2
C. Beato et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Figure 1. Malonate (1), 2-methylmalonate
(2), 2,2-dichloromalonate (3), 1,2-cyclopro-
panedicarboxylate (4).
method is equal to Ki. The IC50 value of selected compounds was
measured by enzymatic assays. Since racemization and elimin-
ation reactions are known to share the same enzymatic active site,
it is possible to study SR activity by analyzing either reaction.
In this case, the b-eliminase activity of hSR in the presence of
selected inhibitors was measured using a coupled assay with
lactate dehydrogenase. The reaction kinetics was measured at
37 ꢀC following the disappearance of NADH at 340 nm using a
Varian CARY400 spectrophotometer (Agilent Technologies,
Santa Clara, CA). The reaction mixture contained 0.4 lM hSR
in 50 mM TEA, pH 8, 5–10 % DMSO, and a concentration of
L-Ser equal to the KM of the enzyme in the absence of
DMSO. IC50 value was calculated, for a competitive inhibition
mechanism as
Figure 2. Superposition of malonate (cyan) and cis-1,2-cyclopropanedi-
carboxylate (green).
½Iꢁ
which can then be further optimized without stepping away from
developability metrics such as the Lipinski rule of five27.
In the present study, also on the basis of our expertise in the
synthesis of cyclopropane derivatives28,29, we present our efforts
to develop a new series of non-covalent competitive SR inhibitors
based on the cyclopropane scaffold (Table 1), together with
synthetic routes, chemical characterization, experimental tests,
and a rational explanation for the observed results.
IC50
¼
ð1Þ
V0
Vj
ð
ꢂ 1Þ
Molecular modeling
Docking studies were carried out using a conformational ensem-
ble of hSR formed by five enzyme conformations. The closed
conformation is represented by crystal structure 3L6B; three
partially closed conformations, namely the half-closed, inter-
mediate, and half-open, were extracted from a Targeted MD
simulation previously performed by us33. Finally, the open hSR
conformation was built by comparative modeling using the rat SR
crystal structure in its open conformation as a template (PDB code
3HMK, model previously reported by us)33. Ligands were
prepared using LigPrep at pH 7 134. The Protein Preparation
Workflow was used to add hydrogen atoms, assign bond orders,
optimize the hydrogen-bonding network and, finally, to refine the
Methods
Synthetic chemistry
The synthesis of cis-cyclopropane dicarboxylic acid 4 and the
alkyl-ester derivatives 6 and 7 started from commercially
available 3-oxabicyclo[3.1.0]hexane-2,4-dione, which was hydro-
lyzed either in water at room temperature (4), or ethyl alcohol (6)
and isopropyl alcohol (7) at reflux, affording the title compounds
in good overall yields (Scheme 1). The hydrolysis of diethyl
(1R,2R)-cyclopropane-1,2-dicarboxylate in the presence of an
excess of potassium hydroxide, followed by acidification of the
medium with HCl 1N, led to the synthesis of the trans-1,2-
cyclopropanedicarboxylic acid 5. Finally, the synthesis of 8 was
achieved through basic hydrolysis of intermediate 1,2-diethyl-1,2-
cyclopropanedicarboxylate, obtained according to the reported
McCoy procedure30,31, reacting the corresponding methylacrylate
and the a-chloro ester. Detailed experimental procedures are
available in the Supporting information.
35
˚
protein structures with a maximum RMSD of tolerance of 0.3 A .
At first, water molecules were removed from all the enzyme
conformations. Then, 3L6B was also prepared for docking studies
retaining structural water molecules W403, W372, and W373. All
docking studies were performed with three different software:
Glide SP 6.1 from Schrodinger suite36,37, Autodock4.238, and
¨
Plants39. For all the three software and for all the enzyme
conformations used, the binding site grid box was centred on the
centroid of the following residues: PLP, Ser84, Ser85, Arg135,
Asn154, Ser242, and Thr285 (3L6B numbering). For the docking
studies using 3L6B retaining the conserved water molecules, we
´
applied the same protocol reported by Vorlova et al., with the
grid centred on malonate25.
Biochemical assays
The six selected compounds were assayed in vitro on recombinant
human SR. The affinity of compounds was measured by
spectrofluorimetry, exploiting the fluorescence emission proper-
ties of the cofactor at 500 nm upon excitation at 412 nm using
Results
The main goal of our study was the identification of a new series
of SR inhibitors based on the cyclopropane scaffold given its 3D
similarity with malonate, the most well-known serine racemase
inhibitor. We prepared and tested as potential SR inhibitors the
cyclopropane derivatives reported in Table 1. Compound 4 was
synthesized on the basis of the similarity with malonate
and compound 5, trans 1,2-cyclopropanedicarboxylic acid, was
synthesized to probe the effect of a trans-configuration of the two
carboxylate moieties, also on the basis of the mild inhibition
a
Spex Fluoromax-2 fluorimeter (HORIBA Jobin Yvon,
Kyoto, Japan)11. Briefly, a solution containing 2.5 lM SR,
expressed in E. coli and purified as previously described32 in
50 mM TEA buffer pH 8, 10% DMSO, was titrated by addition of
ligand at 20 ꢀC. The dependence of the fluorescence emission
intensity of the cofactor on ligand concentration was fitted by
a binding isotherm to calculate the dissociation constant (Kd;
Table 2). For competitive inhibitors, Kd calculated with this