Evaluation of NAD(H) analogues as selective inhibitors for trypanosoma cruzi S-adenosylhomocysteine hydrolase

Article abstract of DOI:10.1080/15257770903044572

S-Adenosylhomocysteine (AdoHcy) hydrolases (SAHHs) from human sources (Hs-SAHHs) bind the cofactor NAD+ more tightly than several parasitic SAHHs by around 1000-fold. This property suggests the cofactor binding site of this essential enzyme as a potential

Full text of DOI:10.1080/15257770903044572

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Evaluation of NAD(H) Analogues as
Selective Inhibitors for Trypanosoma
Cruzi S-Adenosylhomocysteine Hydrolase
Qing-Shan Li a , Sumin Cai b , Jianwen Fang c , Ronald T. Borchardt a
, Krzysztof Kuczera b d , C. Russell Middaugh a & Richard L. Schowen
a b d
a Department of Pharmaceutical Chemistry, The University of
Kansas, Lawrence, Kansas, USA
b Department of Molecular Biosciences, The University of Kansas,
Lawrence, Kansas, USA
c Applied Bioinformatics Laboratory, The University of Kansas,
Lawrence, Kansas, USA
d Department of Chemistry, The University of Kansas, Lawrence,
Kansas, USA
Version of record first published: 29 Jul 2009.
To cite this article: Qing-Shan Li , Sumin Cai , Jianwen Fang , Ronald T. Borchardt , Krzysztof
Kuczera , C. Russell Middaugh & Richard L. Schowen (2009): Evaluation of NAD(H) Analogues
as Selective Inhibitors for Trypanosoma Cruzi S-Adenosylhomocysteine Hydrolase, Nucleosides,
Nucleotides and Nucleic Acids, 28:5-7, 473-484
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Nucleosides, Nucleotides and Nucleic Acids, 28:473–484, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903044572
EVALUATION OF NAD(H) ANALOGUES AS SELECTIVE INHIBITORS
FOR TRYPANOSOMA CRUZI S-ADENOSYLHOMOCYSTEINE
HYDROLASE
Qing-Shan Li,1 Sumin Cai,2 Jianwen Fang,3 Ronald T. Borchardt,1
Krzysztof Kuczera,2,4 C. Russell Middaugh,1 and Richard L. Schowen1,2,4
1Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas,
USA
2Department of Molecular Biosciences, The University of Kansas, Lawrence, Kansas, USA
3Applied Bioinformatics Laboratory, The University of Kansas, Lawrence, Kansas, USA
4Department of Chemistry, The University of Kansas, Lawrence, Kansas, USA
S-Adenosylhomocysteine (AdoHcy) hydrolases (SAHHs) from human sources (Hs-SAHHs) bind
2
the cofactor NAD+ more tightly than several parasitic SAHHs by around 1000-fold. This property
suggests the cofactor binding site of this essential enzyme as a potential anti-parasitic drug target,
e.g., against SAHH from Trypansoma cruzi (Tc-SAHH). The on-rate and off-rate constants
and the equilibrium dissociation constants were determined for NAD+/NADH analogues and
suggested that NADH analogues were the most promising for selective inhibition of Tc-SAHH. None
significantly inhibited Hs-SAHH while S-NADH and H-NADH (see Figure 1) reduced the catalytic
activity of Tc-SAHH to < 10% in six minutes of exposure.
Keywords S-adenosylhomocysteine hydrolase; enzyme inhibitors; anti-parasitic drugs;
cofactor-site targeting
INTRODUCTION
During his long and productive career in chemistry, biochemistry, and
medicinal chemistry, Professor Morris J. Robins has created novel nucleo-
sides and other structures, often with fascinating properties, particularly in
Received 10 March 2009; accepted 15 April 2009.
This research was supported by Grant GM-29332 from the National Institute of General Medical
Sciences and by the K-INBRE Bioinformatics Core, NIH Grant P20 RR016475.
This paper is dedicated to Professor Morris J. Robins, in honor of and in celebration of his 70th
birthday.
Address correspondence to R. L. Schowen, Department of Pharmaceutical Chemistry, 2095
Constant Avenue, The University of Kansas, Lawrence, KS, 66047, USA. E-mail: rschowen@ku.edu
473

474
Q.-S. Li et al.
their interactions with enzymes. It is our ambition in this paper to report
brief studies in which we attempt to follow in his footsteps.
The parasites Trypanosoma cruzi, Leishmania donovani and Plasmodium
falciparum, the agents of Chagas disease, Leishmaniasis, and malaria, re-
spectively, infect tens of millions in the less-developed world. Effective,
safe, and cheap anti-parasitic medicines are acutely required. S-adenosyl-
L-homocysteine hydrolase[1] SAHH, EC 3.3.1.1), which catalyzes the re-
versible conversion of AdoHcy to adenosine (Ado) and homocysteine
(Hcy), has emerged as a potential molecular target for design of anti-
parasitic drugs.[2–4] In general, SAHH inhibition raises the cellular con-
centration of AdoHcy, a potent product inhibitor of all S-adenosyl-L-
methionine (AdoMet)-dependent methyltransferases. The resulting inhibi-
tion of AdoMet-dependent methylation reactions[1] produces toxic effects.
Parasites express their own SAHHs[2–4] and anti-parasitic effects of known
SAHH inhibitors are observed in vitro and in vivo for a variety of parasites
including L. donovani,[5] P. falciparum,[6,7] and Trypanosoma species.[8,9] Ob-
viously, only inhibitors specific for the parasitic enzymes have the potential
for medical use.
X-ray crystallographic structures for Hs-SAHH,[10,11] Pf-SAHH (from P.
falciparum,[12]) and Tc-SAHH (from T. cruzi,[13]) are available. All these
enzymes are highly conserved homotetrameric proteins[12,14,15] with one
NAD+ molecule bound into the active site of each subunit. All residues
that interact directly with substrate or cofactor are conserved among these
three enzymes. Such observations and other data[16] suggest that selective
inhibition of parasitic enzymes poses a difficult challenge.
However, it has been reported that Ld-SAHH (from L. donovani) and
Tc-SAHH bind NAD+ much less tightly than Hs-SAHH,[17] although these
parasitic SAHHs bind the reduced form NADH very tightly and persist in
the inactive form containing reduced cofactor.[17] Therefore, the possibility
exists to develop highly selective inhibitors for parasitic SAHHs through
designing NAD+ analogues that bind to the cofactor binding site. In this
study, we have investigated NAD+ analogues obtained by modification of
the nicotinamide group and analogues obtained by modification of adenine
part (Figure 1) and compared their inhibitory effects on Hs- and Tc-SAHHs,
to provide information for eventual development of anti-parasitic drugs.
MATERIALS AND METHODS
NAD(H) Analogues (Figure 1)
Thionicotinamide adenine dinucleotide (S-NAD), 3-pyridinealdehyde
adenine dinucleotide (H-NAD), 3-acetylpyridine adenine dinucleotide (C-
NAD) and its reduced form (C-NADH), nicotinic acid adenine dinucleotide
(O-NAD), nicotinamide hypoxanthine dinucleotide (NHD) and its reduced

Selective Inhibition of T. cruzi SAHH
475
Name
Structure
Left: Thionicotinamide adenine dinucleotide
(S-NAD)
Right: 3-Pyridinealdehyde adenine dinucleotide
(H-NAD)
Left: Nicotinic acid adenine dinucleotide (O-NAD)
Right: 3-Acetylpyridine adenine dinucleotide
(C-NAD)
Left: Nicotinamide hypoxanthine dinucleotide
(NHD)
Right: Nicotinamide guanine dinucleotide (NGD)
Nicotinamide 1, N6-ethenoadenine dinucleotide
(etheno-NAD)
FIGURE 1 Structures of NAD+ and its analogues.

476
Q.-S. Li et al.
form (NHDH), nicotinamide guanine dinucleotide (NGD), and nicoti-
namide 1, N 6-ethenoadenine dinucleotide (etheno-NAD) were bought
from Sigma (St. Louis, MO, USA). The reduced forms of thionicotinamide
adenine dinucleotide (S-NADH) and 3-pyridinealdehyde adenine dinu-
cleotide (H-NADH) were prepared from the oxidized forms and ethanol
with catalysis by alcohol dehydrogenase (Sigma, A-3263) as follows. A
solution (usually 4 ml) containing 80 U/mL alcohol dehydrogenase, 40
mM ethanol, 4 mM of the oxidized analogue and 1 mM EDTA in 50 mM
phosphate buffer, pH 8.4, was incubated at 25◦C for 30 minutes and then
filtered on a Centricon column (30k, Millipore, Billerica, MA, USA) to
remove alcohol dehydrogenase. Reduction of the oxidized forms of H-NAD
and S-NAD was analyzed by HPLC by use of the procedure for enzyme-
activity assay described below. H-NAD was completely reduced, and S-NAD
was 90% reduced. Purity was in general determined by HPLC.
Expression and Purification of Hs-SAHH and Tc-SAHH
The expression and purification of Hs-SAHH and Tc-SAHH were the
same as previously described.[16–18]
Preparation of apo Forms of Hs-SAHH and Tc-SAHH
The apo forms of Hs-SAHH and Tc-SAHHs were prepared by the same
method as previously described.[17,18]
Enzyme Activity Assay
SAHH activity was assayed in the synthetic direction by measuring the
rate of formation of AdoHcy from Ado and Hcy using HPLC as previously
described.[19,20] The enzyme activity in the hydrolytic direction was deter-
mined by coupling the AdoHcy hydrolysis reaction to Ado deamination
catalyzed by Ado deaminase as previously described.[20]
Determination of the Degree of Occupancy of Enzymes
Reconstituted with NAD(H) Analogues
A solution (1 mL) containing 50 µM apo enzyme (all enzyme concen-
trations used are subunit concentrations), 500 µM of an NAD(H) analogue,
0.4 M (NH4)2SO4 and 1 mM EDTA in 50 mM phosphate buffer, pH
7.4, was incubated for 5 hours at 22◦C. The free analogue that remained
was then removed by passage through a PD10 column (GE Healthcare,
Uppsala, Sweden) which had been equilibrated with 50 mM phosphate
buffer at 4◦C. The enzyme-analogue complex was further concentrated by
Centricon treatment (30K, Millipore) at 4◦C and the filtrate was collected

Selective Inhibition of T. cruzi SAHH
477
to determine the amount of analogue bound. The concentrated solution of
enzyme-analogue complex was mixed with 2 volumes of ethanol, followed by
centrifugation. The precipitated enzyme was re-dissolved and treated with 2
volumes of ethanol again as above. The supernatants were combined and
dried in vacuum. The residue was then dissolved in water and applied to the
HPLC column using the same procedure as for the enzyme-activity assay.
The concentration of each compound was determined by comparison with
a calibration curve obtained from authentic samples.
Determination of the Rate Constant kon for Binding to apo
Enzyme of NAD+, NADH and NAD Analogues
Typical procedures for determination of the rate constant for binding
of NAD+, NGD, NHD, and etheno-NAD to Hs-SAHH and Tc-SAHH are as
follows.[17,18] One µM of Hs-SAHH apo enzyme was mixed with 5 µM NAD+
or 100 µM NAD+ analogue and the resulting solution was pre-incubated at
37◦C for 5 minutes. For binding to the Tc-SAHH apo enzyme of NAD+ and
its analogues, the procedure was the same except that the concentrations
of NAD+ or its analogues were 25 µM and 500 µM, respectively. Samples
were taken for activity assay after selected time periods. The measured
activities A were either converted to the amount of enzyme:NAD+ complex
or enzyme:analogue complex or used directly in Equation (1).
A = Af + (Ao − Af)[exp(−kappt)]
(1)
Here, A is the activity measured at time t, Ao is the activity at the apparent
time zero (reflecting reaction during the dead-time of the experiment), Af
is the activity at the end of the experiment, and kapp is a first-order rate con-
stant for association that generally will be a function of [NAD+] or [NAD+
analogue]. Under the above measurement conditions, Ao is quite small and
kapp is approximately proportional to [NAD+] or [NAD+ analogue]. An
apparent second-order rate constant kon for the association reaction was
therefore calculated as kon = kapp/[NAD+] or kon = kapp/[NAD+ analogue]
for the analogue and kon was used in the calculation of the equilibrium
dissociation constant.
For measurement of kon for NADH, the bound NADH was monitored
by fluorescence as previously described.[19]
Determination of the Rate Constants koff for Dissociation of NAD+
and NAD(H) Analogues from Their Complexes with Hs-SAHH and
Tc-SAHH
Determination of koff for the dissociation of NAD+, NGD, NHD, and
etheno-NAD from complexes with either Hs-SAHH or Tc-SAHH was carried
out as follows. A solution (usually 4 ml) containing 4 µM SAHH, 80 U/ml

478
Q.-S. Li et al.
alcohol dehydrogenase, 30 mM 2-propanol, 2 mM NADH, and 1 mM EDTA
in 50 mM phosphate buffer, pH7.4, was incubated at a chosen temperature.
Samples were taken after selected time periods for determination of enzyme
activity and NADH content. NADH concentration did not change during
all these experimental periods. As a control, the stability of SAHH was also
determined under the same conditions as above except in the absence of
NADH and alcohol dehydrogenase. The enzyme activities relative to initial
activity were plotted against time and fitted to A/Ao = exp(-kt), where A/Ao
is the relative activity and k is the enzyme inactivation rate constant. Enzyme
thermal inactivation rate constants were obtained by the same method. The
dissociation rate constants for NAD+ and NAD(H) analogues were obtained
by subtracting the enzyme thermal inactivation rate constants from the value
of k. For determination of koff for NADH analogues, the enzyme:(NADH
analogue) complex was mixed with 1 mM of NAD+ and the enzyme-activity
recovery rate indicated the rate of the analogue dissociation.
Dissociation Constant (Kd)
Equilibrium dissociation constants of NAD+, NGD, NHD, and etheno-
NAD were calculated from the relationship Kd = koff/kon. Dissociation
constants of NADH, NADH analogues, S-NAD, H-NAD, O-NAD and C-NAD
were calculated by fitting (Microcal Origin 7.0 software) the enzyme activity
as a function of ligand concentration in the presence of NAD+ to the
following equation, previously derived.[17]
Activity0/Activity = 1 + ([Ligand]/KLdigand)(KdNAD/(KdNAD + [NAD+])
“Activity0” refers to the enzyme activity in the absence of ligands other
NAD
than NAD+ and Kd
is the known dissociation constant of NAD+.
Inhibition of Tc-SAHH and Hs-SAHH by NAD(H) Analogues
The inhibition of Hs-SAHH and Tc-SAHH activity by the NAD(H)
analogues was investigated by incubation of 20 µM of each analogue with
Tc-SAHH and Hs-SAHH in 50 mM phosphate buffer, pH 7.4, containing
50 µM NAD+ at 37◦C for 6 minutes (refer to Table 3). 50 µM NAD+ was
included because the concentration of NAD+ inside cells was estimated to
be in this general range.[21]
RESULTS
Table 1 reports that NAD+ analogues can be reconstituted into the active
sites of both Hs-SAHH and Tc-SAHH with 82–98% of the expected unit

Selective Inhibition of T. cruzi SAHH
479
TABLE 1 Fraction of occupancy and cofactor activities of NAD+ analogues as ligands of Hs-SAHH
and Tc-SAHHa
Hs-SAHH
Amount bound
Tc-SAHH
Amount bound
(mol/mol
subunit)
Relative activity
(%)
(mol/mol
subunit)
Relative activity
(%)
Compound
NAD+
0.98 0.04
100
0.96 0.05
100
Modifications in the carboxamide side-chain of the nicotinamide ring
S-NAD
H-NAD
O-NAD
C-NAD
0.91 0.06
0.94 0.08
0.87 0.09
0.88 0.09
1.4 1.1
0.93 0.06
0.98 0.04
0.83 0.08
0.85 0.08
1.3 0.7
0
0
0
0
21.4 5.2
20.5 3.7
Modifications in the adenine ring
NGD
0.84 0.08
0.89 0.07
0.84 0.07
46.5 6.8
69.0 5.3
38.6 6.1
0.73 0.09
0.88 0.09
0.82 0.08
58.5 7.2
80.6 6.4
35.2 4.2
Etheno-NAD
NHD2
aErrors calculated from three independent measurements.
occupation of one active site in each of the four monomeric subunits of
the homotetramer. Modifications of the adenine ring lead to occupancies
in the lower range and generate catalytic activities at about 40–70% of that
for NAD+. Modifications of the nicotinamide lead to occupancies in the
higher range and catalytic activities at 0–20% of that for NAD+.
Table 2 reports values of kon, the second-order rate constant for as-
sociation of a ligand with Hs-SAHH or Tc-SAHH, koff, the first-order rate
constant for dissociation of a ligand from its complex with the enzyme, and
Kd, the equilibrium thermodynamic dissociation constant of a ligand from
its complex with the enzyme. The values of kon range from less than 102 to
over 104 M−1s−1, the values of koff from about 10−5 s−1 to nearly 10−1s−1,
and the Kd values from near-nanomolar to millimolar.
Table 3 identifies five compounds that exhibit some selectivity in trap-
ping the cofactor sites of Tc-SAHH in preference to those of Hs-SAHH
during a 6-minutes incubation period in the face of a 2.5-fold excess of
NAD+.
DISCUSSION
Occupancy and Catalytic Activity of NAD+ Analogues with
Hs-SAHH and Tc-SAHH
The data in Table 1 show that NAD+ analogs complex extensively with
Hs-SAHH or Tc-SAHH, the numerical values of occupancy being 4 equiv-
alents of ligand per equivalent of homotetramer, thus being equal to the
expectation for cofactor-site binding. The fact that five of the seven analogs
produce measurable catalytic activity also is consistent with occupancy of

480

Selective Inhibition of T. cruzi SAHH
481
TABLE 3 Inhibition of Hs-SAHH and Tc-SAHH by NAD(H) analoguesa,b
Analogue
Remaining Activity (%)
Hs-SAHH Tc-SAHH
S-NAD
H-NAD
C-NAD
O-NAD
NGD
NHD
Etheno-NAD
S-NADH
H-NADH
C-NADH
NHDH
97
98
>99
1
1
74
77
94
96
96
96
95
8
3
4
4
3
4
3
3
2
2
3
2
>99
>99
>99
>99
96
96
>99
>99
1
1
9
52
98
aErrors calculated from three independent measurements.
bAfter incubation with 20 µM analogue, 50 µM NAD+ to simulate
intracellular conditions, at pH 7.4 (phosphate buffer) for a period of 6
minutes.
the cofactor site. Further evidence favoring this view is provided by the
fact that modifications of the adenine ring give higher activities and lower
occupancies than modifications of the nicotinamide ring. This combination
is consistent with the long-held view[22] that the adenine “anchor” is more
important for binding than for catalysis. The fact that H-NAD (with an
aldehyde side chain) and O-NAD (with a carboxylate side chain) are the
only NAD+ analogues that do not support measurable catalytic activity with
Tc-SAHH renders them the only compounds in this part of the set worthy of
further consideration for anti-parasitic agents.
Off-Rates, On-Rates, and Affinities for NAD+ and NADH
Analogues
Table 2 presents these data for all compounds studied. A useful baseline
for comparison is provided by the data for the oxidized cofactor NAD+ and
the reduced cofactor NADH with Hs-SAHH and Tc-SAHH. As previously
noted,[17,18] the affinity of both forms of the cofactor is in the nanomolar
range for Hs-SAHH, the result of an off rate constant in the range of 10−6 s−1
and an on-rate constant in the range of 103 M−1s−1; in contrast, the affinity
of both forms of the cofactor is in the micromolar range for Tc-SAHH, the
result of an off-rate constant in the range of 10−3 to 10−4 s−1 and an on-rate
constant in the range of 103 to 102 M−1s−1. The contrast in cofactor affinities
between Hs-SAHH and Tc-SAHH thus arises mainly from a much faster off-
rate constant for the latter, and it is therein one may seek the opportunity
for selective inhibition of Tc-SAHH over Hs-SAHH.

482
Q.-S. Li et al.
Among the NAD+ analogues, the most promising seemed to be H-NAD,
with an aldehydic side-chain, and O-NAD, with a carboxylate side-chain,
which gave no detectable activity when reconstituted into the active sites of
either Hs-SAHH or Tc-SAHH (Table 1). H-NAD shows an on-rate constant
10-fold larger and an off-rate constant 12-fold larger than NAD+ with Hs-
SAHH so that the affinity for Hs-SAHH of H-NAD is not much different
from its affinity for NAD+. With Tc-SAHH, more or less the same thing
is observed. The close similarity to the kinetics for NAD+ does not bode
well for successful inhibition in the presence of NAD+. O-NAD presents no
better picture. Its affinity for both Hs-SAHH and Tc-SAHH is smaller than
their affinity for NAD+ by factors of 35–40 and their on-rate constants are
smaller than those for NAD+.
The NADH analogs show generally poorer affinity than NADH with
Hs-SAHH (factors of 3–600) but affinities with Tc-SAHH that, except for
NHDH, are weaker than that for NADH by smaller factors (3–40) and are
greater in all cases except NHDH than the affinity of Tc-SAHH for NAD+,
the probable competitor for inhibitor binding in vivo. We conclude that
NADH analogues may offer the best opportunities for selective inhibition of
Tc-SAHH.
Inhibition of Hs-SAHH and Tc-SAHH by NAD+ and NADH
Analogues
Table 3 shows for each analogue with Hs-SAHH and Tc-SAHH the re-
maining levels of catalytic activity after a 6-minutes exposure of the enzyme
to 20 µM analogue in the presence of 50 µM NAD+ to simulate competition
by intracellular cofactor. Under these conditions, no significant inhibition
of Hs-SAHH occurred with any analogue. Tc-SAHH was reasonably strongly
inhibited only by the NADH analogues S-NADH and H-NADH, with a bit
less than 10% activity remaining. These are the only NADH analogues
for which the on-rate constants for Tc-SAHH exceed (S-NADH) or nearly
match (H-NADH) the on-rate constant. This feature may in part enable the
effective competition they display.
CONCLUSION
We would recommend NADH analogues as a possible starting point
for the design of anti-trypanosomal drugs. Although the set of compounds
studied herein does not in any sense approach a thorough exploration of
structures in any part of the cofactor, we hope it serves to support the further
study of cofactor analogs. Clearly the modification of the carboxamide side
chain of the nicotinamide ring was more successful than modification of

Selective Inhibition of T. cruzi SAHH
483
the adenine ring, in agreement with previous indications of a more intimate
binding relationship at the latter center.[1,22]
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