(Received for publication, November 2, 1995; and in revised form, December 28, 1995)
From the
Aggregation of physiologically produced soluble amyloid
protein (A
) to insoluble, neurotoxic fibrils is a crucial step in
the pathogenesis of Alzheimer's disease. Aggregation studies with
synthetic A
1-40 peptide by the thioflavin T fluorescence
assay and electron microscopy and cytotoxicity assays using rat
pheochromocytoma PC12 cells showed that an antibiotic, rifampicin, and
its derivatives, which possess a naphthohydroquinone or naphthoquinone
structure, inhibited A
1-40 aggregation and neurotoxicity in
a concentration-dependent manner. Hydroquinone, p-benzoquinone, and 1,4dihydroxynaphthalene, which represent
partial structures of the aromatic chromophore of rifampicin
derivatives, also inhibited A
1-40 aggregation and
neurotoxicity at comparable molar concentrations to rifampicin.
Electron spin resonance spectrometric analysis revealed that the
inhibitory activities of those agents correlated with their
radical-scavenging ability on hydroxyl free radical, which was shown to
be generated in cell-free incubation of A
1-40 peptide. These
results suggest that at least one mechanism of rifampicin-mediated
inhibition of A
aggregation and neurotoxicity involves scavenging
of free radicals and that rifampicin and/or appropriate hydroxyl
radical scavengers may have therapeutic potential for Alzheimer's
disease.
Amyloid protein (A
), (
)a 39-43 amino
acid peptide, is a primary component of the amyloid that is deposited
in the brains of patients with Alzheimer's disease (AD). A
is physiologically produced as a soluble form by enzymatic cleavage of
the larger precursor, termed amyloid precursor protein (1, 2, 3) . Soluble A
is not toxic and
its physiological function is not known; however, it has been shown
that aggregation of A
to insoluble fibrils causes neurotoxic
change of the peptide(4, 5, 6) . Therefore,
inhibition of this process would seem to be an effective therapeutic
strategy for AD.
The mechanisms of A aggregation and
neurotoxicity are not completely known. Recently, it was suggested that
free radical generation may be involved in the processes of A
aggregation and/or neurotoxicity (7, 8, 9) .
Those hypotheses imply that appropriate radical scavengers could
inhibit A
aggregation and/or neurotoxicity.
It was previously
reported that non-demented elderly leprosy patients showed an unusual
absence of senile plaques in their brains compared with age-matched
controls(10) . Although that finding itself is still a matter
of controversy(11) , we surmised that some drug being used for
leprosy might be preventing A aggregation, resulting in the
absence of amyloid deposition. Thus, we tested two well known
anti-leprosy drugs, dapsone and rifampicin, and found that rifampicin
inhibited A
1-40 aggregation and neurotoxicity in
vitro(12) . Rifampicin is a semisynthetic derivative of
the rifamycins, a class of antibiotics that are fermentation products
of Nocardia mediterranei (for a review, see (13) ).
The common structure of rifamycins is a naphthohydroquinone or
naphthoquinone chromophore spanned by an aliphatic ansa chain. Taken
together with the above free radical hypotheses, this structural
feature of rifampicin suggests that this drug may function as a radical
scavenger with its naphthohydroquinone ring in inhibiting A
aggregation and neurotoxicity.
In the present study, we confirmed
the published finding that free radicals are generated in cell-free
incubation of A1-40 peptide (7) and show that at
least the hydroxyl radical is involved in this process. Also, we show
that the inhibitory activities of rifampicin and its derivatives
against A
aggregation and neurotoxicity correlate with their
radical-scavenging ability on hydroxyl radical, which function arises
from their naphthohydroquinone or naphthoquinone structure. These
results implicate therapeutic potential of rifampicin for AD and
provide useful information for developing new compounds for the
treatment of AD.
Figure 1:
Structures and preparation of
rifampicin derivatives. a, MnO,
CH
Cl
, room temperature, 15 min; b, 1) N-(2,4,6-trimethylbenzyl)piperazine, dioxane, 70 °C, 3 h;
2) ascorbic acid, room temperature, 30 min; c, 1)
MnO
, CH
Cl
, room temperature, 15
min; 2) ClCOC(CH
)
, pyridine, room temperature,
30 min; 3) Zn powder, tetrahydrofuran, 1 N HCl, room
temperature, 15 min; d, ClCOC(CH
)
,
pyridine, 50 °C, 30 min; e,
CH
OCH
CH
OH, reflux, 130 °C, 5 h; f, 1) H
, Pd/C, EtOH, room temperature, 3 days; 2)
ascorbic acid, room temperature, 30 min.
To identify the structure required for the inhibitory
activities of rifampicin, we synthesized a panel of rifampicin
derivatives (Fig. 1) and tested them for effects on
A1-40 aggregation and neurotoxicity (Table 1). Fig. 2shows the time course of aggregation of A
1-40
peptide without any test agent. Rifampicin inhibited A
aggregation
and neurotoxicity in a concentration-dependent manner. This inhibitory
effect of rifampicin against A
aggregation was confirmed by
electron microscopy ( Table 1and Fig. 3). A control
peptide solution showed apparent amyloid-like fibrils of
A
1-40, while very few fibrils were observed in the presence
of 100 µM rifampicin. Rifamycin SV and rifamycin S, which
possess a naphthohydroquinone and naphthoquinone ring, respectively,
also inhibited A
aggregation and neurotoxicity, without any
functional group at position C-3 of their aromatic ring. From these
observations and the free radical
hypotheses(7, 8, 9) , we speculated that the
active structure of rifampicin responsible for the inhibition of
A
's activities might be the naphthohydroquinone (or
naphthoquinone) ring. In support of this, hydroquinone, p-benzoquinone, and 1,4-dihydroxynaphthalene, which represent
partial structures of the aromatic chromophore of rifampicin
derivatives, inhibited A
aggregation and neurotoxicity at
comparable molar concentrations to rifampicin. The hydroxyl group at
position C-1 of the naphthohydroquinone ring must be essential for the
inhibitory activities of rifampicin, because substitution of the
hydroxyl group at this position, or cyclization between the hydroxyl
group at position C-1 and the carbon at position C-15 of the ansa
chain, considerably reduced the activities, as demonstrated with
RFM-007 and RFM-008, respectively. In addition, p-methoxyphenol also showed only weak inhibition, suggesting
that both of the hydroxyl groups at positions C-1 and C-4 are equally
required for the inhibitory activities. On the contrary, the hydroxyl
group at position C-8 and the double bonds of the ansa chain, which are
essential for the antibacterial activity of rifampicin(13) ,
were not necessary for the inhibitory activities against A
aggregation and neurotoxicity, as shown with RFM-005 and RFM-030,
respectively. As well, the functional group at position C-3, which
modulates the antibacterial activity of rifampicin probably by
influencing transport of the drug molecule through the bacterial wall
and membrane(13) , appeared not to affect the anti-A
activities, as shown with rifamycin SV and RFM-002. Thus, we concluded
that the inhibitory activities of rifampicin against A
aggregation
and neurotoxicity arise from its naphthohydroquinone (or
naphthoquinone) structure.
Figure 2:
Time course of A1-40 peptide
aggregation. A
1-40 peptides at 20 µM in
phosphate-buffered saline were incubated at 37 °C. Peptide
aggregation was monitored by the ThT fluorescence assay. Each point represents the mean ± S.D. for triplicate
determinations.
Figure 3:
Electron micrographs of A1-40
peptide aged in the presence of test agents. A
1-40 peptides
at 20 µM were incubated at 37 °C for 7 days in the
presence of 100 µM test agents. The control peptide
solution containing 1% Me
SO showed apparent fibrils of
A
1-40 peptide (A). Rifampicin (B),
hydroquinone (C), and 1,4-dihydroxynaphthalene (D)
inhibited the fibril formation, whereas RFM-008 (E) and p-methoxyphenol (F) did not. The scale bar is 0.2 µm.
As a reference for evaluating the
activity of rifampicin, three natural radical scavengers,
-tocopherol (vitamin E), ascorbic acid (vitamin C), and
-carotene (provitamin A), were also examined. These vitamins were
all effective in inhibiting A
aggregation, but their activities
were weaker than that of rifampicin. Thus, 10-100-fold higher
concentrations were necessary for the vitamins to achieve the same
degree of inhibition of A
aggregation as shown by rifampicin.
As mentioned already, the naphthohydroquinone (or naphthoquinone)
structure of rifampicin is speculated to function as a radical
scavenger. Thus, using ESR spectrometry, we examined the ability of
rifampicin and the related agents to quench free radicals (Fig. 4). Initially we confirmed the published finding that free
radicals are generated in cell-free incubation of A1-40
peptide(7) . With PBN as a spin trapping agent, an
A
1-40 solution showed an obvious three-line spectrum
(
= 17.1 G) after 3-day incubation. When the
peptide was incubated in the presence of Me
SO, it showed a
different ESR spectrum (
= 17.2 G and
= 16.0 G,
= 3.5 G) partly due to new species of free radicals. Since
Me
SO is known to react with the hydroxyl radical to produce
the methyl radical(17) , and actually, the ESR spectrum of
hydroxyl radical in the presence of Me
SO (
= 16.4 G,
= 3.6
G) corresponded to some portion of the ESR spectrum of an
A
1-40 solution containing Me
SO, the above ESR
result suggests that at least the hydroxyl radical is involved in the
radical-generating process in A
solution. Based on this
observation and because of the simplicity of the radical-generating
system, we focused our experiments on the radical-quenching ability of
agents in relation to the hydroxyl radical generated by the Fenton
reaction(16) . With DMPO instead of PBN, a typical 1:2:2:1
four-line spectrum (
=
= 14.9 G) due to hydroxyl
radical (18) was detected in the control solution. As expected
from the molecular structure, rifampicin quenched the hydroxyl radical
in a concentration-dependent manner. The two analogs, hydroquinone and
1,4-dihydroxynaphthalene, also diminished the hydroxyl radical, while
RFM-008 and p-methoxyphenol showed no quenching ability at
concentrations up to 1 mM. These results suggest that the
inhibitory activities of the agents against A
aggregation and
neurotoxicity correlate with their radical-quenching ability on
hydroxyl radical and that at least one mechanism by which rifampicin
and its analogs inhibit A
aggregation and neurotoxicity involves
scavenging of free radicals.
Figure 4:
ESR spectra of A1-40 peptide
and hydroxyl radical in the presence of test agents. A, ESR
spectrum with PBN of free radicals generated in cell-free incubation of
A
1-40 peptide in the absence (a) or presence (b) of Me
SO. c, ESR spectrum with PBN of
hydroxyl radical generated by the Fenton reaction in the presence of
Me
SO. B, ESR spectra with DMPO of hydroxyl radical
generated by the Fenton reaction in the presence of test agents. The
control solution without any test agent showed a 1:2:2:1 four-line
spectrum (a). Rifampicin (b), hydroquinone (c), and 1,4-dihydroxynaphthalene (d) quenched free
radicals, whereas RFM-008 (e) and p-methoxyphenol (f) did not.
Neuronal degeneration is a significant pathological feature
of AD brains, and the toxicity of A has been implicated in the
neuronal damage. Although the molecular mechanisms of A
neurotoxicity are not completely known, there is general agreement that
the neurotoxicity of A
correlates with its state of
aggregation(4, 5) . Furthermore, it was shown that
fibril formation by A
is definitely necessary for its
neurotoxicity(6) . These observations suggest that drugs that
inhibit A
aggregation may be able to protect neurons from A
toxicity and hence may have therapeutic potential for AD. Here we have
shown that rifampicin and its derivatives, which inhibit A
aggregation, also inhibit A
neurotoxicity.
It was recently
proposed that the A1-40 peptide, in aqueous solution,
spontaneously fragments into free radical peptides, which may react
with one another to generate covalently bonded aggregates and may also
attack nerve cell membranes to induce neuronal
degeneration(7) . We confirmed that free radicals are generated
in cell-free incubation of A
1-40 peptide, and our findings
that radical scavengers inhibit A
aggregation and neurotoxicity
may support this hypothesis. However, the possibility cannot be ruled
out that free radicals are generated independently of A
peptide
and then trapped and stabilized by the peptide to be detected in ESR
analysis. Oxidation of A
by free radicals was shown to cause
peptide aggregation, which was prevented by radical
scavengers(8) .
It was also demonstrated that A induces
increased intracellular H
O
accumulation, which
may cause oxidative damage on neurons probably via hydroxyl radical
generation(9) . A number of antioxidants and the
H
O
-degrading enzyme, catalase, protected cells
from H
O
accumulation and also A
neurotoxicity(9) . Our findings that hydroxyl radical
scavengers inhibited A
neurotoxicity may also support this model.
They did not refer to any relationship between A
aggregation and
A
-induced H
O
production. It may be that
aggregated A
has more potent activity to induce
H
O
production than soluble A
.
In
addition to AD, there are numerous other human amyloidoses. Although
those amyloids contain different proteins, all amyloidogenic peptides
are characterized by the antiparallel -sheet
conformation(19) . It was recently shown that these
amyloidogenic peptides may share a common cytotoxic
mechanism(20, 21) . For example, three amyloidogenic
peptides, i.e. amylin, calcitonin, and atrial natriuretic
peptide, are all toxic to clonal and primary neurons and increase the
intracellular H
O
level(20) . The
cytotoxicity of these peptides is suggested to be mediated through a
free radical pathway indistinguishable from that of
A
(20) . These observations imply that rifampicin could
also inhibit the cytotoxicity of other amyloidogenic peptides besides
A
and that agents that inhibit amyloid fibril formation or
cytotoxicity may have therapeutic potential for several different
amyloidoses.
In summary, we have shown that rifampicin and its
derivatives inhibit A aggregation and neurotoxicity, and their
inhibitory activities are attributed to the naphthohydroquinone or
naphthoquinone structure, which possibly functions as a radical
scavenger. Although the ansa chain appears not to be essential for the
inhibitory activities, its lipophilicity may contribute to transport of
the drug molecule into the brain in vivo(22) . Our
data presented here provide useful information for investigating the
mechanisms of A
aggregation and neurotoxicity and developing new
compounds for the treatment of AD.