Synthesis of lithocholic acid derivatives as proteasome regulators

Article abstract of DOI:10.1021/ml3001962

Accumulation of aberrant protein aggregates, such as amyloid β peptide (Aβ), due to decreased proteasome activities, might contribute to the neurodegeneration in Alzheimer's disease. In this study, lithocholic acid derivatives 3α-O-pimeloyl-lithocholic acid methyl ester (2) and its isosteric isomer (6) were found to activate the chymotrypsin-like activity of the proteasome at an EC₅₀ of 7.8 and 4.3 μM, respectively. Replacing the C24 methyl ester in 2 with methylamide resulted in a complete devoid of proteasome activating activity. Epimerizing the C3 substituent from an α to β orientation transformed the activator into a proteasome inhibitor. Unlike the cellular proteasome activator PA28, proteasome activated by 2 was not inhibited by Aβ. Furthermore, 2 potently antagonized the inhibitory effect of Aβ on the proteasome. In summary, compound 2 represents a novel class of small molecules that not only activates the proteasome but also antagonizes the inhibitory effect of Aβ on the proteasome.

Full text of DOI:10.1021/ml3001962

Letter
pubs.acs.org/acsmedchemlett
Synthesis of Lithocholic Acid Derivatives as Proteasome Regulators
Zhao Dang,† Kathy Jung,† Keduo Qian,‡ Kuo-Hsiung Lee,‡,§ Li Huang,*,† and Chin-Ho Chen*,†
†Surgical Science, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, United States
‡Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina
27599, United States
§Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
S
* Supporting Information
ABSTRACT: Accumulation of aberrant protein aggregates, such as amyloid β
peptide (Aβ), due to decreased proteasome activities, might contribute to the
neurodegeneration in Alzheimer's disease. In this study, lithocholic acid
derivatives 3α-O-pimeloyl-lithocholic acid methyl ester (2) and its isosteric
isomer (6) were found to activate the chymotrypsin-like activity of the
proteasome at an EC50 of 7.8 and 4.3 μM, respectively. Replacing the C24
methyl ester in 2 with methylamide resulted in a complete devoid of proteasome
activating activity. Epimerizing the C3 substituent from an α to β orientation
transformed the activator into a proteasome inhibitor. Unlike the cellular proteasome activator PA28, proteasome activated by 2
was not inhibited by Aβ. Furthermore, 2 potently antagonized the inhibitory effect of Aβ on the proteasome. In summary,
compound 2 represents a novel class of small molecules that not only activates the proteasome but also antagonizes the
inhibitory effect of Aβ on the proteasome.
KEYWORDS: proteasome activator, lithocholic acid, Alzheimer's disease, amyloid β
neurodegenerative diseases such as Alzheimer's, Parkinson's,
and Huntington's diseases.6 Proteasome dysfunction could be
due to structural alteration of the proteasome during the aging
or disease process. It could also result from proteasomal
inhibition by misfolded proteins. For example, the amyloid β
(Aβ) aggregates can be degraded and cleared by the
proteasome.7−9 However, higher concentrations of Aβ were
found to inhibit the proteasome function.10−12 Inhibition of the
proteasome by Aβ may create a vicious cycle that results in
accumulation of not only more Aβ but also other protein
aggregates, such as phosphorylated tau, which are also
implicated in the pathogenesis of Alzheimer's disease
(AD).13−15 There was evidence that Aβ-induced proteasome
inhibition increased the levels of reactive oxygen species, tau
tangles, and amyloid plaques, all of which are associated with
AD pathogenesis.13−15
Enhancement of proteasome activity has been tested as a
strategy to clear the unwanted proteins accumulated due to
proteasome dysfunction. Overexpression of the proteasome
activator PA28 was shown to enhance the survival of
Huntington's disease (HD) neuronal model cells.16 Dysfunc-
tion of the ubiquitin-proteasome system was indicated in
patients with HD. The fact that overexpression of PA28
exhibited beneficial effects on the HD cell model raised the
he proteasome is the cellular machinery responsible for
Tintracellular degradation of damaged, unwanted, or
misfolded proteins.1,2 Arranged in a cylindrical structure, the
proteasome (20S) contains four rings that are stacked on top of
each other, with each ring composed of seven subunits. The
two outer rings contain α subunits and do not have enzymatic
activity. The two inner rings are comprised of β subunits where
the proteolytic activities reside. There are three major
proteolytic activities in the β subunits: a chymotrypsin-like
(β5), a trypsin-like (β2), and a caspase-like (β1) activity. These
proteolytic activities enable the proteasome to cleave proteins
into small peptides, 8−9 amino acids in length. The 20S
proteasome is normally associated with 19S components to
form the 26S proteasome. The 19S components, also termed
PA700, regulate the entry of proteins into the 20S proteasome
and activate proteasome degradation of ubiquitin-conjugated
proteins.3 In addition to PA700, there are two other
intracellular protein complexes, PA28 and PA200, that can
activate 20S proteasome using peptide substrate as models.4,5
The evolutionary conservation of multiple cellular proteasome
activators underlines the importance of proteasome activators
in regulating cellular function. The proteasome is involved in
many essential cellular functions, such as regulation of cell
cycle, cell differentiation, signal transduction pathways, antigen
processing for appropriate immune responses, stress signaling,
inflammatory responses, and apoptosis.1,2
Special Issue: Alzheimer's Disease
The importance of the proteasome in multiple cellular
functions predicates the cells for abnormalities in conditions of
proteasomal dysfunction. The dysfunction and impairment of
the proteasome have been linked to the aging process and
Received: July 13, 2012
Accepted: August 15, 2012
Published: August 15, 2012
© 2012 American Chemical Society
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Letter
Figure 1.
a
Scheme 1. Synthesis of LA Derivatives 4 and 5
a
Reagents and conditions: (a) NH2CH3HCl, DCC. (b) Pimelic acid, DCC, DMAP, pyridine, microwave heat. (c) DEAD/Ph3P/HCOOH. (d)
KOH, MeOH.
possibility that small molecule proteasome activators could
have the potential to become useful therapeutics. However,
druglike small molecules, such as betulinic acid (BA) that can
activate or enhance proteasome activity, are rare and not well
studied.17,18 Other proteasome activators, such as SDS at
0.05%, polylysine at 1 mg/mL, and peptide-based molecules at
100 μM, were reported.19 These compounds are either too
weak or lack druglike properties to be considered as good leads.
One exception is oleuropein, a polyphenol compound isolated
from the leaf extract of Olea europaea (Figure 1). Oleuropein
was reported to enhance proteasome activities in vitro at 0.5
μg/mL and expand the proliferative life span of cultured human
embryonic fibroblasts.20 In contrast to the scarcity of
proteasome activator, many proteasome inhibitors have been
identified from either natural products or synthetic peptide
mimetics.21,22 A peptide boronate, PS341 (Velcade),22,23 was
shown to inhibit the proteasome at low nanomolar
concentrations and was approved by the FDA for treating
relapsed multiple myeloma.24
In light of the central role of proteasome dysfunction in aging
or neurodegeneration, we were searching for small molecules
that could activate the proteasome. As a result, BA was found to
activate the proteasome with moderate activity (Figure 1).18 In
a continued effort to synthesize more potent proteasome
activators, lithocholic acid (LA) was used as a scaffold for
chemical synthesis because it has a lower molecular mass and
shares some structural similarity when compared to BA.
Because Aβ was reported to inhibit the proteasome,10−12 the
proteasome activators identified in this study were also tested
for their ability to overcome the inhibitory effect of Aβ on the
proteasome. Among the LA derivatives that we synthesized
with a variety of C3, C24, and C3/C24 dual modifications, the
majority of them were found to inhibit instead of activate the
proteasome.25 However, the LA derivative 2 with a C3 pimeloyl
side chain, which was inactive in proteasome inhibition in a
previous study, was found active in proteasome activation.
Thus, the current study was focused on LA derivatives with C3
pimeloyl group. The synthesis of compounds 1−3 was
described previously.25 Compound 4 with the same C3
pimeloyl as 1−3 but with a methylamide side chain at C24
was synthesized by sequentially coupling LA at the C24
position with methyl amine hydrochloride and then at the C3
position with pimelic acid (Scheme 1). Compound 5, a
diastereoisomer of 2 with a C3-β side chain instead of α, was
synthesized from 3-β-lithocholic acid methyl ester (10),26,27
which was converted from 3-α LA methylester through a
Mitsunobu reaction with the treatment of formic acid−diethyl
azodicarboxylate (DEAD)−triphenylphosphine followed by
KOH/MeOH-catalyzed hydrolysis (Scheme 1).
To synthesize the C3 amide isosteric isomers 6 and 7, LA
was oxidized with Jones reagent, followed by esterification at
C24 with ethanol/HCl to form the C3 ketone/C24 ethyl ester
intermediate 11. Compound 11 was heated to reflux in
formamide to give an α/β mixture of C3 formylamino-
substituted 12a and 12b. The mixture of 12a and 12b can be
easily separated by partially dissolving the mixture in ethyl ether
or methanol, in which the 3α-fomylamino-lithocholic acid ethyl
ester (12a) remained solid as it has a much lower solubility
than its β isomer (12b).28 Further treatment of the pure 12a
and 12b with methanol/HCl under reflux resulted in
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a
Scheme 2. Synthesis of LA Derivatives 6−9
a
Reagents and conditions: (a) Jones reagent, acetone. (b) EtOH, HCl. (c) Formamide, heat. (d) MeOH or Et2O partial precipatation. (e) MeOH/
HCl, reflux. (f) Pimelic acid, DCC, DMAP, pyridine, microwave heat.
Table 1. Regulation of Proteasome Activity by LA Derivatives
a
EC50 is the median effective activation concentration for chymotrypsin-like activity of human 20S proteasome. EC50 was obtained using the
b
nonlinear regression program of CalcuSyn. IC50 is the concentration that inhibited the chymotrypsin-like proteasome activity by 50% in the
presence of PA28. The EC50 and IC50 in the table represent the mean SD of three independent experiments. EC50 for activation of trypsin-like
activity. EC50 for activation of caspase-like activity. −, Inactive.
c
d
e
corresponding C3-α (8) and C3-β (9) aminolithocholic acid
methyl ester, respectively. The configurations of the C3 isomers
were confirmed by identifying 9 as the same compound in
literature obtained by Mitsunobu conversion of the 3-α-
lithocholic acid methyl ester to 3-β-amino-lithocholic acid
methyl ester.29 Compounds 6 and 7 were subsequently
obtained by coupling pimelic acid to 8 or 9, respectively
(Scheme 2).
(for inhibitor) or absence (for activator) of PA28. Cleavage of
the substrates by the proteasome resulted in emission of
fluorescence, which was recorded and used for calculating the
potency of the compounds. The detailed methodology is
described in the Supporting Information and in previous
reports.18,25 The activating or inhibitory effects of the LA
derivatives with C3 pimeloyl substituent on the chymotrypsin-
like activity of the proteasome are summarized in Table 1.
Oleuropein, a natural product previously reported to have
proteasome activation activity, is also included in the study;20
however, it did not show any significant activity in activating
The effect of the compounds on the proteasome was
determined by incubating the compounds with the 20S
proteasome and fluorogenic peptide substrates in the presence
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Letter
the chymotrypsin-like activity of the proteasome under our
assay conditions. Unmodified LA did not activate the
proteasome and is a weak proteasome inhibitor.
In our previous study, we found that certain C3-modified LA
derivatives inhibited proteasome activity with moderate
potency, but none of the C24-esterified LA derivatives inhibited
the proteasome including compounds 1−3.25 However, further
study revealed that compound 2 with C3 pimeloyl group and
C24 methyl ester can activate the proteolysis activity of the
proteasome (Table 1). The concentration required for 2 to
activate the chymotrypsin-like activity of the proteasome to
50% of its maximum level (EC50) was 7.8 μM. Compound 2
also activated the trypsin-like and caspase-like activities of the
proteasome at 8.6 and 11 μM, respectively. The proteasome
activation activity of 2 was quite sensitive to minor changes in
the C24 side chain. A change from CH3O− to C2H5O− or
CH3NH− at C24 as seen in compounds 3 and 4 completely
abrogated the proteasome activation activity. Modification of
the C3 side chain could also affect the potency of the LA
derivatives. An isosteric change of the C3 ester linkage into an
amide linkage resulted in compound 6 and increased the
proteasome activation activity by approximately 2-fold.
Compound 6 activated the chymotrypsin-like activity to 50%
maximal at 4.3 μM.
One particularly interesting finding is that the stereo-
chemistry of the C3 side chain of compounds 2, 6, and 8
dictated whether the compound was an activator or inhibitor.
The C3 side chains of compounds 2, 6, and 8 have an α
orientation, whereas their corresponding stereoisomers 5, 7,
and 9 have a β orientation. In contrast to the α stereoisomers
discussed above, 5, 7, and 9 did not activate the proteasome.
Instead, these three compounds all inhibited the proteasome
(Table 1). The stereospecific proteasome regulatory activity
suggests that these three compounds specifically interact with a
critical motif on the proteasome to modulate its proteolytic
activity.
To determine if LA derivatives could overcome the inhibitory
effect of Aβ on the proteasome, we designed and carried out
the experiments using Aβ1−42, the likely pathogenic form of
Aβ.30 As shown in Figure 2a, Aβ1−42 inhibited the proteasome
in a dose-dependent manner. The slope of each line in the
figure represents the reaction rate and is positively correlated
with the activity of the proteasome. The top line (line 1) in the
figure represents the proteasome activity without the presence
of Aβ1−42. Line 4 in Figure 2a shows that Aβ1−42 inhibited the
proteasome by greater than 90% at 10 μg/mL. These results
confirmed that Aβ1−42 is a proteasome inhibitor, which allowed
us to further test our hypothesis as to whether a small molecule
proteasome activator, such as 2, can antagonize the inhibitory
effect of Aβ1−42 on the proteasome. Aβ1−42 was used at a
concentration of 10 μg/mL in further experiments to study the
relationship between 2 and Aβ1−42 in terms of proteasome
activity.
In evaluating whether compound 2 could activate the
proteasome in the presence of Aβ1−42, 2 was used at 18 μM,
a concentration at which it potently activate the proteasome.
Compound 2 at 18 μM potently activated the chymotrypsin-
like activity of the proteasome (line 2, Figure 2b). The effect of
Aβ1−42 on compound 2-activated proteasome was determined
in the presence of various concentrations of Aβ1−42. At
concentrations ranging from 1.1 to 30 μg/mL, Aβ1−42 did not
inhibit 2-activated chymotrypsin-like activity of the proteasome
(Figure 2b). In contrast, at 10 μg/mL, Aβ1−42 inhibited PA28-
Figure 2. Aβ1−42 inhibited PA28-activated but not compound 2-
activated proteasome. (a) Inhibition of PA28-activated proteasome.
The cellular proteasome activator PA28 (1.8 μg/mL) was used to
activate the proteasome (line 1). Aβ1−42 at various concentrations was
added to inhibit the PA28-activated proteasome activity. The
concentrations of Aβ1−42 used in the assay were 1.1 (line 2), 3.3
(line 3), 10 (line 4), and 30 μg/mL (line 5). Line 6 was background
control (the proteasome and the substrate Suc-LLVY-AMC). R.F.U.,
relative fluorescence units. (b) Aβ did not inhibit compound 2-
activated proteasome. Compound 2 (18 μM) was used to activate the
proteasome (line 2). Aβ1−42 at various concentrations was added to
inhibit the compound 2-activated proteasome activity. The concen-
trations of Aβ1−42 used in the assay were 30 (line 1), 10 (line 3), 3.3
(line 4), and 1.1 μg/mL (line 5). Line 6 was the background control.
activated proteasome activity by more than 90% (Figure 2a).
These results indicated that compound 2 could activate the
proteasome and that Aβ1−42 was ineffective at inhibiting
compound 2-activated proteasome.
The results shown in Figure 2b were derived from assays in
the absence of the cellular proteasome activator PA28. To
determine whether compound 2 could reverse the inhibitory
effect of Aβ1−42 on the PA28-activated proteasome, the PA28-
activated proteasomal activity was tested in the presence of
Aβ1−42 and various concentrations of compound 2. The results
indicated that PA28-activated proteasome activity was com-
pletely inhibited by Aβ1−42 at 10 μg/mL (line 5, Figure 3). The
Figure 3. Compound 2 antagonized the inhibitory effect of Aβ1−42 on
PA28-activated proteasome. The cellular proteasome activator PA28
(1.8 μg/mL) was used to activate the proteasome (line 1). Aβ1−42 (10
μg/mL) was added to inhibit the PA28-activated proteasome activity
(line 5). Compound 2 at various concentrations was added to
antagonize the inhibitory effect of Aβ1−42 on the proteasome. The
concentrations of 2 used in the assay were 56 (line 2), 28 (line 3), and
14 μM (line 4). Lines 6 and 7 were background (the proteasome and
the substrate Suc-LLVY-AMC) and solvent control, respectively.
addition of compound 2 antagonized the Aβ1−42-mediated
proteasome inhibition in a dose-dependent manner (line 2−4,
Figure 3). Compound 2 was able to reverse the inhibitory
activity of Aβ1−42 by over 50% at 14 μM (line 4, Figure 3). This
result strongly suggested that 2 could antagonize the inhibitory
effect of Aβ1−42 on PA28-activated proteasome.
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In this study, we have successfully identified several novel LA
derivatives that can activate the proteasome and demonstrated
that one of the most active compound (2) could activate the
proteasome in the presence of Aβ1−42 and antagonize the
inhibitory effect of Aβ1−42 on the proteasome. Small molecule
proteasome activators are rare. BA and oleuropein are two small
molecules that have been found to activate the proteasome.18,20
Oleuropein was previously described to activate the proteasome
to approximately 1.5-fold over background proteasome activity
in the absence of cellular proteasome activators.20 However,
this compound did not activate the proteasome under our
experimental conditions. Our experimental data suggested that
LA has the potential to serve as an ideal scaffold to derive novel
proteasome activators (Table 1). LA exhibits an unusual
toxicity profile in that it is safe for humans but toxic to small
animals and other primates.31
The results shown in Figure 2b indicated that Aβ1−42 was
unable to inhibit the proteasome in the presence of the
proteasome activator 2. Compound 2 may have activated the
proteasome by a different mechanism from the cellular
proteasome activator, PA28. The fact that PA28 and 2 act by
different mechanisms of action, such as binding to distinct
allosteric sites on the proteasome, might explain why PA28-
activated proteasome was sensitive to Aβ1−42 inhibition,
whereas compound 2-activated proteasome was not. Recently,
a study has shown that methylene blue can slightly enhance
proteasome activity, which might correlate with a reduction of
Aβ1−42 in the brains of an AD mouse model.32 However, it is
not clear whether methylene blue can directly activate the
proteasome or antagonize Aβ1−42. Thus, this study is the first to
show that a proteasome activator can directly counter the
inhibitory effect of Aβ1−42 on the proteasome. The ability of 2
to antagonize Aβ1−42 was further demonstrated by the fact that
2 could completely reverse the inhibitory effect of Aβ1−42 on
PA28-activated proteasome (Figure 3). This result suggested
that compound 2 has the potential to rescue the proteasome
from the inhibitory effects of Aβ1−42 by recovering the clearance
function of the proteasome. The novel proteasome activators
discovered in this study may become useful tools to study how
the proteasome plays a role in so many vital cellular processes,
such as cell cycle regulation and apoptosis. Future studies will
include determining the effectiveness of these compounds in
intracellular milieus.
ABBREVIATIONS
■
LA, lithocholic acid; DCC, N,N′-dicyclohexylcarbodiimide;
DMAP, 4-(dimethylamino)pyridine; DEAD, diethyl azodicar-
boxylate
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ASSOCIATED CONTENT
* Supporting Information
Methods of organic synthesis, proteasome assays, and
spectroscopic data of synthesized compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: 919-684-2952. Fax: 919-684-3878. E-mail: li.huang@
duke.edu (L.H.). Tel: 919-684-3819. Fax: 919-684-3878. E-
mail: chc@duke.edu (C.H.C.).
■
Funding
This work was supported by the National Institute of General
Medical Sciences (NIGMS) Grant AI-84337 awarded to
C.H.C. and the Alzheimer's Drug Discovery Foundation
(ADDF) Grant 20110802 to L.H.
Notes
The authors declare no competing financial interest.
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