Inhibition of inosine monophosphate dehydrogenase (IMPDH) by the antiviral compound, 2-vinylinosine monophosphate

Article abstract of DOI:10.1016/S0968-0896(02)00247-X

A new enzyme-mediated synthesis of 2-vinylinosine, a compound with broad-spectrum RNA antiviral activity, is described. In order to understand the mechanism of action of this compound, we synthesized its monophosphate and investigated the behavior of that compound toward the enzyme, inosine monophosphate dehydrogenase (IMPDH), a key enzyme involved in the biosynthesis of nucleotides. 2-Vinylinosine monophosphate is a potent inhibitor of IMPDH with a K_i of 3.98 μM (k_inact=2.94×10^-2 s^-1). The antiviral activity of 2-vinylinosine may be explained by its cellular conversion to the monophosphate through the sequential action of PNP and HGPRT and subsequent inhibition of IMPDH by the cellularly produced 2-vinylinosine 5′-monophosphate.

Full text of DOI:10.1016/S0968-0896(02)00247-X

Bioorganic & Medicinal Chemistry 10 (2002) 3615–3618
Inhibition of Inosine Monophosphate Dehydrogenase (IMPDH) by
the Antiviral Compound, 2-Vinylinosine Monophosphate
Suresh Pal, Bindu Bera and Vasu Nair*
Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA
Received 22 January 2002; accepted 1 May 2002
Abstract—A new enzyme-mediated synthesis of 2-vinylinosine, a compound with broad-spectrum RNA antiviral activity, is
described. In order to understand the mechanism of action of this compound, we synthesized its monophosphate and investigated
the behavior of that compound toward the enzyme, inosine monophosphate dehydrogenase (IMPDH), a key enzyme involved in
the biosynthesis of nucleotides. 2-Vinylinosine monophosphate is a potent inhibitor of IMPDH with a Ki of 3.98 mM
(kinact=2.94ꢀ10ꢁ2
50-monophosphate.
s
ꢁ1). The antiviral activity of 2-vinylinosine may be explained by its cellular conversion to the monophosphate
through the sequential action of PNP and HGPRT and subsequent inhibition of IMPDH by the cellularly produced 2-vinylinosine
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
by transferring hydride to NAD+ and then undergoing
hydration at the 2-position which is followed by ejection
of the enzyme. IMP, the natural substrate of IMPDH,
appears to protect the active site from inactivation by
inhibitors.11 X-ray crystallographic data suggest that a
covalent adduct may be formed between Cys-331 of
IMPDH and IMP during catalysis.13
Inosine 50-monophosphate dehydrogenase (IMPDH;
EC 1.2.1.205) catalyzes the NAD+-dependent oxidation
of inosine 50-monophosphate (IMP) to xanthine
50-monophosphate (XMP). Its role at the metabolic
branch point in the de novo purine nucleotide biosyn-
thetic pathway makes it a useful target in the develop-
ment of drugs for antiviral and anticancer
chemotherapy and in the immunosupressant area.1ꢁ8
The human enzyme exists in two isoforms: Type I which
is expressed in normal cells and Type II which pre-
dominates in neoplastic and fast replicating cells.1,3,4,9
The activity of the enzyme increases markedly in rapidly
proliferating cells such as leukemic cells and hepatoma
cells. Thus, inhibitors of IMPDH have been suggested
as being of considerable interest in cancer chemo-
therapy.4,7,8 The mechanism of action of IMPDH
involves interaction of the enzyme and coenzyme
(NAD+) complex at the 2-position of IMP, which is of
significance in the design of inhibitors of the
enzyme.10ꢁ12 It has been shown that IMPDH contains a
cysteine residue at the active site that attacks the
2-position of substrate IMP and forms a covalent com-
plex (E-IMP). This intermediate (E-IMP) forms XMP
Several compounds with antiviral activity have been
found to be inhibitors of IMPDH. For example, riba-
virin, a competitive inhibitor of IMPDH,14 has broad-
spectrum antiviral activity against DNA and RNA
viruses.15 Ribavirin has been approved as an inhaled
antiviral agent for the treatment of respiratory syncytial
infection and, orally in combination with alpha inter-
feron (IFN-a), for the treatment of chronic hepatitis C
virus (HCV) infection.15ꢁ17 3-Deazaguanosine mono-
phosphate, also a competitive inhibitor of IMPDH,16
has been discovered to be inhibitory towards several
RNA viruses.18 5-Ethynyl-1-b-d-ribo-furanosylimidazole-
4-carboxamide or EICAR, another inhibitor of IMPDH
as its MP, is a potential antiviral and antileukemic
agent.19,20 This paper reports on an enzyme-mediated
synthesis of 2-vinylinosine monophosphate (2-VIMP)
and a comprehensive study of its inhibition of the
enzyme, IMPDH. 2-Vinylinosine monophosphate
appears to be the cellularly active form of 2-vinylino-
sine, a compound discovered by us and found to have
broad-spectrum RNA antiviral activity.21
*Corresponding author. Tel.: +1-319-335-1364; fax: +1-319-353-2621;
e-mail: vasu-nair@uiowa.edu
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00247-X

3616
S.Pal et al./ Bioorg.Med.Chem.10 (2002) 3615–3618
Results
of time, the reaction was started by addition of the
substrate (IMP) and the enzymatic activity was deter-
mined by measuring the absorbance at 340 nm. During
the data collection less than 10% of the substrate was
consumed.
Materials
IMP, NAD+, and DTT were purchased from Sigma Che-
mical Co. (St. Louis, MO, USA). Escherichia coli cells were
grown at the University of Iowa Biocatalysis and Biopro-
cessing Center (Iowa City, IA, USA). Green A-Sepharose
gel was procured from Millipore (Bedford, MA, USA).
All other chemicals used were of analytical grade.
Another experiment of inhibition was also performed
separately where the enzyme was incubated with inhi-
bitor in presence of substrate (IMP) for various time
intervals to determine the extent of protection of the
enzyme by the substrate.
Purification of IMPDH
The enzyme was purified from E.coli B3 cells using a
modification of the procedure previously described.22
Bacterial cells (80 g) were harvested and disrupted (French
Press) in 200 mL of 50 mM Tris–HCl (pH 8.0), 0.15M
KCl, 2 mM 2-mercaptoethanol, 1 mM EDTA and 0.1
mM PMSF (buffer A). The resulting solution was cen-
trifuged at 7000 rpm in order to remove cell debris. The
supernatant was collected and saturated with solid
ammonium sulfate and the resulting solution was cen-
trifuged at 20,000 rpm for 30 min. The pellet was collected,
dissolved in buffer A and dialyzed first against buffer A
and then against buffer B (20 mM Tris–HCl buffer pH 8.0
containing 0.15 M KCl, 0.1 mM EDTA and 0.8 M urea).
The pellet, which contained IMPDH activity, was further
purified by loading on a Green A-Sepharose (Matrix
Green Resin, Millipore) column. The column was devel-
oped in buffer B. The bound enzyme was eluted with 0.15–
1.0 M KCl linear gradient in buffer B. The fractions with
IMPDH activity were concentrated and dialyzed against
50 mM Tris–HCl (pH 8.0), 0.1 M KCl and 1 mM EDTA.
A total of 80 mmol/ min activity units were recovered.
Inactivation, denaturation and renaturation of E. coli
IMPDH
E.coli IMPDH was incubated with 25 mM of 2-VIMP
for 4 h at 25 ꢂC. After this, urea was added to a final
concentration of 8 M and the resulting solution was
incubated for another 1 h at this temperature. The
denatured enzyme was dialyzed overnight against the
assay buffer [50 mM Tris–HCl (pH 8.0), 0.15 M KCl, 1
mM EDTA containing 1 mM DTT] with 3–4 inter-
mittent changes of buffer. A control sample of E.coli
IMPDH (in the absence of inhibitor 2-VIMP) was trea-
ted likewise following the same procedure. Enzymatic
activity for both control and treated enzyme was mon-
itored at each step (Fig. 1).
Synthesis of 2-vinylinosine and its 5-monophosphate.
6-Chloro-2-vinyl-9-(b-d-ribofuranosyl)purine (1) was
synthesized from 6-chloro-2-iodopurine ribonucleoside
triacetate by palladium-mediated coupling [with
Pd(CH3CN)2Cl2, tributylvinyltin, DMF, 90–95 ꢂC] fol-
lowed by careful deprotection of the acetate groups with
methanolic ammonia. The general procedure for the
coupling reaction has been previously described by us.23
The product was purified by HPLC on a C18 column
and eluted with 35% MeOH/H2O: mp 201–202 C
(decomp); UV (MeOH) lmax 277 (e 11,614), 228 (e 17,631);
1H NMR (CD3OD) d 3.86 (m, 1H), 3.90 (m, 1H), 4.15
(m, 1H), 4.38 (m, 1H), 4.72 (m, 1H), 5.70 (dd, J=1.4,
10.2 Hz, 1H), 6.11 (d, J=5.3 Hz, 1H), 6.64 (m, 1H),
6.81 (m, 1H), 8.73 (s, 1H); 13C NMR (CD3OD) d 62.8,
72.0, 75.8, 87.4, 90.8, 125.1, 131.4, 136.1, 147.0, 151.3,
153.1, 160.1.
Assay procedure for IMP dehydrogenase
The assays were typically performed with appropriate
amounts of enzyme in 50 mM Tris–HCl buffer pH 8.0
containing 0.15 M KCl, 1 mM DTT and 1 mM EDTA
using substrate concentrations of 0.5 mM IMP and 1.5
mM NAD+ at 25 C. The production of NADH was
monitored by the change in absorbance at 340 nm using
a Cary 3 UV spectrophotometer. One unit of enzyme
has been defined as the amount of enzyme that produces
1 mmol of NADH per min at 25 ꢂC under the assay
conditions.
2-Vinyl-9-(ꢀ-D-ribofuranosyl)hypoxanthine (2). To
a
solution of compound 1 (20 mg) in 0.1 M potassium
phosphate buffer pH 7.0 (3.0 mL) was added adenosine
deaminase (calf spleen, 250 units). The reaction mixture
was left for 4 days at 25 ꢂC. Progress of reaction was
followed by monitoring the bathochromic shift of the
Kinetics of IMPDH inhibition by 2-vinylinosine
monophosphate (2-VIMP)
Purified IMPDH was incubated with various con-
centrations of 2-VIMP at 25 ꢂC. After various intervals
Figure 1. Chemoenzymatic synthesis of IMPDH inhibitor, 2-vinylinosine monophosphate (2-VIMP). Reagents: (i) adenosine deaminase, 0.1 M
phosphate buffer; (ii) POCl3, triethyl phosphate.

S.Pal et al./ Bioorg.Med.Chem.10 (2002) 3615–3618
3617
lmax at 277 nm to a final value of about 300 nm. The
enzyme was filtered using an Amicon Ultra Filtration
device with YM-10 membrane and the solution was
purified by HPLC on a C18 column with water/metha-
nol. Pure product was eluted with 30% methanol/water
to give 13 mg (70% yield) of compound 2:23 mp 225–
230 ꢂC (decomp); UV (MeOH) lmax 300 (e 6286), 256 (e
6345); 1H NMR (CD3OD) d 3.30 (m, 1H), 3.83 (m, 1H),
4.11 (m, 1H), 4.34 (m, 1H), 4.65 (m, 1H), 5.83 (m, 1H),
6.03 (d, J=5.5 Hz, 1H), 6.57 (m, 2H), 8.30 (s, 1H); 13C
NMR (DMSO-d6) d 62.9, 72.0, 75.9, 87.3, 89.6, 124.7,
126.5, 130.8, 141.1, 150.2, 153.6, 158.7.
2-Vinyl-9-(ꢀ-D-ribofuranosyl) hypoxanthine 50-mono-
phosphate (3). To a mixture of compound 2 (52 mg,
0.17 mmol) and anhydrous triethyl phosphate (0.03 mL)
at 0 ꢂC was added anhydrous POCl3 (0.1 mL).24 The
reaction mixture was stirred at 0 ꢂC for 6 h and neu-
tralized with NaHCO3. Ether was added and the result-
ing precipitate was separated by centrifugation. The
white precipitate was dissolved in water and was pur-
ified by HPLC on a C18 column with water–methanol.
Pure product was eluted with 100% water. Removal of
the solvents under reduced pressure afforded compound
(3) (28 mg) in 43% yield: mp 228–230 ꢂC (decomp); UV
(H2O) lmax 290 (e 4937), 256 (e 7826); 1H NMR
(CD3OD) d 3.94 (m, 3H), 4.24 (m, 1H), 4.40 (m, 1H),
5.82 (m, 1H), 6.04 (d, J=5.8 Hz, 1H), 6.42 (m, 1H), 6.52
(m, 1H), 8.34 (s, 1H); 13C NMR (D2O) d 65.2, 71.7,
75.6, 85.4, 88.3, 123.5, 127.7, 129.6, 141.1, 150.7, 154.2,
159.6; 31P NMR (D2O): d 1.16.
Figure 2. Inhibition of E.coli IMPDH by 2-VIMP. Enzyme assay:
IMP=0.5 mM, NAD+=2.5 mM, (*) no inhibitor, horizontal line,
2-VIMP=(*) 0.71 mM, (!) 1.0 mM, (!) 1.67 mM, (*) 3.5 mM, (&)
5.0 mM.
Discussion
Chemoenzymatic synthesis of 2-vinylinosine and its
monophosphate
Figure 3. Plot of apparent rate constant (kobs
(2-VIMP) concentration.
) versus inhibitor
Our new synthesis of 2-vinylinosine (2) from the hydro-
lytic dechlorination of 6-chloro-2-vinyl-9-(b-d-ribofur-
anosyl) purine (1) with adenosine deaminase represents
an excellent approach to the synthesis of this and rela-
ted compounds through this chemoenzymatic approach.
Compound 2 was phosphorylated using standard phos-
phorylation methodology and the monophosphate was
purified by HPLC on a C18 column.
k1
kinact
ÀÀÀ*
E þ I )ÀÀÀ EÁI ÀÀÀ! E À I
kÀ1
.
where E I is a reversible complex and EꢁI is the irre-
versibly inactivated enzyme. Support for this type of
mechanism comes from the work on inactivation of
E. coli and human type-2 IMPDHs by 6-chloropurine
ribonucleoside monophosphate.25
Kinetics of inhibition/inactivation of IMPDH
The inactivation/inhibition of IMPDH followed psuedo
first-order kinetics as demonstrated by the exponential
loss of activity (Fig. 2). The data were analyzed using
the following equation:
The values of kobs for VIMP were then fitted into the
following equation:
kinact½Iꢀ
kobs
¼
lnðVt=V0Þ ¼ Àkobs
t
Ki þ ½Iꢀ
where Vt is the activity at time t and V0 is the activity at
time t=0. The replots of apparent rate constants (kobs
versus inhibitor (2-VIMP) concentration displayed
biphasic hyperbolic behavior (Fig. 3). This behavior
indicates that the inhibition/inactivation caused by
2-VIMP on IMPDH follows a two-step mechanism:
)
where [I] is the inhibitor concentration, kinact is the rate
constant for inactivation and Ki is the apparent dis-
.
sociation constant of E I. The values of kinact and Ki
were then calculated by plotting 1/kobs versus 1/[I]
values (Fig. 4).

3618
S.Pal et al./ Bioorg.Med.Chem.10 (2002) 3615–3618
cellular conversion to the monophosphate may be
through the sequential action of PNP and HGPRT.
Acknowledgements
We thank the National Institutes of Health (NIAID)
for support of these investigations. The authors express
their gratitude to Dr. R. C. Kamboj for technical
assistance.
References and Notes
1. Jackson, R. C.; Weber, G.; Morris, H. P. Nature 1975, 256,
331.
Figure 4. Plot of 1/Kobs versus 1/[I] for calculation of kinact and Ki.
2. De Clercq, E. Rev.Med.Virol. 1995, 5, 149.
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The data thus obtained show that 2-vinylinosine mono-
phosphate is a potent inhibitor of IMPDH with a Ki of
3.98 mM and a kinact of 2.94ꢀ10ꢁ2
s
ꢁ1. A side-by-side
comparison with the well-known IMPDH inhibitor,
6-chloropurine ribonucleoside monophosphate, gave
values for Ki and kinact indicative of a weaker inhibitor
(Ki=62.0 mM and kinact=7.60ꢀ10ꢁ2 sꢁ1).
Attempts made to recover the activity of VIMP-inacti-
vated enzyme were unsuccessful. On the other hand, a
control experiment of urea denaturation followed by
renaturation, showed almost 80% recovery of activity.
When IMPDH was incubated with various concentra-
tions of inhibitor, 2-VIMP, in the presence of excess of
its normal substrate, IMP, the enzyme inhibition/inac-
tivation again followed psuedo first-order kinetics, and
the data were as follows: Ki=163.0 mM and
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kinact=3.30ꢀ10ꢁ2
s
ꢁ1. These experiments suggest that
the binding of IMPDH with 2-VIMP may involve a
reversible competitive step which is followed by the
inactivation process. The results indicate that the inhi-
bition caused by 2-VIMP may be due to the formation
of an apparently irreversible covalent bond between the
inhibitor and IMPDH where the vinyl group conjugated
to the purine ring of VIMP acts as a Michael acceptor
presumably for Cys-331 of the enzyme.
2-Vinylinosine is not a substrate for adenosine kinase
but it is a cleaved slowly by purine nucleoside phos-
phorylase (PNP, calf spleen, 30% cleavage)26 and the
resulting base appears to be a substrate for hypoxan-
thine–guanine phosphoribosyl transferase (HGPRT)
(10% conversion).27
Conclusion
2-Vinylinosine has been found to possess broad-spec-
trum antiviral activity against a number of exotic RNA
viruses including JEV, PIC, PT, VEE and YF.21 The
mechanism of this antiviral activity may be associated
with the ability of the cellularly produced monophos-
phate of this compound to be an inhibitor of IMPDH.
Preliminary results from our laboratory suggest that its
26. Pal, S.; Nair, V. Biocatalysis and Biotransformation 1997,
15, 147.
27. Ali, L. Z.; Sloan, D. L. J.Biol.Chem. 1982, 257, 1149.