(Received for publication, September 20, 1995; and in revised form, January 11, 1996)
From the
Benzimidazoles (BZ) are broad spectrum anthelmintics thought to
exert their effects by interacting with and disrupting the functions of
microtubules. However, direct biochemical evidence for binding between
BZ and tubulin has not been shown nor is it known what sequences in
tubulin interact with BZ. In this study, a photoactive analogue of
2-acetamido-5-(3-aminophenoxy)benzimidazole that has biological
activity similar to other benzimidazoles was synthesized and used to
photoaffinity label cell lysates from the parasitic nematode of sheep Haemonchus contortus. The photoactive analogue,
2-acetamido-5-[3-(4-azido-3-I-salicylamido)phenoxy]benzimidazole
or
I-ASA-BZ, was shown to photolabel a 54-kDa protein
that was specifically immunoprecipitated with anti-tubulin monoclonal
antibodies. Tubulin photoaffinity labeling by
I-ASA-BZ
was also inhibited with molar excess of various BZ analogues and
colchicine. Interestingly,
I-ASA-BZ photoaffinity-labeled
the
- and not the
-subunits of tubulin. Proteolytic digestion
of
I-ASA-BZ-labeled tubulin with Staphylococcus
aureus V8 proteinase revealed one major peptide with an apparent
molecular mass of 3.5 kDa. Exhaustive digestion of
I-ASA-BZ-labeled
-tubulin with trypsin resulted in
two fractions containing radioactive peptides. Protein sequencing of
the high performance liquid chromatography-purified tryptic
ASA-BZ-photolabeled peptides identified the N-terminal 63-77 and
78-103 sequences as the BZ binding domain.
Benzimidazoles (BZ) ()are broad spectrum
anthelmintics that display excellent activity against parasitic
nematodes and to a lesser extent against cestodes and
trematodes(1) . Recently, BZ were shown to be very effective
anti-protozoal agents (2) in addition to their anti-tumoral and
anti-fungal activity(3, 4, 5) . It is
presently believed that BZ exert their cytotoxic effects by binding to
and disrupting the functions of the microtubule
system(6, 7, 8, 9) . The implication
of tubulin as target for BZ has been supported by drug binding studies
using enriched extracts for helminth and mammalian
tubulin(6, 7, 8) . Moreover, competitive
drug-binding studies using mammalian tubulin have shown that BZ compete
for colchicine binding and inhibit the growth of L1210 tumor cells in vitro(10, 11, 12) . However, BZ
display selective toxicity toward nematodes when administered as
anthelmintics and are not toxic to the host(1) . Such selective
toxicity is in contrast to the effect of BZ on the in vitro polymerization of mammalian tubulin and the growth of tumor
cells(3, 4, 5) . Differences in both the
affinity between host and parasite macromolecules for BZ (13, 14) and the pharmacokinetics of BZ within the
host and the parasite have been suggested as responsible for BZ
selective toxicity(15) . Therefore, the nature of BZ selective
toxicity remains unclear. The direct identification of BZ receptor(s)
in nematodes would help clarify this uncertainty.
The frequent
application of BZ in the control of parasitic nematodes has led to the
rapid selection of BZ-resistant populations(16, 17) .
Recently, mutations in tubulin genes have been correlated with the
development of BZ resistance(18, 19, 20) ,
and mutations conferring BZ resistance have been mapped to the locus
encoding the -tubulin (21, 22, 23, 24, 25, 26, 27) .
However, without direct biochemical evidence for binding between
tubulin and BZ, it is not clear if the mutations in the
-tubulin
genes that confer resistance to BZ affect direct BZ binding to
-tubulin. In addition, we have shown recently (5) that BZ
are substrates for the P-glycoprotein drug efflux pump that mediates
the multidrug resistance phenotype in tumor cells (28, 29) and in some
parasites(30, 31) . Therefore, an enhanced drug efflux
mechanism in resistant H. contortus could confer resistance to
BZ.
To further characterize the biochemical and molecular basis for
the action of BZ, it was of interest to show directly the receptor(s)
of BZ using a photoaffinity labeling assay. This approach has been used
to identify the receptor and the ligand binding domain on such a
receptor for many ligands including several antimitotic drugs.
Photoactive analogues of vinblastine, colchicine, rhizoxin, and taxol
have been used previously to demonstrate their direct binding to
tubulin and to identify their photolabeled
sequences(32, 33, 34, 35) .
Moreover, and in support of the latter approach, a recent study (36) using high resolution images of taxol-bound tubulin has
provided structural evidence for taxol binding domain that is
consistent with earlier results using a photoactive analogue of
taxol(35) . In this report, we describe the synthesis of the
photoactive-radioactive analogue of
2-acetamido-5-(3-aminophenoxy)benzimidazole, I-ASA-BZ,
and its use in a photoaffinity labeling assay. The results of this
study show for the first time the direct binding of BZ to tubulin. In
addition, we show that the
I-ASA-BZ binding domain is
localized to a 36-amino acid sequence
(Ala
-Lys
) in the N-terminal of
-tubulin. The implication of these findings with respect to
tubulin drug binding sites and BZ species selectivity will be
discussed.
Figure 1:
Organic structures of
2-acetamido-5-(3-aminophenoxy)-benzimidazole (Amino-BZ) and
its iodinated photoactive derivative
2-acetamido-5-[3-(4-azido-3-I-salicylamido)phenoxy]benzimidazole ([
I]ASA-BZ).
Figure 2:
Photoaffinity labeling of cytosolic
extracts from H. contortus with I-ASA-BZ.
Cytosolic fractions (100 µg) were photoaffinity-labeled with 50
nM
I-ASA-BZ and resolved on SDS-PAGE. Lane 1 of A shows crude extract of H. contortus stained
with Coomassie Blue. B, lane 1, shows an
autoradiograph of crude extract from H. contortus incubated
with
I-ASA-BZ but not exposed to UV light. B also shows
I-ASA-BZ-photolabeled proteins in the
absence (lane 2) or presence of 1, 5, 10, and 50 µM amino-BZ (lanes 3, 4, 5, and 6, respectively). The positions of molecular mass markers are
shown on the left of lane 1, A. C shows photoaffinity
labeling of H. contortus fractions with increasing
concentrations (10-200 nM)
I-ASA-BZ (inset). The 54-kDa photoaffinity-labeled protein was excised,
and the radioactivity in each gel slice was determined by
counting. A plot of the ASA-BZ incorporation in the 54-kDa protein versus the concentration of
I-ASA-BZ is shown in C.
To confirm the identity of the
54-kDa ASA-BZ-photolabeled protein, I-ASA-BZ-photolabeled
cytosolic fractions were incubated with anti-tubulin monoclonal
antibodies (mAbs), and the immunoprecipitated proteins were resolved on
SDS-PAGE. The results in Fig. 3show a 54-kDa protein
immunoprecipitated from H. contortus with anti-tubulin mAbs (lane 3), but not with an irrelevant IgG
(lane 1). Similar results were also obtained when a cell
extract from E. coli, which overexpress the H. contortus
12-16 tubulin gene, was photoaffinity-labeled with
I-ASA-BZ and immunoprecipitated with anti-tubulin mAbs or
an irrelevant IgG
(Fig. 3, lane 4 or 2, respectively). These results confirm the identity of the
54-kDa protein as tubulin and provide the first direct evidence for BZ
binding to tubulin.
Figure 3:
Immunoprecipitation of I-ASA-BZ photoaffinity-labeled proteins. The cell
extracts from E. coli expressing recombinant
-tubulin and
from adult H. contortus were photoaffinity-labeled with
I-ASA-BZ and immunoprecipitated with antibodies to
tubulins as described under ``Experimental Procedures.'' A
54-kDa
I-ASA-BZ-photolabeled protein immunoprecipitated
with anti-
/
-tubulins from H. contortus cytosolic
extracts (lane 3) and from E. coli expressing the
recombinant
-tubulin (lane 4). Immunoprecipitations from
the above tubulin-containing fractions (H. contortus cytosolic
extracts or E. coli) with an irrelevant IgG
are
shown in lanes 1 and 2,
respectively.
The above results in Fig. 3(lane
4) suggest that -tubulin expressed in E. coli is
photoaffinity-labeled with ASA-BZ; hence, BZ can interact with
recombinant
-tubulin monomer(s). However, it is unclear whether
-tubulin is also photolabeled by
I-ASA-BZ. To
determine if one or both tubulin subunits are photoaffinity-labeled
with ASA-BZ, cytosolic extracts from H. contortus were
photolabeled with
I-ASA-BZ, separated on gradient
SDS-PAGE (4 to 14%), and transferred to nitrocellulose membrane for
Western blot analysis with anti-
- and/or
-tubulin mAbs. The
results in lane 3 of Fig. 4show a single polypeptide
detected with anti-
-tubulin mAb. Lane 4 of Fig. 4shows the two subunits of tubulin when the sample
identical with lane 3 is probed with both anti-
- and
-tubulin mAbs. These results demonstrate that the photolabeled
protein (Fig. 4, lanes 1 and 2) co-migrates
with
-tubulin subunit (lane 3). In addition, the
photoaffinity-labeled subunit in H. contortus (lane
1) co-migrated with the recombinant
-tubulin from E. coli extracts. Similar results were also obtained when purified
mammalian brain tubulin was photoaffinity-labeled and fractionated on
gradient SDS-PAGE to separate
- and
-tubulin subunits (data
not shown) except that the mammalian
- migrates slower than the
-tubulin as reported previously(43) .
Figure 4:
Separation of - and
-tubulin
subunits. Cytosolic extracts from H. contortus and E. coli were photoaffinity-labeled with
I-ASA-BZ, separated
on a 4-16% gel, and transferred to nitrocellulose membrane. The
nitrocellulose was exposed to an x-ray film to locate the
photoaffinity-labeled proteins. Lanes 1 and 2 show
I-ASA-BZ photoaffinity-labeled tubulin in cytosolic
extracts from H. contortus and E. coli, respectively.
The same nitrocellulose membrane strip that contains only the
I-ASA-BZ-photolabeled tubulin from H. contortus (lane 1) was later probed sequentially with an
anti-
-tubulin monoclonal antibody alone (lane 3) and then
with an equal mixture of anti-
- and anti-
-tubulin monoclonal
antibodies (lane 4). For Western blot, the signals for
-tubulin or
- and
-tubulins (lane 3 or 4, respectively) were detected following a 30-s exposure using
a peroxidase-conjugated goat anti-mouse second antibody and the
chemiluminescent substrate, luminol (see ``Experimental
Procedures''). Under the latter exposure times (30 s), the
presence of
I-photolabeled
-tubulin on the
nitrocellulose membrane from lane 1 did not contribute to the
signal seen by Western blotting (lanes 3 and 4).
Figure 5:
Inhibition of I-ASA-BZ
photoaffinity labeling of H. contortus tubulin by BZ analogues
and colchicine. Cytosolic extracts from H. contortus were
photoaffinity-labeled with
I-ASA-BZ in the absence or in
the presence of molar excess of amino-BZ and other drug analogues. Lanes 1-6 show photolabeling of tubulin in the absence (lane 1) or the presence of 50 µM amino-BZ (lane 2), mebendazole (lane 3), oxfendazole (lane
4), colchicine (lane 5), and thiabendazole (lane
6), respectively. The bar graph shows an estimate of
I radiolabel in the tubulin protein bands when cytosolic
extracts of H. contortus were photolabeled with
I-ASA-BZ in the absence (lane 1) or presence of
amino-BZ and other drug analogues (lanes 2-6). The
amount of bound
I-ASA-BZ is shown to the right of the figure. The positions of the molecular mass markers are
shown to the left of the figure.
Figure 6:
Photoaffinity labeling of recombinant
-tubulin and inhibition by BZ analogues and colchicine. Aliquot (5
µg) of
12-16 tubulin recovered from E. coli transfected with H. contortus
12-16 gene was
photolabeled with 50 nM
I-ASA-BZ in the absence (lane 1) or in the presence of 50 µM amino-BZ,
colchicine, or thiabendazole (lanes 2, 3, and 4, respectively).
To determine if I-ASA-BZ interacts with similar domain(s) in native and
recombinant
12-16 tubulin, photolabeled tubulins were
subjected to Cleveland mapping using S. aureus V8 proteinase. Fig. 7shows a V8 digest of recombinant
12-16 tubulin (lane 1) and that of native tubulin from H. contortus (lane 2). These results show a similar peptide map for
native and recombinant
-tubulin with one major
photoaffinity-labeled peptide migrating with an apparent molecular mass
of
3.5 kDa. In addition to the 3.5-kDa photoaffinity-labeled
peptide, a minor photoaffinity-labeled peptide (
5.3 kDa) was also
detected in the above V8 map. However, the latter peptide may represent
an incomplete digest of the 3.5-kDa peptide or a different
photoaffinity-labeled site. The other major radioactivity signal in Fig. 7co-migrates with the SDS-PAGE loading dye and may contain
smaller peptides and free
I-ASA-BZ. Furthermore, partial
cleavage of
I-ASA-BZ-photolabeled native and recombinant
-tubulin using increasing concentrations of V8 protease (0.001
µg-10.000 µg/gel slice) did not reveal differences in the
number or the electrophoretic mobility of the photolabeled peptides
(data not shown). Taken together, these results suggest that
I-ASA-BZ binding to recombinant and native tubulin is
similar. In addition, the identity of the 54-kDa photoaffinity-labeled
protein as
-tubulin in H. contortus is further confirmed
since identical peptide maps were obtained when recombinant
12-16 tubulin and the 54-kDa (unresolved
- and
-tubulin) photoaffinity-labeled proteins were digested with V8
proteinase.
Figure 7:
Cleveland map of I-ASA-BZ
photoaffinity-labeled tubulin. Protein extracts from E. coli expressing the H. contortus
12-16 tubulin and
from adult H. contortus were photoaffinity-labeled with
I-ASA-BZ and resolved on SDS-PAGE. Gel slices containing
photoaffinity-labeled tubulin were digested with 10-20 µg of S. aureus V8 protease in wells of a 15% Laemmli gel. Lanes
1 and 2 show peptide maps obtained for the recombinant
12-16 tubulin and H. contortus,
respectively.
Figure 8:
Separation of tryptic peptides from I-ASA-BZ-labeled recombinant tubulin by reverse phase
hplc. Recombinant
12-16 tubulin (2 mg) was
photoaffinity-labeled and digested with trypsin as described under
``Experimental Procedures.'' Tryptic peptides were separated
on a C18 reverse phase column using 0-80% gradient of
acetonitrile. A shows the absorbance at 214 nm of fractions 1
to 80. Two fractions eluting at 45 and 54 min in the gradient (see arrowheads in A) were further purified by hplc and
processed for sequencing by N-terminal Edman degradation. B (panels a and b or c and d)
shows the absorbance at 214 nm and the radiolabel within the resolved
tryptic peptides in fractions 45 and 54.
In this study we show that a photoactive analogue of BZ
interacts directly and specifically with tubulin from H.
contortus. The specificity of I-ASA-BZ toward
tubulin was confirmed by the inhibition of photoaffinity labeling in
the presence of BZ analogues. These results are consistent with earlier
predictions that the high affinity BZ binding to nematode homogenate is
due to tubulin (6, 7, 8, 14, 50) .
Furthermore, the observations that thiabendazole, which lacks the
methyl carbamate and the sulfoxide BZ analogue, oxfendazole, competes
poorly for BZ binding to tubulin agree with previous drug binding
studies(6, 7, 8, 49) . Colchicine,
which is structurally unrelated to BZ, was less inhibitory to the
photolabeling of tubulin with
I-ASA-BZ. An earlier study (6) has shown that colchicine reduces the binding of BZ to both
mammalian tubulin and nematode homogenate. Moreover, BZ binding to
fungal extracts was competitively inhibited by colchicine and
oncodazole and not by other unrelated anti-microtubule
agents(51) . However, binding of colchicine to tubulin is
characteristically different from that of BZ compounds, and the
selectivity of the latter for lower eukaryotic protein has not been
demonstrated for colchicine. Several studies (6, 51) have suggested that (a) both BZ and
colchicine bind to the same site or (b) the binding of
colchicine induces conformational changes in tubulin that are
unfavorable to BZ binding.
The development of BZ resistance in
nematodes and other BZ-sensitive organisms has often been associated
with changes in the genes encoding -tubulin. Our data provide
direct evidence that indeed
-tubulin is the acceptor protein for
BZ in parasitic nematodes. This conclusion is further bolstered by the
observation, in this study, that recombinant
12-16 tubulin
monomers are specifically photoaffinity-labeled by ASA-BZ. Thus,
-tubulin alone appears to bind BZ while the
-subunits are
less essential for BZ binding to microtubule. This finding is not
exclusive to BZ, as photoactive analogues of several antimitotic drugs
(taxol, colchicine, and rhizoxin) that bind to and inhibit the
functions of microtubule have been shown to bind
- and not
-tubulin(33, 34, 35) . This is in
contrast with other anti-tubulin drugs (e.g. vinblastine) that
photoaffinity label both
- and
-tubulin(32) . The
significance of the observed difference in antimitotic drug
interactions with tubulin subunits is currently not clear.
The
cleavage of I-ASA-BZ-photolabeled
-tubulin with
trypsin yielded two labeled peptides. N-terminal sequencing of
I-ASA-BZ-photolabeled tryptic peptides has localized the
BZ binding domain to a span of 36 amino acids (Ala
to
Lys
) in
-tubulin. The assignment ASA-BZ binding to
this region of
-tubulin is consistent with our V8 protease mapping
results which showed one major
I-ASA-BZ-photolabeled
peptide of
3.5 kDa on SDS-PAGE. Analysis of
-tubulin amino
acid sequence for all possible V8 cleavage sites revealed a 34-amino
acid peptide (Ser
to Glu
) with a calculated
molecular mass of
3.7 kDa. This peptide would contain a few amino
acids from the first tryptic photolabeled peptide and the complete
sequence of the second. Other V8 peptides that contain the
I-ASA-BZ-photolabeled amino acid (e.g. Leu
) from the first tryptic peptide in fraction 45
would be too small to detect on SDS-PAGE and would migrate with the dye
front.
The photolabeling of two tryptic peptides by I-ASA-BZ was of the same intensity as determined from the
radiolabel associated with each peptide. Moreover, comparison of the
amino acid sequences of the two
I-ASA-BZ-photolabeled
peptides showed no apparent sequence identity to support the
possibility of two similar binding domains. Thus, the photoaffinity
labeling of
-tubulin at two sites is likely due to rotational
freedom about the ASA moiety that allows the photoreactive group in
ASA-BZ to cross-link more than one sequence in native protein. In this
respect, it was shown recently that two different sequences in
-tubulin are photolabeled by different photoactive analogues of
taxol (p-azidobenzoyl- or m-azidobenzoyltaxol; (34) and (52) ). The photolabeling of the two domains (i.e. amino acids 1-31 and 217-231) in
-tubulin by m-azidobenzoyl- and p-azidobenzoyltaxol is thought to be due to differences in the
position of the photoreactive groups(52) . Although further
characterization of the taxol binding domain is required, in general,
the photolabeling of several distant sites in a protein is compatible
with the three-dimensional nature of a drug binding site. Consequently,
it is conceivable that other photoactive analogues of BZ could
cross-link different sites in
-tubulin. Future studies using
molecular and structural approaches to define the BZ binding domain are
required to determine if the above photolabeled peptides are part of
the BZ binding site in
-tubulin.
The binding of BZ to the
N-terminal of -tubulin brings to three the number of
anti-microtubule agents whose binding maps to this region (33, 35) . However, both taxol and colchicine interact
with two regions of
-tubulin (i.e. amino acids 1-46
and 214-247). These two domains although far removed from each
other in the primary sequence, are thought to come together in the
folded protein to form the drug binding site(33) . It is
interesting that although taxol stabilizes while colchicine
depolymerizes microtubule, these two drugs appear to interact with
common domains (i.e. amino acids 1-46 and
214-247). Thus, the location of a BZ binding site to N-terminal
quarter suggests that this region may be critical for the assembly of
microtubule. Accordingly, the efficacy of major classes of
anti-microtubule drugs may be dependent on their ability to interact
with this region. It is important to note that GTP, required for
microtubule assembly, interacts with tubulin at the N-terminal half and
the requirement of GTP for BZ binding has been suggested although not
clearly shown(6) . One of the GTP binding sites (amino acids
63-69) is contained within the
I-ASA-BZ
photoaffinity-labeled domains, and this may explain the GTP requirement
for BZ binding to tubulin. The binding of taxol is known to reduce the
need for GTP during microtubule assembly. The poor ability of
colchicine to inhibit photoaffinity labeling of tubulin by
I-ASA-BZ could be explained by the fact that these two
drugs bind adjacently but not the same domains.
Genetic analyses of
BZ resistance in H. contortus have identified three possible
amino acid substitutions, Phe
Val, Phe
Tyr, and Ile
Val, that could lead to
resistance(53, 54) . Similarly, four amino acid
substitutions at His
, Val
, Glu
,
and Phe
in benA
-tubulin gene of Aspergillus nidulans are thought to confer resistance to
BZ(25, 27) . The change at position 200 was strongly
favored as the one most likely candidate for causing BZ resistance
since tyrosine is observed in other BZ-resistant organisms and in
mammalian tubulin. Our study, however, identified amino acids
63-103 as the BZ binding domain that spans one of the mutations
implicated in drug resistance (i.e. Phe
Val). Further biochemical evidence will be required to confirm the
effects of these three amino acid changes in BZ-tubulin interactions.
As amino acid changes that may lead to resistance need not be found on
the BZ binding domain or photolabeled peptides in the linear sequence,
it is conceivable that any of these mutations could be important in
conferring BZ resistance. We (
)show that Phe
Tyr substitution leads to reduced photoaffinity labeling
of
12-16 tubulin by
I-ASA-BZ. However, since
several
-tubulin genes exist in parasitic nematodes, drug
resistance in vivo may involve a combination of several
isoforms.
BZ show a remarkable safety when used as anthelmintics in
the treatment of many veterinary and human helminthiases. This is
surprising since BZ also inhibit mammalian microtubule formation in
vitro(6, 55) . In fact, the efficacy of different
BZ analogues correlates well with their microtubule inhibitory
potencies(25) . Thus, the molecular basis for BZ selectivity is
unknown. However, several factors may contribute to the selective
toxicity of BZ. The binding of BZ to parasite tubulin is stable to
charcoal adsorption and is stronger than that of mammalian tubulin that
is readily removed by charcoal
adsorption(7, 13, 14) . A comparison of the
amino acid sequence of ASA-BZ-photolabeled peptides (N-terminal
63-103) of H. contortus to bovine or human -tubulin
show two different amino acid residues (Ala
Gln and
Leu
Ile) that could confer weaker binding of BZ to
mammalian tubulin. Alternatively, the observed safety of BZ as
anthelmintics may be unrelated to BZ-tubulin binding but due to
differences in metabolism or detoxification pathways. For example, the
rapid and extensive metabolism of BZ into less toxic metabolite (e.g. sulfoxides and sulfones) by the liver microsomal enzymes (56, 57) may account for some lack of host toxicity.
Parasites, on the other hand, lack these metabolic pathways and are
killed by BZ. In addition, we have shown recently that BZ are
substrates for the P-glycoprotein transporter in multidrug-resistant
tumor cells(5) . It may be speculated that P-glycoprotein which
is overexpressed in several normal tissues and organs (58, 59) could mediate the transport of BZ. Thus,
P-glycoprotein rather than differences in tubulin amino acid sequence
may mediate the observed safety of BZ to the host. The latter
speculation is interesting since ivermectin, a potent anthelmintic
agent, is also remarkably safe to the host(60) , and its
accumulation in normal tissues is affected by the presence of
P-glycoprotein(61) . The latter results were elegantly
demonstrated using homologous recombination to inactivate the class I
P-glycoprotein gene in mice(61) . Consequently,
P-glycoprotein-deficient mice showed a dramatic increase in ivermectin
accumulation and toxicity in comparison to mice with normal
P-glycoprotein expression(61) .