Inhibition of human α-methylacyl CoA racemase (AMACR): A target for prostate cancer

Article abstract of DOI:10.1002/cmdc.201300179

The enzyme α-methylacyl CoA racemase (AMACR) is involved in the metabolism of branched-chain fatty acids and has been identified as a promising therapeutic target for prostate cancer. By using the recently available human AMACR from HEK293 kidney cell cultures, we tested a series of new rationally designed inhibitors to determine the structural requirements in the acyl component. An N-methylthiocarbamate (K_i=98nM), designed to mimic the proposed enzyme-bound enolate, was found to be the most potent AMACR inhibitor reported to date. Enolate mimicry: A range of inhibitors were designed and synthesized to determine the structural requirements for inhibition of the prostate cancer target racemase AMACR using the recently available human enzyme from HEK293 kidney cell cultures. An N-methylthiocarbamate (K_i=98nM), designed to mimic the proposed enzyme-bound enolate, is the most potent AMACR inhibitor reported to date.

Full text of DOI:10.1002/cmdc.201300179

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DOI: 10.1002/cmdc.201300179
Inhibition of Human a-Methylacyl CoA Racemase
(AMACR): a Target for Prostate Cancer
Andrew J. Carnell,*[a] Ralph Kirk,[a] Matthew Smith,[a] Shane McKenna,[a] Lu-Yun Lian,[b] and
Robert Gibson[b]
The enzyme a-methylacyl CoA racemase (AMACR) is involved
in the metabolism of branched-chain fatty acids and has been
identified as a promising therapeutic target for prostate
cancer. By using the recently available human AMACR from
HEK293 kidney cell cultures, we tested a series of new rational-
ly designed inhibitors to determine the structural requirements
in the acyl component. An N-methylthiocarbamate (Ki =98 nm),
designed to mimic the proposed enzyme-bound enolate, was
found to be the most potent AMACR inhibitor reported to
date.
Introduction
Prostate cancer (PCa) is the
second leading cause of cancer
mortality among men in the US,
with 28088 deaths in 2009.[1] An-
drogen deprivation is the most
common treatment for PCa.
However, despite a rapid initial
response, PCa often progresses
to a hormone-refractory stage
and ultimately metastatic dis-
ease. Abiraterone can add valua-
ble months of survival time for
men with advanced metastatic
Figure 1. AMACR racemization reaction, substrates 1–3, and AMACR inhibitor 4.
PCa, although there are still no
curative therapies.[2]
The enzyme a-methylacyl CoA racemase (AMACR)[3,4] plays
a gate-keeping role in the b-oxidation of such branched-chain
fatty acid CoA esters as pristanoyl CoA 1, catalyzing the race-
mization of the 2-R-configured CoA ester to the S-configured
stereoisomer just prior to b-oxidation (Figure 1). This is an obli-
gatory step, as the first stage of b-oxidation is catalyzed by an
oxidase that accepts only S-configured 2-methylalkanoic CoA
esters.
The AMACR gene is consistently up-regulated,[5] and the
enzyme is overexpressed in prostate cancer[6] and other can-
cers;[7] recent data have shown that AMACR is functionally im-
portant in the growth of PCa cells. AMACR is an interlocked
dimer in which the active site is at the interface, with key cata-
lytic residues contributed by each monomer.[8,9] Ten isoforms
of AMACR have been reported,[10–12] although only the main
isoforms, AMACR1A and AMACR1Adel, are likely to be catalyti-
cally active, as the others are C-terminally truncated and lack
critical active site residues or cannot dimerize.[4] siRNA or
shRNA knockdown of the AMACR gene decreases or eliminates
the expression of AMACR and significantly impairs the prolifer-
ation of the androgen-responsive PCa cell line LAPC-4. Simulta-
neous inhibition of androgen signaling and siRNA knockdown
suppresses the growth of LAPC-4 cells to a greater extent than
either treatment alone. These results suggest that the enzyme
is a potential drug target that is complementary to androgen
ablation therapy.[13] AMACR overexpression starts early in onco-
genesis, with 70% of high-grade prostatic intra-epithelial neo-
plasia staining positive. Once overexpressed in PCa, AMACR
levels remain elevated during progression to higher grades
and stages and even metastases, although some studies have
suggested lower levels of AMACR in advanced PCa. However,
recent results suggest a link between androgen dependence
and AMACR expression and that inhibition of AMACR may
revert the phenotype of some cell lines that have become an-
[a] Dr. A. J. Carnell, R. Kirk, M. Smith, S. McKenna
Department of Chemistry, Robert Robinson Laboratories
University of Liverpool, Liverpool L69 7ZD (UK)
E-mail: acarnell@liv.ac.uk
[b] Prof. L.-Y. Lian, Dr. R. Gibson
Institute of Integrative Biology
Biosciences Building, University of Liverpool
Crown Street, Liverpool L69 7ZB (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300179.
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Results and Discussion
drogen-independent.[14] This may allow treatment of patients
with advanced hormone-resistant prostate cancer to be treat-
ed with AMACR inhibitors and androgen ablation.
Chemistry
Prospects for drug treatment with AMACR inhibitors are
good, because although AMACR is present in many organs,
the major clinical manifestation of congenital racemase defi-
ciency is adult-onset sensory motor neuropathy, which is
caused by the slow and prolonged accumulation of branched-
chain fatty acids.[15] This effect could be prevented during
treatment with AMACR inhibitors with a diet low in phytanic
acid. AMACR is also involved in bile acid biosynthesis; however,
an alternative pathway that does not rely on AMACR has re-
cently been demonstrated in AMACR-deficient mice, which are
healthy and fertile.[16] Bile acid intermediates di- and trihydrox-
ycoprostanoyl CoA (DHT- and THC-CoA 2)[17] and various
NSAID profenoyl CoA esters such as ibuprofenoyl CoA 3 are
also known unnatural substrates for isomerization by
AMACR.[18]
We previously reported a series of AMACR inhibitors, some of
which are substrate analogues containing 2-trifluoromethyl
groups or b-fluorine-a-methyl analogues.[22] We also reported
that both R and S isomers of ibuprofenoyl CoA are inhibitors.
We hypothesized that the presence of the b-fluorine atoms or
indeed the presence of an aryl group lowered the pKa of the
a-proton, making these analogues better substrates. This
would imply a slower release from the active site because pro-
tonation of the enzyme-bound enolate would be slower for
these inhibitors. The competitive assays were carried out with
THC-CoA as the substrate, and the best inhibitor, 2-trifluorome-
thyltetradecanoyl CoA 4, had a Ki value of 0.9 mm. For practical
reasons we have now switched to (S)-ibuprofenoyl CoA as the
substrate, as it is much less expensive and quicker to assay by
HPLC.
In our previous work,[21] when using ibuprofenoyl CoA 3 as
an inhibitor against the substrate THC-CoA 2, R and S isomers
were found to have a ~4.5-fold difference in Ki. This result is
likely to have reflected a difference in inhibition early on in rac-
emization, or that the crude liver enzyme was not effective for
racemization of this substrate, as we now know that ibuprofe-
noyl CoA is a substrate for racemization when using pure
hAMACR.
The bacterial homologue (MCR) from Mycobacterium tuber-
culosis has 43% sequence identity with human AMACR, and
the MCR crystal structure shows the enzyme as an interlocked
dimer with the active site at the interface between the large
and small subunits of each monomer.[8] The active site geome-
try with bound ligands suggests a 1,1-proton transfer mecha-
nism in which the acid/base-pair residues are His126 and
Asp156.[9] The substrate binds such that the a-position sits be-
tween the active site bases and undergoes reversible deproto-
nation to from an enzyme-stabilized enolate.[19] A large methio-
nine-rich hydrophobic pocket accommodates a range of acyl
groups. Minimum substrate requirements for turnover appear
to be a linear chain length of at least eight carbon atoms and
an a-methyl group,[20] although low-level activity has recently
been demonstrated without an a-methyl group.[21]
We synthesized a new series of inhibitors (5–9, Figure 2) de-
signed to test requirements for inhibition, particularly in rela-
tion to the requirement for the a-proton, the a-methyl group,
We previously reported a series of fluorinated substrate ana-
logues as inhibitors of AMACR.[22] We demonstrated that the
free acid corresponding to the best inhibitor, 2-trifluoromethyl-
tetradecanoyl CoA 4, inhibits cancer cell proliferation in vitro
and correlates with AMACR expression levels. More recently,
Wilson et al. developed and applied a high-throughput screen
based on tritium release from [2,3-3H]pristanoyl CoA to discov-
er the first non-substrate-based inhibitors.[23] However, the best
inhibitors to date still have modest Ki values of ~1 mm, and
there is a need to discover more potent inhibitors to further
validate AMACR as a druggable target and to better under-
stand interactions with the active site to optimize inhibitors.
For initial kinetic studies, we previously used rat liver micro-
somes as a source of AMACR. Others have used AMACR puri-
fied from human[20] and rat liver, an E. coli recombinant
AMACR–MBP fusion[23] or recombinant MCR.[24] The use of vari-
ous enzyme sources and various assays and substrates by dif-
ferent groups has made comparison of kinetic data difficult. In
this study we used human AMACR (hAMACR) isolated from
HEK293 kidney cell cultures (Origene), as we have found this
enzyme to possess much higher activity than recombinant
hAMACR from E. coli (see Supporting Information S16 for
a comparison of activities for hAMACR, rAMACR, and MCR).
Figure 2. New inhibitors designed to test structural requirements for the in-
hibition of hAMACR.
and the possibility to include groups for stabilization of the
proposed enzyme-bound enolate. Although we were con-
scious that separate diastereomers of inhibitor (Æ)-5 may pos-
sess different potencies, individual isomers were not tested.
Given the similarities in Ki values obtained for ibuprofenoyl
CoA isomers,[22] inhibitor 6, which is also likely to undergo race-
mization, was tested as a mixture of isomers.
To determine the requirement for an a-proton we synthe-
sized a-fluoroibuprofenoyl CoA 5, which is non-enolizable
(Scheme 1). This was made by treatment of ibuprofen methyl
ester 11 with lithium diisopropylamide followed by fluorination
with N-fluorobenzenesulfonimide (NFSI). The a-fluoro methyl
ester 12 was then hydrolyzed prior to CoA ester formation by
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each of the inhibitors, support
a two-base mechanism, similar
to that proposed for MCR/
AMACR. We therefore designed
inhibitor 7 and its desmethyl an-
alogue 8, based on the amido-
carboxymethyldethia CoA 20,
that might mimic the AMACR-
bound enolate intermediate 21
in an AMACR substrate. In both
CS mimetics the CoA sulfur atom
is replaced by CH2, whereas in
our inhibitors 7 and 8 we re-
tained the sulfur atom to facili-
tate synthesis. Retention of the
Scheme 1. Synthesis of inhibitors (Æ)-5 and (Æ)-6. Reagents and conditions: a) MeOH, cat. H2SO4, D, 4 h, 99%;
b) nBuLi, DIPEA, À788C then NFSI, warmed to RT, 1 h, 66%; c) 5n NaOH, MeOH/THF, RT, overnight, 96%; d) CDI,
THF; e) CoA-SH·Li3, THF/H2O, RT, 24 h; f) AlCl3, ClC(O)C(O)OEt, CH2Cl2, 08C, 1 h, 71%; g) NaBH4, MeOH, 08C, 1 h,
89%; h) SOCl2, 808C, 2 h, 90%; i) HCl, H2SO4, H2O, D, 2 h, 73%; j) CDI, THF, RT, 2 h.
using the CDI coupling method to give CoA ester (Æ)-5, which
was purified by RP-HPLC.
sulfur in these thiocarbamates, relative to the CS inhibitors, is
also supported by DFT calculations at the B3LYP/6-31G** level
that indicate minimal perturbation by sulfur of the Mulliken
charges on the polarization of the amide carbonyl group.
Starting with the requisite long-chain amines 22/25, we
made the acyl imidazoles and then the more reactive methyli-
midazolium salts 24/27. Initial attempts to form the thiocarba-
mates 7/8 via the carbamoyl chlorides failed due to the sensi-
tivity of the latter under the aqueous conditions required for
reaction with CoA. However, coupling of the methylimidazoli-
um salts with CoA proceeded smoothly to give 7 and 8 in rea-
sonable yields after purification by RP-HPLC.
The a-chloroibuprofen analogue (Æ)-6, in which the a-
methyl group is replaced by an isosteric chlorine atom, was
made by starting with a Friedel–Crafts reaction of isobutylben-
zene with ethyl 2-chloro-2-oxoacetate to give the aryl oxalate
ester 15. This underwent borohydride reduction to the a-hy-
droxy ester before conversion into chloride 16 using excess
thionyl chloride. Hydrolysis of the ester was carried out with
a mixture of hydrochloric and sulfuric acids in order to avoid
displacement of the chloride, which occurred under basic con-
ditions, prior to formation and purification of the CoA ester 6.
In 1994 Druekhammer and co-workers published two very
potent inhibitors of citrate synthase (CS), carboxymethyldethia
CoA 19 (Ki =1.6 nm) and amidocarboxymethyldethia CoA 20
(Ki =28 nm), both designed to mimic acetyl CoA enolate 18
(Scheme 2).[25] CS catalyzes the formation and electrophilic
attack of this enolate on oxaloacetate to give citrate. In inhibi-
tor 19, it was proposed that the carboxylate ion mimics the
enolate oxyanion; in compound 20, the amide is isoelectronic
with the enolate. The ternary crystal structures, containing
Studies have shown that placement of an a-amide substitu-
ent on an enolizable ketone such as N-acetylamino-4-methyla-
cetophenone 29 (pKa a-proton=14.5) results in a four-unit de-
crease in the pKa value of the a-protons, compared with the
parent ketone 4-methylacetophenone 28 (pKa a-proton=18.7)
(Scheme 3).[26,27] This stabilization is proposed to result from in-
tramolecular electrostatic interactions between the enolate
anion and the partial positive charge at the amide nitrogen
atom. We therefore synthesized (R)-N-dodecanoylalanine 9
from carboxylic acid 30 using
modified
Schotten–Baumann
conditions with the addition of
DMAP. This a-amido-2-methyl
CoA ester might be expected to
behave in a similar way to an a-
amidoketone and thus facilitate
enolization within the enzyme
active site.
Inhibition studies
As a benchmark, we re-ran the
assay using inhibitor
4 with
hAMACR and obtained a Ki value
of 156 nm (Table 1). This is 6.5-
fold lower than that obtained
with the crude rat liver microso-
mal preparation as previously re-
ported by us (Ki =1 mm).[22] Such
variation is to be expected,
Scheme 2. Design and synthesis of inhibitors 7 and 8. Reagents and conditions: a) CDI, CH2Cl2, RT, overnight, 88%;
b) excess MeI, MeCN, RT, overnight, 87%; c) CoA·Li3, THF/H2O, RT, 4 h, 29%.
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hibitor does not need to be enolizable. The isosteric replace-
ment of hydrogen by fluorine clearly renders the inhibitor
a stronger binder, as it is able to outcompete the substrate
ibuprofenoyl CoA 3, perhaps due to polarization of the CÀF
bond. The isosteric replacement of the methyl group in ibu-
profen with the chlorine atom in inhibitor 6 gave a Ki value of
575 nm, showing that methyl can be replaced by a similarly
sized group and that the electronegative chlorine may assist
enolization.
More interesting was the low Ki value of 98 nm obtained for
the enolate mimic 7, the most potent AMACR inhibitor report-
ed to date. This clearly validates our proposal that the N-meth-
ylamide component of the thiocarbamate provides an isoelec-
tronic and isosteric mimic for the natural substrate and per-
haps the enzyme-bound enolate. The requirement for the a-
methyl group is clearly shown by the 10-fold less potent des-
methyl compound 8 (Ki =1 mm).
The N-acylalanyl derivative (R)-9 was the least effective of
the inhibitors tested, despite containing the a-methyl group.
This result again suggests that inhibition does not rely on the
ability of the inhibitor to enolize. It also suggests that this
compound perhaps cannot adopt the correct conformation for
the electrostatic stabilization proposed.
Scheme 3. Design and synthesis of a-amidothioester 9. Reagents and condi-
tions: a) SOCl2, CH2Cl2, reflux, overnight, 92%; b) l-Ala, K2CO3, DMAP THF,
H2O, RT, overnight, 64%; c) CDI, THF, RT, 2 h, then CoA·Li3, THF/H2O, RT, 4 h.
Table 1. Inhibition of hAMACR by compounds 4–9.
Inhibitor
Ki[a]
156Æ70 nm
Conclusions
AMACR is a potential therapeutic target that may be compli-
mentary to androgen ablation therapy. Using this enzyme and
(S)-ibuprofenoyl CoA as substrate, we have synthesized and
measured Ki values for a new series of AMACR inhibitors de-
signed to test structural requirements in the acyl component.
We have shown that inhibitors need not be enolizable and
that an a-chlorine atom can replace the a-methyl group. Most
notably, the novel N-methyl thiocarbamate 7 is the most
potent inhibitor reported thus far (Ki =98 nm); it was designed
to mimic the proposed enzyme-bound substrate enolate and
validates this strategy for designing inhibitors for other en-
zymes known to catalyze the formation and stabilization of
thioester enolates. The desmethyl analogue 8 was 10-fold less
potent, underscoring the requirement for an a-methyl (or
chloro) substituent. Future work will focus on bioisosteric re-
placement of the CoA moiety with the aim of developing
more drug-like inhibitors.
324Æ30 nm
575Æ76 nm
98Æ27 nm
1.0Æ0.26 mm
2.3Æ0.45 mm
[a] Assays were carried out using hAMACR (Origene). Inhibitor concentra-
tion was varied (50 nm to 2 mm) against a constant concentration of (2S)-
ibuprofenoyl CoA (80 mm–120 mm) as assay substrate. A final reaction
volume of 200 mL was pre-incubated at 378C for 5 min and initiated with
an appropriate amount of enzyme. Aliquots (50 mL) were quenched every
3 min with TFA (final concentration 1% v/v). The relative rate of (2S)-ibu-
profenoyl CoA conversion (50 mm–460 mm) was analyzed by HPLC (Chiral-
Pak AGP column, 100 mm potassium phosphate pH 6.1, 5% IPA) at a flow
rate of 0.7 mLminÀ1 and monitored at l 260 nm (see Supporting Informa-
tion for details).
Abbreviations
AMACR: a-methylacyl CoA racemase, CoA: coenzyme A, MBP:
maltose binding protein, MCR: AMACR homologue from Myco-
bacterium tuberculosis.
Acknowledgements
given the change in enzyme preparation and that the Ki values
reported herein are likely to be more accurate given that we
are using a pure enzyme.
We thank Dr. S. Ferdinandusse, University of Amsterdam, for
kindly supplying us with the plasmid for MBP–AMACR. We thank
Dr. David Cooper, Department of Chemistry, University of Liver-
pool, for DFT calculations for inhibitors 7 and 8. We thank the
The non-enolizable a-fluoroibuprofenoyl CoA 5 was a good
competitive inhibitor (Ki =254 nm), demonstrating that the in-
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Keywords: AMACR · coenzyme A · inhibitors · MCR · a-
methylacyl CoA racemase
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Published online on August 8, 2013
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