Examination of N-hydroxylation as a prerequisite mechanism of nitric oxide synthase inactivation

Article abstract of DOI:10.1016/S0960-894X(00)00171-2

L-N⁵-(1-Hydroxyiminoethyl)-ornithine (L-NHIO) and L-N⁶-(1-hydroxyiminoethyl)-lysine (L-NHIL) were synthesized and tested as potential intermediates in the mechanism-based inactivation of nitric oxide synthase (NOS) by L-N⁵-iminoethyl-ornithine (L-NIO) and L-N⁶-iminoethyllysine (L-NIL). Although these compounds were determined to be competitive inhibitors, mechanism-based inactivation was not observed. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00171-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1077±1080
Examination of N-hydroxylation as a Prerequisite Mechanism of
Nitric Oxide Synthase Inactivation
Tristan S. Maurer,y Jianping Pan,{ Brian P. Booth,x Thomas I. Kalman
and Ho-Leung Fung*
Department of Pharmaceutics and Department of Medicinal Chemistry, School of Pharmacy, University at Bu€alo,
State University of New York, Bu€alo, NY 14260-1200, USA
Received 26 January 2000; accepted 10 March 2000
AbstractÐl-N5-(1-Hydroxyiminoethyl)-ornithine (l-NHIO) and l-N6-(1-hydroxyiminoethyl)-lysine (l-NHIL) were synthesized
and tested as potential intermediates in the mechanism-based inactivation of nitric oxide synthase (NOS) by l-N5-iminoethyl-
ornithine (l-NIO) and l-N6-iminoethyllysine (l-NIL). Although these compounds were determined to be competitive inhibitors,
mechanism-based inactivation was not observed. # 2000 Elsevier Science Ltd. All rights reserved.
Amino acid-based inhibitors of nitric oxide synthase
(NOS) have shown promise in the treatment of many
in¯ammatory diseases.1 4 However, insucient potency
and selectivity for the inducible isoform (NOS II) limits the
therapeutic potential of many NOS inhibitors.5 Although
binding selectivity to the NOS II isoform has been
improved,6 8 overall inhibitory selectivity is frequently
confounded by mechanism-based inactivation.9 12 Cur-
rently, very little is known about the fundamental
mechanism for NOS II inactivation. The natural sub-
strate, l-arginine, and the mechanism-based inactivator,
l-NG-methylarginine (l-NMMA), are similarly N-
hydroxylated by NOS II to the intermediates l-NG-
hydroxyarginine and l-NG-hydroxy-NG-methylarginine,
respectively.13 Both l-NG-hydroxyarginine and l-NG-
hydroxy-NG-methylarginine display lower anities for
NOS II, but are metabolized more rapidly than their
non-hydroxylated parent compounds.13 Unlike l-NG-
hydroxyarginine, further metabolism of l-NG-hydroxy-
NG-methylarginine results in mechanism-based inacti-
vation.13 This inactivation is associated with both cova-
lent modi®cation of the NOS protein and loss of NOS
associated heme.11,14 These e€ects on the NOS protein
have been postulated to arise from the release of formal-
dehyde or nitrosomethane radical cation during the
metabolism of l-NG-hydroxy-NG- methylarginine.9,12 14
Unlike l-NMMA, inactivation by l-N5iminoethyl-orni-
thine (l-NIO) and l-N6-iminoethyl-lysine (l-NIL) is not
associated with covalent modi®cation of the NOS pro-
tein, but instead, is associated only with heme loss.11,12
In addition, the release of formaldehyde or nitro-
somethane radical cation that follows N-hydroxylation
is not likely to occur in the case of l-NIO and l-NIL,
because it would require a C±C instead of a C±N bond
cleavage. Herein, we examined whether N-hydroxyla-
tion is a prerequisite for the inactivation of NOS by l-
NIO and l-NIL.
Results and Discussion
Using eq 1 and PCNONLIN 4.2, we were able to
describe the observed inhibition kinetics of l-NIO and
l-NIL (Fig. 2A and B). The concentration dependence
and saturability of enzyme inactivation, typical of
mechanism-based inactivation, is apparent. Inactivation
of NOS by l-NIO and l-NIL was dependent upon the
presence of NADPH (data not shown). Based upon this
analysis, the KI=kinact parameters of l-NIO and l-NIL
*Corresponding author. Tel.: +1-716-645-2842, ext. 222; fax: +1-716-
645-3693; e-mail: hlfung@acsu.bu€alo.edu
1
were estimated to be 1.42 Æ 0.34 mM/0.16 Æ 0.01 min
yPresent address: P®zer Central Research, Department of Drug Meta-
bolism. Groton, CT 06340, USA.
and 2.16 Æ 0.27 mM/0.35 Æ 0.01 min 1, respectively. These
parameters are in close agreement with those previously
reported for these compounds.12 Unlike l-NG-hydroxy-
NG-methylarginine, the presumed metabolic intermediates
of NOS inactivation by l-NIO and l-NIL, l-NHIO
{Present address: Schering-Plough Research Institute, Chemical
Research, Kenilworth, NJ 07033, USA.
xPresent address: Food and Drug Administration, Center for Drug
Evaluation and Research. Rockville, MD 20857, USA.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00171-2

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T. S. Maurer et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1077±1080
and l-NHIL, did not inactivate NOS. Kinetic studies
revealed that l-NHIO (Fig. 3A) and l-NHIL (Fig. 3B)
are competitive antagonists of NOS. The KI parameters
of l-NHIO and l-NHIL were estimated to be 37.7 Æ
8.12 mM and 41.1 Æ 13.6 mM, respectively.
TFA was removed in vacuo. The residue was dissolved
in 3 mL water and the pH was adjusted to 7 with 2 N
NaOH. This solution was applied to a Dowex 50W
cation ion exchange column and the product was eluted
with 200 mL of 1.5 M NH4OH. The eluate was con-
centrated to 20 mL and adjusted to pH <2 with 2 N
HCl. The solvent was removed in vacuo and the residue
was crystallized from 90% ethanol to yield G (48 mg,
As previously reported for l-NG-hydroxyarginine and
l-NG-hydroxy-NG-methylarginine20,21, these association
constants are signi®cantly higher than that observed for
the non-hydroxylated counterparts (ANOVA p<0.05).
We conclude that, despite their ability to bind to the
active site, l-NHIO and l-NHIL cannot inactivate the
enzyme. These observations suggest that, unlike l-
NMMA, N-hydroxylation is not a prerequisite mechan-
ism of NOS inactivation for these analogues. This dif-
ferent interaction with the NOS enzyme coincides with
the dissimilar mechanisms of inactivation reported for
l-NMMA and these inhibitors.9 12 This work is also
consistent with the recent hypothesis that, in the pre-
sence of dioxygen, binding of l-NIO and l-NIL may,
instead, induce the formation of reactive oxygen species
that irreversibly modify the NOS protein.12
1
44.1%). H NMR (D2O): d 1.45±1. (m, 2H), 1.68 (t,
2H), 1.91 (s, 3H), 3.19 (t, 2H), 3.5 (t, 1H). MS m/z 190
+
.
(MH ). Anal. C7H15N3O3 HCl. Calcd: C, 37.26; H,
7.15; N, 18.62. Found: C, 37.12; H, 7.16; N, 18.38.
L-N6-(1-hydroxyiminoethyl)-lysine (H). l-N1-Boc-N6-(1-
hydroxyiminoethyl)-lysine tert-butyl ester (F, 180 mg,
0.5 mmol) was mixed with 1.0 mL TFA and 2 mL
CH2Cl2. The reaction mixture was stirred in an ice bath
for 2 h, and the product was isolated as described above
for G. Recrystallization from aqueous ethanol yielded
1
pure H (42 mg, 35.0%). H NMR (D2O): d 1.45±1.50
(m, 2H), 1.65±1.70 (m, 2H), 1.91±1.94 (m, 2H), 2.10 (s,
3H), 3.36 (t, 2H), 3.78 (t, 1H). MS m/z 204 (MH+).
Anal. C8H17N3O3HCl. Calcd: C, 40.09; H, 7.57; N,
17.53. Found: C, 40.06; H, 7.60; N, 17.64.
Experimental
Synthesis of (G), L-N5-(1-hydroxyiminoethyl)-ornithine,15
and (H), L-N6-(1-hydroxyiminoethyl)-lysine16 (Fig. 1).
Compounds G and H were prepared from commercial
starting materials A and B (Fig. 1). The protected orni-
thine and lysine derivatives C and D were reacted with
ethyl N-hydroxyacetimidate15 and the resulting hydro-
xyiminoethyl amino acids E and F were isolated by
preparative TLC. After deprotection, using tri-
¯oroacetic acid (TFA), the ®nal products (G and H)
were puri®ed by ion exchange chromatography16 and
recrystallized from aqueous ethanol.
L-N5-(1-hydroxyiminoethyl)-ornithine (G). l-N1-Boc-N5-
(1-hydroxyiminoethyl)-ornithine tert-butyl ester (E, 167
mg, 0.5 mmol) was mixed with 1.5 mL TFA. The reac-
tion mixture was stirred in an ice bath for 2 h, then the
Figure 1. Synthesis of l-N5-(1-hydroxyiminoethyl)-ornithine (G) and l-
N6-(1-hydroxyiminoethyl)-lysine (H). (a) H2, Pd/C; (b) t-BuOH, EDCI,
DMAP, CH2Cl2; (c) ethyl N-hydroxyacetimidate, 90 ꢀC; (d) TFA.
Figure 2. Inactivation kinetics of (A) l-N5-iminoethylornithine and (B) l-N6-iminoethyllysine for murine macrophage nitric oxide synthase.
Symbols (*) represent the observed data of triplicate experiments. Line represents the ®t obtained through these data using PCNONLIN 4.2 and
eq 1.

T. S. Maurer et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1077±1080
1079
Figure 3. Dixon plots for the competitive antagonism of murine-macrophage nitric oxide synthase by (A) l-N5-(1-hydroxyiminoethyl)-ornithine and
(B) l-N6-(1-hydroxyimino-ethyl)-lysine. Symbols represent the mean data of duplicate experiments obtained at Arg concentrations of (~) 1.25, (&)
2.5, (*) 5.0 mM. Lines represent the best-®t linear regression through the observed data.
Mechanism-based inactivation studies
At 0, 10, 20 and 30 min, the remaining enzyme activity
was assessed by addition of 2 mM l-arginine, as
described above.
l-NIO, l-NIL, l-NHIO, and l-NHIL were evaluated
for mechanism-based inactivation of puri®ed murine
macrophage nitric oxide synthase (NOS II, Cayman
Chemical Co. Ann Arbor, MI). In a gas-tight reaction
vial, NOS (0.1 units / 200 mL) was pre-incubated for 10
min at 37 ꢀC with l-NIO, l-NIL (0, 1.25, 2.5, 5.0, 10, 15
and 20 mM), l-NHIO or l-NHIL (0, 1.25, 2.5, 5.0, 10,
15 and 20 and 30 mM) in 800 mL of 15 mM HEPES
bu€er, pH 7.4, containing 1 mM magnesium acetate, 0.1
mM NADPH, 60 mM tetrahydrobiopterin, 833 mM
dithiothreitol. Following preincubation, 0.1 units of the
enzyme (200 mL of the solution) was added to 800 mL of
15 mM HEPES bu€er, pH 7.4, containing a ®nal con-
centration of 2 mM l-arginine, 1 mM magnesium ace-
tate, 0.15 mM NADPH, 24 mM tetrahydrobiopterin,
333 mM dithiothreitol, 100 units/mL superoxide dis-
mutase (bovine erythrocyte). Following a 15-min incu-
bation period, the amount of NO in the headspace of
the reaction vial was quanti®ed by chemiluminescence.17
The inhibition constant (KI) and the pseudo-®rst order
inactivation rate constant (kinact) were estimated using
eq 1 and the nonlinear parameter estimation program
PCNONLIN 4.2 (Statistical Consultants Inc. Lex-
ington, KY). Equation 1 is a nonlinear form of an
equation previously described by Kitz and Wilson.18
The reported values of these parameters represent the
mean Æ S.D. of triplicate experiments.
Competitive inhibition studies
l-NHIO and l-NHIL were evaluated for competitive
inhibition of NOS. In a gas-tight reaction vial, murine
macrophage NOS (0.1 units) was incubated at 37 ꢀC
with l-NHIO or l-NHIL (5, 10, 20, 40 or 80 mM) and
l-arginine (1.25, 2.5, or 5.0 mM) in 1 mL of 15 mM
HEPES bu€er, pH 7.4, containing 1 mM magnesium
acetate, 0.1 mM NADPH, 12 mM tetrahydrobiopterin,
100 units/mL SOD (bovine erythrocyte) and 170 mM
dithiothreitol. At 15 min, the headspace NO was deter-
mined by chemiluminescence17. Data were analyzed by
the Dixon method.19 The KI value reported represents
the mean Æ SD of the value obtained by the three dif-
ferent substrate regression lines.
Acknowledgements
This research was supported by NIH grants CA 35212
and HL 22273. T.S.M is a recipient of the American
Foundation for Pharmaceutical Education predoctoral
fellowship.
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