mebendazole ([‘HIMBZ. 33 GBq mmol ‘) was prepared and purified as described by Lacey, Dawson. Long & Than (1989). [‘H]MBZ solutions were prepared in either DMSO or 20% (v/v) DMSO in 0.025 M-MES buffer, pH 6.5. H. contortus krrvcre. The BZ-susceptible McMaster H. con/or/us strain was routinely maintained by passage in 446 month old Merino wethers. L3 larvae were isolated from faecal cultures by standard Baermann filtration and stored at 10°C in tap-water prior to use. Prepration of‘ H. contortus tuhulin. A cytosolic fraction was obtained by homogenizing packed L3 larvae in 0.025 MMES buffer, pH 6.5, in a mechanically driven. tapered glassglass tissue grinder (Wheaton Scientifc, Millville, NJ, U.S.A.). then centrifuging the homogenate at lOO.OOOg for 45 min in a Kontron TGA-75 Ultracentrifuge (Zurich. Switzerland) to

give a clear supernatant. To prepare partially purified H. wntor/a~ tubulin. the 100,000~ supernatant from L3 larvae homogenzied in 0.45 M-MES buffer, pH 6.5, was chromatographed on poly-L-lysine agarose using a modification of the procedure described by Lacey & Prichard (1986). The crude supernatant was applied to a 3 ml bed volume of the gel. pre-equilibrated with 0.45 M-MES buffer. pH 6.5. and protein was eluted sequentially with 2 x I4 ml 0.45 M-MES butrer,pH6.5. I x l4ml I% (w’v)(NH,),SO,. I y 7ml 1.4% (w/v) (NH&SO, and 2 x 7 ml 5% (w/v) (NH&SO,. All (NH,),SO, solutions were prepared in 0.025 M-MES bultir. pH 6.5. EGTA (I mM) and magnesium sulphate (0.5 mb!) were included in all MES buffers. All operations wcrc performed at 4°C. Prot& c~sscr,~‘. Protein concentrations were mcdsured using the Bio-Rad Protein Assay (Bio-Rad. Sydney. Australia), which is based on the method of Bradford (1976). BSA was used as the standard. [‘H]MBZ hiruling rrs.vrc). Duplicate samples were incubated with [‘HIMBZ (final volume 100 /)I, 2% (v/v) DMSO) at 37”C, then unbound drug was absorbed onto activated charcoal (2 mg ml ’ in I% (w/v) BSA. 0.5 ml; 5 min incubation). After sedimentation of the charcoal by ccntrifugation, bound radioactivity in the supcrnatant wab determined by liquid scintillation spectroscopy as previouslq described (Lacey & Snowdon, 1988). For equilibrium binding experiments. association constants (K,$) and the amount ofdrug bound at infinite free ligand concentration (B,,,,,)were calculated by relating the binding. y, to the drug concentration. .\, according to Equation (1) after hquarc root transformation of the data (Laces c/ trl.. 19X7).

Ru/r o/m.voc'iu/ion. To obtain the association rate constant (k,. Fig. I), the amount of [‘H]MBZ bound to nematode tubulin was assayed as a function of time at 37°C. The initial concentration of protein capable of binding MBZ w’as determined by incubation of the protein preparation with a saturating concentration of [‘H]MBZ for 20 min at 37°C. On the basis of the assumption that the reverse rate was negligible during the short time in which the binding data were obtained (/, 2 for the dissociation of MB2 from nematode tubulin at 37°C was shown to be 12.4 h), data up to 85% of saturation were plotted according to Equation (2). where P,, is the initial concentration of sites competent IO bind MBZ, M,, is the initial concentration of MBZ and PM is the concentration of complex at time /

k,t = ~

I

PC,~ MC,

In

M,,(P,,

- PIN)

p,, CM,, - P‘W

Rurr of rfissocicr/ion. The rate constant for the dissociation of the MBZ-tubulin complex (k II Fig. I) was determined by monitoring the loss of [‘HIMBZ from the [‘H]MBZ-tubulin complex and was corrected for the irreversible loss of the MBZ binding activity of the tubulin. Tub&n was incubated with [‘HIMBZ (I .2. or 3 /IM. total volume 100 {II. 2% (\ \) DMSO) for 30 min at 37°C. Unbound [‘HIMBZ was remoted

Mebendazole

binding

by incubation with activated charcoal (10 mg ml ’ in 1% (w/ v) BSA. 100 ~1, 5 min) followed by centrifugation. A sample (90 ~1) of the supernatant containing [‘H]MBZ~tubulin complex was then reincubated with either(i) [‘H]MBZ of the same concentration and specific activity as that used in the initial incubation or (ii) the same concentration of unlabelled MBZ. Samples were assayed for bound [‘HIMBZ at various times up to 5.5 h. The loss of bound [‘HIMBZ in the presence of added [‘H]MBZ results only from the irreversible loss of MBZ binding activity by conversion of the MBZXubulin complex to inactive tubulin and MBZ; when graphed according to I&order kinetics, the slope of the data equals k,‘. The rate of loss of bound rH]MBZ in the presence of added unlabelled MBZ is the sum of the rate of dissociation of MBZ from the complex and the rate of irreversible loss of the MBZ binding activity of the complexed tubuhn; when these data are graphed according to 1st.order kinetics, the slope equals k , + lid’. The rate constant for the dissociation (k ,) was calculated as the difference between the two slopes. The rate constant for the denaturdtion of MBZ binding site in the absence of MBZ (k,) was determined by incubating H. wflf~rtu.~ cytosolic fractions (0.83 mg ml ‘) or partially purified tubulin preparations (0.35 mg ml ‘) at 4 or 37°C in the presence or absence of 2% (v/v) DMSO and reincubating samples removed at various time intervals with [‘H]MBZ for 30 min at 37°C. Bound [‘H]MBZ was determined as described above. k, was calculated as the slope of the data plotted according to 1st.order kinetics. lnhihition srudies. For inhibition studies, samples (90 ~1) of crude supernatant were incubated in duplicate for 30 min at 37°C with [‘HIMBZ (10 ~1 of a 5, 2.5 or 1.25 P’M solution in 20% (v/v) DMSO) and inhibitor (2 ~1 in 50% (v/v) DMSO). Inhibition constants (K,) for competitive inhibitors were estimated by simultaneous solution of all pairs of regression lines from plots of l/(MBZ binding) against inhibitor concentration for three concentrations of [‘H]MBZ (Dixon. 1953). The mean of these determinations and the range of the solutions obtained are quoted for each inhibitor. The data were replotted as (MBZ concentration):(MBZ binding) against inhibitor concentration according to Comish-Bowden (1974) to confirm the competitive nature of the binding. RESULTS Pur$cution

@‘H. contortus tuhulin The charcoal-stable MBZ binding activity in crude cytosolic fractions of H. contortus L3s was purified IO- to ISfold by chromatography on poly-L-lysine agarose. Greater than 90% of the charcoal-stable MBZ binding activity was eluted from the column when 5% (w/v) (NH&SO, was introduced into the elution buffer. Further purification of this fraction on hydroxyapatite indicated that nematode tubulin comprises some 15520% of the protein present in this fraction (Gill and Lacey, unpublished results). The nuture ofthe

MBZ-H. contortus tuhulin comp1e.u Analogous to the colchicine-mammalian tubulin binding complex (Wilson, 1970; Sherlinc, Bodwin & Kipnis. 1974), the BZ-parasite tubulin complex

941

to tubulin

TABLEl-STABILITYOFTHEMBZ-H.

CO~/OY/USTUBUL~NCOMPLEX

%control CF

Treatment

binding

1.4 18 4.0 86

Boiling? SDS1 2-MercaptoethanoQ Ethyl acetate extraction/i

activity* F5 0 1.8 0.6 88

*The crude cytosolic fraction (CF, 0.85 mg ml ‘) or partially purified tubulin (FS, 0.35 mg ml ‘) were incubated with [‘H]MBZ (lp~) for 30 min at 37°C. Control binding activity was determined after charcoal extraction of unbound [‘HIMBZ. Samples were treated before charcoal extraction, except as indicated. i- Boiled 5 min. $ Incubated with SDS (I % (w/v), 37°C. I5 min). 9 Incubated with 2-mercaptoethanol (10% (v/v), 37”C, I5 min). I/ After sedimentation of the charcoal. a sample (0.3 ml) of the supernatant was extracted with ethyl acetate (0.5 ml) and the radioactivity extracted determined as a percentage of control binding.

appears action

to involve which

is stable

a tight

pseudo-irreversible

to charcoal

extraction

inter(Lacey

&

The binding is, however, non-covalent and quantitiative dissociation occurs when the protein is denatured either by boiling, treatment with detergent or sulphydryl reagents, or solvent extraction (Table I). The mixture of proteins present in the crude cytosolic fraction provided the MBZ binding material greater protection from the effects of these denaturing conditions than was found in partially purified preparations. The tertiary structure of the native protein is necessary to allow the binding of MBZ to H. contortus tubulin. Greater than 90% of the MBZ binding activity in crude cytosolic fractions was abolished by heating at 100°C for 4 min or by incubation with 2mercaptoethanol (I 0% (v/v). 15 min). Prichard,

Rate

1986).

of dwuy of the MBZ binding uctivitj,

The stability of the MBZ binding activity was measured in both crude cytosolic fractions and partially purified preparations. At 4°C the MBZ binding activity of the H. contortus crude cytosolic fraction was lost according to Ist-order kinetics with a half-life of 20.5 h (k, = 5.63 x 10ml mini ‘, Table 2); at 37°C its half-life was reduced to 5.04 h (k, = 2.24 x IO-’ mini’). In the presence of 2% DMSO. inactivation of the MBZ binding site at 37°C was further accelerated (kd = 2.77 x IO-’ min-‘). Rates of MBZ binding site inactivation in partially purified preparations (Table 2) were very similar to those obtained under the same

942

J. H. GILL and E. LACEY TABLE 2-THEEFFECTOFTEMPERATURE, DMSO* AND MBZ~N STABILITYOFTHE MBZ BINDINGSITEON H. CO?ZfOrIUST"B"LIN

k,‘t

k,+

4°C

THE

No DMSO

37°C No DMSO

37°C DMSO

37°C DMSO

Cytosolic fraction

0.56

2.24

2.77

I .22

Partially purified

0.61 0.51

I .68 1.52

1.26: I .04 0.95

0.56

I .60

I .oo

Preparation

1.30 2.98

* 2% (v/v) DMSO. t Ist-order rate constants (IO’ min ‘). $ Mean of two independent determinations. conditions with the crude cytosolic fraction. It should be noted that the partially purified preparations were stored in a buffer containing 0.19 M-(NH&SO,; no (NH,),SO, was present in the buffer in which the crude cytosolic fractions were stored. The effect of (NH&SO, on the stability of the MBZ binding site was not examined. It has been observed that bound colchicine protects the colchicine binding site on mammalian tubulin, reducing the rate of irreversible denaturation/degradation by an order of magnitude (Sherline, Leung & Kipnis, 1975; Garland, 1978). In the presence of MBZ, the stability of the MBZ binding activity in crude cytosolic fractions of H. contortus at 37°C was increased two-fold (kd’= 1.26 x IO-‘min-‘; T, 2 = 9.17 h, Table 2); in partially purified preparations the halflife of the MBZ binding activity was increased threcfold (T,.? = 1I .6 h) in the presence of MBZ. Rate of MBZ association Figure 2 depicts a time course for the binding of MBZ to partially purified H. contortus tubulin. A linear 2nd-order plot was obtained for these data (see inset) from which k, (Fig. 1) was determined. (The association of MBZ to nematode tubulin is rapid and could not be monitored under pseudo- I St-order conditions using our assay technique.) The association rate constant, k,, determined for the crude cytosolic fraction, 1.4 X IO5 M-'In&', was similar to that obtained with partially purified preparation (2.6 x 10’ Mm'ITin-').

Rate of MBZ dissociation The binding of MBZ to nematode tubulin was shown to be slowly reversible. The rate constant for the dissociation of MBZ-tubulin complex (k- ,, Fig. 1) was determined in an experiment designed to correct

1.4

,

lima (min)

0

5

10 Incubation

15 time

20

25

(min)

FIG. 2. Rate of association of MBZ with H. ~~n/or~u.rtubulin. The column-purified tubulin fraction (0.40 mg ml ‘) was incubated with [‘HIMBZ (2 PM) at 37°C. Inset: the same data graphed as a 2nd-order rate process.

for the irreversible loss of the MBZ binding activity. Data from a typical experiment with partially purified nematode tubulin are shown in Fig. 3. For the cytosolic fraction k_, was 9.5 x IO-” min-’ (Table 3). which was slightly less than k,‘, 1.26 x IO-’ min ’ (Table 2). For the partially purified preparation. k_, was slightly higher, 1.58 x IO-‘min-‘. Equilibrium studies The binding of [‘H]MBZ (0.039 ~ IO ,UM) to partially purified H. contortus tubulin was measured after a 30 min equilibration time (Fig. 4). The B,,, for this preparation was determined to be 780 i 100 pmol rng-’ protein (_U f s U. n= 3). The ‘equilibrium’ K,, obtained, (5.3 f 1.6) x lOh (.? f SI), n=3) (Table 3), was similar to the ‘equilibrium’ K,, of (8.6 31 2. I) x IO”

Mebendazole

binding

943

to tubulin

TABLE 3-KINETLCCONSTA~TS FORTHEBINDINGOF MBZ TO H. contortus TUBULLN k (min

K, (kinetic)*

,

Preparation

k, (M ’ min

Cytosolic fraction

1.25 x 10’ 1.53 X 10’

1.08 x 10 0.82 x 10

Partially purified

1.39 x 10’1 2.35 x 10’ 2.82 x IO’

0.95 x 10 1.56 x 10 1.59 x 10

2.58 x 10’

1.58 x IO

‘)

‘)

’ ’ ’ ’ i ’

(M ‘)

(150 * 50) x IO6

(8.6 f 2.1) x lO”g:

(163 f 21) x IO’

(5.3 f

* Calculated from the kinetic constants, K, = k,/k ,. t Determined from equilibrium binding measurements conditions. 1 Mean of two independent determinations # From Lacey & Snowdon (1988). 1Mean f S.D (n = 3).

1.6) x lOhi

under the same experimental

M-’ reported -1

K, (equil.)?

(M ‘)

by

Lacey

& Snowdon

(1988)

for

the

to a H. contortus L3 cytosolic fraction, indicating that partial purification of the parasite tubulin did not alter its affinity for MBZ. H . I-d . . -a- 5 Both ‘equilibrium’ K,s are about 30 times less than the _ . a- _ ; _ . :: -e _ _ ‘kinetic’K,sof(l.5 f 0.5) x 10XM-‘and(l.63 f 0.21) n --L G x 10’ M-’ (Table 3) obtained for the crude and I Y partially purified preparations respectively. Lacey et al. (I 987) reported that the ‘equilibrium’ K, for the binding of a series of BZs, although not MBZ, to H. contortus cytosolic fractions increased with -3 1 0 50 100 150 200 250 decreasing protein concentration. The effect ofprotein Time (min) concentration on the ‘equilibrium’ K, for the binding FIG. 3. Rate of dissociation of MBZ from H. NVZ~O~/US of MBZ to partially purified H. contortus tubulin was therefore investigated. As the charcoal extraction tubulin. Partially purified tubulin (0.69 mg ml ‘) was incubassay has been reported to be less reliable when protein ated with [‘HIMBZ (3 FM) for 30 min. Unbound [‘H]MBZ was removed by charcoal extraction and the samples (0.31 concentrations below 0.2 mg ml-’ are used (Lacey & mg ml ‘) were reincubated in the presence of either [‘H]MBZ Snowdon, 1988), these experiments were carried out in (3 PM, 0 -- 0, slope = k,‘) or unlabelled MBZ(3 PM, Q -- C, the presence of BSA (1 mg ml-‘). At this concentration slope = k,’ + k ,). BSA had no measurable effect on the binding of [‘HIMBZ to nematode tubulin. Under these conditions, the K, for the binding of MBZ to H. contortus tubulin increased from 4.9 to 15 PM-’ as the concentration of nematode protein decreased from 0.4 to 0.05 mg ml-’ (Fig. 5). charcoal-stable

binding

of MBZ

I

0.01 0

’

2

4

6

MBZ concentration

8

1

10

@A)

FIG. 4. Binding of [‘HIMBZ to H. c’~n/or/u.~ tubulin. The column-purified tubulin fraction (0.42 mg ml ‘) was incubated with [‘HIMBZ for 30 min at 37°C. The solid line is a fit of the data according to Equation (1).

Calcium and calciumlcalmodulin act to modulate tubulin polymerization (Berkowitz & Wolff, 1981; Lee & Wolff, 1982). The binding of MBZ (2 PM) to H. contortus crude cytosolic preparations and partially purified tubulin was unaffected by calcium (1.5 mn) with or without calmodulin (I PM). The concentrations of tubulin competent to bind MBZ in these preparations were estimated to be 0.06 and 0.3 PM. respectively, assuming one binding site per tubulin dimer. These results indicate that no aggregation of tubulin occurred in either preparation.

J. H. GILL and E.

944

LACEY

_

01 0.0

0.1

0.2 Protein

Fro. 5. Effect of parasite

0.3

’

0.4

J

-500

0.5

(mg/ml)

protein

concentration on the K, for the binding of MB2 to H. ~ontortu.s tubulin. Partially purified H. contortus tubulin was incubated with [‘H]MBZ (0.00244 10 PM) for 30 min at 37°C in the presence (0) or absence (0) of BSA (1 mg ml ‘).

Inhibition studies

The binding of MBZ to H. contortus tubulin in crude cytosolic fractions was inhibited by colchicine and podophyllotoxin (Fig. 6). This inhibition was competitive, indicating that MBZ binds to the colchicine binding site on H. contortus tubulin. The affinity of H. contortus tubulin for MBZ (K, = 0.21 f 0.06 PM) is 60-fold higher than its affinity for podophyllotoxin (K, = 17.5 f I.1 PM) and about IOOO-fold higher than its affinity for colchicine (K, = 413 f 21 ,uM). Neither /%lumicolchicine, an inactive colchicine analogue (Wilson & Friedkin, 1967) nor vinblastine, which binds to a site on the tubulin dimer distinct from the colchicine binding site (Wilson, 1970) inhibited the binding of 0.5 PM MBZ to H. confortus tubulin at 500 or 200 ,UM, respectively. DISCUSSION

The binding of M BZ to H. contortus tubulin, which is stable to charcoal extraction, is characterized by a rapid rate of association and a slow rate of dissociation; the latter is of the same order of magnitude as the rate of irreversible denaturatiomdegradation of the complexed protein. The slow dissociation of MBZ from nematode tubulin may reflect conformational changes that have been induced in the ligand and/or the protein as the drug binds, similar to the conformational changes believed to be responsible for the near irreversibility of colchicine binding to mammalian brain tubulin (k_, = 3 x IO-” min-‘) (Garland, 1978). The ‘equilibrium’ K, values obtained for larval tubulin in both partially purified preparations (5.3 x IOh rv-’ ) and crude cytosolic fractions (8.6 x 1OhM- ‘, Laccy & Snowdon, 1988) lie within the range ofvalues, (I ~ 2) x lOh K’ (Laccy & Prichard, 1986; Lacey rt rd.. 1987) to 1.2 x IO’ k4-l (Lubega & Prichard, 1989).

I

-i-,

3

0.15

% E 4

0.10

-

-300

-100

100

500

300

B

-

F ‘E Lj

0.05_ k! 5 - 0.00 -30

-20

-10

0

10 Inhibitor

20

30

40

50

(pt.4)

FIG. 6. Inhibition of MB2 binding to H. u~n~o~~u.~tubulin by colchicine (A) and podophyllotoxin (B). The cytosolic fraction (0.93 mg ml ‘) was incubated with [‘H]MBZ at 0.5 (V ). 0.25 (0) or 0.125 pi (A) and various concentrations ot inhibitor for 30 min at 37°C.

reported for the charcoal-stable binding of MBZ to tubulin in cytosolic fractions prepared from adult II. contortus. (It should be noted that the value of 1.2 x IO’ M-’ was obtained only after correction for a high level of low affinity/non-specific binding; others working with similar preparations have not reported such high levels of low affinitynon-specific binding.) The characteristics of the binding of MBZ to larva1 tubulin thus appear similar to those of MBZ binding to tubulin from adult H. contortus. The K,, values calculated from the kinetic constants obtained for the binding of MBZ to H. contortus tubulin are mom than an order of magnitude higher than the K, values determined in equilibrium binding studies. Similar discrepancies have been observed between ‘kinetic’ and ‘equilibrium’ K,,s for the binding of colchicine to porcine (Garland, 1978) and rat (Sherline et a/., 1975) brain tubulin. In these cases, the lower K,, estimate from equilibrium binding studies was attributed to the decay of free tubulin (T, 2 = 9 or 5 h) during the lengthy incubation time (3 or 4.5 h) necessary to achieve equilibrium. However. as the association of MBZ with nematode tubulin is rapid, and hence the time required to achieve equilibrium relatively short, decay of the binding site would not account for the present discrepancy. Higher affinities

Mebendazole binding to tubulin are generally found when K, values are calculated by the method of comparing rates of complex formation and dissociation, particularly when the concentrations of ligand or receptor used in equilibrium binding experiments are much greater than the dissociation constant (K,,) for the reaction; such conditions tend to result in data that underestimate the true affinity of the complex (Cuatrecasas & Hollenberg, 1976). The Ku for the binding of MBZ to H. contortus tubulin calculated from kinetic parameters, 6 x IO-” M. is considerably lower than the concentrations of tubulin competent to bind MBZ present in the equilibrium binding experiments (estimated as (0.03 to 0.3) x 10eh M from B,,,, values). Hence the ‘equilibrium’ K, values obtained in this study probably underestimate the true affinity as a result of the high protein concentrations used to achieve measurable binding. The ‘equilibrium’ KG1for the binding of MBZ to partially purified H. contortus tubulin increased as the protein concentration was reduced. Two factors which may contribute to this effect arc (I) the presence of higher oligomers of tubulin, such as rings and spirals (Gethner, Flynn, Berne & Gaskin, 1977). which dissociate on dilution, or (2) the dissociation of tubulin heterodimers to yield LY-and psubunits. The binding of MBZ to parasite tubulin was insensitive to both calcium and calcium/calmodulin, suggesting that the dissociation of tubulin oligomers did not contribute to the protein concentration dependency of the ‘equilibrium’ K,, observed for this interaction. Detrich & Williams (1978) demonstrated a reversible, concentration-dependent dissociation of the heterodimer of bovine brain tubulin using equilibrium ultracentrifugation. If individual LY-and Psubunits cannot bind MBZ. or if they bind the drug, but do so with an affinity that differs from that of the heterodimer, then the increasing proportion of monomers which would result from dimer dissociation as protein concentration is reduced may affect the overall K,, value obtained. The extent of the dimer dissociation under the conditions used in this study is unknown. Binding of colchicine or BZs to individual tubulin subunits has yet to be demonstrated. BZs have been demonstrated to be competitive inhibitors of colchicine binding to mammalian brain tubulin (Friedman & Platzer. 1978; Kiihler & Bachmann. 1981). However. results for tubulin from nematode sources are equivocal. Friedman & Platzer (1980a) reported that MBZ and fenbendazole inhibited colchicine binding to embryonic tubulin from As~uris suutn non-competitively. while Kohler & Bachmann (1981) found that colchicine binding to intestinal tubulin from A. ~uurn was competitively inhibited by MBZ. In the present study both colchicine and podophyllotoxin, which bind to the colchicine

945

binding site (Bryan, 1972), were found to be competitive inhibitors of MBZ binding to H. contortus tubulin. The affinity of H. contortus tubulin for MBZ was much greater than its affinity for podophyllotoxin or colchitine. The low affinity of H. contortus tubulin for colchicine is consistent with the low affinities for colchicine reported for the tubulins from other invertebrate species (Russell & Lacey, 1989 and references therein). Friedman & Platzer (1980a,b) and Lacey (1988) have suggested that differences between the affinities of host and parasite tubulin for the BZ anthelmintics may be the basis of their selective toxicity, although other workers (Kiihler & Bachmann, 198 I ) have attributed the selective toxicity of these drugs to differences in their pharmacokinetic behaviour in host and parasite tissues. The results of this study support the former hypothesis. The rate of dissociation of MBZ from H. contortus tubulin, 1.6 x IO-’ min-‘, is much slower than the rate of dissociation of MBZ from ovine brain tubulin, 2.6 min-’ (Russell, Gill & Lacey. 1992). Hence, the levels of MBZ bound to tubulin in parasite cells will change more slowly in response to decreases in the intracellular concentration of MBZ compared to the situation in host cells. An effective anthelmintic need not necessarily have a lethal effect per se, but the parasite must be sufficiently affected that it can no longer maintain itself at its predeliction site. Thus, the tightness of BZ binding and the dynamics of parasite elimination will be the major determinants of the in V&Oefficacy of these drugs. A reduction in the amount of high affinity BZ-tubulin binding, as is observed in BZ-resistant isolates of parasitic nematodes (Lacey & Snowdon, 1988), will lead to a more transient disruption of microtubule polymerization in the parasite. hence favouring its retention in the host. Acknoll./c,c/Rnlmr.r~The authors wish to acknowledge the assistance of MS Elizabeth Barnes in the analysis of the binding data and to thank Mr Greg Russell and Dr Michael Morris for many helpful discussions. The work reported here was supported by a grant from the Australian Wool Corporation (USPZO).

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