Rational Design, Synthesis, and Preliminary Structure-Activity Relationships of α-Substituted-2-Phenylcyclopropane Carboxylic Acids as Inhibitors of Salmonella typhimurium O-Acetylserine Sulfhydrylase

Article abstract of DOI:10.1021/acs.jmedchem.5b01775

Cysteine is a building block for several biomolecules that are crucial for living organisms. The last step of cysteine biosynthesis is catalyzed by O-acetylserine sulfydrylase (OASS), a highly conserved pyridoxal 5′-phosphate (PLP)-dependent enzyme, present in different isoforms in bacteria, plants, and nematodes, but absent in mammals. Beside the biosynthesis of cysteine, OASS exerts a series of moonlighting activities in bacteria, such as transcriptional regulation, contact-dependent growth inhibition, swarming motility, and induction of antibiotic resistance. Therefore, the discovery of molecules capable of inhibiting OASS would be a valuable tool to unravel how this protein affects the physiology of unicellular organisms. As a continuation of our efforts toward the synthesis of OASS inhibitors, in this work we have used a combination of computational and spectroscopic approaches to rationally design, synthesize, and test a series of substituted 2-phenylcyclopropane carboxylic acids that bind to the two S. typhymurium OASS isoforms at nanomolar concentrations.

Full text of DOI:10.1021/acs.jmedchem.5b01775

Article
pubs.acs.org/jmc
Rational Design, Synthesis, and Preliminary Structure−Activity
Relationships of α‑Substituted-2-Phenylcyclopropane Carboxylic
Acids as Inhibitors of Salmonella typhimurium O‑Acetylserine
Sulfhydrylase
Marco Pieroni,†,■ Giannamaria Annunziato,†,■ Claudia Beato,† Randy Wouters,†,¶ Roberto Benoni,‡
Barbara Campanini,*,‡ Thelma A. Pertinhez,§,△ Stefano Bettati,⊥,∥ Andrea Mozzarelli,‡,∥,#
and Gabriele Costantino*,†
†P4T group, ‡Laboratory of Biochemistry and Molecular Biology, Department of Pharmacy, and §Centro Interdipartimentale Misure
(CIM)’G. Casnati’, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy
⊥Department of Neurosciences, University of Parma, Via Volturno, 39, 43125 Parma, Italy
∥National Institute of Biostructures and Biosystems, Viale delle Medaglie d’Oro 305, 00136 Rome, Italy
#Institute of Biophysics, CNR, /o Area di Ricerca San Cataldo, Via G. Moruzzi N° 1, 56124 Pisa, Italy
S
* Supporting Information
ABSTRACT: Cysteine is a building block for several biomolecules that are crucial for living organisms. The last step of cysteine
biosynthesis is catalyzed by O-acetylserine sulfydrylase (OASS), a highly conserved pyridoxal 5′-phosphate (PLP)-dependent
enzyme, present in different isoforms in bacteria, plants, and nematodes, but absent in mammals. Beside the biosynthesis of
cysteine, OASS exerts a series of “moonlighting” activities in bacteria, such as transcriptional regulation, contact-dependent
growth inhibition, swarming motility, and induction of antibiotic resistance. Therefore, the discovery of molecules capable of
inhibiting OASS would be a valuable tool to unravel how this protein affects the physiology of unicellular organisms. As a
continuation of our efforts toward the synthesis of OASS inhibitors, in this work we have used a combination of computational
and spectroscopic approaches to rationally design, synthesize, and test a series of substituted 2-phenylcyclopropane carboxylic
acids that bind to the two S. typhymurium OASS isoforms at nanomolar concentrations.
INTRODUCTION
■
Cysteine is the amino acid precursor of all sulfur-containing
biomolecules, including methionine, CoA, biotin, Fe−S
clusters, penicillin, and glutathione, that are vital for the
majority of living organisms.1 Mammals lack the biosynthetic
machinery that leads to the de novo synthesis of cysteine from
inorganic sulfur, whereas bacteria and plants possess redundant
and highly conserved enzymes enabling cysteine biosynthesis
(Figure 1A). In enteric bacteria the last two steps of cysteine
biosynthesis (Figure 1B) are catalyzed by serine acetyltransfer-
ase (SAT) and O-acetylserine sulfhydrylase (OASS).2
Figure 1. (A) Uptake and reduction of inorganic sulfur by
microorganisms. (B) Cysteine biosynthesis from serine.
The two enzymes form the so-called “cysteine synthase
complex” that is stabilized by the interaction of the C-terminal
portion of SAT with the substrate binding pocket of OASS.
The first identified OASS isoform, named OASS-A, was
originally isolated and characterized from Salmonella typhimu-
Received: November 13, 2015
© XXXX American Chemical Society
A
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rium. Later, it was noticed that many organisms possess two
isoforms of the enzyme, that show homology although with
peculiar functional and structural properties: O-acetylserine
sulfhydrylase (OASS-A, encoded by cysK)3 and O-phosphoser-
ine sulfhydrylase (OASS-B, encoded by cysM). In S.
typhimurium, both isozymes use O-acetylserine as first substrate
and bisulfide as sulfur source. In addition to bisulfide, OASS-B
can also use thiosulfate. OASS-A and OASS-B are differently
expressed under aerobic/anaerobic conditions2,4 but their
specific role is still partly unclear. Both enzymes are pyridoxal
5′-phosphate (PLP)-dependent and share a similar Bi−Bi ping-
pong type reaction mechanism. In the first half-reaction, the β-
elimination of the β-substituted L-serine external aldimine takes
place with the accumulation of the α-aminoacrylate inter-
mediate Schiff base. In the second half-reaction, the α-
aminoacrylate is attacked by sulfide or thiosulfate with
formation of cysteine.5 The three-dimensional structures of
OASS-A and OASS-B6−20 invariably show a dimeric assembly
with each subunit organized into two domains and the active
sites, each carrying a molecule of PLP, facing the solvent
(Figure 2A). The two domains are flexible and close upon
coli,27 from swarming motility in Proteus mirabilis28 and S.
typhimurium29 to antibiotic resistance.30,31
Considering the multifaceted functions of this enzyme, and
considering that the biosynthesis of cysteine is not shut down
unless both enzymes are inhibited,4 the discovery of molecules
that interact with both isoforms of OASS and inhibit their
activity would be a valuable tool to investigate the physiological
role of these enzymes in unicellular organisms.32 Since SAT
competitively inhibits OASS-A, the structural features of OASS-
SAT interaction have paved the way to the rational design of
the first sulfhydrylase inhibitors. Starting from the structure of
the physiological inhibitor of OASS, Salsi et al. have analyzed at
the molecular level the interaction of SAT C-terminal peptide
with OASS active site33 and have studied the kinetics of SAT-
OASS complex formation from Haemophilus influenzae.34 These
studies supported the view that the last five amino acids of the
carboxy-terminal portion of HiSAT (MNLNI) (Figure 3) are
Figure 3. Structure of the carboxy-terminal portion of HiSAT in the
active site of HiOASS as inferred from the three-dimensional structure
of the complex between HiOASS and the MNLNI peptide.6
Figure 2. (A) Homodimer of OASS-A taken from 1OAS crystal
structure; the two monomers are highlighted with different colors. (B)
Superposition of OASS-A (1OAS chain A, in light gray) and OASS-B
(2JC3 chain A, in blue). The major structural difference concerning
the loops above the binding site is highlighted; the sequences of the
loops of the two isoforms, aligned as in the superposition of the two
crystal structures, are reported at the bottom of the figure. (C) Zoom
on the loops highlighted in panel B: OASS-A in light gray with
residues labeled in italics; OASS-B in blue with residues labeled in
bold. The structure-based amino acid sequence alignment of the loops
of the two isoforms is reported at the bottom of the figure.
predominantly responsible for the interaction with OASS, and,
in particular, the C-terminal isoleucine is essential for this
interaction.6,33,35 Indeed, although the primary structure of
SAT C-terminal peptide is species-specific in bacteria, it
invariantly carries a terminal isoleucine.36
A previous attempt to identify OASS inhibitors was carried
out by exploiting a small library of pentapeptides, leading to the
identification of molecules with affinities in the micromolar
range.33,37 In order to circumvent the chemical liabilities of
peptides such as low stability and low bioavailability, while
maintaining the enzyme inhibition, we carried out an in silico
campaign21 and also prepared a number of small molecules
inspired by the structures of these peptides.38 We reasoned that
the acid moiety of the isoleucine was crucial for the activity,
since it establishes a dense network of hydrogen bonds with the
backbone of several residues belonging to the catalytic pocket,
namely, Asn72, Thr73, Thr69, and Gln143 (Figure 4).
An alkyl moiety mimicking the side chain of isoleucine was
attached to the carboxylic acid moiety through a suitable spacer
that could maintain the two anchoring arms of isoleucine into
the favorable trans configuration. For this purpose, a cyclo-
propane scaffold was selected. Despite the good affinity toward
HiOASS-A, the majority of the prepared compounds suffered
from several weaknesses, such as poor chemical feasibility and
stability. Therefore, we have initiated a program directed to the
design and synthesis of novel substituted cyclopropane-
carboxylic acids, aimed at improving their stability and their
potency toward both the OASS-A and OASS-B isoforms. The
substrate binding, thus allowing diffusion into the active site
only of small molecules and protecting the highly reactive α-
aminoacrylate intermediate from attack by water or other
nucleophiles. OASS-A and OASS-B exhibit 40% sequence
identity and 56% sequence similarity21 and a very similar
structural fold, belonging to the fold type II family of the PLP-
dependent enzymes22,23 (Figure 2B). However, major differ-
ences can be detected in the region of the flexible loop located
at the entrance of the active site (Figure 2C). In particular, in
the B isoform the motif Gly230-Ala231-Gly232 is replaced by
Arg210-Arg211-Trp212 (S. typhimurium numbering scheme),
increasing the overall polarity and causing a variation in the size
of the binding site.
Since its discovery, OASS was thought to be involved only in
the biosynthesis of cysteine, but recently a growing number of
additional “moonlighting” activities have been identified.24
Indeed, OASS-A activities span from transcriptional regulation
in some bacteria and nematodes25,26 to toxin activation in E.
B
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Figure 4. (A) Rational design of cyclopropane carboxylic acids. (B) Expansion of the series from compound 17.
results are reported herein and, to the best of our knowledge,
some of the newly prepared compounds represent the most
potent binders of the B isoform reported so far. Furthermore,
insights into the mechanism of ligand/protein interaction were
obtained with the integration of computational and spectro-
scopic methods, providing the basis for further chemical
optimization of these chemical tools.
Scheme 1. Preparation of Compounds 12a−c, 13a−c, 14a−c,
a,b
15a−c, and 16a
CHEMISTRY
■
The 2-phenylcyclopropane carboxylic acids (12a−c, 13a−c,
14a−c, 15a−c, and 16a) were prepared through a straightfor-
ward protocol already reported,39−41 reacting the appropriate
styrene oxides a−c with the suitable phosphonates in
dimethoxyethane at 130 °C using butyllithium as deprotonating
agent. This procedure has the advantage to direct in the trans
position the carboxylic moiety and the phenyl ring, with
retention of configuration. This allows synthesizing the
enantiopure 2-phenylcyclopropane carboxylic acids simply
starting from the commercially available enantiopure styrene
oxides. Subsequent basic hydrolysis with LiOH·H2O under
MW irradiation afforded the title compounds in good overall
yields (Scheme 1). When not commercially available,
phosphonates were obtained by reacting triethyl phosphonoa-
cetate with the proper alkyl bromide, in the presence of NaH as
the base, from 0 °C to room temperature.42,43 Compound 18
was commercially available.
a
Reagents and conditions: (a) R-Br, NaH, DME, 60 °C, 4 h, 54−75%;
(b) n-BuLi, DME, 90 °C, 18 h, 67−76%; (c) LiOH·H2O, THF/
b
MeOH/H2O (3:1:1); 10 min, MW, 86−98%. For details see Table 1.
solution after a few days, hampering sometimes the
reproducibility of experiments. In addition, since both the
isoforms of OASS must be inhibited to obtain cysteine
auxotrophs,4 the lack of activity toward HiOASS-B (unpub-
lished observation) was considered furtherly detrimental for the
development of these compounds. Taking these considerations
into account, we thought that a feasible starting point for
further modification of 17 was to embody the vinyl moiety into
a phenyl ring, maintaining the trans stereorelationship with the
carboxylic moiety. Besides providing an improved synthetic
feasibility and stability, the insertion of a phenyl ring
significantly expands the scope for further decoration and
functionalization aimed at better modulating both activity and
drug-likeness. When tested against OASS-A and OASS-B from
S. typhimurium, a well characterized facultative intracellular
pathogen for which both the enzyme isoforms have been
thoroughly characterized,3,5,10,12,17,44−60 this modification
proved to be effective, as compound trans-2-phenylcyclopro-
RESULTS AND DISCUSSION
■
In a previous communication we have reported several small
molecules capable of inhibiting HiOASS-A.38 These structures
were rationally designed based on the HiSAT native C-terminal
pentapeptide MNLNI, which is the natural inhibitor of OASS
(Figure 3). The general structure of these inhibitors relied on
the presence of a carboxylic acid moiety linked through a
cyclopropane linker to a side chain mimicking that of
isoleucine, in a trans configuration. Analogues of these β-
substituted cyclopropane carboxylic acids were prepared, and
the most active compound trans-2-(prop-1-enyl)-
cyclopropanecarboxylic acid (17, Figure 4) showed an
encouraging Kd of 1.45 μM toward HiOASS-A.
However, this derivative suffered from several issues that
make it particularly unsuitable for further investigation: harsh to
synthesize, highly volatile, prone to decomposition in the stock
C
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Figure 5. Binding of 18 to StOASS-A (A) and StOASS-B (B) as probed by the increase in the fluorescence emission of the cofactor PLP.
Table 1. Dissociation Constants of the Title Compounds for StOASS-A and StOASS-B
Kd (μM)
b
cmpd
stereochemistry
R
R1
OASS A
OASS B
∼3000
SI
c
MNLNI
18
na
na
H
120 12
trans
OH
OEt
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
9.2 0.9
148 16
16
na
48
71
na
21
25
4
e
f
8a
trans
Bn
Et
no bind
nd
12a
trans
15
1
720 100
860 90
12b
12c
1R,2S
1S,2R
trans
Et
12.1 0.5
1200 300
0.20 0.02
0.067 0.007
Et
>2000
d
13a
Bn
Bn
Bn
4.1 0.2
1.66 0.07
197 27
g
13b
1S,2S
1R,2R
trans
13c
14a
14b
14c
15a
15b
15c
16a
50
8
4-CH3−Bn
4-CH3−Bn
4-CH3−Bn
4-Cl-Bn
0.077 0.011
0.028 0.005
81 17
1.2 0.2
16
18
1S,2S
1R,2R
trans
0.49 0.05
121 12
1.5
5
0.156 0.007
0.054 0.008
84 10
0.76 0.10
0.42 0.06
152 19
1S,2S
1R,2R
trans
4-Cl-Bn
8
4-Cl-Bn
1.8
1.9
OH
PhEt
14
3
27
2
a
b
c
d
All the compounds are in trans configuration. SI: Selectivity index is intended as the ratio Kd,OASS‑A/Kd,OASS‑B. Not attainable. Stoichiometry
e
calculated for this compound: 2.36. No binding at concentrations <20 μM. The compound is not soluble in the assay buffer with 1% DMSO at
concentrations >20 μM. Not determined. Stoichiometry calculated for this compound: 1.05.
f
g
panecarboxylic acid (18, Figure 4) showed moderate affinity
toward StOASS-A, with a Kd of 9.2 μM (Figure 5A, Table 1)
although toward StOASS-B the binding was still not
statisfactory (Kd = 148 μM, Figure 5B, Table 1). Thus, with
the aim of understanding which structural modification can lead
to an increased affinity toward OASS-B while retaining good
potency at OASS-A, 18, along with its parent compound 17,
was docked into the active site of the two enzyme isoforms.
As expected, we observed that the hydrogen bond network
established by the isoleucine of the carboxy-terminal portion of
SAT (NLNI) in the crystallographic complex with H. influenzae
OASS-A (PDB code 1Y7L) is maintained by the carboxylic
group of 17 and 18 in the docking poses obtained by using the
crystal structure 1OAS from S. typhymurium (Figure 6A). In
addition, the alkyl portion of 17 and the aromatic ring of 18 are
located in the small lipophilic pocket occupied by the
hydrophobic portion of isoleucine. As can be appreciated in
Figure 6B, a considerable portion of the binding pocket is
empty. This region is characterized by the presence of a small
lipophilic area surrounded by mildly polar residues.20,33,61
Therefore, we reasoned that the functionalization of the α-
carbon of the cyclopropane ring might exploit this portion of
the binding site, thus establishing new interactions and
increasing the binding affinity. Moreover, also in the OASS-B
isoform there is space to add substituents in this direction, even
if the cavity is slightly more polar (Figure 6C). Substituents at
the β position of the cyclopropane ring or on the phenyl ring
were not investigated, since it seems that in this region of the
pocket there is no space to add other substituents, in particular
considering the shape of the binding pocket of the B isoform.
Based on these observations, we designed compound 12a,
bearing an ethyl moiety at the α-carbon, which was initially
prepared as racemic mixture. The affinity to StOASS-A did not
change significantly with respect to that of 18, whereas the
affinity toward the B isoform was considerably lower (Kd,OASS‑A
= 15 μM; Kd,OASS‑B = 720 μM). Since for optically active
compounds the stereochemistry is usually crucial for the
interaction with the enzymes, we deemed of interest to
synthesize as reported in Scheme 1 the two enantiomers of 12a.
Interestingly, we found that while the 1R,2S enantiomer 12b
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Figure 6. Binding of 18 to StOASS-A. (A) binding mode of 18 in StOASS-A binding site (1OAS crystal structure, in magenta), superposed to the
previously identified compound 17 (in green) and to the C-terminal portion of CysE as solved in 1Y7L crystal structure from H. influenzae. (B) Map
of the features of StOASS-A binding site with compound 18 as a reference; hydrogen bond donor areas are reported in blue meshes, the hydrogen
bond acceptor in red and the hydrophobic ones in yellow. A second small hydrophobic area can be seen at the bottom of the picture, surrounded by
polar areas. (C) Map of the features of StOASS-B binding site with compound 18 as a reference; colors are the same as in panel B).
Figure 7. Stereochemical preferences for the binding of cyclopropane carboxylic acid derivatives to StOASS-A. (A) Two possible binding modes for
compound 12b (1R,2S); the one represented in green seems to be the preferred one (see text). (B) Docking pose of the inactive compound 12c
(1S,2R, in cyan) compared to the active enantiomer 12b: the carboxylic group establishes fewer hydrogen bonds and the position of the aromatic
ring causes some steric clashes with the residues in proximity to the compound. (C) Docking pose for compound 13b (1S,2S); the additional
aromatic moiety is located in the small lipophilic region. (D) Docking poses of derivatives 14b (1S,2S, blue) and 15b (1S,2S pink). (E) Docking
poses of one of the enantiomers of 16a (in slate blue and lime green), superposed to 13b (in pink, transparent): the ethyl spacer is too long to allow
a perfect matching of the aromatic ring with the small hydrophobic area; therefore, the derivative seems to adopt a different conformation inside the
binding site, in agreement with the loss of affinity observed for this compound.
E
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was barely as active as the racemic mixture, the 1S,2R
enantiomer 12c was inactive.
shielded from the solvent inside the binding pocket.
CORCEMA-ST (complete relaxation and conformational
exchange matrix analysis of saturation transfer)65 theory was
used to assess the agreement between the docking poses
selected for compound 12b and the corresponding STD
spectrum. CORCEMA-ST calculates the predicted STD-NMR
intensities for a proposed molecular model of a ligand−protein
complex. The predicted STD intensities are expressed as GEM
values and compared with the experimental data (Figure 8B).
The first proposed binding mode (Figure 7A, in green)
shows that predicted GEM values are more consistent (75% of
the analyzed protons, Pose 1, Figure 8A) with the
experimentally measured GEM values with respect to the
second binding mode (Pose 2, Figure 8A). First of all, the GEM
value obtained for the hydrogen atom bound to the β-carbon of
the cyclopropane ring (Hc) reveals that the docking pose is fully
in agreement with the experimental value, predicting this region
as being very close to the binding site surface with stable van
der Waals interactions. Moreover, similar intermediate GEM
values are predicted for the ethyl moiety. Small differences are
found for the aromatic portion and the hydrogen atoms in γ
position of the cyclopropane ring. In particular, concerning the
aromatic ring, docking results predict a more buried pose
compared to the experimental findings, in particular for the
para position. It can be hypothesized that these differences are
due to the proximity of the two portions of the ligand to the
flexible loop delimiting the binding pocket. This loop can adopt
different conformations in solution, and the observed STD
signals are realistically an average of the different conforma-
tions, whereas in the docking experiments the conformation of
this loop is fixed. To take into account accurately the behavior
of this loop and the influence of protein dynamics on the
predicted STD values is beyond the aim of this work and will be
tackled separately. The predicted GEM values for the second
possible binding mode (Figure 7A, purple) are substantially
different from the experimental ones, with a GEM value of only
55% for the hydrogen atom of the β-carbon of the
cyclopropane ring, suggesting that this binding mode is
probably not the one adopted in the formation of the
protein−ligand complex.
The orientation of 12c in the binding site, as obtained by
docking of the compound into StOASS-A active site, is slightly
turned compared to the active enantiomer 12b (Figure 7A and
B). This leads to a less efficient interaction of the carboxylic
acid with the surrounding residues and to some steric clashes in
the hydrophobic pocket occupied by the phenyl ring.
Therefore, we can assume that this enantiomer is not properly
accommodated inside the binding site, partially explaining the
huge difference measured in the Kds for the two compounds.
With regard to 12b, given the small size of the ligand and the
big cavity of the binding site, docking studies found two equally
possible binding modes (Figure 7a). In both cases, the
carboxylic group is involved in a hydrogen bond network and
van der Waals interactions with the phenyl ring and the ethyl
group. Although it can be speculated that the first interaction is
the most likely, as it resembles that of the parent pentapeptide,
we took advantage of the fairly high binding constant of 12b to
carry out saturation transfer difference (STD) measurements
(see details in Supporting Information). STD is a NMR
spectroscopic method allowing transfer of transient energy
from a protein to its ligand. The distribution of saturation
transferred to the ligand protons reveals the relative spatial
proximity of moieties of the ligand to the protein. This
information is used to determine which part of the ligand
molecule is responsible for binding, assuming that the higher
the STD effect, the closer the ligand proton is to the receptor
surface, and, therefore, the stronger the ligand−protein
interaction.62,63 To simplify the STD data analysis, the
saturation received by the different ligand protons is expressed
as group epitope mapping (GEM), i.e., percentage of STD
signals normalized with respect to the most saturated signal.
The higher the value, the more intimate the recognition of the
ligand portion is by the protein binding pocket.64 From
experimental GEM values for compound 12b (Figure 8A), it
can be seen that the β-carbon of the cyclopropane ring
represents the portion of the ligand that is more deeply buried
inside the cavity and the ring hydrogens exhibit the highest
saturation. Ethyl and phenyl groups seem to be only partially
With the aim of gaining further insight into the correct
position of the 2-phenylcyclopropane carboxylic acid backbone
and to further exploit the available volume inside the binding
site to increase the binding affinity, we synthesized a compound
bearing a benzyl group at the α-carbon. This substituent might
stabilize the binding mode in the most favorable pose,
occupying the portion of the binding site that is left empty
by smaller ligands. We reasoned that an aromatic group at the
α-carbon could be well allocated in this region characterized by
a small hydrophobic area surrounded by polar residues, defining
mildly polar areas. Moreover, the aromatic group leaves space
for easily adding further substituents and maximizing the
complementarity with the binding site. Therefore, compounds
13a and its enantiomeric pure analogues 13b (1S,2S) and 13c
(1R,2R) were synthesized and tested. The racemic mixture
exhibited a very good activity compared to the ethyl derivative
and, of the two enantiomers, 13b was found to show high
affinity to both StOASS-A and StOASS-B with Kds in the
nanomolar and low micromolar ranges, respectively (Table 1).
On the other hand, 13c, as expected, was found to be several-
fold (700-fold) less potent than the corresponding enantiomer
for StOASS-A. The enantiomeric preferences were further
confirmed by calculation of the binding stoichiometry to
Figure 8. (A) GEM values for compound 12b (1R,2S) from the
experimental STD experiments. (B) The histogram compares the
experimental STD values of 12b (1R,2S) (in orange) to those
predicted for the docking poses (in red and blue).
F
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StOASS-A. In the case of the racemic mixture the binding
stoichiometry is about 2 and decreases to about 1 when
compound 13b is used for the titration. In fact, one molecule of
13b binds for each active site of StOASS-A, whereas 2
molecules of the racemic mixture 13a are needed, since it is a
50:50 mixture of 13b and 13c. The docking of 13b into the
binding pocket of StOASS-A gave us other useful information
(Figure 7C). First, it allowed identifying the binding mode of
the 2-phenylcyclopropane carboxylic acid backbone within the
binding site. This is helpful in order to carry out further ligand
optimization via decoration of the 2-phenyl ring with various
functional groups. Second, the analysis of the pose of 13b led
us to speculate that there is further room for additional small
functional groups in the para position of the α-benzyl ring.
Therefore, compounds bearing at the para position of the
benzyl ring either an electron donor group, such as the methyl,
or an electron withdrawing group, such as chlorine, were
synthesized and tested. First, we synthesized the 1S,2S
enantiomers 14b and 15b, assuming that also in this case this
stereochemistry would have been the preferred one. However,
we deemed it of interest to also prepare the racemic derivatives
14a and 15a and the enantiomers 1S,2S 14c and 15c, in order
to further corroborate, with additional data, the most favorable
stereochemistry (Table 1, Figure 7D). Moreover, since the
pocket accommodating the benzyl ring seemed to be large, we
also synthesized the α-phenylethyl derivative 16a, in which the
aromatic ring is connected to the α-carbon of the cyclopropane
through an ethyl spacer. We found that, compared to 13b, the
affinities of 14b and 15b toward StOASS-A were almost the
same. However, both compounds showed a more than 3-fold
improved affinity toward StOASS-B, with Kds in the low
nanomolar range. To our knowledge, this represents the
strongest binder reported so far for the B isoform of StOASS.
In fact, a previously identified small molecule compound active
on StOASS-B21 exhibited dissociation constants for the B
isoform of about 30 μM, about 70-fold higher than the best
compound here identified. As expected, for what concerned
StOASS-A, the activity can be ranked based on the absolute
configuration as follows: 1S,2S > rac ≫ 1R,2R. Compound 16a
was only marginally active. It can be speculated that the
phenylethyl substituent affects the interaction of the whole
molecule since the ethyl linker is too long to allow the
accommodation of the phenyl ring in the small lipophilic area
occupied by the benzyl group. Therefore, for both the
enantiomers of 16a the phenylethyl moiety points toward the
other side of the binding site, where the C-terminal residues of
CysE in the 3IQG crystal structure from HiOASS-A bind
(Figure 7E). The importance of the acid group and its
interactions in the pocket of the binding site is further
corroborated by the lack of activity of the ester derivative 8a.
Preliminary Structure Activity Relationships (SAR).
Although the set of compounds synthesized is relatively small,
some preliminary hints of SAR can be described. The vinyl
moiety of 17 can be embodied in a phenyl ring and kept in a
trans conformation with respect to the carboxylic acid moiety.
Trapping the acid group into an ester leads to a severe decrease
or even loss of the activity. Substitution at the α-carbon of the
cyclopropane not only is well tolerated, as in the case of the
compound 12a, but it also remarkably enhances the affinity
toward both StOASS-A and StOASS-B, as in the case of the α-
benzyl substitution. Elongating the spacer between the phenyl
ring and the α-carbon of cyclopropane from one to two
methylene units leads to a loss of activity (16, Kd,OASS‑A = 14
μM). The same effect is recorded with the deletion of the
aromatic ring (12a, Kd,OASS‑A = 15 μM; 12b, Kd,OASS‑A = 12.1
μM). The benzyl ring, along with positioning the 2-phenyl-
cyclopropane carboxylic acid into its most favorable pose for
interaction, is probably responsible for the π−π interaction.
Moreover, the benzyl ring can be further decorated with small
functional groups, such as methyl and chlorine, regardless of
their electron-donor or electron-withdrawing properties. In this
first round of modifications, only the para position was
investigated. However, a medicinal chemistry campaign aimed
at refining the SAR for these derivatives is currently underway
in our laboratory. Stereochemistry was found to be pivotal for
ensuring high affinity. In the case of the α-ethyl substituted
derivatives, the levorotatory enantiomer 1R,2S was found to be
more than 1000-fold less active than the counterpart. In the
case of the α-benzyl substitution, the dextrorotatory enantiomer
1S,2S shows consistently an affinity higher than that of other
isomers, according to the following trend: 1S,2S > rac ≫ 1R,2R.
Therefore, the most active compounds are those showing a 2S
stereochemistry in their structure. This is an important finding
to consider in the planning of further derivatives. Indeed, since
the synthetic method of cyclopropanation maintains the
configuration of the starting epoxides, it can be speculated
that (S)-styrene oxides will yield the active derivatives.
Selectivity of the Compounds toward the Two
StOASS Isoforms. As described above, inhibition of both
isoforms of StOASS is important for granting a complete
inhibition of cysteine biosynthesis in bacteria. In this work, we
have obtained an excellent activity toward StOASS-B, especially
with compounds 15b and 14b. However, considering that the
ultimate target of our work is a compound showing the same
activity toward both isoforms, we have rationalized the
information collected in this work on the basis of the ratio
between StOASS-B and StOASS-A affinities (Table 1). First, it
must be noticed that, in general, the affinity toward StOASS-B
is proportional to that for StOASS-A, although always weaker
(Figure S2). Therefore, we focused on the increase in the
activity toward the A isoform. When the α-carbon is not
substituted, the ratio between the activity to StOASS-B and
StOASS-A is around 16 (see compound 18). Substitution with
an ethyl group, although maintaining the activity toward the A
isoform, led to a sharp decrease in the affinity toward the B
isoform (see 12a and 12b, with an affinity ratio of around 50
and 70, respectively). Substitution with the α-benzyl group
reduces the activity ratio compared to the α-ethyl, but such a
ratio still remains around 20 in the case of the racemic mixture
and 25 for the more active enantiomer. What seems to
remarkably improve the affinity ratio is the insertion of a
lipophilic substituent at the para position of the benzyl ring.
Indeed, for those compounds bearing a para-chloro sub-
stitution, that is 15a and 15b, the Kd for StOASS-B is only 5-
and 8-fold higher than that for StOASS-A, respectively.
Although a higher number of derivatives will shed more light
into the SAR of these inhibitors, we can conclude that the α-
benzyl substitution leads not only to the strongest compounds
toward StOASS-A and StOASS-B so far identified, but also, if
properly substituted, to the loss of selectivity between the two
isoforms.
Inhibitory Activity of Compounds 15b and 15c
toward the Two StOASS Isoforms. After characterizing
the compounds with regard to their SAR, we investigated
whether there was a direct correlation between the binding
affinities and the enzyme inhibitory activity. Indeed, it is
G
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Journal of Medicinal Chemistry
Article
expected that for compounds that act as competitive inhibitors
Kd coincides with Ki. Compound 15b was selected for the
inhibition assay because of its high binding activity and its low
selectivity toward the two enzyme isoforms StOASS-A and B.
The dependence of the fractional activity on 15b concentration
is reported in SI Figure S3 and the calculated IC50s are reported
in Table 2. As expected for a compound that binds to the
extremely important in conferring high potency, and the
enantiomer 1S,2S is more potent than the 1R,2R counterpart by
an average 1000-fold factor. Finally, to the best of our
knowledge, 14b and 15b are the most active compounds
described so far toward StOASS-B. Altogether, these findings
provide a solid base to further investigate this series of 2-
phenylcyclopropane carboxylic acids as a valuable tool to tune
the events associated with the biosynthesis of cysteine in
unicellular organisms, such as bacterial virulence and drug
resistance.
Table 2. IC50s of Representative Inhibitors of StOASS-A and
StOASS-B
OASS-A
OASS-B
EXPERIMENTAL SECTION
■
cmpd
Kd (μM)
IC50 (μM)
Kd (μM)
IC50 (μM)
General Information. All the reagents were purchased from
Sigma-Aldrich, Alfa-Aesar, and Enamine at reagent purity and, unless
otherwise noted, were used without any further purification. Dry
solvents used in the reactions were obtained by distillation of technical
grade materials over appropriate dehydrating agents. MCRs were
performed using CEM Microwave Synthesizer-Discover model.
Reactions were monitored by thin layer chromatography on silica
gel-coated aluminum foils (silica gel on Al foils, SUPELCO Analytical,
Sigma-Aldrich) at 254 and 365 nm. Where indicated, intermediates
and final products were purified by silica gel flash chromatography
(silica gel, 0.040−0.063 mm), using appropriate solvent mixtures.
1H NMR and 13C NMR spectra were recorded on a BRUKER
AVANCE spectrometer at 400 and 100 MHz, respectively, with TMS
as internal standard. For STD experiments please refer to the
15b
15c
0.054 0.008
84 10
0.099 0.004
0.42 0.06
152 19
0.50 0.03
14
1
48
2
enzyme active site and competes with the substrate OAS,
compound 15b was found to inhibit both isoforms of StOASS
with an IC50 comparable to the dissociation constants
calculated by the direct fluorimetric titration (Kd StOASS-A =
0.054 μM 0.008 vs IC50 StOASS-A = 0.099 μM 0.004; Kd
StOASS-B = 0.42 μM 0.06 vs IC50 StOASS-B = 0.50 μM
0.03). A similar conclusion can be drawn for the 1R,2R isomer,
which exhibits a lower affinity for the enzyme. Therefore, we
have demonstrated that there is a direct correlation between
ligand binding and inhibition for both the isoforms.
1
Supporting Information. H NMR spectra are reported in this order:
multiplicity and number of protons. Standard abbreviation indicating
the multiplicity were used as follows: s = singlet, d = doublet, dd =
doublet of doublets, t = triplet, q = quadruplet, m = multiplet, and br =
broad signal. HPLC/MS experiments were performed with an Agilent
1100 series HPLC apparatus, equipped with a Waters Symmetry C18,
3.5 μm, 4.6 mm × 75 mm column and an MS: Applied Biosystem/
MDS SCIEX instrument, with API 150EX ion surce. HRMS
experiments were performed with an LTQ ORBITRAP XL THERMO
apparatus.
CONCLUSIONS
■
Inhibition of cysteine biosynthesis may strongly affect the life
cycle of many unicellular microorganisms and plants. O-
Acetylserine sulfhydrylase catalyzes the last step of cysteine
biosynthesis, allowing the transfer of sulfide to acetylserine.
Since mammals are not equipped with this enzyme, supplying
cysteine through the reverse transsulfuration pathway from
methionine, inhibitors of OASS would be highly selective and
safe. Taking into account the data from our previous efforts,38
and combining computational and spectroscopic approaches
such as STD NMR, we have rationally designed and
synthesized a series of 2-phenylcyclopropane carboxylic acids
to be tested against StOASS-A and StOASS-B. Overall, this
medicinal chemistry campaign led to derivatives about 4000-
fold and 7000-fold more active on StOASS-A and StOASS-B,
respectively, than the peptidic inhibitor MNLNI, and to a
reduction of the selectivity toward the two isoforms. Moreover,
we have demonstrated that compounds that bind to the
enzyme active site are inhibitors of both the isoforms efficiently
competing with the substrate. Given the various range of Kds, a
preliminary hint of structure−activity relationships could be
outlined. The carboxylic acid moiety is crucial for the activity, as
its substitution with an ester led to loss of activity. The
cyclopropane ring, that maintains in the suitable trans
configuration the carboxylic acid moiety and the phenyl ring,
proved to be a valuable structural solution, confirming the
results of our previous investigations. Substitution at the α-
carbon of the cyclopropane strongly affects both the potency of
the compounds and their selectivity toward the two isoforms
StOASS-A and StOASS-B. Bulky groups such as the para
substituted and unsubstituted benzyl moieties allow to obtain
potent inhibitors. However, a substituent such as chlorine at the
para position of the benzyl ring allows a strong affinity to be
coupled with a reduction of selectivity toward the two isoforms
of StOASS, with the Kd toward OASS-B only 8 times higher
than that toward OASS-A. Interestingly, the stereochemistry is
All compounds were tested as 95% purity or higher (by HPLC/
MS).
General Procedure for the Synthesis of Phosphonates (2−
6). Ethyl 2-(diethoxyphosphoryl)acetate (1 equiv) was added
dropwise to a cooled suspension of NaH (1.1 equiv) in dry DME
(2 mL/mmol). After stirring at 25 °C for 2 h the appropriate halide
(1.1 equiv) was added, and the mixture was heated at 60 °C for 2 h.
The reaction mixture was poured into ice water and extracted with
ethyl acetate and the combined organic layers were washed with H2O
and brine, dried over Na2SO4, and evaporated under reduced pressure.
The crude material was purified through flash chromatography eluting
with dichloromethane/diethyl ether 95:5 to give the desired
compounds as colorless oil in yields ranging from 54% to 68%.
Analytical data for compounds 5a, b, and e matched the data already
published.41−43
General Procedure for the Cyclopropanation. To a solution of
the appropriate phosphonate (2 equiv) in dry DME (5 mL/mmol) at
25 °C, n-buthyllithium (2.5 M in hexane, 2 equiv) was added dropwise
over 5 min. After stirring at the same temperature for 30 min, the
appropriate styrene oxide (1 equiv) was added portionwise and the
mixture heated at 90 °C for 18 h. After cooling, saturated aqueous
solution of NH4Cl was added and the organic layers were extracted
with ethyl acetate (3 × 10 mL), washed with brine, dried over
anhydrous Na2SO4, and evaporated under reduced pressure. The oil
obtained was purified through flash column chromatography eluting
with petroleum ether/ethyl acetate (95:5), to give the desired product
as yellowish oil in yields ranging from 65% to 78%.
General Procedure for the Ester Hydrolysis: Synthesis of
Compounds 12a−c, 13a−c, 14a−c, 15a−c, and 16a. Esters 7a−
c, 8a−c, 9a−c, 10a−c, and 11a (1 equiv) and LiOH·H2O (4 equiv)
were dissolved in a solution of THF/MeOH/H2O (3/1/1, 1 mL/
mmol) and heated under stirring in a microwave oven at 100 °C for 7
min. The reaction mixture is then evaporated in vacuo, and the crude is
H
DOI: 10.1021/acs.jmedchem.5b01775
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
taken up with H2O, acidified with HCl 1 N, and extracted with ethyl
acetate, that is in turn washed with brine and dried over anhydrous
Na2SO4. After the evaporation of the solvent the crude material was
purified through flash column chromatography eluting dichloro-
methane/methanol (95:5), to give the desired product as a white
solid in yields ranging from 56% to 67%.
CHCl3), HRMS (ESI) calculated for C18H18O2 ([M-H]−) 266.3343;
found 266.3288.
trans-1-(4-Chlorobenzyl)-2-phenylcyclopropanecarboxylic acid
1
(15a). H NMR (300 MHz, CDCl3): 7.39−7.11 (m, 9H); 3.20 (d, J
= 15.72, 1H); 3.02 (t, J = 8.34, 1H); 2.00−1.93 (m, 2H); 1.51−1.47
(m, 1H). 13C NMR (100.6 MHz, CDCl3): 181.64; 139.98; 136.22;
129.39; 128.73; 128.49; 128.21; 127.26; 126.04; 34.09; 32.93; 30.73;
18.49; HRMS (ESI) calculated for C17H15ClO2 ([M-H]−) 286.7528;
found 286.8022.
trans-1-Ethyl-2-phenylcyclopropanecarboxylic acid (12a). 1H
NMR (300 MHz, CDCl3): 7.43−7.36 (m, 5H); 3.20 (d, J = 15.72,
1H); 3.02 (t, J = 8.34, 1H); 2.13−1.97 (m, 2H); 1.51−1.47 (m, 1H);
0.80 (t, J = 7.32 Hz, 3H).
(1S,2S)-1-(4-Chlorobenzyl)-2-phenylcyclopropanecarboxylic acid
(15b). 1H NMR (300 MHz, CDCl3): 7.39−7.11 (m, 9H); 3.20 (d, J =
15.72, 1H); 3.02 (t, J = 8.34, 1H); 2.00−1.93 (m, 2H); 1.51−1.47 (m,
1H). 13C NMR (100.6 MHz, CDCl3): 181.64; 139.98; 136.22; 129.39;
128.73; 128.49; 128.21; 127.26; 126.04; 34.09; 32.93; 30.73; 18.49.
[α]20D − 40.77 (c 1, CHCl3), HRMS (ESI) calculated for C17H15ClO2
([M-H]−) 286.7528; found 286.7686.
(1R,2R)-1-(4-Chlorobenzyl)-2-phenylcyclopropanecarboxylic acid
(15c). 1H NMR (300 MHz, CDCl3): 7.39−7.11 (m, 9H); 3.20 (d, J =
15.72, 1H); 3.02 (t, J = 8.34, 1H); 2.00−1.93 (m, 2H); 1.51−1.47 (m,
1H). 13C NMR (100.6 MHz, CDCl3): 181.64; 139.98; 136.22; 129.39;
128.73; 128.49; 128.21; 127.26; 126.04; 34.09; 32.93; 30.73; 18.49.
[α]20D + 40.77 (c 1, CHCl3), HRMS (ESI) calculated for C17H15ClO2
([M-H]−) 286.7528; found 286.7682.
13C NMR (100.6 MHz, CDCl3): 129.29; 128.21; 126.87; 33.32;
31.17; 21.48; 18.49; 11.59. HRMS (ESI) calculated for C12H14O2 ([M-
H]−) 190.0994; found 190.1002.
(1R,2S)-1-Ethyl-2-phenylcyclopropanecarboxylic acid (12b). 1H
NMR (300 MHz, CDCl3): 7.43−7.36 (m, 5H); 3.20 (d, J = 15.72,
1H); 3.02 (t, J = 8.34, 1H); 2.13−1.97 (m, 2H); 1.51−1.47 (m, 1H);
0.80 (t, J = 7.32 Hz, 3H). 13C NMR (100.6 MHz, CDCl3): 129.29;
128.21; 126.87; 33.32; 31.17; 21.48; 18.49; 11.59. [α]20 − 100.77 (c
D
1, CHCl3), HRMS (ESI) calculated for C12H14O2 ([M-H]−) 190.0994;
found 190.0998.
(1S,2R)-1-Ethyl-2-phenylcyclopropanecarboxylic acid (12c). 1H
NMR (300 MHz, CDCl3): 7.43−7.36 (m, 5H); 3.20 (d, J = 15.72,
1H); 3.02 (t, J = 8.34, 1H); 2.13−1.97 (m, 2H); 1.51−1.47 (m, 1H);
0.80 (t, J = 7.32 Hz, 3H). 13C NMR (100.6 MHz, CDCl3): 129.29;
trans-1-Phenethyl-2-phenylcyclopropanecarboxylic acid (16a).
1H NMR (300 MHz, CDCl3): 7.39−7.20 (m, 10H); 3.02 (t, J =
128.21; 126.87; 33.32; 31.17; 21.48; 18.49; 11.59. [α]20 + 100.77 (c
D
8.34, 1H); 3.01−2.89 (m, 1H); 2.78−2.68 (m, 1H); 2.63−2.53 (m,
1H); 2.37−2.21 (m, 1H); 2.20−2.10 (m, 1H); 1.51−1.47 (m, 1H).
13C NMR (100.6 MHz, CDCl3): 181.64; 139.98; 136.22; 129.39;
128.73; 128.49; 128.21; 127.26; 126.04; 34.09; 32.93; 30.73; 18.49;
15.23. HRMS (ESI) calculated for C18H18O2 ([M-H]−) 266.1307;
found 266.2286.
1, CHCl3), HRMS (ESI) calculated for C12H14O2 ([M-H]−) 190.0994;
found 190.1010.
trans-1-Benzyl-2-phenylcyclopropanecarboxylic acid (13a). 1H
NMR (300 MHz, CDCl3): 7.39−7.20 (m, 10H); 3.20 (d, J = 15.72,
1H); 3.02 (t, J = 8.34, 1H); 2.00−1.93 (m, 2H); 1.51−1.47 (m, 1H).
13C NMR (100.6 MHz, CDCl3): 181.64; 139.98; 136.22; 129.39;
128.73; 128.49; 128.21; 127.26; 126.04; 34.09; 32.93; 30.73; 18.49.
HRMS (ESI) calculated for C17H16O2 ([M-H]−) 252.3077; found
252.3202.
Molecular Modeling. For docking studies, chain A of the crystal
structures of OASS-A (pdb 1OAS) and OASS-B (pdb 2JC3) from
Salmonella typhimurium were used. Protein structures were prepared
using the wizard tool available from the Schrodinger suite. Ligands
were prepared using the LigPrep tool of the Schrodinger suite at pH
7.5 1. Docking studies were performed using PLANTS induced fit
approach, allowing some selected residues of the binding pocket to
move during ligand binding. The following residues were selected: for
OASS-A: E66, T68, N69, N71, T72, R99, M119, Q142, P143, V175,
T177, I229; for OASS-B: E66, T68, S69, N71,T72, R99, M119, Q140,
F141, M173, T175, I209, R210, R211, W212.
NMR Measurements for STD Experiments. NMR spectra were
acquired on a Varian (Agilent Technologies) Inova 600 NMR
Spectrometer at 20 °C, on samples with a molar excess of 1:300
(OASS-A: 1R,2S-(−)-12b). Ligand concentration was kept at 3 mM in
the presence of 10 μM protein and were dissolved in phosphate buffer
pH 8.0 (5 mM K2PO4, 3% DMSO-d6).
Saturation transfer difference (STD) experiments were collected
with 64 K data points in the direct dimension and 512 scans in a
spectral window of 7000 Hz. The water signal was suppressed by the
excitation sculpting method (dpfgse_water). Selective saturation of the
protein resonances (on resonance spectrum) was performed by
irradiating at −0.9 ppm using Gaussian-shaped pulses for a saturation
time of 3 s. For the reference spectrum (off resonance), the samples
were irradiated at 26.6 ppm. NMR data were processed and analyzed
using MestReNova 8.1 software. Group epitope mapping (GEM) was
calculated setting the highest STD intensity to 100% and all other
STD signals were calculated accordingly.
All CORCEMA calculations were carried out using CORCEMA SX
3.8.65−67 Protein residues within 7 Å distance of the ligand were
included for the calculation of theoretical STD effects. Ligand and
protein concentrations, their equilibrium constant, and spectrometer
frequency were all set according to experimental values. The free
ligand rotational correlation time was estimated to be 0.3 ns. The
bound ligand rotational correlation time was 32 ns. Because protein
signals around 0 ppm were selectively irradiated for the STD
experiment, all methyl protons in the enzyme were assumed to be
saturated. STD intensities of nonexchangeable protons on ligand were
calculated at a saturation time of 3 s.
1
(1S,2S)-1-Benzyl-2-phenylcyclopropanecarboxylic acid (13b). H
NMR (300 MHz, CDCl3): 7.39−7.20 (m, 10H); 3.20 (d, J = 15.72,
1H); 3.02 (t, J = 8.34, 1H); 2.00−1.93 (m, 2H); 1.51−1.47 (m, 1H).
13C NMR (100.6 MHz, CDCl3): 181.64; 139.98; 136.22; 129.39;
128.73; 128.49; 128.21; 127.26; 126.04; 34.09; 32.93; 30.73; 18.49.
[α]20 − 25.88 (c 1, CHCl3), HRMS (ESI) calculated for C17H16O2
D
([M-H]−) 252.3077; found 252.3158.
(1R,2R)-1-Benzyl-2-phenylcyclopropanecarboxylic acid (13c). H
1
NMR (300 MHz, CDCl3): 7.39−7.20 (m, 10H); 3.20 (d, J = 15.72,
1H); 3.02 (t, J = 8.34, 1H); 2.00−1.93 (m, 2H); 1.51−1.47 (m, 1H).
13C NMR (100.6 MHz, CDCl3): 181.64; 139.98; 136.22; 129.39;
128.73; 128.49; 128.21; 127.26; 126.04; 34.09; 32.93; 30.73; 18.49.
[α]20 + 25.88 (c 1, CHCl3), HRMS (ESI) calculated for C17H16O2
D
([M-H]−) 252.3077; found 252.3200.
trans-1-(4-Methylbenzyl)-2-phenylcyclopropanecarboxylic acid
1
(14a). H NMR (300 MHz, CDCl3): 7.39−7.20 (m, 5H); 7.11 (d, J
= 8.43, 4H); 3.20 (d, J = 15.72, 1H); 3.02 (t, J = 8.34, 1H); 2.25 (s,
3H); 2.00−1.93 (m, 2H); 1.51−1.47 (m, 1H). 13C NMR (100.6 MHz,
CDCl3): 181.64; 139.98; 136.22; 129.39; 128.73; 128.49; 128.21;
127.26; 126.04; 34.09; 32.93; 30.73; 18.49; 11.59. HRMS (ESI)
calculated for C18H18O2 ([M-H]−) 266.3343; found 266.3206.
(1S,2S)-1-(4-Methylbenzyl)-2-phenylcyclopropanecarboxylic acid
(14b). 1H NMR (300 MHz, CDCl3): 7.39−7.20 (m, 5H); 7.11 (d, J =
8.43, 4H); 3.20 (d, J = 15.72, 1H); 3.02 (t, J = 8.34, 1H); 2.25 (s, 3H);
2.00−1.93 (m, 2H); 1.51−1.47 (m, 1H). 13C NMR (100.6 MHz,
CDCl3): 181.64; 139.98; 136.22; 129.39; 128.73; 128.49; 128.21;
127.26; 126.04; 34.09; 32.93; 30.73; 18.49; 11.59. [α]20D − 110.9 (c 1,
CHCl3), HRMS (ESI) calculated for C18H18O2 ([M-H]−) 266.3343;
found 266.4008.
(1R,2R)-1-(4-Methylbenzyl)-2-phenylcyclopropanecarboxylic acid
(14c). 1H NMR (300 MHz, CDCl3): 7.39−7.20 (m, 5H); 7.11 (d, J =
8.43, 4H); 3.20 (d, J = 15.72, 1H); 3.02 (t, J = 8.34, 1H); 2.25 (s, 3H);
2.00−1.93 (m, 2H); 1.51−1.47 (m, 1H). 13C NMR (100.6 MHz,
CDCl3): 181.64; 139.98; 136.22; 129.39; 128.73; 128.49; 128.21;
127.26; 126.04; 34.09; 32.93; 30.73; 18.49; 11.59. [α]20D + 110.9 (c 1,
I
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Journal of Medicinal Chemistry
Article
Ligand Binding Affinity to OASS. The binding affinity of ligands
was determined by monitoring the increase in fluorescence emission of
the bound coenzyme following excitation at 412 nm.21,36 Emission
spectra were recorded as a function of ligand concentration in a
solution containing 0.5−1.0 μM OASS, 100 mM Hepes buffer, pH 7.0,
at 20 °C. Fluorescence measurements were carried out using a
FluoroMax-3 fluorometer (HORIBA), equipped with a thermostated
cell-holder. The dependence of fluorescence intensity at the emission
peak, 500 nm, on ligand concentration was fitted to a binding isotherm
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I
max[L]
I =
Kdiss + [L]
where I is the fluorescence intensity at 500 nm in the presence of the
ligand, after subtraction of the fluorescence intensity at 500 nm in the
absence of the ligand, Imax is the maximum fluorescence change at
saturating [L], [L] is the ligand concentration, and Kdiss is the
dissociation constant of the OASS-ligand complex. This measurement
is a direct determination of ligand dissociation constant in the absence
of substrate. For competitive inhibitors Kd coincides with Ki.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01775.
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AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: barbara.campanini@unipr.it. Phone: +39 0521906613.
*E-mail: gabriele.costantino@unipr.it. Phone: +39
0521905054.
Present Addresses
¶Laboratory of Medicinal Chemistry, Rega Institute for Medical
Research, KU Leuven, Leuven, Belgium
△Dipartimento Oncologico e Tecnologie Avanzate - IRCCS-
ASMN, Reggio Emilia, Italy
Author Contributions
■Marco Pieroni and Giannamaria Annunziato contributed
equally to this work.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work described in this paper was partly carried out under
the MSCA-ITN-2014-ETN project INTEGRATE (grant
number 642620).
ABBREVIATIONS
■
CoA, Coenzyme A; GEM, group epitope mapping; MW,
microwave; OASS, O-acetylserine sulfhydrylase; PDB, protein
data bank; PLP, pyridoxal 5′-phosphate; SAR, structure−
activity relationship; SAT, serine O-acetyltransferase; STD,
saturation transfer difference
J
DOI: 10.1021/acs.jmedchem.5b01775
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