Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics

Article abstract of DOI:10.1021/jm060202r

Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k_cat = 21 s^-1 and K_M = 5 μM) and the molar enthalpy (ΔH_app = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (K_i) were determined to be 12.4 μm and 29 μM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.

Full text of DOI:10.1021/jm060202r

J. Med. Chem. 2006, 49, 4937-4945
4937
Design of Inhibitors of Orotidine Monophosphate Decarboxylase Using Bioisosteric
Replacement and Determination of Inhibition Kinetics
†
†
†
‡,§
‡
,†,|
Ewa Poduch, Angelica M. Bello, Sishi Tang, Masahiro Fujihashi, Emil F. Pai, and Lakshmi P. Kotra*
Molecular Design and Information Technology Center, Leslie Dan Faculty of Pharmacy, UniVersity of Toronto, Toronto, Ontario M5S 2S2,
Canada, Departments of Biochemistry, Medical Biophysics, and Molecular & Medical Genetics and DiVision of Cancer Genomics &
Proteomics, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario M5G 2M9, Canada, and DiVision of Cell and Molecular
Biology, Toronto General Research Institute, UniVersity Health Network, Toronto, Ontario, M5G 2C4, Canada
ReceiVed February 21, 2006
Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic,
and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP),
producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed
on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric
acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was
developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring
the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters
-
1
(
k
cat ) 21 s and K
decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the
inhibition constants (K ) were determined to be 12.4 µM and 29 µM for 6-aza-UMP and 6-cyano-UMP,
M
) 5 µM) and the molar enthalpy (∆Happ ) 5 kcal/mol) were determined for the
i
respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition
constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism
and a novel method of using isothermal calorimetry for enzyme inhibition studies.
Introduction
Scheme 1. ODCase-Catalyzed Synthesis of Uridine
Monophosphate (UMP) from Orotidine Monophosphate (OMP)
Orotidine monophosphate decarboxylase (ODCase, EC
1
4.1.1.23) is among the most proficient enzymes known. In
human, ODCase is a part of the bifunctional enzyme UMP
2
synthase. In bacteria and parasites, ODCase is a monofunctional
enzyme.3 It is involved in the catalytic decarboxylation of
,4
orotidine monophosphate (OMP, 1) to uridine monophosphate
(UMP, 2), which in its triphosphate form is a constituent of
RNA as well as a precursor for the synthesis of other pyrimidine
nucleotides (Scheme 1). ODCase accomplishes the decarbox-
ylation of OMP without the help of any cofactors and metal
ions. This is a remarkable achievement in light of the fact that
ODCase (from yeast) exhibits extraordinary rate enhancement
of over 17 orders of magnitude compared to the uncatalyzed
decarboxylation of orotidine monophosphate in water and at
West Nile virus, a recent thread to humans and birds in the
1
3
United States and Canada. Plasmodia, including the malaria-
causing Plasmodium falciparum, are another class of pathogens
sensitive to the inhibition of this enzyme’s activity.14 ODCase
has also been evaluated as an anticancer target. Inhibitors of
ODCase such as 6-azauridine and pyrazofurin exhibited good
anticancer activities against a number of clinical tumor mod-
1
neutral pH, at 25 °C. ODCase is among those few special
enzymes that have developed a very high level of sophistication
in catalyzing decarboxylation and thus has been a subject of
intense investigation.1 Several analogues of OMP have been
investigated extensively to understand the catalytic mechanism
1
5,16
els.
Moreover, the structures, kinetic profiles, and affinities
,5
toward various inhibitors are quite different for ODCase from
different sources including yeast, Escherichia coli, mouse, P.
falciparum, and human among others. Thus, ODCase presents
itself as a good potential drug target.
6
,7
of ODCase. Among these analogues, 6-aza-UMP (3) and
-hydroxy-UMP (or BMP, 4) are widely known as potent
6
8
inhibitors of ODCase.
On the basis of the mechanistic understanding of ODCase
that a carbon dioxide molecule is eliminated and the resulting
anionic center at C-6 is protonated to produce UMP, we
designed 6-cyano-UMP and 6-amino-UMP (5 and 6, respec-
tively) as potential inhibitors of ODCase (Scheme 1, Chart 1).
These two compounds possess small substituents at the C-6
position of the uracil and their size is comparable to that of
carboxylate (Figure 1).
ODCase has been identified as a target for drugs directed
against RNA viruses such as poxviruses and flaviviruses; the
former are causing increasing concern as a potential bioterrorist
9-12
weapon.
ODCase inhibitors have also been effective against
*
Corresponding author: present address #5-356, Toronto Medical
Discoveries Tower, 101 College St., Toronto, Ontario, M5G 2L7 Canada;
tel (416) 581-7601; fax (416) 581-7621; e-mail lkotra@uhnres.utoronto.ca.
†
University of Toronto.
Ontario Cancer Institute/Princess Margaret Hospital.
Present address: Graduate School of Science, Kyoto University, Sakyo,
Recently in a communication, we revealed that 6-cyano-UMP
‡
(
5) undergoes hydrolysis in the active site of ODCase and
§
1
7,18
generates BMP (4).
The conversion of compound 5 into 4
Kyoto, 606-8502, Japan.
|
Toronto General Research Institute.
by ODCase is an unprecedented biochemical event that estab-
1
0.1021/jm060202r CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/19/2006

4
938 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Poduch et al.
instruments attracted more attention to this technique as a means
2
0-22
to study enzyme inhibition kinetics.
Isothermal calorim-
etry’s advantages over other commonly used techniques is its
ability to detect heat changes during enzymatic reactions under
conditions such as poor transparency of reaction solution and
free of any chromogenic, fluorogenic, or radioisotope-enriched
(labeled) ligands. Heat changes are fundamental in any bio-
chemical event, both during the binding events as well as
biochemical reactions; thus ITC is an ideal method for the
characterization of enzyme reactions.
Here, we present the details of the design of novel nucleotide
inhibitors of ODCase and the determination of enzyme inhibition
kinetics by isothermal titration calorimetry assays for the
compounds 3, 5, and 6. Findings revealed here set a stage for
the design of novel inhibitors of ODCase for probing further
details on this enzyme’s mechanism as well as novel means to
explore enzyme inhibition kinetics in the absence of suitable
labeled substrates.
Figure 1. Models of 1-methylorotate (A), 6-hydroxy-1-methyl uracil
B), 6-cyano-1-methyluracil (C), and 6-amino-1-methyluracil (D).
Isodensity surface on each model was rendered by use of electrostatic
potential (negative charge is represented in red and positive charge in
blue). Various substitutions at the C-6 position are shown by the arrow
at 5 o’clock position.
(
Experimental Section
General. Computer modeling was performed with Spartan
version 5.0 software, on an Octane dual processor computer or an
Onyx supercomputer at the Molecular Design and Information
Technology Center (http://www.phm.utoronto.ca/∼mdit). All syn-
thetic reactions were performed under nitrogen atmosphere. All
solvents and reagents were obtained from commercial sources.
Chromatographic purifications were performed on silica gel (60
Chart 1
-
Å, 70-230 mesh) or Dowex (OH ) ion-exchange resin. NMR
spectra were recorded on a Varian spectrometer (recorded at 300
1
13
and 400 MHz for H, 75 and 100 MHz for C, and 121.46 MHz
for 31P). Chemical shifts are reported in δ (parts per million, ppm)
with tetramethylsilane as a reference for the H and C spectra,
and phosphoric acid as an external standard for P spectra.
-Azauridine was purchased from Aldrich, and other chemicals were
1
13
3
1
6
either purchased or synthesized from commercially available starting
materials. Mass spectra for organic compounds were obtained on
a Q-Star mass spectrometer by either the ESI or EI technique.
Mononucleotide derivatives were converted into the corresponding
lished its “catalytic” promiscuity for this fascinating enzyme.
ODCase converted 5 into 4 very slowly over a period of 24-
3
6 h. It remained unclear if compound 5 itself functions as an
inhibitor of ODCase. Here, we reveal the complete details of
inhibition kinetics for 6-cyano-UMP (5) for the first time. In
an effort to conduct enzyme inhibition studies, we discovered
that there are no reliable nonradiological assays for ODCase to
investigate inhibition profiles. Enzyme assays using 1 C-labeled
OMP have been used, but the availability of this substrate is an
issue for routine enzyme assays. Spectrophotometric assays have
been used for ODCase characterization, but when the substrate,
inhibitor, and product have overlapping spectral absorption
properties (λmax), the spectrophotometric assay is not a method
of choice. An alternative nonradioactive method to study
ODCase-catalyzed reaction has been proposed by Krungkrai et
ammonium salts by neutralization with 0.5 M NH
°C and freeze-dried to obtain the ammonium salts as powder.
4
OH solution at
0
For enzymology, orotidine monophosphate trisodium salt (OMP,
1) and Tris buffer were purchased from Sigma. DL-Dithiothreitol
(DTT) was obtained from Fluka Chemicals. Methanobacterium
thermoautotrophicum ODCase (EC 4.1.1.23) was expressed in E.
coli and purified as described previously.23 All ligands used for
the calorimetric measurements were prepared as concentrated stock
solutions and stored at -20 °C.
4
Computer Modeling. Models of 6-carboxy-1-methyluracil,
6
1
-hydroxy-1-methyluracil, 6-cyano-1-methyluracil, and 6-amino-
-methyluracil were used as mimics of OMP, BMP (protonated
form), 6-cyano-UMP, and 6-amino-UMP, respectively, to under-
stand the effects of the C-6 substitution (Figure 1). Position 1 in
these nucleic bases were substituted with a methyl group (in place
of a ribos-1-yl 5-monophosphate) and were geometry-optimized
1
9
19
al. using high-performance liquid chromatography (HPLC).
High detection sensitivity (picomolar range) and no need for
costly radiolabeled reagents make this method appealing,
although the preparation of the sample for HPLC analysis is
quite lengthy. The applicability of this method to kinetic
investigations of ODCase is limited to monitoring the synthesis
of UMP only. The HPLC technique may not be easily adaptable
to study enzyme inhibition kinetics. A simple and sensitive
method with limited sample preparation and a universal detec-
tion system was needed.
Thus, we used isothermal titration calorimetry (ITC) to
develop a reliable assay and to study the inhibitory properties.
Although ITC is used extensively to study thermodynamic
properties of biological systems, including enzymes, this
technique has not been explored in-depth to investigate enzyme
inhibitions. The recent development of ultrasensitive ITC
2
4
with AM1 Hamiltonian as implemented in Spartan version 5.0.
The electrostatic potential surfaces were constructed after the
geometry optimization of the compounds by mapping the electro-
static property on the isodensity surface. The isovalue was set at
3
0
.002 electron/Bohr . The volumes for 1-methyluracil, 6-amino-1-
methyluracil, 6-hydroxy-1-methyluracil (protonated species), 6-cy-
ano-1-methyluracil, 6-hydroxy-1-methyluracil (deprotonated spe-
cies), and 1-methylorotate were 113.59, 141.13, 122.17, 132.68,
3
1
6
14.81, and 140.11 Å , respectively. Substructure volumes for
-carboxylate, 6-hydroxyl, 6-cyano, and 6-amino groups were
derived by subtracting the volume of 1-methyluracil from those of
the appropriate nucleic base derivatives.
Syntheses: 6-Cyanouridine 5′-O-Monophosphate (5). Com-
pound 5 was synthesized as reported earlier.17

Inhibitors of Orotidine Monophosphate Decarboxylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4939
experiment:25
6
-Azido-5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene-
uridine (8). Compound 7 (0.25 g, 0.48 mmol) was dissolved in
dry N,N-dimethylformamide (DMF) (3 mL), and NaN (0.034 g,
.53 mmol) was added. The reaction mixture was stirred at room
dQ
dt
3
power (P) )
(1)
0
temperature for 1 h in the dark. Organic solvent was evaporated
under vacuum and the crude residue was dissolved in ethyl acetate
If one were to measure the heat from a biochemical reaction, the
amount of net heat (Qnet, simply shown here as Q) generated or
absorbed during the conversion of n moles of substrate into product
is expressed by
(
15 mL), washed with brine, and dried (Na
were evaporated and the crude was purified by silica gel column
chromatography (1% EtOH/CHCl ). Purification of the compound
2 4
SO ). Organic layers
3
and solvent evaporation were performed in the dark to yield the
compound 8 (0.19 g, 0.44 mmol) in 92% yield as a light brown
Q ) n∆Happ ) [P] V ∆H
app
(2)
t
0
1
solid. H NMR (CDCl
1
1
3
) δ 0.06 (s, 6H), 0.89 (s, 9H), 1.34 (s, 3H)
where, ∆Happ is molar enthalpy, [P]
t
is the concentration of the
product generated, V is the volume of the ITC cell (1.3 mL). Upon
.54 (s, 3H), 3.74-3.85 (m, 2H), 4.08-4.15 (m, 1H), 4.80 (dd,
H, J ) 4.8 and 6.3 Hz), 5.14 (dd, 1H, J ) 1.5 and 6.3 Hz), 5.50
s, 1H), 6.09 (dd, 1H, J ) 1.5 Hz), 9.12 (br s, 1H).
-Azidouridine (9). A stirred solution of compound 8 (0.300 g,
.683 mmol) was treated with 50% aqueous trifluoroacetic acid (3
0
rearrangement of eq 2, a relationship between the rate of change
in the product concentration, the molar enthalpy, and the thermal
power is obtained:
(
6
0
mL) at 0 °C. The reaction mixture was then brought to room
temperature and was stirred for an additional hour. Evaporation of
the solvent and purification of the crude residue by column
dQ
dt
d[P]t
power (P) )
)
V ∆H
(3)
0
app
dt
chromatography (10-15% EtOH in CHCl
3
) gave the product 9
0.17 g, 0.61 mmol) in 89% yield as a light brown solid. UV (H O)
O) δ 3.77 (dd, 1H, J ) 5.4 and 12.0
(
2
To determine ∆Happ, the total heat (Q) generated by the reaction is
divided by the number of moles of substrate (n) converted into
product22 (assuming complete substrate depletion). Reaction rate
at time t is calculated from eq 4 and is plotted as a function of [S]:
1
λ
max ) 285 nm; H NMR (D
2
Hz), 3.89-4.00 (m, 2H), 4.43 (t, J ) 6.9 Hz, 1H), 4.77 (dd, 1H, J
)
3.6 and 6.9 Hz), 5.76 (s, 1H), 6.07 (d, 1H, J ) 3.6 Hz). HRMS
+
(
9 11 5 6
ESI) calcd for C H N O Na (M + Na ) 308.0601, found
3
08.0597.
-Aminouridine 5′-O-Monophosphate (6). A stirred solution
of water (0.03 g, 1.89 mmol) and POCl (0.28 mL, 2.97 mmol) in
anhydrous acetonitrile (3 mL) was treated with pyridine (0.26 mL,
.24 mmol) at 0 °C and stirred for 10 min. Compound 9 was added
0.25 g, 0.68 mmol) and the mixture was stirred for an additional
h at 0 °C. The reaction mixture was quenched with 25 mL of
d[P]t
1
dQ
Rt )
)
(4)
6
dt
V ∆H dt
0
app
3
V0 is the volume of the sample cell, which is a constant, and Q and
t are values measured during the experiment. ∆Happ is calculated
3
(
5
as described above. Substrate concentration [S] at any instance t
t
is determined from
cold water and the stirring was continued for another hour.
Evaporation of the solvent and purification of the crude by column
chromatography (Dowex ion-exchange basic resin, 0.1 M formic
t
P dt
∫
0
[
S] ) [S]
-
t)0
(5)
t
acid) gave the mononucleotide (0.23 g, 0.63 mmol) in 60% yield
V ∆H
0
app
1
as syrup. UV (H
(
1
2
O) λmax ) 283 nm; H NMR (D
2
O) δ 3.78-3.85
m, 1H), 3.89-4.00 (m, 2H), 4.34 (t, J ) 6.9 Hz 1H), 4.80 (m,
where [S]t)0 is the initial substrate concentration.26
By use of the enzyme concentration [E], the catalytic rate kcat
and the Michaelis constant KM can be determined by fitting [S]t
and R to the Michaelis-Menten equation:
H), 5.73 (s, 1H), 6.04 (br s, 1H). 3 P NMR (D
1
O) δ (ppm) 2.47.
2
-
HRMS (ESI, negative) calcd for C
9
H
11
N
O
5 9
P (M ) 364.0299, found
364.0307.
t
The mononucleotide (0.06 g, 0.15 mmol) was then dissolved in
0% aqueous methanol and 10% Pd/C (10 mg) was added. The
5
kcat[E][S]t
V )
(6)
reaction mixture was stirred for 2 h under hydrogen atmosphere at
room temperature. The mixture was filtered through Celite and the
KM + [S]t
solvent was evaporated to dryness to give compound 6 as syrup in
In the presence of inhibitor, the rate is calculated by fitting the
data to the competitive inhibition equation:
1
8
5% yield (43 mg, 0.13 mmol). UV (H
NMR (D
J ) 6.6 Hz, 1H), 4.81 (s, 1H), 6.20 (d, J ) 6.6 Hz, 1H). HRMS
2
O) λmax ) 270 nm; H
2
O) δ 3.96-4.05 (m, 2H), 4.12-4.24 (m, 2H), 4.51 (t,
kcat[E][S]
t
-
(
ESI, negative) calcd for C
38.0403.
-Azauridine 5′-O-Monophosphate (3). A stirred solution of
water (0.034 g, 1.89 mmol) and POCl (0.28 mL, 2.97 mmol) in
anhydrous acetonitrile (3 mL) was treated with pyridine (0.261 mL,
.24 mmol) at 0 °C and stirred for 10 min. Compound 10 (0.200
9
H
13
N
3
O
9
P (M ) 338.0394, found
V )
(7)
[
Ki
I]
3
KM 1 +
+ [S]t
(
)
6
3
The inhibition constant (K ) is the only fitting parameter, and the
i
3
values of k , K , ∆Happ, and [I] (determined from separate
cat
M
experiments) are used as variables.26
g, 0.81 mmol) was added and the mixture was stirred for an
additional 5 h at 0 °C. The reaction mixture was quenched with 25
mL of cold water and stirring was continued for an additional hour.
Evaporation of solvent and purification of the crude by column
chromatography (Dowex ion-exchange basic resin, 0.1M formic
Enzymology. The substrate OMP (1) solution was prepared with
dialysis buffer (vide infra) as a concentrated 20 mM stock solution
and diluted as required. The OMP (1) stock solution was stored in
aliquots at -20 °C. Concentrated solutions of all inhibitors were
prepared in the same buffer as the substrate and the enzyme.
The enzyme was dialyzed against Tris buffer (50 mM Tris, pH
7.5) by use of DiaLyzer dialysis cassettes (MW cutoff 10 000) from
Pierce. The reaction buffer for ITC experiments was prepared with
triple-distilled water. ODCase samples for each assay were prepared
by diluting enzyme stock solution with Tris buffer supplemented
with DTT (1 mM final concentration). Substrate and inhibitors were
dissolved in the buffer collected from the dialysis, to maintain
homogeneity between the buffers used for the enzyme and the
ligands. Prior to the injections in the microcalorimeter, enzyme and
acid) gave the mononucleotide 3 (0.17 g, 0.51 mmol) in 63% yield
1
as a syrup. UV (H
3
2
O, pH ) 6) λmax ) 260 nm; H NMR (D
2
O) δ
.85-4.07 (m, 3H), 4.33 (t, J ) 5.7, 1H), 4.46 (dd, 1H, J ) 3.0
31
and 5.1 Hz), 5.96 (d, 1H, J ) 3.0 Hz), 7.46 (s, 1H). P NMR
-
(
(
2 4
D O) δ 0.55. MS (ESI, negative) m/z 342 ([MNH ] , 100), 324
-
-
M , 62), 404 ([M2K] , 30).
Isothermal Titration Calorimetry and Enzyme Kinetics. The
thermal power (microcalories/second) is directly proportional to the
rate of the reaction (R ). This relationship allows derivation of kinetic
parameters (kcat and K ), and molar enthalpy (∆Happ) in a single
t
M

4
940 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Poduch et al.
ligand samples were degassed with stirring for 5 min by use of a
vacuum degasser (ThermoVac, MicroCal). The degassed solution
was transferred to the sample cell via a gastight Hamilton syringe.
Experimental Conditions. All enzyme reactions were performed
at 55 °C, the optimal growth temperature of M. thermoautotrophi-
cum. Titrations were performed on an isothermal titration calorim-
eter, VP-ITC instrument from MicroCal (Northampton, MA). In
all cases, the calorimetric cell contained the enzyme solution while
the ligand(s) were loaded into the automatic injection syringe. The
reference cell was filled with degassed reaction buffer. In the case
of control reaction (without inhibitor), the syringe contained the
substrate OMP (1) only. Substrate and inhibitor mixtures were
prepared and loaded into the injection syringe to monitor enzyme
activity in the presence of inhibitor. The concentrations of the
substrate and inhibitor in the syringe were calculated to give the
desired, final concentration of each component in the calorimetric
cell after one injection. Single injection method was used in all
studies. Substrate was injected into the enzyme solution in a single
injection, and change in heat was measured as the reaction
progressed until the heat returned to baseline, indicating the end
of the reaction. After the preliminary equilibration period when
the instrument reached the final temperature of 55 °C, an additional
incubation mixtures was 50 µM, and the concentration of compound
5 was either 25 or 62.5 mM. The final enzyme concentration in
the reaction mixture was 20 nM, and the inhibitor concentrations
were 10 and 25 µM. Separate control samples without the inhibitor
were prepared for each of the two experiments. Reaction mixtures
were prepared in 50 mM Tris buffer containing 10 mM DTT and
80 mM NaCl. Enzyme activity was measured after 1-µL aliquots
of each sample were diluted to 2.5 mL with the reaction buffer (50
mM Tris and 1 mM DTT, pH 7.5). Each reaction was initiated by
the addition of 20 µL of 2.86 mM OMP. Enzyme activity remaining
in the samples containing inhibitor was calculated as a percentage
of control activity. The activities in each sample were derived from
the slopes of the linear portion of the reaction progress curves.
Data Analysis. Analyses of the raw data were performed with
Origin version 7.0 software.26 Baseline data points before the
injection and after the completion of reaction were removed prior
to any calculations. The data representing the heat of substrate
dilution and mixing were not considered in the data analyses. The
remaining data represent the reaction progress curve (Figure 2A).
These curves were analyzed in Enzyme Assay mode in Origin
software. The reaction progress curves at specific substrate
concentrations were considered for further analyses and data fitting.
60 s delay period was allowed to generate the baseline used in the
Kinetic parameters (KM and k ) as well as the enthalpy for the
cat
subsequent data analyses. After a 60 s delay, each reaction was
automatically initiated by a 20 µL injection of ligand into the
enzyme-containing cell. The stirring rate of the injection syringe
was set to 300 rpm. Heat flow (microcalories/second) was recorded
as a function of time. Data were collected every 2 s until the signal
reached the baseline and continued to be recorded for an additional
reaction of OMP decarboxylation were calculated by use of Origin
version 7.0 software. Direct determination of K values with this
i
software was performed by nonlinear squares fit of the data to the
competitive inhibition equation (eq 7) with K , k , and ∆H as
M
cat
app
fitting variables determined in the absence of inhibitor.
i
Another method employed to derive the inhibition constant (K )
6
0-100 s to generate the final baseline. The time of reaction varied
is by converting raw data from inhibited reactions to Michaelis-
Menten curves by use of the Substrate Only mode in the Origin
software to fit the raw data. This allowed for the computation of
the reaction rates at each substrate concentration in the absence
and presence of inhibitor. Since the reaction times ranged from 5
to 25 min, there was a large number of data points (collected every
from 5 to 25 min depending on the type of assay (i.e., enzyme
activity or inhibition assay).
Substrate Catalysis. The rate of OMP decarboxylation at 55
°
C was measured in 50 mM Tris buffer at pH 7.5. The reaction
was initiated by an injection of 20 µL of substrate (OMP, 1) into
the enzyme solution, and the change in thermal power was
monitored until the signal returned to baseline values. The enzyme
concentration in the cell was 20 nM and the substrate concentration
in the syringe was 2.86 mM (after injection, the final concentration
of the substrate was 40 µM in the reaction cell). The reaction rate
was also determined at 25 °C with 20 nM and 100 nM enzyme.
The effective substrate concentration was the same as that in the
assay above at 55 °C.
2
s). Several substrate concentrations, spanning the length of the
Michaelis-Menten curve (Figure 3B vs 4B), were selected for
subsequent data analysis (Figure 3B). The same set of substrate
concentrations and the corresponding reaction rates were selected
from each experiment. The inhibition constant was then derived
from a Dixon plot by plotting the reciprocal of the reaction rate
versus inhibitor concentrations. The value of the inhibition constant,
i
K , was determined as the point on the negative x-axis directly below
the intersection of the slopes.
Enzyme Inhibition. Inhibitory properties of the compounds 3,
5, and 6 were evaluated by the competitive inhibition method.
Mixtures of substrate and each inhibitor were prepared as a
concentrated solution to obtain desired dilutions in the calorimetric
cell after a 20 µL injection of the mixture. The final OMP
concentration was always 40 µM, unless specified otherwise. Final
concentrations of each inhibitor in the reaction cell were 10, 25,
and 50 µM for compound 3; 25, 50, 75, and 100 µM for compound
Results and Discussion
A survey of known inhibitors of ODCase revealed that BMP
(6-hydroxy-UMP or barbituric acid monophosphate) is the most
potent inhibitor known to date with a picomolar inhibition
8
constant against yeast ODCase. BMP inhibits yeast ODCase
5; and 0.5, 1.0, 1.5, and 2.5 µM for compound 6. Enzyme activity
-12
8
with a Ki of 8.8 × 10
M. We were interested in exploring
was monitored after the addition of the mixture of substrate and
inhibitor. The velocity of the reaction without the inhibitor (control)
was also measured. Reactions were performed in triplicate. The
inhibition constant for 6-aza-UMP (3) was calculated by two
methods. First, direct fitting of the data from inhibition assays was
performed by use of the fitting function in Origin software. In the
alternate substitutions at the C-6 position using the principles
of bioisosteric replacement and the size of the substitution. Thus,
we used the structures of OMP, the substrate for ODCase with
a carboxyl substitution at the C-6 position, and BMP, the most
potent inhibitor of ODCase with a hydroxyl substitution, to
compare the characteristics of these substitutions. Both sub-
structures carry a net negative charge and are comparable in
size. Upon investigation of the binding site of ODCase, it
appears that the space available for a C-6 substitution is not
large and should be within the volume limits dictated by the
known substrates and inhibitors of ODCase. We decided to use
the volumes of the substructures at the C-6 position as a means
to design novel inhibitors.
i
second method, K was derived from the Dixon plot for competitive
inhibition (Figure 3C).27 Fitting of data from the inhibition assay
with 6-cyano-UMP (5) with Origin software did not yield a good
fit. The Dixon method was used instead to derive the inhibition
i
constant for this compound (Figure 4C). K for 6-amino-UMP (6)
was determined by nonlinear least-squares fit of the experimental
data to the competitive inhibition equation in Origin 7.0 software.26
Compound 5 was also evaluated by ITC against ODCase (M.
thermoautotrophicum) for its potential time-dependent, tight binding
inhibition due to the conversion of 5 into 4.17 Samples of ODCase
1-Methyluracil, 6-hydroxy-1-methyluracil (representing the
protonated form of orotate), 6-cyano-1-methyluracil, 6-amino-
1-methyluracil, and 6-carboxy-1-methyluracil were used as
model systems for UMP (2), BMP (4), 6-cyano-UMP (5),
with and without compound 5 were incubated at room temperature,
and the enzyme activity was measured by ITC at different time
increments as described above. The enzyme concentration in the

Inhibitors of Orotidine Monophosphate Decarboxylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4941
Scheme 2a
3
volumes were 19.1 and 27.5 Å , respectively. Thus, compounds
5
and 6 were synthesized as potential inhibitors of ODCase.
Compound 5 was synthesized from uridine according to the
1
7
previously published report. Compound 6 was synthesized
from the 6-iodo derivative 7 (Scheme 2). Introduction of the
iodo moiety at the C-6 position of fully protected uridine was
achieved through LDA and iodine, and further substitution of
the iodo by the azido group produced the 6-azido derivative
2
8
8
.
Deprotection of the isopropylidene and tert-butyldimeth-
ylsilyl groups by use of trifluoroacetic acid yielded compound
. Monophosphorylation of 9 with phosphorus oxychloride to
9
afford its mononucleotide followed by the reduction of the azido
group with Pd/C gave the target compound 6 in good yield.2
The 6-azauracil derivative 3 was synthesized by subjecting 10
to phosphorus oxychloride conditions generating the target
mononucleotide. Mononucleotides (free forms) of compounds
9-31
3
and 6 were transformed into the corresponding ammonium
salts by neutralizing with 0.5 M NH4OH solution followed by
freeze-drying to obtain the corresponding ammonium salt.
ITC has been rarely used to investigate enzyme inhibition
profiles, although it found extensive applications in enzyme
kinetics and binding studies. Typically, in an isothermal
calorimetry experiment, the energy (∆H) for the association of
a protein and its ligand are in the range of 1-25 kcal/mol
3
2-35
depending on the experimental conditions.
(
Since the heat
Q) is directly related to the concentration of protein (Mtot),3
6
the assumption made above to use the linear region of the
thermogram to interpret enzyme kinetics is reasonable:
a
Reaction conditions: (a) NaN3, DMF, room temperature; (b) 50% TFA,
room temperature; (c) POCl3, pyridine, H2O, CH3CN, 0 °C; (d) H2, Pd/C,
MeOH, room temperature.
Q ) ∆HMtotV
(8)
6-amino-UMP (6), and OMP (1), respectively, to compare the
volumes of the substructures at the C-6 position. The substruc-
ture volumes for the 6-carboxyl and 6-hydroxyl (protonated
form) moieties were 26.5 and 8.6 Å , respectively, and these
where Q is heat (microcalories), ∆H is enthalpy (calories/mole),
Mtot represents total concentration of protein in the cell, and V
is the volume of the calorimetric cell. The average enthalpy
(∆H) produced by various ligands binding to ODCase was in
the range of 0.5-1.0 kcal/mol, in our experiments, and the
concentrations of the ligands and enzymes are in either
micromolar or millimolar range. 6-Aza-UMP (3, 35 µM final
concentration) produced 0.98 kcal/mol corresponding to 49 µcal
of heat/injection in the presence of equimolar ODCase (35 µM
final concentration) (Figure S-1A, Supporting Information).
Compound 5 (final concentration 122 µM after first injection)
upon titration with ODCase (50 µM final concentration)
produced a total of 0.62 kcal/mol (∆H), and the corresponding
heat (Q) for this reaction was 111 µcal (Figure S-1B, Supporting
Information). This enthalpy and the corresponding heat are a
result of the first injection in the thermogram. It should be noted
that the binding enthalpies (∆H) for 6-aza-UMP and 6-cyano-
3
substitutions represent the volumes in the substrate OMP and
the most potent inhibitor, BMP. In fact, the volume of the
deprotonated 6-hydroxyl substitution, which is the most abun-
3
dant form, is merely 1.2 Å larger than that of the unsubstituted
1
-methyluracil. In terms of the inhibitory potency, UMP is a
very poor inhibitor of ODCase compared to BMP, despite such
small differences in the volume of substitutions. This led to the
reasoning that the negative charge at the C-6 position on BMP
plays a very important role in enhancing its affinity to ODCase.
Thus, our focus has been to incorporate these tight volume limits
in the design of a substitution at the C-6 position to create
inhibitors of ODCase. On the basis of this information, we
decided to investigate 6-cyano and 6-amino groups as potential
substitutions on UMP, and their corresponding substructure
Figure 2. (A) Thermal power change as a function of time during the conversion of 40 µM OMP to UMP catalyzed by ODCase. Reaction was
initiated by injection of 20 µL of 2.86 mM OMP into 20 nM enzyme. (B) Nonlinear least-squares fit (solid line) of experimental data (dotted line)
to Michaelis-Menten equation to determine kinetic parameters of the ODCase reaction.

4
942 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Poduch et al.
Table 1. Kinetic Parameters of ODCase
ODCase
source
Km
kcat
-1
kcat/Km
-1 -1
6
compound
method
(×10- M)
(s
)
(M
s
)
ref
6
OMP at 55 °C
OMP at 25 °C
OMP at 25 °C
OMP at 25 °C
ITC
ITC
Mta
Mt
5 ( 1
21
2
42
7.5
4.2 × 10
6
2
1 × 10
b
7
UV, radioactivity
UV
Sc
Pf
0.7*
13 ( 1
6 × 10
40, 41
14
c
5
5.6 × 10
a
Methanobacterium thermoautothropicum. b Saccharomyces cereVisiae. c Plasmodium falciparum.
UMP were obtained in the presence of 14 and 50 µM ODCase,
while enzyme kinetics experiments required only 20 nM enzyme
in the reaction cell, or the protein concentration was lower by
approximately 3 orders of magnitude.
Considering the sensitivity of the VP-ITC instrument (0.1
µcal) and the direct relation between the binding heat and the
protein concentration (eq 8), the heat generated by ligand
binding to 20 nM ODCase enzyme would be well below the
36
instrument’s detection limits. In fact, we were unable to record
any heat exchange due to the presence of inhibitors 3 and 5,
when the enzyme concentration was less than 1 µM. Energies
involved in bond breaking/making are several orders of mag-
nitude higher; thus such heats could be used in enzyme kinetics.
Monitoring enzyme kinetics and inhibitions as reported here is
a feasible means to study various enzyme inhibitors by ITC.
Initially, optimal concentrations of the enzyme and the
substrate were determined for the conversion of OMP to UMP
by ODCase via ITC. Figure 2A shows a typical thermogram
representing heat flow as a function of time for the ODCase-
catalyzed reaction. The initial trace from 0 to 60 s represents
the baseline, followed by a small peak formed due to substrate
dilution effect after the injection of the substrate (Figure 2A).
Negative thermal power (P) after the substrate injection indicated
that the reaction is exothermic. Maximum heat was released at
about 120 s and the signal returned gradually to the baseline
almost in a linear fashion after the substrate was consumed,
indicating the end of the reaction. The linear portion of the curve
was used in the subsequent data analyses (defined by two arrows
in Figure 2A). The average time required to catalyze the
conversion of 40 µM OMP into UMP at 55 °C was 5 min.
On the basis of the profile in Figure 2A, rates of the reaction
at various substrate concentrations were calculated by nonlinear
least-squares fit to the Michaelis-Menten equation (eq 6). The
output of this calculation is shown in Figure 2B. The Michaelis
constant KM for the M. thermoautotrophicum ODCase reaction
was 5 ( 1 µM and the catalytic rate constant (kcat) was 21 ( 2
-1
s
(Table 1). The enthalpy (∆Happ) of conversion of 40 µM
OMP to UMP was 5.0 ( 0.5 kcal/mol. The rate of catalysis
Figure 3. (A) Reaction progress curves in the absence and presence
of different concentrations of 6-Aza UMP. (B) Rate of reaction at
different substrate concentrations in the presence of 10 and 25 µM
(
kcat) for ODCase from M. thermoautotrophicum at 25 °C was
-1
2
s , noticeably lower than that at 55 °C (Table 1). The reaction
6
-Aza UMP. (C) Determination of inhibition constant for 6-Aza UMP
took significantly longer to complete (25 min) but enthalpy
∆Happ) and the KM were similar to those at 55 °C, as expected.
from Dixon plot.
(
The increase of enzyme concentration from 20 to 100 nM was
required to achieve a comparable reaction time (5 min) at 25
measurements with ITC and other known methodologies
imparted confidence into this method, and we proceeded to
investigate the inhibition kinetics.
-1
°
C to the one at 55 °C and the kcat remained at 2 s .
Kinetic parameters determined with the thermophilic ODCase
by ITC conform to those obtained by a standard UV-based assay
Competitive inhibition experiments were performed by inject-
ing substrate and each inhibitor (3, 5, or 6) simultaneously into
the enzyme solution. The inhibitor concentration was varied
while the concentrations of enzyme and substrate were kept
constant. Figures 3A, 4A, and 5A depict the thermograms
generated during OMP decarboxylation in the presence of the
inhibitors 6-aza-UMP (3), 6-cyano-UMP (5), and 6-amino-UMP
(6), respectively. As the inhibitor concentration was increased,
the thermograms exhibited a shallow elongated shape, indicating
the presence of fewer enzyme molecules available to catalyze
the reaction (thus taking a longer time to complete the reaction).
-
1
-1
where kcat was established to be 42 s and 5.4 s at 55 °C
and 25 °C, respectively.23 The Michaelis-Menten constant KM
for the same protein was determined as 10 µM. The KM (5
µM) assessment by the ITC method is also in good agreement
with the KM values determined for ODCase originating from
2
3
37-39
other organisms (Table 1).
Comparison of the ODCase
reaction rates at 25 and 55 °C yielded significantly different
values. The discrepancy is characteristic to thermophilic organ-
isms.4 Thus, the congruence between the ODCase kinetics
0

Inhibitors of Orotidine Monophosphate Decarboxylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4943
Table 2. Inhibition Constants for Various Inhibitors of ODCase
replotted against substrate concentration at various concentra-
tions of the inhibitors to extract inhibition constants (Figure 3B
and 4B). On the basis of these assumptions and observations,
we interpreted the isothermal calorimetry data to understand
the enzyme inhibition profiles.
ODCase
compound
source
Ki (M)
ref
a
-6
-9
-6
6
6
6
-aza-UMP (3) at 55 °C
-aza-UMP (3) at 25 °C
-aza-UMP (3) at 25 °C
Mt
Sc
Pf
Sc
12.4 × 10
64 × 10
b
40, 41
14
c
1.0 × 10
Initially, we considered a known inhibitor of ODCase, 6-aza-
UMP (3), and investigated its inhibition of M. thermoau-
totrophicum ODCase. The interactions of 6-aza-UMP with the
thermophilic ODCase were studied at 55 °C in a competitive
binding assay. Figure 3A shows the profiles of the heat flow
-
12
BMP (4) at 25 °C (radiolabeled)
8.8 × 10
8
-
6
6
6
-cyano-UMP (5) at 55 °C
-amino-UMP (6) at 55 °C
Mt
Mt
(29 ( 2) × 10
-
9
(840 ( 25) × 10
a
Methanobacterium thermoautothropicum. b Saccharomyces cereVisiae.
c
Plasmodium falciparum.
(P) during the enzymic reaction as a function of time at different
More heat was needed to balance the temperature differences
between the sample and reference cells, resulting in less negative
power with increasing inhibitor concentrations.
concentrations of 6-aza-UMP. Upon replotting of the linear
regions from the thermograms in terms of the rate of the reaction
versus substrate concentration, a decrease in the enzymic
reaction rate was observed as the inhibitor concentration was
increased (Figure 3B). By the method of Dixon, the inhibition
constant Ki was estimated to be 12.4 ( 0.7 µM for 6-aza-UMP
against M. thermoautotrophicum ODCase at 55 °C (Figure 3C,
The “intersecting” nature of the linear regions of thermograms
after the completion of the injection prompted us to take a closer
look at these curves and explore their use for the interpretation
of enzyme inhibition kinetics (Figures 3A, 4A, and 5A). If one
assumes that the power compensation imparted by ITC (y-axis)
during the enzymic reaction is dominated by the heat generated
from the enzymatic reaction, and the linear regions in the
thermograms reflect the “steady-state” conditions, these curves
depict the response of the reaction rate due to various inhibitor
concentrations as a function of time. Rate of reaction can be
2
7
Table 2). We also used a nonlinear fitting function in the
Origin software and arrived at a Ki of 11 ( 2 µM, confirming
2
6
the value from the Dixon plot. 6-Aza-UMP is a well-known
-
8
inhibitor of ODCase with Ki ) 6.4 × 10 M (yeast enzyme,
Table 2). There is a difference of 2 orders of magnitude
in the inhibition constant Ki for ODCase from M. thermoau-
8
,41,42
Figure 4. (A) Thermograms for the enzymatic conversion of OMP to UMP in the presence of different concentrations of 5. (B) Michaelis-Menten
curves representing the reaction rates at various substrate concentrations in the presence of 5. (C) Determination of the inhibition constant, K . (D)
i
Time-dependent enzyme activity as a function of time, when the enzyme (20 nM) was incubated in the presence of 5. (E, F) Time required to
inactivate 50% of ODCase activity (t1/2) by 5 via its conversion into BMP (4) by ODCase.

4
944 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Poduch et al.
Figure 5. (A) Thermograms of the ODCase reaction in the presence of different concentrations of 6-amino-UMP (6) as a function of time. (B)
Reaction rate at various substrate concentrations in the absence and presence of inhibitor.
totrophicum and that from Saccharomyces cereVisiae. The
difference in inhibition constants might be due to temperature
differences for the assay as well as difference in the susceptibil-
ity to the inhibitors by ODCase from different sources (Table
used by enzymologists and several orders of magnitude less
than a typical ITC thermodynamics experiment. The measure-
ment of the reaction rate is direct and does not require additional
coupled reactions. Additionally, these are undisturbed by
overlapping absorption characteristics of substrate and inhibitor
molecules.
2
).
We reported recently that 6-cyano-UMP is a substrate for
ODCase, undergoing very slow conversion into BMP (24-48
h or longer).17 We were interested in understanding its binding
profile to ODCase and its behavior as a competitive inhibitor
based on the structural characteristics of the inhibitors. 6-Cyano-
UMP indeed exhibited competitive and reversible inhibition of
ODCase enzyme activity during the initial phases of the
incubation (<1 h). The inhibition was concentration-dependent,
with an observable loss of enzyme activity showing character-
istic profiles in the thermograms (Figure 4A). Subsequent
analyses of the linear regions revealed the inhibition constant
as Ki 29 ( 2 µM (Figure 4B,C). A time-dependent assay,
measuring the residual enzyme activity as a function of
incubation time by isothermal calorimetry, showed an “irrevers-
ible” pattern of loss of enzyme activity at two different
concentrations of 6-cyano-UMP, 10 and 25 µM (Figure 4D).
Using ITC, one can determine the inhibition constants for
various ODCase inhibitors, and further refinements will make
this method a very useful addition to the tool set available to
perform enzyme inhibition studies. Additionally, the principles
outlined here can be applied to other enzyme systems. In some
experiments relatively high concentrations of the inhibitor were
used without compromising the quality of measurements. This
is not detrimental as long as the energies of interaction between
the ligand and the enzyme are negligible in comparison to those
of the biochemical reaction. We also report for the first time
that 6-amino-UMP (6) acts as a nanomolar inhibitor of ODCase
and 6-cyano-UMP as a competitive inhibitor. The principles
and methods outlined here pave the way for the design of novel
ODCase inhibitors and provide new means to investigate
enzyme inhibition kinetics.
1
7
This pattern was similar to that in our earlier observations.
The enzyme lost more than 90% of its activity after 30 h of
incubation and did not recover the activity even after 4 days
Acknowledgment. We thank Dr. David Clarke for gener-
ously sharing the VP-ITC calorimeter for these experiments.
We are grateful to Ms. Wanda Gillon for her expert help with
enzyme purification. This work was supported by a grant from
the Canadian Institutes of Health Research (E.F.P. and L.P.K.).
L.P.K. is a recipient of an Rx&D HRF-CIHR research career
award and Premier’s Research Excellence award. An infra-
structure grant from the Ontario Innovation Trust provides
support for the Molecular Design and Information Technology
Centre. E.F.P. acknowledges support through the Canada
Research Chairs program. M.F. was a recipient of an Overseas
Fellowship and a Research Fellowship for young scientists from
the Japan Society for the Promotion of Science (JSPS).
(up to 108 h) of incubation. Conversion of 6-cyano-UMP to
BMP is independent of the concentration of the inhibitor in the
incubation mixture, and the rates are very similar at two different
concentrations, as expected if the concentration of the inhibitor
is well above the Ki value (Figure 4E). The time required to
inhibit 50% of the enzyme activity (t1/2) was estimated to be 5
h (Figure 4E). It should be noted that the “substrate” nature of
the 6-cyano-UMP was ignored during the interpretation of
6
-cyano-UMP as a competitive inhibitor and it was assumed
that the conversion of 6-cyano-UMP to BMP during the 600 s
of the assay was negligible. Thus, the above inhibition constant
Ki was computed with the assumption that 6-cyano-UMP (5)
interacted purely as a competitive inhibitor.
Supporting Information Available: Thermograms and iso-
therms illustrating the titration curves for 5-aza-UMP (3) and
6
-Amino-UMP (6) was similarly assayed as a competitive
6-cyano-UMP (5) by use of ODCase enzyme; HPLC and purity
inhibitor by coinjecting the inhibitor and substrate (Figure 5).
This compound showed a typical competitive inhibition profile.
The inhibition constant for 6-amino-UMP was determined as
data for compounds 5 and 6; and isotherms of titration with UMP
and multiple injections of OMP. This material is available free of
charge via the Internet at http://pubs.acs.org.
840 ( 25 nM. This molecule does not show any time-dependent
loss of activity, eliminating the possibility of covalent interac-
tions.
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