(Received for publication, July 20, 1995)
From the
Pharmacological intervention using selective tyrosine kinase
inhibitors has been shown to be an effective approach to inhibit
osteoclast function. Here, we report on the structure-activity
relations of benzoquinone ansamycins isolated from Streptomyces
rishirensis, which form a new class of potent inhibitors of
osteoclast-mediated bone resorption. Parathyroid hormone-stimulated
bone resorption was inhibited concentration dependently by both
mycotrienin I and mycotrienin II, showing half-maximal inhibition in
the low nanomolar range in fetal rat long bones in vitro.
Structure-activity relation studies indicate that position 19 contained
within the quinone/hydroquinone element and the double bonds in
position 4, 6, and 8 are crucial for full bioactivity. In contrast,
substitutions in position 22 are well tolerated. The lack of a similar
effect of 2,6-dimethyl-p-benzoquinone and vitamin K signifies
that the mechanism of action is not solely due to the oxygen scavenger
capacity of the quinone/hydroquinone moiety. The inhibition of
osteoclastic bone resorption is in line with the diminished activity of
immunopurified pp60 from bone suggesting that
pp60
is a possible target of mycotrienins in
the organ culture. Thus, mycotrienins may be useful as pharmacologic
inhibitors of osteoclastic bone resorption.
Osteopetrosis, often referred to as ``marble bone
disease,'' is a sclerosing bone dysplasia mainly characterized by
impaired bone resorption(1) . Over the last few years more
insight was gained on the genetic defects involved in the onset of the
disease pointing to several different disease mechanisms. The op mouse, a well characterized animal model of the human disease, is
unable to develop osteoclasts (2) due to a point mutation in
the coding region of macrophage colony stimulation factor(3) .
In another animal model, osteopetrosis is manifested after homologous
recombination following targeted disruption of the src
protooncogene(4) . Detailed examination revealed that
osteoclasts express high levels of pp60comparable to the levels expressed in brain and
platelets(5) . In the pp60
minus
model, multinucleated cells are formed on bone surfaces but neither
ruffled border formation nor bone resorption occurs(6) . Thus,
in contrast to the op mouse, the inherent defect in the src
knock-out mouse is in the mature osteoclast and is autonomous of the
bone microenvironment(7) .
pp60 is
a non-receptor protein tyrosine kinase(8) , which is expressed
ubiquitously but with elevated levels in neurons (9) and
platelets(10) . Its function seems to be the transduction of
signals arising from the stimulation of cells by growth
factors(8, 9, 10, 11) . The
pp60
-deficient mouse model suggests that src
kinase is essential for osteoclast function, but not for osteoclast
formation. Pharmacological intervention using relatively selective
pp60
tyrosine kinase inhibitors, e.g. herbimycin A, have been shown to be effective inhibitors of
osteoclastic bone resorption in vitro and are able to block
hypercalcemia in vivo(12, 13) . Mycotrienins,
belonging to the class of benzoquinone ansamycins, are structurally
related to herbimycin A in that they also contain a
quinone/hydroquinone system. Starting from natural-derived mycotrienin
I and II, isolated from Streptomyces rishirensis, we prepared
a series of benzoquinone ansamycins derivatives and determined their
activity to inhibit bone resorption in fetal rat long bones (radii and
ulnae) in vitro. We further analyzed their inhibitory effect
on an immunopurified pp60
preparation from
bone. The observed structure activity relationship allowed for
conclusions about a possible mechanism of action for inhibition of bone
resorption by this class of compounds.
Figure 1: Structure of mycotrienin analogues: A1, representing the basic structural element of mycotrienin I; A2, basic structural elements of mycotrienin II. Mycotrienin I, mycotrienin II, and hexadehydromycotrienin II have been prepared, isolated, and purified from S. rishirensis. Details on the derivatization processes are provided under ``Experimental Procedures.''
Figure 2:
Concentration dependence of the inhibitory
effect of mycotrienin I and mycotrienin II on bone resorption
stimulated in the presence of parathyroid hormone in 19-day-old fetal
rat long bones in vitro. Fetal rat long bones were cultured
with 10M hPTH-(1-34) in the presence
or absence of the mycotrienin analogues over the concentration range of
10
to 10
M. Bone
resorption was assessed as the percentage release of the total
Calcium that is released into the culture medium at day 5
of culture. Results are presented as the treated/control (T/C) ratio
and represents means ± S.E. (n = 6) for six long
bones/group. Results of a representative experiment (n = 3) are shown.
Figure 3:
Concentration dependence of the inhibitory
effect of herbimycin A on bone resorption stimulated in the presence of
parathyroid hormone in 19-day-old fetal rat long bones in
vitro. Fetal rat long bones were culture in the presence of
10M hPTH-(1-34) in the presence or
absence of herbimycin A over the concentration range of 10
to 10
M. Bone resorption was assessed
as the percentage release of the total
Calcium that is
released into the culture medium at day 2 and day 5 of culture. Results
are presented as the treated/control (T/C) ratio and represent means
± S.E. (n = 6) for six long bones/group. Results
of a representative experiment (n = 2) are
shown.
The acylation at position 22 with benzyol chloride
resulted in a 4-fold loss in activity. The natural compound SDZ
220-542, in which the cyclohexane ring of mycotrienin is replaced
by a phenyl group and with a methylether at position 22, showed only
little change in the activity. Hydrogenation of the double bonds in
positions 4, 6, 8, and 14 with or without the removal of the hydroxyl
group in position 13 (see Z in Fig. 1) was
carried out. The resulting analogues showed no inhibition of bone
resorption. Removing the bulky side chain in position 11 as in SDZ
224-957 (structure not shown) also abolished the antibone
resorptive activity.
To test for any activity of the
quinone/hydroxyquinone structure in other molecules, we assayed
2,6-dimethyl-p-benzochinon and vitamin K1 for inhibition of
bone resorption. None of the two compounds had any influence on the
release of Calcium from long bones over the concentration
range tested (see Table 2), showing that the quinone moiety by
itself was not sufficient for antiresorptive activity.
Figure 4:
Effect of two potent mycotrienin analogues
on the parathyroid hormone-induced [H]thymidine
incorporation into the acid-precipitable bone DNA fraction in
4-6-day-old neonatal mouse calvariae. After a preincubation
period (18 h), calvarial halves were stimulated with 10
M hPTH-(1-34) in the presence or absence of
different concentrations of mycotrienin II for 24 h with
[
H]thymidine for the last 2 h of culture. Bones
were washed in phosphate-buffered saline, extracted with
trichloroacetic acid, aceton, and ether.
[
H]Thymidine incorporation into the
acid-precipitable bone DNA fraction is expressed as
counts/min/milligram bone dry weight, and is presented as the means
± S.E. for six half-calvariae. *, significantly different from
PTH (p < 0.01).
In addition we have tested possible toxic effects of the
potent mycotrienin 115-962 in the long bone organ cultures by
[H]proline incorporation into the de novo synthesized bone proteins. This compound did not significantly
impair [
H]proline incorporation into the fetal
rat long bones at the concentration range of
10
-10
M (see Fig. 5).
Figure 5:
Effect of mycotrienin II on the
parathyroid hormone-induced [H]proline
incorporation into fetal rat long bones. After a preincubation period
of 24 h, bone explants were labeled with 5 µCi of
[
H]proline for 48 h. At the end of the culture
period, the amount of [
H]proline in the
acid-insoluble bone protein fraction was determined.
[
H]Proline incorporation into the
acid-precipitable bone protein fraction is expressed as counts/min/four
bone explants and is presented as the means ± S.E. (n = 4). *, significantly different from PTH (p <
0.01).
Figure 6:
Influence of mycotrienin II and herbimycin
A on pp60 kinase activity in fetal rat long
bones in vitro. Panel A, fetal rat long bones were
cultured for 24 h with hPTH-(1-34), mycotrienin II, and
herbimycin A at concentrations indicated in the figure. Total long bone
lysates were immunoprecipitated with pp60
antibody GD-11 and assayed for kinase activity using enolase
as a substrate in the presence of [
-
P]ATP. Panel B, quantitative analysis of the phosphorylation of
enolase by the pp60
oncoprotein. After
separation on 10% SDS-PAGE, radioactivity of the band corresponding to
enolase was determined using a PhosphorImager (Molecular Dynamics,
model PI400S). *, significantly different from PTH (p <
0.01),**, significant different from control (p <
0.01).
Figure 7:
Influence of mycotrienin II and herbimycin
A on the total protein amount of pp60 in fetal
rat long bones in vitro. Fetal rat long bones were cultured
for 24 h with hPTH-(1-34), mycotrienin II and herbimycin A as
indicated in Fig. 6. Total bone lysates were immunoprecipitated
with pp60
antibody GD-11. Immunoprecipitates
were subjected to SDS-PAGE and immunoblotted by a sheep antibody to the
pp60
oncoprotein
(OA-11-863).
Figure 8:
Structure activity relations for a
selective series of mycotrienin analogues on pp60 kinase activity in fetal rat long bones in vitro.
Fetal rat long bones were cultured for 24 h with hPTH-(1-34)
(10
M), in the presence or absence of the
mycotrienin analogues at concentrations indicated in the figure. Total
long bone lysates were immunoprecipitated with pp60
antibody GD-11 and assayed for kinase activity using enolase
as a substrate in the presence of [
-
P]ATP.
Radioactivity of the band corresponding to enolase was determined using
a PhosphorImager (for details see legend to Fig. 6). *,
significantly different from PTH (p <
0.01).
Next, we were interested to
see whether mycotrienin I inhibits pp60 kinase directly
or whether it interferes with an earlier step in the signaling cascade.
For this purpose pp60
was immunoprecipitated from fetal
rat long bone homogenates. To obtain optimal pp60
activity, bone explants were stimulated with
10
M hPTH-(1-34) during 24 h in
culture prior to homogenization. The immunocomplexed enzyme was
preincubated with the different compounds for 30 min before the
addition of the substrate and [
-
P]ATP. As
shown in Fig. 9, both herbimycin A and mycotrienin I inhibited
the kinase activity by 50% at a concentration of 0.5 µM.
Figure 9:
Direct inhibitory effect of mycotrienin II
and herbimycin A on the pp60 kinase. Fetal rat
long bones were cultured with hPTH-(1-34) (10
M) for 24 h and lysed. Total bone lysates were
immunoprecipitated with pp60
antibody GD-11.
Immunoprecipitates were treated for 30 min at room temperature with the
compounds as indicated, washed, and assayed for kinase activity using
enolase as a substrate in the presence of
[
-
P]ATP. Quantification of the
radioactivity within the band corresponding to enolase was carried out
as described in the legend of Fig. 6B. *, significantly
different from control (p <
0.01).
The results which we obtained in the fetal rat long bone
resorption assay indicate that mycotrienins form a potent class of
inhibitors of osteoclastic bone resorption. Both mycotrienin I and
mycotrienin II inhibited the release of Calcium into the
culture medium by fetal rat long bones with apparent half-maximal
inhibition (IC
) values of 64 and 21 nM,
respectively. The inhibition was complete and persisted during the
entire culture period of 5 days. In contrast, no significant change in
the basal bone resorption rate was observed at the concentrations
tested (data not shown). Our results are in agreement with a previous
study by Yoneda et al.(12, 22) who
demonstrated that the structurally related herbimycin A inhibited
osteoclast formation in bone marrow cultures, diminished the function
of matured osteoclast in the pit assay, and prevented hypercalcemia by
interleukin-1 or tumor-induced hypercalcemia.
From an analysis of
the structure relationship of the mycotrienins, it is concluded that
substitution in position 19 is essential for potent inhibition of bone
resorption. Methylation of the hydroxyl group in position 19 resulted
in a significant loss in activity to an apparent IC value of 2.88
10
M. Despite a
10-fold drop in activity the compound retained full intrinsic activity
(intrinsic activity of mycotrienin II is 1 by definition). Further
structural elements for the inhibition of bone resorption are the
double bonds in position 4, 6, 8, and 14, the hydroxyl group at
position 13, and the amide function at position 25. In distinction to
the elements required in position 19, substitutions in position 22 are
well tolerated indicating that this position is not crucial for
bioactivity. Methylation even led to a small increase of the inhibitory
effect of the analogue on bone resorption.
It has been hypothesized
that oxygen-derived free radicals produced by the osteoclast serve as
intermediates in the recruitment and activation of
osteoclasts(23, 24, 25) . Depletion of
superoxide anions in tissue by superoxide dismutase led to an
inhibition of stimulated bone resorption, e.g. by parathyroid
hormone or interleukin 1 (25) . Vitamin K, a
quinone/hydroquinone derivative, abolished the induction of interleukin
1 by the phorbol ester phorbol myristate acetate, an effect which
has been related to the radical scavenging properties of vitamin
K(26) . As the quinone/hydroquinone moiety is a common element
of the different mycotrienin analogues tested, we have investigated
whether the inhibition of bone resorption was related to the radical
scavenging capacity of these analogues. Results obtained with
2,6-dimethyl-p-benzoquinone and vitamin K do not support this
notion. A further argument against the scavenger theory may be seen in
the observation that structural modifications within the quinone
moiety, at position 22, are well tolerated. Second, esterification of
both positions 19 and 22 in the quinone ring preserved the bone
antiresorptive activity. We therefore conclude that the ability of the
mycotrienins to inhibit bone resorption is not explained by the oxygen
radical scavenging capacity of these analogues. The bioeffect/toxicity
ratios of these compounds indicates that the inhibition of bone
resorption in the low nanomolar range probably is not just a
consequence of their nonspecific toxic effects. Inhibition of cell
proliferation and protein synthesis, markers for general toxicity, was
only seen at high micromolar concentrations for two of our potent
analogues.
Mycotrienins which belong to the benzoquinone ansamycin
class of compounds were first isolated from different strains of
Streptomyces and described to have antifungal and antitumor activity.
Related compounds in this group include geldanamycin (27) and
herbimycin A (28) . Both compounds have been reported to revert
the morphology of fibroblasts transformed by many oncogenic tyrosine
kinases e.g. src, fyn, bcr-abl, and erbB2(29) . Our findings indicate that pp60 kinase may serve as a possible target for mycotrienins in the
fetal rat long bones. The structure activity relations for the
different mycotrienin analogues to inhibit bone resorption closely
resembles the ability of these compounds to inhibit
pp60
. Fetal rat long bones treated with parathyroid
hormone showed a 2-fold increase in pp60
kinase
activity. Our observation that parathyroid hormone increased
pp60
kinase activity rather than to increase the total
content of pp60
at the protein level seems to be at
variance with results previously reported by Yoneda et
al.(12, 22) . However, the observation period of
24 h in our studies was probably to short to detect any significant
increase in protein expression.
It has been speculated that
herbimycin A irreversibly binds to active SH groups of target proteins
resulting in the sterical hindrance of the active site of the
kinase(21) . Recent evidence suggests that benzoquinone
ansamycins may inhibit pp60 activity in a more indirect
fashion via inhibition of the heat shock protein HSP90-pp60
heterocomplex formation(30, 31) . From our
results it is evident that herbimycin A as well as the mycotrienins can
exert a direct inhibitory effect on pp60
itself as
demonstrated in the in vitro kinase assay.
The half-maximal
inhibition (IC) of mycotrienin I and mycotrienin
II detected in the pp60
kinase assay differ from the IC
found in the fetal rat long bone assay in that
a
2-5-fold higher concentration is needed for the inhibitory
effect in the kinase assay. Several possible explanations could explain
this apparent discrepancy. If the mycotrienin binds reversible to the
kinase, events during several washing steps and the immunopurification
procedure could result in the loss of inhibitor. This loss could
explain the difference in half-maximal inhibition values obtained in
the bone resorption assay and kinase assay. In addition our results do
not exclude that mycotrienins might have further targets in the fetal
rat long bones which potentiate their antibone resorptive capacity in vitro.
The polypeptide hormone calcitonin plays an
important role in the physiological regulation of bone resorption.
Calcitonin exerts its inhibitory effect on bone resorption, at least in
part, via an inhibition of
pp60(22, 32) . Short term exposure of
murine bone marrow cells to the hormone led to an inhibition of
pp60
kinase activity while long term exposure also
diminished the total amount of the pp60
protein. In the
fetal rat long bone resorption assay salmon calcitonin is a powerful
inhibitor of osteoclast-mediated bone resorption (IC
value around 7.4
10
M,(33) ) at the initial phase of culture. After
an extended culture time (up to day 5), the system becomes less
sensitive to calcitonin and bone resorption returns to the parathyroid
hormone-stimulated resorption level (``escape phenomenon'').
In this respect it is interesting to note that the mycotrienin-induced
inhibition was complete and persisted for the entire culture period of
5 days. Thus, inhibiting the osteoclast downstream of the calcitonin
receptor would be an approach to overcome the escape phenomenon that is
associated with a calcitonin.
Parathyroid hormone has been shown to
stimulate both osteoclast activity and osteoclast
recruitment(34) . Bone resorption, as assessed by the fetal rat
long bone system, is almost entirely dependent on the activation of
mature osteoclasts. Thus, this particular system does not allow
discriminating whether the mycotrienins inhibit either one or both,
parathyroid hormone-stimulated osteoclast recruitment and osteoclast
function. Results obtained with the pp60 knock-out
mouse indicate that osteoclast formation is normal but that the
osteoclasts are not functional in the animals as indicated by the
absence of the ruffled borders(6) . However, previous findings
using bone marrow cultures indicated that the total amount of
tartrate-resistant acid phosphatase-positive multinucleated cells in
1,25-dihydroxyvitamine D
-treated cultures is significantly
inhibited in the presence of herbimycin A(22) . Taking these
findings into consideration it cannot be excluded that the mycotrienins
interfere with cellular processes distinct from the pp60
signaling cascade which lead to an inhibition of osteoclast
formation as well.
In conclusion, our results are consistent with
the observation that pp60 is essential for normal
osteoclastic bone resorption and point to a potential for pharmacologic
intervention in the bone resorption process at the level of
pp60
. Therefore, the mycotrienins may have a
therapeutic potential as bone resorption inhibitors in diseases where
bone resorption is increased.