Chiral inversion of 2-arylpropionyl-CoA esters by human α-methylacyl-CoA racemase 1A (P504S) - A potential mechanism for the anti-cancer effects of ibuprofen

Article abstract of DOI:10.1039/c1cc10763a

Metabolic chiral inversion of 2-arylpropanoic acids (2-APAs; 'profens'), such as ibuprofen, is important for pharmacological activity. Several 2-APA-CoA esters were good racemisation substrates for human AMACR 1A, suggesting a common chiral inversion pathway for all 2-APAs and an additional mechanism for their anti-cancer properties.

Full text of DOI:10.1039/c1cc10763a

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COMMUNICATION
Chiral inversion of 2-arylpropionyl-CoA esters by human
a-methylacyl-CoA racemase 1A (P504S)—a potential
mechanism for the anti-cancer effects of ibuprofenw
Timothy J. Woodman,* Pauline J. Wood, Andrew S. Thompson, Thomas J. Hutchings,
Georgina R. Steel, Ping Jiao, Michael D. Threadgill and Matthew D. Lloyd*
Received 9th February 2011, Accepted 26th April 2011
DOI: 10.1039/c1cc10763a
Metabolic chiral inversion of 2-arylpropanoic acids (2-APAs;z
‘profens’), such as ibuprofen, is important for pharmacological
activity. Several 2-APA-CoA esters were good racemisation
substrates for human AMACR 1A, suggesting a common chiral
inversion pathway for all 2-APAs and an additional mechanism
for their anti-cancer properties.
Although the acyl groups of AMACR substrates are known
to be structurally diverse,16 no systematic study of substrate
structure has been reported. This communication reports the
first such systematic study and shows that all of the
tested 2-APA-CoA esters are good substrates. High doses of
ibuprofen reduce the risk of developing prostate cancer26 and
these results suggest that the interaction of 2-APA-CoA esters
with AMACR may contribute to this chemopreventive effect.
Initially, a number of 2-APA-CoA esters were synthesized
for evaluation. Ibuprofenoyl-CoA 2 was chosen, as a known
substrate for native rat AMACR14 and the homologous
MCR from M. tuberculosis.21 ꢀ-Fenoprofenoyl-CoA 7,
ꢀ-flurbiprofenoyl-CoA 8, and S-ketoprofenoyl-CoA 9 were
synthesized, as these 2-APAs undergo chiral inversion
in vivo.2,3,8,10 S-Naproxenoyl-CoA 10 was also synthesized,
as it is structurally related but its chiral inversion in vivo is
reported to be extremely limited.11 The 2-APAs reacted
with carbonyl diimidazole20,22 to form the acyl-imidazole
intermediates, the presence of which was confirmed by
1H NMR. These intermediates were immediately treated with
CoA-SH, to afford the desired esters on B10 mg scales (ESI,w
Scheme S1). S-2-Methyldecanoyl-CoA 11 was also synthesized
as a known substrate of human recombinant AMACR 1A.17,23
These acyl-CoA esters were incubated at ca. 400 mM with
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely
used in human and veterinary medicine for their analgesic and
anti-inflammatory properties.1 They inhibit cyclo-oxygenase-1
and -2 (COX-1 and -2), reducing production of prostaglandins
and other inflammatory mediators. COX-1 is present in all
tissues, while COX-2 levels are normally low but increase in
response to tissue injury.1 Common examples include aspirin,
indomethacin, diclofenac and the 2-arylpropanoic acids
(2-APAs; a.k.a. profens), including ibuprofen 1. 2-APA drugs
contain a chiral centre and their S-enantiomers potently
inhibit COX activity, while the R-enantiomers are much less
active.2,3 These drugs are often given as racemic mixtures.
Ibuprofen 14–7 fenoprofen 3,3,8 flobufen,9 flurbiprofen 4,10
ketoprofen 52 and naproxen 611 undergo uni-directional
conversion to the single S-enantiomer in vivo. R-Ibuprofen 1
is converted to R-ibuprofenoyl-CoA 212,13 and chirally
inverted by an epimerase.7,14,15 Finally, hydrolysis of the
CoA ester gives S-ibuprofen 1. Other 2-APAs are assumed
to be inverted by the same enzymes as for ibuprofen 1, but this
has not been proven.
2
human AMACR 1A in the presence of H2O for 416 h and
2
activity was monitored by exchange of the a-proton for H by
1H NMR.17 Exchange of the a-proton for 2H at ca. 70–90%
conversion was observed for all 2-APA-CoAs. The signal for the
methyl group on C-2 appears as an asymmetrical doublet (Fig. 1),
Cloning of ‘ibuprofenoyl-CoA epimerase’ showed that it
was identical to a-methylacyl-CoA racemase (AMACR),
which also catalyses chiral inversion of other 2-methyl-fatty
acyl-CoA esters.16 The reaction involves removal of the substrate
a-proton followed by non-stereoselective reprotonation.17
Protein levels and activity are increased in prostate, some
colon and other cancers,16 and AMACR has attracted interest
as a novel marker18 and drug target.19,20
2
composed of the unresolved triplet of the H-labelled product
superimposed on the doublet of unreacted substrate. There was
also a reduction in the intensity of the signal for 2-H. The known
substrate, S-2-methyldecanoyl-CoA 11, was converted 495%
under the same conditions.17 No exchange was observed in
negative controls containing heat-inactivated enzyme.
Experiments were then performed to determine whether chiral
inversion of the 2-APA-CoA had also occurred in addition to
a-proton exchange. Thus, ca. 10 mg of S-ketoprofenoyl-CoA 9
and S-naproxenoyl-CoA 10 were separately incubated with
AMACR in 1H2O-containing buffer. The product CoA
esters were hydrolysed and the 2-APAs were converted to the
Medicinal Chemistry, Department of Pharmacy & Pharmacology,
University of Bath, Claverton Down, Bath BA2 7AY, UK.
E-mail: M.D.Lloyd@bath.ac.uk; Fax: +44-1225-386114;
Tel: +44-1225-386786
w Electronic supplementary information (ESI) available: Synthetic
methods; kinetic plots. See DOI: 10.1039/c1cc10763a
c
7332 Chem. Commun., 2011, 47, 7332–7334
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Table 1 Substrate kinetic parameters for AMACR 1A. The para-
meters shown are derived using the Direct Linear Plot24,25
Fig. 1 2H-Exchange of ꢀ-flurbiprofenoyl-CoA 8 by AMACR 1A,
showing change in methyl group splitting at d 1.45 p.p.m. (A)
Heat-inactivated enzyme; (B) Active enzyme.
diastereomeric N-(S-1-phenylethyl)amides 12 and 13. The
1H NMR spectra showed two overlapping series of peaks
corresponding to the 2-H, the 2-Me, the NCHMe and the amide
NH (Fig. 2B), showing that chiral inversion had occurred. The
ratio of the two epimeric products in both cases was close to 1 : 1,
consistent with observations for 2-methyldecanoyl-CoA 11
(Keq = 1.09 ꢀ 0.14; S/R).17 Similar treatment of the 2-APAs
without exposure to the enzyme gave single sets of signals,
confirming that the configuration of the chiral centre was
maintained during derivatization (Fig. 2A). Exchange of the
a-proton is obligatory for chiral inversion and, since no exchange
is observed with heat-inactivated enzyme, this demonstrates that
chiral inversion is enzyme-catalysed.
Vmax
kcat/Km
Substrate Km (mM) (nmol minꢁ1 mgꢁ1
)
kcat (sꢁ1
)
(Mꢁ1 sꢁ1
)
2
7
74
2.3
9.36
4.16
5.12
19.2
12.2
39.3
0.0073
0.0033
0.0040
0.0150
0.0095
0.0310
99
1426
156
290
140
8
9
26
52
10
11
68
277
112
due to the very low estimated Km value (2.3 mM cf. 26–74 mM for
other substrates). Examination of the kinetic plots for 7 showed
near-saturation even at the lowest substrate concentrations and it
is possible that the Km value is significantly lower than estimated
by this assay. It was not possible to use significantly lower
substrate concentrations, as a tight binding situation would occur,
i.e. the concentrations of substrate and enzyme would be similar.
Interestingly, the Km for 7 is of similar magnitude to the Ki values
for the best inhibitors of the rat enzyme (ca. 0.9 mM).20
Kinetic parameters for the 2-APA-CoA and S-2-methyl-
decanoyl-CoA esters were then determined (Table 1). All the
2-APA-CoA esters were good substrates of human AMACR 1A.
Flurbiprofenoyl-CoA 8, ibuprofenoyl-CoA 2 and naproxenoyl-
CoA 10 were converted about as efficiently as S-2-methyl-
decanoyl-CoA 11 (as judged by kcat/Km). The results also
suggest that the limited chiral inversion of flurbiprofen10
in humans is due to low conversion to its CoA ester 8
in vivo rather than it not being a chiral inversion substrate.
Ketoprofenoyl-CoA 9 was converted ca. twice as efficiently
as S-2-methyldecanoyl-CoA 11. Fenoprofenoyl-CoA 7 was by
Literature inhibitors of AMACR20 increase the acidity of
the a-proton using electron-withdrawing groups, to facilitate
deprotonation and mimic the deprotonated intermediate.
The aliphatic side-chain of S-2-methyldecanoyl-CoA 11 is
electron-donating and replacement with any aryl side-chain
will make the a-proton more acidic. 2-APA-CoA substrates
are expected to be more tightly bound based on this argument
and lower Km values are observed. Substrates with aromatic
rings substituted with electron-withdrawing groups (e.g. 8) are
expected to have particularly acidic a-protons and this may
explain its tight binding.
far the best substrate (kcat/Km = ca. 1400 Mꢁ1 ꢁ1), largely
s
There is no reported structure for AMACR, but the
structure of the M. tuberculosis homologue with a number of
acyl-CoA has been reported.21 These structures show the CoA
moiety locked into the active site by a network of hydrogen
bonds and ionic interactions. The carboxylate thioester and
methyl groups of the substrate are close to the catalytic
residues. The substrate side-chains are directed to the methionine-
rich surface at the active site entrance. AMACR is expected to
bind substrates in broadly similar manner and this explains the
diverse structures of known substrates.16 However, these factors
do not explain the low Km value of fenoprofenoyl-CoA 7.
Post facto molecular modelling studies were undertaken to
try to rationalize the binding of 2-APA-CoAs to AMACR.
Fig. 2 Chiral inversion of naproxenoyl-CoA 10 by AMACR 1A. The
spectra show the signals for the a-proton of 1-phenylethylamine and
the NH of the amide linkage. (A) Without exposure to enzyme; (B)
Following exposure to AMACR 1A. The signals appear as two
superimposed doublets at 5.55 p.p.m. and two superimposed quintets
at 5.10 p.p.m.
c
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l, R. Kral, L. Skalova, B. Szotakova, F. Trejtnar and
´ ´ ´ ´ ´ ´
This work was supported by Cancer Research UK. Part of
this work was performed for a Nuffield Summer Student
Bursary (TJH), a Nuffield Science Bursary (GRS) and a
Bath-Shandong Pharmacy
& Pharmacology Scholarship
project (PJ). This work is dedicated to Mr David K. Lloyd
(1936 to 2010). TJW, PJW, AST, MDT and MDL are
members of the Cancer Research @ Bath network.
Notes and references
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z Abbreviations used: AMACR 1A, a-methylacyl-CoA racemase,
splice variant 1A; 2-APA, 2-arylpropionic acid; CoA, coenzyme A;
COX, cyclooxygenase; MCR, a-methylacyl-CoA racemase
(M. tuberculosis).
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and J. R. Vane, Proc. Natl. Acad. Sci. U. S. A., 1993, 90,
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7334 Chem. Commun., 2011, 47, 7332–7334
This journal is The Royal Society of Chemistry 2011