Inactivation of S-adenosyl-L-homocysteine hydrolase by 6′-cyano- 5′,6′-didehydro-6′-deoxyhomoadenosine and 6′-chloro- 6′-cyano-5′,6′-didehydro-6′-deoxyhomoadenosine. Antiviral and cytotoxic effects

Article abstract of DOI:10.1021/jm051023x

6′-Cyano-5′,6′-didehydro-6′-deoxyhomoadenosine (E)-1, (Z)-1, and 6′-chloro-6′-cyano-5′,6′-didehydro- 6′-deoxyhomoadenosine (E)-2 were synthesized and tested as new mechanism-based inhibitors of AdoHcy hydrolase. Nucleoside (E)-1 was identified as a type I

Full text of DOI:10.1021/jm051023x

© Copyright 2006 by the American Chemical Society
Volume 49, Number 4
February 23, 2006
Letters
Chart 1. Structures of Type II Irreversible Inhibitors of
AdoHcy Hydrolase and Structures of the Targeted Compounds 1
and 2.
Inactivation of S-Adenosyl-L-homocysteine
Hydrolase by 6′-Cyano-5′,6′-didehydro-
6′-deoxyhomoadenosine and 6′-Chloro-6′-
cyano-5′,6′-didehydro-6′-deoxyhomoadenosine.
Antiviral and Cytotoxic Effects
Georges Guillerm,§ Murielle Muzard,§,* Ce´dric Glapski,§
Serge Pilard,† and Erik De Clercq‡
Laboratoire de Chimie bioorganique, UMR 6519, UFR Sciences,
B.P. 1039, 51687 Reims Cedex 2, France and Plate-Forme
Analytique, UniVersite´ de Picardie Jules Verne, 33 rue Saint-Leu,
80039 Amiens, France, and Rega Institute for Medical Research,
Katholieke UniVersiteit LeuVen, B-3000 LeuVen, Belgium
ReceiVed October 12, 2005
in its inactive NADH form (type I inhibitor)3 and/or they use
its 5′/6′- hydrolytic activity (type II inhibitor).4
Abstract: 6′-Cyano-5′,6′-didehydro-6′-deoxyhomoadenosine (E)-1, (Z)-
1, and 6′-chloro-6′-cyano-5′,6′-didehydro-6′-deoxyhomoadenosine (E)-2
were synthesized and tested as new mechanism-based inhibitors of
AdoHcy hydrolase. Nucleoside (E)-1 was identified as a type I inhibitor
of the enzyme, whereas inactivation of the enzyme by nucleosides (Z)-1
and (E)-2 was accompanied by the formation of a covalent labeling of
AdoHcy hydrolase.
Among them, AdoHcy hydrolase was irreversibly inactivated
by the (E)-5′,6′-didehydro-6′-chlorohomoadenosine (EDDClHA,
Chart 1).5 The type II inactivation observed in this case involved
a rapid addition of water to the 5′,6′ double bond of EDDClHA
and was concomitant with the reduction of the enzyme-bound
NAD+ to NADH. Dihalogenated analogues of EDDClHA were
also recognized as substrates for the hydrolytic activity of the
enzyme.6 On the other hand, we have shown that 5′-S-cyano-
5′-thioadenosine7 (Chart 1) caused inactivation of the enzyme
with only partial depletion of the enzyme’s cofactor NAD+.
The proposed mechanism consisted of enzyme-mediated addi-
tion of water to the thiocyano group and subsequent formation
of 5′-thioadenosine, a nucleoside which is known to induce a
specific covalent linkage of the enzyme.8
S-Adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1)
catalyzes the reversible hydrolysis of AdoHcy into adenosine
(Ado) and homocysteine (Hcy) and thus controls intracellular
levels of the two esssential metabolites, AdoHcy and Hcy.
Intracellular accumulation of AdoHcy provokes feedback inhibi-
tion of S-adenosylmethionine-dependent methylation reactions
which are essential for viral replication,1 whereas Hcy levels
appear to be a risk factor for cardiovascular diseases.2
A number of irreversible inhibitors of the enzyme, analogues
of Ado, have been identified: they act as substrates for the 3′-
oxidative activity of AdoHcy hydrolase and keep the enzyme
These results led us to consider that nucleosides 1 and 2
(Chart 1) might also function as type II inhibitors by affecting
the 5′/6′-hydrolytic activity of AdoHcy hydrolase. In particular,
the 2-cyanovinyl conjugated group, which bears multiple
electrophilic centers, might be easily susceptible to nucleophilic
attack by the activated water molecule, or a nucleophilic residue,
at the active site of the enzyme.
Synthesis of 6′-cyano-5′,6′-didehydro-6′-deoxyhomoadenos-
ine 1 and 6′-chloro-6′-cyano-5′,6′-didehydro-6′-deoxyhomoad-
enosine 2 was accomplished via Wittig reaction of 5′-aldehyde
* To whom correspondence should be addressed. Phone: +33 3 26 91
32 38. Fax: +33 3 26 91 31 66. E-mail: murielle.muzard@univ-reims.fr.
§ Laboratoire de Chimie bioorganique, UMR 6519, UFR Sciences, Reims,
France.
† Plate-Forme Analytique, Universite´ de Picardie Jules Verne, Amiens,
France.
‡ Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Belgium.
10.1021/jm051023x CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/27/2006

1224 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Letters
Scheme 1a
Table 1. Ki and kinact Values and t1/2 for the Inhibitory Effect of 1 and 2
on AdoHcy Hydrolase
compound
Ki (µM)
kinact (min-1
)
t1/2 (min)
(E)-1
(Z)-1
(E)-2
-
-
17
-
10 [I]a ) 3 µM
6 [I]a ) 100 µM
164 [I]a ) 5 µM
-
0.007
a Concentrations of inhibitor used to determine t1/2
.
Table 2. Effect of (E)-1, (Z)-1, and (E)-2 on NAD+ Content of
AdoHcy Hydrolase
% NAD+ a
native enzyme
100
34
73
a Reagents: (a) DCC, DMSO, CHCl2COOH; (b) (CN)HCdPPh3; (c)
(CN)ClCdPPh3.
enzyme inactivated by (E)-1 (100)b
enzyme inactivated by (Z)-1 (60)b
enzyme inactivated by (E)-2 (100)b
72
Scheme 2a
a AdoHcy hydrolase (10 µM) was incubated with 4 mM of inhibitor in
assay buffer at 37 °C until significant or total inactivation was observed.
The NAD+ content was determined by HPLC as described in Supporting
Information. b % inactivation.
Table 3. Mass Excess Observed for Each Subunit of AdoHcy
Hydrolase
∆M observed, Da ( 2
enzyme inactivated by (E)-1, FW ) 288
enzyme inactivated by (Z)-1, FW ) 288
enzyme inactivated by (E)-2, FW ) 322.5
none
290
321.5
The nonseparable mixture of (E)-5 and (Z)-5 was treated with
acetic acid/water (4/1) to remove the methoxytrityl protecting
group (Scheme 2). Isomers (E)-7 and (Z)-7 thus obtained were
separated by silica gel chromatography.
Final deprotection of the isopropylidene group was achieved
with formic acid/water (1/1) and afforded the (E)-6′-cyano-5′,6′-
didehydro-6′-deoxyhomoadenosine (E)-1 and (Z)-6′-cyano-5′,6′-
didehydro-6′-deoxyhomoadenosine (Z)-1.
Accordingly, the nucleoside (E)-6 was isolated in a pure form
by careful chromatography after the Wittig reaction and then
deprotected in one step to obtain (E)-6′-chloro-6′-cyano-5′,6′-
didehydro-6′-deoxyhomoadenosine (E)-2. However, all our
attemps to isolate pure (Z)-6 failed, even after N6 deprotection.
In consequence, (Z)-2 could not be obtained as a pure compound
and thus was not tested.
a Reagents: (a) acetic acid/water 4/1 (b) formic acid/water 1/1.
4 (Scheme 1). Key intermediate 4 resulted from Moffatt
oxidation9 of compound 3, which was obtained by N6 protection
of 2′,3′-O-isopropylideneadenosine10 by a monomethoxytrityl
group. 4 was used without purification in the next step.
Treatement of 4 with commercial cyanomethylenetriphenylphos-
phorane gave 5 in good yield (91%). Analogue 6 was obtained
by the same procedure using cyanochloromethylenetriphe-
nylphosphorane11 (87%).
Incubation of purified recombinant placental AdoHcy hy-
drolase13 with nucleosides (E)-1, (Z)-1, and (E)-2 resulted in
time- and concentration-dependent inactivation of the enzyme
(Figure 1).
For each compound, the inactivation observed was irreversible
since enzyme activity could not be restored after dialysis (24
h) against assay buffer.
Compounds 5 and 6 were obtained as diastereoisomeric Z/E
mixtures, with E as the major diastereoisomer. Configuration
of the double bond for compound 6 was determined after
deprotection of a (Z+E) mixture and comparison of δH-5′ for
each isomer, in agreement with Tronchet et al. results for the
analogous thymidine12a and carbohydrate12b derivatives: δH-5′
(Z-2) ) 7.41 ppm > δH-5′ (E-2) ) 7.18 ppm.
Figure 1. Time-dependent inactivation of AdoHcy hydrolase with (E)-1 (panel a), (Z)-1 (panel b), and (E)-2 (panel c). AdoHcy hydrolase was
incubated with inhibitors at indicated concentrations in assay buffer at 37 °C, and remaining activity was determined in the synthetic direction as
described in Supporting Information.

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1225
Table 4. Antiviral Activity and Cytotoxicity for Compounds (E)-1, (Z)-1, and (E)-2 and Controls
antiviral activity EC50 (µg/mL)a
cytotoxicity (µg/mL)
TK+ VZV
OKA strain
TK- VZV
07/10 strain
cell morphology
cell growth
(CC50)c
compound
AD-169 strain
Davis strain
(MCC)b
(E)-1
(Z)-1
(E)-2
ganciclovir
cidofovir
acyclovir
brivudin
>20
>20
>20
3.2
>20
>20
>20
3.2
>20
>20
11
>20
>20
17
g20
100
100
g400
g400
>400
>400
11
>20
1.2
34
23
118
>200
0.49
0.64
0.22
0.0051
14
149
a Effective concentration required to reduce virus plaque formation by 50%. b Minimun cytotoxic concentration that causes a microscopically detectable
alteration of cell morphology. c Cytotoxic concentration required to reduce cell growth by 50%.
Scheme 3. Proposed Mechanisms for Irreversible Inactivation
Inactivation of AdoHcy hydrolase by (E)-2 followed classical
of AdoHcy Hydrolase by Compounds (Z)-1 and (E)-2
pseudo-first-order kinetics (Figure 1, panel c); Ki and kinact values
were determined using the Kitz and Wilson method (Table 1).14
(E)-2 proved to be a potent AdoHcy hydrolase inhibitor
displaying Ki ) 17 µM but with a low value of 0.007 min-1
for kinact. Compounds (E)-1 and (Z)-1 led to a biphasic system
(Figure 1, panels a and b), showing pseudo-first-order kinetics
only during the first period of inactivation (t < 10 min). For
these two compounds, a t1/2 was determined which allowed
comparison of the inhibitory potency of each inhibitor (Table
1).
Experiments were then conducted in order to investigate the
mechanism of inactivation of the enzyme by inhibitors 1 and
2. In a first set of experiments, compounds (E)-1, (Z)-1, and
(E)-2 were incubated with reconstituted NAD+ form AdoHcy
hydrolase (100% NAD+).13 Significant inhibition of the enzyme
was observed with only partial depletion of its NAD+ content
(Table 2).
molecular weight of the inhibitors concerned. The mechanism
of inactivation seemed to proceed via a direct nucleophilic
addition of an amino acid residue on the inhibitor at the active
site of the enzyme. Crystallographic data on rat liver16 and
human17 AdoHcy hydrolase and site-directed mutagenesis
experiments18 allowed the identification of the amino acids
present at the active site. In particular, 55His and 186Lys (54His
and 185Lys in rat liver AdoHcy hydrolase) were identified as
essential amino acids involved in the substrate binding.18 They
were also assumed to participate as a base in their neutral form
to the catalytic process.19 Consequently, they could add to the
C-5′/C-6′ double bond of inhibitors or to the CN group (Scheme
3).
Interestingly, the reaction occurred only with (Z)-1 and (E)-2
featuring the CN group and nucleoside moiety on the same side
of the double bond. The CN group’s position might be essential
to induce the appropriate binding interactions. It might also act
as hydrogen-bond acceptor and become more reactive toward
enzymatic nucleophiles.
Compounds (E)-1, (Z)-1, and (E)-2 were evaluated for
antiviral activity against cytomegalovirus (CMV) and varicella-
zoster virus (VZV) in human embryonic lung cells (Table 4).
No specific activity antiviral effects (i.e. minimal antivirally
effective concentration g 5-fold lower than minimal cytotoxic
concentration) were noted for compounds against the tested
CMV or VZV strains.
In conclusion, we have identified three new irreversible
inhibitors of AdoHcy hydrolase: nucleoside (E)-1 is a type I
inhibitor, whereas nucleosides (E)-2 and (Z)-1 act as active-
site directed irreversible inhibitors and covalently modify the
enzyme without previous activation (3′-oxidation or 5′/6′-
hydrolytic activity).
These results suggest that the enzyme’s oxidative activity was
not the only pathway by which compounds 1 and 2 inactivated
AdoHcy hydrolase. Indeed nucleoside (E)-1 caused an important
conversion of the enzyme from its E-NAD+ to the E-NADH
form and appeared to be mainly a type I inhibitor. However,
there was clearly another way of inactivation which could use
the hydrolytic activity of the enzyme since the inactivated
AdoHcy hydrolase was not completely reduced to its E-NADH
form. Similar results were previously obtained by Wnuk et al.
with sugar-modified diene15a and enyne15b analogues of adenos-
ine. Inactivation of AdoHcy hydrolase with (Z)-1 and (E)-2 led
to more than 70% NAD+ unchanged.
The mechanism of inactivation was further investigated using
electrospray ionization mass spectrometry (ESI-MS) analysis
of the protein. An accurate molecular weight of AdoHcy
hydrolase subunit for the native or inactivated protein with (E)-
1, (Z)-1, and (E)-2 was determined from the wide distribution
of highly charged states observed in positive ion ESI-MS
analysis under acidic conditions. An algorithm (MaxEnt) based
on the maximum entropy method was used to produce true
molecular mass spectra from multiply charged electrospray
spectra. The native enzyme had a subunit molecular weight of
47586.7 ( 0.4 Da. The mass modifications (∆M) obtained after
total inactivation of enzyme by inhibitors are listed in Table 3.
After total inactivation of AdoHcy hydrolase by compound
(E)-1 only unchanged native enzyme was detected in the mass
spectrum. Thus inhibition of AdoHcy hydrolase occurred
without covalent bonding of (E)-1 or a corresponding metabolite.
This result reinforces the above hypothesis suggesting a type I
inhibitor behavior for (E)-1.
Acknowledgment. The authors thank le Conseil Ge´ne´ral de
la Marne for a doctoral grant for C.G.
On the other hand, inactivation of enzyme with cyanovinyl
nucleosides (Z)-1 and (E)-2 was accompanied by a specific
covalent linkage, the observed ∆M corresponding to the
Supporting Information Available: Experimental procedures
for synthesis of targeted compounds, AdoHcy hydrolase assay,

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