Inhibitors of inosine monophosphate dehydrogenase: Probes for antiviral drug discovery

Article abstract of DOI:10.1081/NCN-200060308

The role of inosine monophosphate dehydrogenase (IMPDH) at the metabolic branch point of de novo purine nucleotide biosynthesis makes this enzyme an attractive probe for the discovery of antiviral compounds. Introduction of unsaturation at the 2-position of IMP, the natural substrate for IMPDH, produces Michael acceptors at that position, which results in these compounds being inhibitors of IMPDH. Consistent with this mechanism-based molecular design, some of the parent nucleosides exhibited antiviral activity. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200060308

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INHIBITORS OF INOSINE MONOPHOSPHATE
DEHYDROGENASE: PROBES FOR ANTIVIRAL DRUG
DISCOVERY
Sherry Story a , Mukta Gupta a , Eric Bonsu a & Vasu Nair a
a Department of Pharmaceutical and Biomedical Sciences , The University of Georgia ,
Athens, Georgia, USA
Published online: 15 Nov 2011.
To cite this article: Sherry Story , Mukta Gupta , Eric Bonsu & Vasu Nair (2005) INHIBITORS OF INOSINE MONOPHOSPHATE
DEHYDROGENASE: PROBES FOR ANTIVIRAL DRUG DISCOVERY, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 717-720, DOI:
10.1081/NCN-200060308
To link to this article: http://dx.doi.org/10.1081/NCN-200060308
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):717–720, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060308
INHIBITORS OF INOSINE MONOPHOSPHATE DEHYDROGENASE:
PROBES FOR ANTIVIRAL DRUG DISCOVERY
5
Sherry Story, Mukta Gupta, Eric Bonsu, and Vasu Nair
Department of
Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, Georgia, 30602,
USA
5
The role of inosine monophosphate dehydrogenase (IMPDH) at the metabolic branch point of de
novo purine nucleotide biosynthesis makes this enzyme an attractive probe for the discovery of antiviral
compounds. Introduction of unsaturation at the 2-position of IMP, the natural substrate for IMPDH,
produces Michael acceptors at that position, which results in these compounds being inhibitors of
IMPDH. Consistent with this mechanism-based molecular design, some of the parent nucleosides
exhibited antiviral activity.
Keywords IMPDH, Michael Acceptor, Antiviral Activity
INTRODUCTION
Inosine monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) catalyzes the
oxidation of inosine 5’-monophosphate (IMP) to xanthosine 5’-monophosphate
(XMP) with the involvement of the coenzyme, nicotinamide adenine dinucleotide
(NAD+), which is reduced to NADH.[1,2] The role of IMPDH at this metabolic
branch point of de novo purine nucleotide biosynthesis renders it a target for the
discovery of antiviral, anticancer, and immunosuppressive agents.[3] Consistent with
this is the observation that some inhibitors of IMPDH have been found to have
anticancer, antiviral, and immunosuppressive activity.[4–6] IMPDH is a sulfhydryl
enzyme in which the active-site Cys-331 residue may act as a nucleophilic
participant in interactions with inhibitors that are Michael acceptors.[7] This is also
consistent with the mechanism of substrate action of IMPDH, which involves
interaction of the enzyme and coenzyme (NAD+) complex at the 2-position of
Received 1 October 2004, accepted 21 January 2005.
This project was supported by Grant No. AI056540 from the NIH. Its contents are solely the responsibility of
the authors and do not necessarily represent the official views of the NIH. We thank Drs. E. Kern and R. Sidwell
for the antiviral screening data and Dr. D. Jung for technical assistance with the molecular modeling representation.
Address correspondence to Vasu Nair, Department of Pharmaceutical and Biomedical Sciences, Room 320,
R. C. Wilson PH, The University of Georgia, Athens, GA 30602, USA; E-mail: vnair@rx.uga.edu
717
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718
S. Story et al.
SCHEME 1 Example of the synthesis of a 2-substituted IMP analog.
IMP.[1] X-ray crystallographic data support the suggestion of a covalent adduct
formation between Cys-331 of IMPDH and IMP during catalysis.[8] In an ongoing
drug discovery program on antiviral compounds in our laboratory, we have utilized
IMPDH as a probe for the initial identification of potential antiviral molecules.
RESULTS AND DISCUSSION
Modification of the 2-position of IMP involving Michael acceptors was key in
the design of IMPDH inhibitors. Synthesis of the 2-substituted IMP analogs used
both chemical and enzymatic methodologies. An example of the synthesis is shown
in Scheme 1.[9] The key steps that were involved included a palladium-catalyzed
cross-coupling reaction, a modified Wittig reaction and a hydrolytic elimination
reaction with the enzyme adenosine deaminase.[10–12] For IMPDH studies, 5’-
phosphorylated compounds were needed and these were synthesized by standard
phosphorylation methods.
The IMPDH used in this study was isolated from E. coli B3 strain on our
laboratory.[7] A comparison of the kinetic parameters of this IMPDH with others is
shown in Table 1. IMPDH activity was measured by UV spectral methods by
monitoring the formation of NADH at 340 nm. In inhibition studies, the inhibition
reaction was initiated by the addition of various concentrations of inhibitor to the
appropriate enzyme solution.[7,13]

S. Story et al.
719
TABLE 1 Kinetic Parameters of Various IMPDHs
Enzyme
V (sÀ1
)
IMP (Km) mM
NAD+ (Km) mM
Human (Type I)a
Human (Type I)a
E. coli ( B3)b
1.8
1.4
6.0
1.9
14.2
9.2
12.0
1.7
42
32
315
150
Trichomonas foetusc
V (sÀ1) is turnover number per second.
aBiochem. Pharmacol 1995, 49, 1323–1329.
bNair lab data.
cBiochemistry 1999, 38, 15388–15397.
2-[2-(Z)-Fluorovinyl]inosine 5’-monophosphate (2-FVIMP) gave kinact and Ki
of 0.0269 sÀ1 and 1.11 mM, respectively, whereas the well-known IMPDH inhibitor,
6-chloropurine ribonucleoside monophosphate gave values of 0.076 minÀ1 and
62.0 mM. The inactivation of IMPDH by 2-FVIMP is time dependent and follows a
two-step mechanism. Another 2-substituted nucleoside monophosphate, 6-chloro-2-
ethynylpurine riboside monophosphate, was also a strong inhibitor of IMPDH with
values of kinact and Ki of 0.75 minÀ1 and 4.25 mM, respectively. The mechanism of
inhibition is similar to FVIMP (Figure 1). Comparisons were made with another
inhibitor of IMPDH, 4-carboxamido-5-hydroxyimidazole riboside monophosphate,
which exhibited a Ki of 0.5 nM.[14]
Antiviral studies of the parent nucleosides of these inhibitors of IMPDH
showed that 6-chloro-2-ethynylpurine riboside was active but toxic against the
vaccinia virus (HFF cells, IC50 > 0.8 mg/mL; CC50 3.0 mg/mL). Both 2-fluoro-
vinylinosine and its 6-chloropurine analog also exhibited activity towards the
vaccinia virus (SI < 4.5 and < 3.7). 4-Carboxamido-5-hydroxy-imidazole riboside
FIGURE 1 Representation of the interaction of FVIMP with IMPDH.

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S. Story et al.
showed good activity against HSV-2 (HFF cells, IC50 0.26 mg/mL; CC50 > 100 mg/
mL) and VZV (HFF cells, IC50 2.8 mg/mL; CC50 > 100 mg/mL). Other examples of
the use of IMPDH as a probe for antiviral drug discovery are in progress.
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