Rational design and synthesis of substrate-product analogue inhibitors of α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis

Article abstract of DOI:10.1039/c5cc08096g

2,2-Bis(4-isobutylphenyl)propanoyl-CoA and 2,2-bis(4-t-butylphenyl)propanoyl-CoA are rationally designed, gem-disubstituted substrate-product analogues that competitively inhibit α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis with K

Full text of DOI:10.1039/c5cc08096g

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DOI: 10.1039/C5CC08096G
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COMMUNICATION
Rational Design and Synthesis of Substrate-Product Analogue
Inhibitors of α-Methylacyl-coenzyme A Racemase from
Mycobacterium tuberculosis†
Received 00th January 20xx,
Accepted 00th January 20xx
Mohan Pal,a Mandar Khanal,a Ryan Marko,a Srinath Thirumalairajan,a
and Stephen L. Bearnea,b,*
DOI: 10.1039/x0xx00000x
www.rsc.org/chemcomm
2,2-Bis(4-i-butylphenyl)propanoyl-CoA and 2,2-bis(4-t-butylphen- with capacious and plastic active sites. To test this hypothesis,
yl)propanoyl-CoA are rationally designed, gem-disubstituted sub- we rationally designed substrate-product analogue inhibitors
strate-product analogues that competitively inhibit α-methylacyl- of α-methylacyl-coenzyme A racemase from Mycobacterium
coenzyme A racemase from Mycobacterium tuberculosis with Ki tuberculosis (EC 5.1.99.4, MtMCR), employing gem-
values of 16.9 ± 0.6 and 21 ± 4 µM, respectively, exceeding the disubstitution on the carbon-2 stereocenter of a substrate by
enzyme’s affinity for the substrate by approximately 5-fold.
the group undergoing motion (i.e., the acyl groups of MtMCR
substrates) during epimerization. Herein, we report that the
novel, gem-disubstituted, substrate-product analogues 5 (Fig.
1 and Scheme 2) competitively inhibit MtMCR with binding
affinities commensurate with the affinities of inhibitors
identified for the human homologue (vide infra), thereby
demonstrating the utility of our inhibitor design strategy.
gem-Disubstituted inhibitors are reminiscent of bisubstrate
analogue inhibitors. Such analogues combine structural fea-
tures of the two substrates required by a target enzyme,1,9 and
thereby often yield enhanced specificity and binding, the latter
arising because of entropic effects.10 Similarly, substrate-
product analogues for racemases and epimerases combine
structural attributes of two ligands and enhanced binding
affinity is anticipated due to the additional binding determin-
ants, provided that the additional steric bulk is tolerated.
The X-ray crystal structure of MR complexed with the sub-
strate analogue (S)-atrolactate revealed that the carboxylate,
hydroxyl group, and ipso carbon of the phenyl ring of the
substrate bind in the same plane (Fig. 1A),11 so that the
enantiomers of mandelate are likely bound in a mirror-image
orientation (Fig. 1B).12,13 X-ray crystallographic studies4 and
site-directed mutagenesis experiments5 suggest that the
phenyl ring moves between two sub-sites (i.e., R- and S-
pockets, Fig. 1B) during catalysis. MR is competitively
inhibited by benzilate (Ki = 0.6 mM, cf. KS = 1.0 mM; Fig. 1C),14
indicating that the active site can accommodate the two
phenyl rings of this substrate-product analogue. Thus gem-
disubstituted inhibitors such as benzilate may be designed to
present binding determinants simultaneously to the R- and S-
pockets. Indeed, we subsequently showed that 3,3,3-trifluoro-
2-hydroxy-2-(trifluoromethyl)propanoate is a highly potent
substrate-product analogue inhibitor of MR based on the
substrate 3,3,3-trifluorolactate (Ki = 27 µM).15,16
With the exception of utilizing compounds that mimic the
electronic and/or geometric characteristics of the substrate(s)
or product(s), or the intermediate(s) or transition state(s)
formed during catalysis,1-3 there are few general strategies for
designing enzyme inhibitors. Our observation that mandelate
racemase (MR, Scheme 1) is inhibited by benzilate4-6 suggested
a new strategy for designing inhibitors of racemases and
epimerases. This strategy utilizes substrate-product analogues
that incorporate structural features of both the substrate and
the product of an enzyme-catalyzed racemization or
epimerization reaction. However, such analogues proved to
be only modest inhibitors of glutamate, serine, and proline
racemases, which have compact active sites.7,8 That the inhibi-
tion of these enzymes was not as effective as in the case of MR
suggested that this inhibitor design strategy requires enzymes
Scheme 1. Similar reactions catalyzed by MR and MtMCR
R1
R1
R1
H
R3
R3
R3
H
–
O
O
O
R2
R2
R2
+
+
+
+
E–B1:
enzyme
HB2–E
E–B1H
HB2–E
E–B1H
:B2–E
B2
substrate
R1
R2
H
R3
O–
B1
MR
mandelate
OH
Lys 166 His 297
MtMCR
ibuprofenoyl-CoA
CH3
i-Bu SCoA Asp 156 His 126
a. Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax,
NS, B3H 4R2, Canada. E-mail: sbearne@dal.ca
b. Department of Chemistry, Dalhousie University, Halifax, NS,B3H 4R2, Canada.
†Electronic Supplementary Information (ESI) available: Synthetic procedures,
enzyme purification and assays, characterization of new compounds, and
inhibition studies. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 2015
Chem. Commun., 2015, 51, 1-3 | 1
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catalysis (Fig. 1E), while the CoA and α-CH3 grVoieuwpAsrticrleemOnalinine
17
DOI: 10.1039/C5CC08096G
bound at their respective binding sites. Substrate-product
analogues could therefore be designed that are gem-
disubstituted with the hydrophobic group undergoing motion.
Our synthesis of such a substrate-product analogue
inhibitor (5) is outlined in Scheme 2 (also see ESI). For the
para-substituted species (R2 = i-butyl or t-butyl), gem-
disubstitution was accomplished using the general synthesis of
2,2-diarylpropanoates described by Patai and Dayagi.26 Ethyl
pyruvate (1) was reacted with either i- or t-butylbenzene in
concentrated sulfuric acid at –15 °C to afford 2c and 2d,
respectively, after purification using silica gel chromatography.
Vigorous stirring was required since the immiscible mixture of
i- or t-butylbenzene and the ethyl pyruvate/sulfuric acid
solution became quite viscous as the reaction proceeded.
Conversion to 3c and 3d was achieved by hydrolysis using 5 M
KOH in hexanes/MeOH. Finally, thioesters 5a – d were
prepared by activating 3a – d with CDI to form 4a – d in dry
DCM, followed by reaction with the trilithium salt of CoA in an
aqueous bicarbonate solution.18 The resulting product was
purified using semi-preparative solid-phase extraction to
afford thioesters 5a – d in yields of 26 – 48%.
Using a continuous circular dichroism (CD)-based assay and
(2S)-ibuprofenoyl-CoA as the substrate,27 we examined the
inhibitory effect of 3a – d and 5a – d on MtMCR activity.
Recombinant MtMCR was overproduced as a fusion protein
bearing an N-terminal hexahistidine tag and purified using
nickel ion affinity chromatography (see ESI). Thioesters 5a – d
are competitive inhibitors of MtMCR (Table 1), with 5c and 5d
being the most effective inhibitors (Fig. 2) and binding with
affinities ~5-fold greater than that exhibited for the substrate
(2S)-ibuprofenoyl-CoA (assuming Km ≈ KS). Clearly, MtMCR can
simultaneously accommodate the two aryl groups of 5a – d.
The geminal phenyl groups of 5a and 5b afforded a binding
affinity that was weaker than that observed for the substrate
by ~3-fold. The similar binding affinity for these two inhibitors
suggests that the α-CH3 group in 5b does not contribute
significantly to the binding affinity, perhaps because of the less
efficient binding of the phenyl groups relative to larger
hydrophobic acyl groups. However, Lloyd and co-workers
found that (S)-2-methyldecanoyl-CoA and decanoyl-CoA
exhibited Km values of 614 and 225 µM, respectively, also
suggesting the methyl group does not contribute to ground
Fig. 1. Binding of ligands to MR and MtMCR. (A) Superposition of the substrate
analogue (S)-atrolactate (PDB 1MDR)11 and the intermediate analogue BzH (PDB 3UXK)4
bound at the active site of MR. The active site Mg2+ is shown in space-filling
representation. (B) Mirror-image binding of substrate enantiomers (superposed)
results in three substituents on the α-carbon lying in the same plane (magenta dotted
line) with the α-hydrogens being antipodal.12,13 Aryl rings attached to the α-carbon
atom project linearly to define the R- and S-pockets of MR for binding the phenyl ring
of R- and S-mandelate (where R1 = OH, R2 = H, and R3 = O–), respectively. (C) Benzilate
with its two phenyl groups binding at the active site of the C92S/C264S/K166C triple
mutant of MR.15 (D) gem-Diaryl substrate-product analogue inhibitors of MtMCR. (E)
Stereoview showing the superposition of the substrates (2S)- and (2R)-ibuprofenoyl-
CoA bound at the active site of MtMCR (PDB 2GCE).17 The CoA moiety and α-CH3
remain fixed, while the 2-aryl moiety moves over a Met-rich surface (purple) during
catalysis.
To test our hypothesis that enzymes with capacious active
sites might be more amenable to inhibition by substrate-
product analogues, we applied our inhibitor design strategy to
MtMCR. MtMCR is a cofactor-independent enzyme that
catalyzes the 1,1-proton transfer reaction17,18 which effects the
reversible epimerization of the CoA-thioesters derived from a
variety of (2R)- and (2S)-methyl branched fatty acids,19
a
Scheme 2. Synthesis of the gem-diaryl substrate-product analogues.
R2
R2
number of C27 bile acid intermediates,20 and 2-arylpropionic
acids (e.g., ibuprofen, Scheme 1).21,22 The MtMCR-catalyzed
epimerization of ibuprofenoyl-CoA is very similar to the
racemization of mandelate catalyzed by MR (Scheme 1).
Studies using 2-2H-labeled or 2-3H-labeled substrates17-19,22-24
and X-ray crystallographic studies on MtMCR indicate that the
enzyme is a two-base epimerase and epimerization proceeds
via an enol/enolate intermediate.17,23,25 Structural studies
conducted by Wierenga and co-workers23 on MtMCR com-
plexed with (R,S)-ibuprofenoyl-CoA, (2S)-methylmyristoyl-CoA,
and (2R)-methylmyristoyl-CoA revealed that the nonpolar acyl
group moves through a Met-rich hydrophobic pocket during
i- or t-BuC6H5,
O
KOH in MeOH (5.0 M) / hexane
reflux, 16 h
H2SO4 (conc.)
O
–15 °C, 2 h
O
O
OH
R1
R1
O
O
R2
R2
1
2c, 61%; 2d, 87%
3c, 74%; 3d, 79%
R2
R2
R2
CoA-SH / NaHCO3 (0.1 M) / THF
CDI, DCM
rt, 2 h
N
rt, 24 h, SPE
OH
N
SCoA
R1
R1
R1
O
O
O
R2
R2
R2
3 (a – d)
4a, 83%; 4b, 78%;
4c, 91%; 4d, 95%
5a, 42%; 5b, 48%;
5c, 26%; 5d, 43%
a, R1 = R2 = H; b, R1 = CH3, R2 = H; c, R1 = CH3, R2 = i-butyl; d, R1 = CH3, R2 = t-butyl
2 | Chem. Commun., 2015, 51, 1-3
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state binding by human α-methylacyl-CoA racemase
(AMACR).28 The addition of the i- or t-butyl groups in 5c or 5d,
respectively, enhanced the inhibition ~20-fold, consistent with
the presence of additional hydrophobic interactions within the
large hydrophobic cavity that comprises the binding sites for
the acyl moiety of substrate epimers.17 Comparison of the IC50
values for the inhibition of MtMCR by CoA and the free acids
3a – d (Table 1) reveals that the presence of both the acyl and
CoA moieties in the inhibitor are required for better binding.
The surprising exception is 3c, which was a reasonably
effective inhibitor, binding with an affinity similar to that of the
substrate. This observation suggests that inhibitors might be
developed that either lack the CoA moiety or have only part of
the CoA structure.
View Article Online
DOI: 10.1039/C5CC08096G
Since 5a bears an α-proton similar to the substrate,
MtMCR could potentially catalyze exchange of this proton with
deuterium from solvent D2O. Using a concentration of MtMCR
exceeding that typically employed under assay conditions by at
least 1000-fold, and monitoring the signals arising from the α-
proton and phenyl protons of 5a by 1H NMR spectroscopy, no
MtMCR-catalyzed exchange was observed over 24 h (see ESI).
Thus, the bound orientation of 5a places the α-proton in a
location where it is not amenable to exchange. Such a binding
orientation is consistent with the two phenyl rings occupying
the binding sites for the 4-i-butylphenyl groups of the
enantiomers of ibuprofenoyl-CoA (Fig. 1E) with the α-proton
located in the binding pocket for the α-methyl group where it
is not accessible to the Brønsted acid-base catalysts.
Fig 2. Inhibition of MtMCR by 5c and 5d. (A) and (C) Representative Lineweaver-Burk
plots showing the competitive inhibition of MtMCR by 5c and 5d, respectively.
Concentrations (µM): 5c: 0 (), 12 (), 24 (), 36 (); 5d: 0 (), 16 (), 32 (), 48
(); (2S)-ibuprofenoyl-CoA: 40, 80, 160, 360, 600, and 800; and MtMCR: 0.18 nM (7.5
ng/mL). (B) and (D) Representative plots of the apparent Km/Vmax values (determined
from direct fits of Eqn. 1S to initial velocity data (i.e., Michaelis-Menten plots; see Figs.
56S and 57S in the ESI)) vs. concentrations of 5c and 5d, respectively.30 Ki values
(determined in triplicate) are given in Table 1.
MtMCR shares
specificity with its homologue human AMACR (43% amino acid
a similar mechanism and substrate
sequence identity with MtMCR).29 Elevated levels of AMACR
have been associated with a variety of cancers,31 including
prostate cancer (PCa)32,33 for which the enzyme serves as a
TABLE 1. Inhibition of MtMCR
compound
inhibition results
IC50, µMa
biomarker.33-35
Knockdown of AMACR gene expression
Ki, µMb
ndc
suppresses the growth of several PCa cell lines suggesting that
AMACR may be a therapeutic target for PCa.29,36-39 Several
efforts to develop inhibitors of AMACR have been reported.
Analogues of the natural substrate branched chain α-
methylacyl-CoA thioesters possessing one or more β-fluorine
atoms were synthesized as potential suicide substrates.40
These compounds, however, did not covalently modify the
enzyme as anticipated but acted as competitive inhibitors with
Ki values of 0.9 – 20 µM (Km/Ki = 27–1.2). Morgenroth et al.41
synthesized 2-methylenacyl-CoA thioesters as AMACR binding
ligands; e.g., (13E/12)-13-iodo-2-methylentridec-12-enoyl-CoA
acted as a competitive inhibitor with a Ki value of 19 µM (Km/Ki
= 1.5). One of the most potent inhibitors is N-dodecyl-N-
methylcarbamoyl-CoA with a Ki value of 98 nM (Km/Ki ≈ 969).42
Because these inhibitors closely resemble the structure of the
substrates, one might anticipate that they would lack
specificity. A high-throughput screen of a 5,000-compound
library for inhibitors of AMACR activity led to the identification
of several compounds that were not structural mimics of the
substrates but inhibited AMACR with IC50 values between 0.8
and 85 µM (Km/Ki ≈ 107 & 1.0, respectively).29 Some of these
compounds were irreversible inactivators of AMACR.
coenzyme A
(S)-ibuprofend
1.2 (± 0.2) × 103
39 (± 5) × 103
–
nd
(2S)-ibuprofenoyl-CoA
diphenylacetic acid (3a)d
diphenylacetyl-CoA (5a)
2,2-diphenylpropanoic acid
(3b)d
106 ± 15 (Km)
nd
358 ± 39
> 6.3 × 103e
1.1 (± 0.3) × 103
42 (± 5) × 103
nd
343 ± 72
102 ± 9
16.9 ± 0.6
nd
2,2-diphenylpropanoyl-CoA
(5b)
610 ± 73
2,2-bis(4-i-butylphenyl)-
propanoic acid (3c)d
2,2-bis(4-i-butylphenyl)-
propanoyl-CoA (5c)
2,2-bis(4-t-butylphenyl)-
propanoic acid (3d)d
2,2-bis(4-t-butylphenyl)-
propanoyl-CoA (5d)
316 ± 37
39.5 ± 0.9
f
–
44 ± 8
21 ± 4
aIC50 values determined with [(2S)-ibuprofenoyl-CoA] = 100 µM and [MtMCR] =
0.18 nM. bValues reported are the averages of three independent trials and the
errors are the standard deviations. cNot determined. dDetermined in the
presence of 20% DMSO. eValue estimated assuming competitive inhibition with
only 24 (± 3)% inhibition observed and [3a] = 20 mM. fOnly 15 (± 0.5)% inhibition
observed at [3d] = 480 µM; however, it is likely that weak inhibition arises from
solubility problems (see Fig. 50S in the ESI).
In summary, we have demonstrated that utilizing gem-
disubstituted substrate-product analogues furnishes an
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18 D. J. Darley, D. S. Butler, S. J. Prideaux, T. W. Thornton, A. D. Wilson,
effective strategy for designing inhibitors of racemases and
epimerases with capacious active sites. This rational approach
offers an alternative to the well-established strategy of using
β-halo compounds43,44 to generate suicide substrates of
racemases and epimerases catalyzing formation of en-
ol/enolate intermediates, or to the strategies of designing an
inhibitor based on the structure of the substrate(s), product(s),
intermediate(s), or transition state(s) of enzyme-catalyzed
reactions. The inhibitors described in the present work may
furnish tools for studying cholesterol ester metabolism in M.
tuberculosis,45 or lead compounds for the development of
AMACR inhibitors directed against PCa. The additional acyl-
like chain on such gem-disubstituted inhibitors may endow the
inhibitor with specificity, obviating its recognition by other
fatty acyl-CoA-metabolizing enzymes. Of course, in vivo stud-
ies will require structural alterations and prodrug strategies to
circumvent hydrolysis of the thioester and the lack of cell
permeability arising from the CoA moiety, respectively.46,47
Alternatively, the reasonable binding affinity exhibited by gem-
disubstituted diaryl acids such as 3c may offer a means of
overcoming the need for incorporation of the CoA moiety.
This work was supported by a Discovery Grant from the
Natural Sciences and Engineering Research Council (NSERC) of
Canada (S.L.B.), a grant from the Prostate Cancer Research
Foundation (S.L.B.), and a Cancer Research Training Program
studentship award (M.K.) from the Beatrice Hunter Cancer
Research Institute. We thank Dr. Mike Lumsden (NMR-3) for
his assistance in conducting NMR experiments.
T. J. Woodman, M. D. Threadgill and M. D. Lloyd, Org.VBieiowmArotilc.leChOenmlin.e,
DOI: 10.1039/C5CC08096G
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DOI: 10.1039/C5CC08096G
gem-Disubstituted substrate-product analogues competitively
inhibit α-methylacyl-coenzyme A racemase from Mycobacterium
tuberculosis, binding with affinities exceeding that of the substrate
by ~5-fold.
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