Báo cáo khoa học: Relationship between the structure of guanidines and N-hydroxyguanidines, their binding to inducible nitric oxide synthase (iNOS) and their iNOS-catalysed oxidation to NO pptx

These data indicate that a key factor for efficient oxidation of a guanidine by iNOS to NO is the ability of the corresponding N-hydroxyguanidine to bind to the active site without being

Relationship between the structure of guanidines and

N-hydroxyguanidines, their binding to inducible nitric

oxide synthase (iNOS) and their iNOS-catalysed oxidation

to NO

David Lefe`vre-Groboillot1,2, Jean-Luc Boucher1, Dennis J Stuehr2and Daniel Mansuy1

1 Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universite´ Paris 5, France

2 Department of Immunology, Lerner Research Institute, Cleveland, OH, USA

Keywords

binding kinetic; guanidines

N-hydroxyguanidines; nitric oxide synthase;

UV ⁄ Vis difference spectroscopy

Correspondence

J-L Boucher, Laboratoire de Chimie et

Biochimie Pharmacologiques et

Toxicologiques, UMR 8601 CNRS,

Universite´ Paris 5, 45 rue des Saints-Pe`res,

75270 Paris Cedex 06, France

Fax: +33 1 42 86 83 87

Tel: +33 1 42 86 21 91

E-mail: boucher@biomedicale.univ-paris5.fr

(Received 18 February 2005, revised

20 April 2005, accepted 25 April 2005)

doi:10.1111/j.1742-4658.2005.04736.x

The binding of several alkyl- and aryl-guanidines and N-hydroxyguanidines

to the oxygenase domain of inducible NO-synthase (iNOSoxy) was studied

by UV⁄ Vis difference spectroscopy In a very general manner, monosubsti-tuted guanidines exhibited affinities for iNOSoxy that were very close to those of the corresponding N-hydroxyguanidines The highest affinities were observed for the natural substrates, l-arginine and Nx

-hydroxy-l-arginine (Kd at the lm level) The deletion of either the CO2H or the

NH2 function of their amino acid moiety led to dramatic decreases in the affinity However, alkylguanidines with a relatively small alkyl chain exhib-ited interesting affinities, the best being observed for a butyl chain (Kd¼

20 lm) Arylguanidines also bound to iNOSoxy, however, with lower affinit-ies (Kd> 250 lm) Many N-alkyl- and N-aryl-N¢-hydroxyguanidines are oxidized by iNOS with formation of NO, whereas only few alkylguanidines led to significant production of NO under identical conditions, and all the arylguanidines tested to date were unable to lead to the production of NO The kcatvalues of NO production from the oxidation by iNOS of the stud-ied N-hydroxyguanidines were found to vary independently of their affinity for the protein The kcat values determined for the two-step oxidation of alkylguanidines to NO were not clearly related to the Kdof these substrates toward iNOSoxy However, there is a qualitative relationship between these

kcatvalues and the apparent rate constants of dissociation of the complex between iNOSoxy and the corresponding N-alkyl-N¢-hydroxyguanidine (koffapp) that were determined by stopped-flow UV⁄ Vis spectroscopy These data indicate that a key factor for efficient oxidation of a guanidine by iNOS to NO is the ability of the corresponding N-hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation This explains why 4,4,4-trifluorobutylguanidine is so far the best non-a-amino acid guanidine substrate of iNOS with formation of NO, because the koffapp of the corresponding N-hydroxyguanidine is particularly low This suggests that the rational design of guanidines as new NO donors

Abbreviations

BH 4 , (6R)-5,6,7,8-tetrahydro- L -biopterin; BuGua, n-butylguanidine; BuNOHG, N-(n-butyl)-N hydroxyguanidine; BzNOHG, N-benzyl-N ¢-hydroxyguanidine; ClPhNOHG, N-(4-chlorophenyl)-N¢-¢-hydroxyguanidine; FPhGua, 4-fluorophenylguanidine; FPhNOHG,

N-(4-fluorophenyl)-N ¢-hydroxyguanidine; HexGua, n-hexylguanidine; HexNOHG, N-(n-hexyl)-N ¢-hydroxyguanidine; homo- L -Arg, homo- L -arginine; homo-NOHA,

N x -hydroxy-homo- L -arginine; HS, high spin; ImH, imidazole; L -Arg, L -arginine; LS, low spin; NOHA, N x -hydroxy- L -Arginine; Nor- L -Arg, nor- L -Arginine; NOHAgma, Nx-hydroxyagmatine; NOHGPA, Nx-hydroxyguanidinopentanoic acid; NOS, nitric oxide synthase; NOS oxy , oxygenase domain of NOS; PentylGua, n-pentylguanidine; PentylNOHG, N-(n-pentyl)-N¢-hydroxyguanidine; ProGua, n-propylguanidine; ProNOHG, N-(n-propyl)-N ¢-hydroxyguanidine; TFBGua, 4,4,4-trifluorobutylguanidine; TFBNOHG, N-(4,4,4-trifluorobutyl)-N ¢-hydroxyguanidine.

Nitric oxide synthases (NOS) catalyse the oxidation of

l-arginine (l-Arg) into l-citrulline and NO, with the

intermediate formation of Nx-hydroxy-l-arginine

(NOHA) [1–3] This reaction ideally consumes 1.5 mol

of NADPH and 2 mol of O2 It occurs in the

homo-dimeric N-terminal domain of the protein called NOS

oxygenase domain (NOSoxy) that contains two

cofac-tors per monomer, the heme (iron-protoporphyrin IX)

and (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4)

Elec-trons from NADPH are provided to heme by flanking

C-terminal reductase domains NOSs are heme-thiolate

monooxygenases comparable with cytochrome P450

Whereas proteins of the cytochrome P450 family are

known to be able to bind and oxidize a very large

number of compounds of various structures, until

recently NOSs were only known to be able to oxidize

l-Arg and a very small number of its close a-amino

acid analogues Recent reports have shown that NOSs

are able to produce NO from the oxidation of many

non-a-amino acid monosubstituted

N-hydroxyguani-dines, including N-alkyl-N¢-hydroxyguanidines and

N-aryl-N¢-hydroxyguanidines, provided that the alkyl

or aryl substituent is neither too small nor too bulky

[4–7] NOS-catalysed oxidation of some of these

com-pounds showed kcat values as high as 80% that

obtained with NOHA, and some proved to be selective

for one of the three isoforms vs the others [5,7] More

recently, NO production has also been observed from

the oxidation of several non-a-amino acid

alkylguani-dines by purified iNOS or by activated mouse

macro-phages, opening the way to the design of stable

exogenous NOS substrates of pharmacological interest

[8,9]

Apart from some equilibrium and kinetic constants

related to the binding of l-Arg [10–14] and NOHA

[10], nothing is known about the thermodynamics and

kinetics of the binding of guanidines and

N-hydroxy-guanidines to iNOS Removal of the a-amino or

a-carboxylate moiety of l-Arg has important effects on

the ability of the resulting compounds to affect the

heme iron spin equilibrium, and to trigger NADPH

consumption and NO production [14–16] Interestingly,

it has been shown that several binding modes exist for

N-hydroxyguanidines in the heme pocket of NOSs [17–

20] Also, the fact that isoform-selective substrates for

NOS [5,7,9] were characterized is striking, given the

high level of similarity between the crystal structures of

the oxygenase domains of the three isoforms [18,19,21]

This study was undertaken to determine structural fac-tors that are important for a guanidine or N-hydroxy-guanidine to be well recognized by the NOS active site, and to be efficiently oxidized with NO formation For that purpose, the dissociation constants of several com-plexes of the oxygenase domain of iNOS (iNOSoxy) with various alkyl- and aryl-guanidines and N-hydroxygua-nidines were determined by UV⁄ Vis difference spectros-copy, according to a previously described technique [22] The kinetics of the binding of some of these substrates

to iNOSoxy was also studied by UV⁄ Vis spectroscopy using stopped-flow techniques [23,24] The correspond-ing thermodynamic and kinetic bindcorrespond-ing constants were then compared with the kinetic constants of NO forma-tion from iNOS-catalysed oxidaforma-tion of guanidine and N-hydroxyguanidine substrates Our results suggest that

a key factor in the efficient oxidation of a guanidine to

NO by iNOS could be the ability of the corresponding N-hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation Our results may help in the further rational design of guanidines as new NO precursors

Results

Study of the binding of guanidines and N-hydroxyguanidines to iNOSoxyby UV⁄ Vis difference spectroscopy

Purified recombinant iNOSoxy showed a wide Soret band with a maximum absorption wavelength around

400 nm, indicating that the heme-iron existed in equi-librium between a hexacoordinated low-spin (LS) state and a pentacoordinated high-spin (HS) state, the major fraction being in the HS state As previously described, addition of l-Arg leads to conversion of the minor population of heme centres being in the LS state into the HS state and to the appearance of a difference spectrum [22,25–27] However, the intensity of this dif-ference spectrum is small, because the spin state of the major fraction of the protein is not affected Imidazole (ImH) was thus used to completely convert iNOSoxy into a LS state iNOSoxy–Fe(III)–ImH complex allow-ing one to more easily follow the bindallow-ing of guanidines

or N-hydroxyguanidines to the iNOSoxy substrate binding site [12,22,28] iNOSoxy(1 lm) in the presence

of 400 lm ImH was first titrated with l-Arg A differ-ence spectrum displaying a peak at 392 nm and a

upon in situ oxidation by NOSs should take into account both thermody-namic and kinetic characteristics of the interaction of the protein not only with the guanidine but also with the corresponding N-hydroxyguanidine

trough at 430 nm (Fig 1) resulting from the

conver-sion of the LS NOS–Fe(III)–ImH complex to the

HS NOS–Fe(III)–l-Arg complex was observed

Inhibi-tion of the binding of l-Arg to iNOSoxyby ImH, and of

the iNOS-catalysed conversion of l-Arg into l-citrulline

has previously been shown to be competitive [11]

Equa-tion (1) was thus used to calculate corrected

equi-librium constants, Kd, for the iNOSoxy–substrate

complexes from apparent constants Kapp[12–14,23,24]

Kapp=Kd¼ 1 þ ½ImH
=KImH ð1Þ

With the ImH concentration used in this study

(400 lm), Eqn (1) became

Variations in the amplitudes of the difference spectra

with the concentrations of l-Arg were in agreement

with a single binding site model (see Experimental

pro-cedures) and Kapp value of 26 ± 2 lm was found for

the apparent equilibrium constant for the dissociation

of the iNOSoxy–l-Arg complex, in good agreement

with a previously reported value (28 ± 4 lm) obtained

with the same ImH concentration [25] At the end of

the titration, the absolute spectrum of the iNOSoxy

solution containing 400 lm ImH and 1 mm l-Arg

showed a maximum absorption wavelength at

395 ± 3 nm (not shown)

Similar titrations of iNOSoxy in the presence of

400 lm ImH were then performed with a large number

of guanidines and N-hydroxyguanidines previously

evaluated as iNOS substrates [4–9,29] The positions of the peaks and troughs of the difference spectra observed during these titrations were similar to those observed when l-Arg was used (Fig 1) Variation

in the amplitude of the observed difference spectra with the concentration of the studied guanidines or N-hydroxyguanidines was always in reasonable agree-ment with a single binding site model The apparent equilibrium constants derived from these experiments are shown in Table 1

NOHA was found to bind to iNOS with a Kapp value slightly lower than that of l-Arg (18 ± 7 lm, Table 1) Homo-L-Arg and homo-NOHA, the l-Arg and NOHA analogues bearing one extra methylene group in the alkyl side-chain, were found to bind to iNOSoxywith higher Kapp values than l-Arg (80 ± 13 and 150 ± 40 lm, respectively) Finally, a much higher Kapp value (2.4 mm) was found for nor-l-Arg, the analogue bearing one methylene fewer than l-Arg Removal of either the a-COOH or the a-NH2group

of NOHA led to a dramatic decrease in the affinity of the resulting compounds, the Kapp values measured for

Nx-hydroxyagmatine (NOHAgma), and Nx -hydroxy-guanidino-pentanoic acid (NOHGPA), being > 1 mm (2 and > 4 mm, respectively; Table 1) However, the simultaneous removal of both the a-NH2and a-COOH functions of NOHA led to N-(n-butyl)-N¢-hydroxygu-anidine (BuNOHG), which showed a much lower Kapp value of 160 ± 40 lm (Table 1) Replacement of the terminal CH3 group of the n-butyl chain by a CF3 group, leading to N-(4,4,4-trifluorobutyl)-N¢-hydroxy-guanidine (TFBNOHG), resulted in a sixfold increase

in the Kappvalue Shorter nonfunctionalized analogues N-(n-propyl)-N¢-hydroxyguanidine (ProNOHG) and longer ones N-(n-pentyl)-N¢-hydroxyguanidine (Pentyl-NOHG) and N-(n-hexyl)-N¢-hydroxyguanidine (Hex-NOHG) showed higher Kapp values than the N-(n-butyl) compound (270, 900 and >1000 lm, respectively) Finally, N-benzyl-N¢-hydroxyguanidine (BzNOHG) and the three para-substituted aryl-deriva-tives N-(4-fluoro-, 4-methyl- and

ClPhNOHG), showed Kappvalues > 2 mm

A study of the binding of the corresponding non-functionalized alkylguanidines to iNOSoxy led to very similar conclusions In the studied series, the alkylgu-anidine exhibiting the highest affinity for iNOS was n-butylguanidine (BuGua), with a Kapp value of

140 ± 20 lm (Table 1) Trifluorination of the terminal methyl group of the n-butyl chain, leading to 4,4,4-tri-fluorobutylguanidine (TFBGua), increased the Kapp value by 10-fold The longer nonsubstituted n-pentyl-and n-hexylguanidines (PentylGua n-pentyl-and HexGua) also

Fig 1 Difference spectrum obtained upon addition of increasing

concentrations of L -Arg to iNOSoxyin the presence of ImH iNOSoxy

and ImH concentrations were 1 and 400 l M , respectively (Inset)

Plot of 1 ⁄ DA vs 1 ⁄ [ L -Arg].

showed higher Kapp values (600 lm and > 4 mm for

the n-pentyl and n-hexyl derivatives, respectively) The

shorter n-propylguanidine (ProGua) showed a Kapp

value similar to that found for BuGua Finally, the

arylguanidines 4-fluorophenyl- and

4-methylphenyl-guanidines (FPhGua and TolGua) interacted with

iNOS with Kappvalues>2 mm

Relationship between the equilibrium constants

measured for the binding of guanidines and

N-hydroxyguanidines to iNOSoxyand the kinetic

constants measured for their iNOS-catalysed

oxidation to NO

In previous studies, we have identified some

N-alkyl-and N-aryl-N¢-hydroxyguanidines, N-alkyl-and alkylguanidines

as NO donors following their oxidation catalysed by

iNOS containing all its cofactors [4,5,8,9] Table 2 gives the Km and kcat values measured for the oxidation of seven N-hydroxyguanidines leading to the highest pro-duction of NO in the presence of iNOS, together with

Kmand kcatvalues for the oxidation of the correspond-ing guanidines [4,5,8,9] The seven N-hydroxyguani-dines NOHA, homo-NOHA, BuNOHG, TFBNOHG, PentylNOHG, FPhNOHG and TolNOHG were oxid-ized with formation of NO with similar high kcatvalues ranging from 58 to 100% of that found for NOHA They showed widespread Km⁄Kd ratios, generally >1 and that varied from 1 to  20 (Table 2) In that ser-ies, the kcat value for the production of NO from the oxidation of the N-hydroxyguanidines varied by less than a factor 2, whereas the kcatvalue for the produc-tion of NO from the oxidaproduc-tion of the guanidines varied

a great deal from 0 to 100% of the kcatvalue obtained

Table 2 Kinetic constants for the formation of NO from the oxidation of guanidines and N-hydroxyguanidines by recombinant iNOS See Table 1 for the structure of compounds Kmand kcatvalues are taken from previous publications [4,5,8,9,29] kcatvalues are expressed per NOS dimer The corrected dissociation equilibrium constants (K d ) for the binding of guanidines and N-hydroxyguanidines to iNOS oxy were obtained by dividing K app values (taken from Table 1) by 8.7.

Compounds

K m (l M ) N-Hydroxy

k cat (min)1) N-Hydroxy

K m ⁄ K d N-Hydroxy

a

The rates of the production of NO from the oxidation by iNOS of 1 m M FPhGua or TolGua were lower than 2 min)1.

Table 1 Apparent equilibrium constants (Kapp) for the binding of N-hydroxyguanidines R-NH-C(¼ NOH)-NH 2 and guanidines R-NH-C(¼ NH 2

)-NH2to iNOSoxy Titrations were performed by UV ⁄ Vis difference spectroscopy in the presence of 25 l M BH4, 1 m M dithiothreitol and

400 l M ImH K app values were calculated as described in Experimental procedures Values ± SD from three different experiments n.d., not determined.

R

N-Hydroxyguanidines

Guanidines

with l-Arg, with the order l-Arg >

homo-l-Arg TFBGua > PentylGua > BuGua >> FPhGua

 TolGua (Table 2)

The Kmand kcatvalues calculated for NO formation

from iNOS-catalysed oxidation of guanidines are

com-plex parameters as they correspond to a two-step

reaction with intermediate formation of

N-hydroxygu-anidines The data are difficult to correlate with kinetic

or thermodynamic constants clearly describing

individ-ual reactions, such as Kd (or Kapp) The situation

should be less complex for Kmand kcatfor NO

forma-tion from N-hydroxyguanidines that are more closely

related to a one-step enzymatic reaction

It is actually well known that, for enzymes having

high kcatvalues, the Kmvalues can be markedly higher

than the Kd values, as indicated by the classical

rela-tion given here [30]

Km¼ Kdþ kcat=kon or

Km=Kd¼ 1 þ kcat=koff This equation implies that Km⁄ Kd will increase as kcat

increases and koff decreases Because the kcat value

found for these seven N-hydroxyguanidines varied by

less than a factor 2, it was tempting to investigate a

possible relationship between Km⁄ Kd and koff The

fol-lowing experiments were performed as a first approach

to find the variation in koff as a function of the iNOS

substrate structure

Kinetics of the binding of guanidines and

N-hydroxyguanidines to iNOSoxymeasured by

stopped-flow UV⁄ Vis spectroscopy

An iNOSoxysolution containing 400 lm ImH was

rap-idly mixed with a solution of the studied ligand

contain-ing the same concentration of ImH Postmixcontain-ing ligand

concentrations corresponded to pseudo-first-order

con-ditions Absorption variations were monitored at 430

and 392 nm (Fig 2), allowing one to follow,

respect-ively, the disappearance of the NOS–Fe(III)–ImH

com-plex and the appearance of the high-spin NOS–Fe(III)

species The calculated kinetic constants kobswere

plot-ted against the ligand concentration and satisfactorily

fitted with a linear function

kobs¼ koffappþ koffapp½L

where L is the guanidine or N-hydroxyguanidine used

(Fig 3), in agreement with a competitive model for the

interaction between ImH and the studied guanidine or

N-hydroxyguanidine [23,24,28] It has previously been

shown that displacement of ImH from the NOS heme-iron by l-Arg or its analogues is a two-step process [23,28] and might involve an intermediate and transient ternary complex between the protein, ImH and the

l-Arg analogue [23] The konapp and koffapp values are thus apparent association and dissociation rate con-stants of the guanidine or N-hydroxyguanidine with the protein in the presence of 400 lm ImH

Three guanidines and the corresponding N-hydroxy-guanidines were studied l-Arg and NOHA were used

0.33 0.32 430 nm

392 nm

0.31

0.29 0.28 0.27 0.26 0.25

Time (s)

0.3

Wavelength (nm)

A

B

0.063 0.113 0.163 0.213 0.263 0.313

Fig 2 Spectral transitions observed as a function of time upon the fast addition of BuNOHG to iNOS oxy in the presence of ImH ImH concentration was 400 l M Final heme and BuNOHG concentra-tions were 5 l M and 2 m M , respectively (A) Rapid-scanning stopped-flow spectra recorded during the reaction (B) Cross-sec-tion of (A) variaCross-sec-tion in absorbance at 430 and 392 nm as a funcCross-sec-tion

of time.

as reference compounds and two pairs of non-a-amino acid compounds, BuGua⁄ BuNOHG and TFBGua ⁄ TFBNOHG were also studied The determined values

of konapp and koffapp are reported in Table 3 In the studied range of concentrations, the kobs values were higher for a guanidine than for the corresponding N-hydroxyguanidine (Fig 3) The konapp values for the guanidines were found to be 5–10 higher than those for the corresponding N-hydroxyguanidines, and the

koffappvalues for the guanidines were 25–60 times higher than those for the corresponding N-hydroxy-guanidines (Table 3) The konapp values for the non-a-amino acid guanidines BuGua and TFBGua were found to be 7- and 25-fold lower than that for l-Arg, and those for the non-a-amino acid N-hydroxyguani-dines BuNOHG and TFBNOHG were 3- and 12-fold lower than that for NOHA The koffapp values for BuGua and TFBGua were found to be 10 and 6 times higher than that for l-Arg, and those for BuNOHG and TFBNOHG were 20 and 5 times higher than that for NOHA Interestingly, konapp values for the

3.8- and 3.6-fold lower than those for their nonfluori-nated analogues BuGua and BuNOHG, respectively, and the koffapp values for TFBGua and TFBNOHG are 1.6 and 4 times lower than those for BuGua and BuNOHG, respectively

Discussion

Binding of guanidines and N-hydroxyguanidines

to iNOSoxy

In the series of guanidines and N-hydroxyguanidines studied here, the ratio between the Kappor Kd (calcula-ted using Eqn 1¢) of a guanidine and that of its corres-ponding N-hydroxyguanidine was always found to be between 0.5 and 2 (Table 1) This was true for pairs

of compounds showing dissociation constants in the micromolar range (l-Arg⁄ NOHA) and pairs of compounds showing Kd in the millimolar range (FPhGua⁄ FPhNOHG) The difference between the

Kapp values for the guanidines and those for the cor-responding N-hydroxyguanidines was, in most cases, small and barely significant However, we found that the Kapp value for NOHA is slightly lower than that for l-Arg (Table 1), and because such an observation has also been previously reported by several authors with nNOS [12,22,23] and iNOS [10], this difference is probably significant By contrast, we found that the

Km value for NOHA is higher than that for l-Arg (Table 2), also in accordance with the literature data

on the three isoforms [29,31,32] In the studied series,

Fig 3 Plots of the rates of spectral transitions observed upon the

addition of guanidines or N-hydroxyguanidines to iNOSoxy in the

presence of ImH vs the postmixing concentration of the studied

guanidine or N-hydroxyguanidine Best linear fits are shown.

(A) L -Arg and NOHA, (B) N-hydroxyguanidines BuNOHG and

TFBNOHG, (C) guanidines BuGua and TFBGua See Table 1 for the

structure of compounds.

binding of an N-hydroxyguanidine moiety in the iNOS

heme pocket thus roughly involves the same binding

energy as binding of the guanidine moiety, and the

equilibrium constants are mainly determined by the

alkyl or aryl substituents of the compounds

The crystal structures of mouse iNOSoxy–l-Arg and

bovine eNOSoxy–l-Arg complexes [33,34] have shown

that the guanidine moiety of l-Arg makes a salt bridge

with the side chain of a conserved glutamate residue

(E371 in mouse iNOS), and H-bonds with a backbone

carbonyl oxygen atom (W366 in mouse iNOS) The

crystal structures of iNOSoxy–NOHA complexes

showed identical positioning of the

N-hydroxyguani-dine moiety of NOHA, with additional contacts

between the N-hydroxyguanidine hydroxy group and

the amide nitrogen of a conserved glycine (G365 in

mouse iNOS) [18,19,21] The crystal structures of

eNOSoxy–ClPhNOHG and nNOSoxy–BuNOHG

com-plexes showed similar positionings of the

N-hydroxy-guanidine moiety of ClPhNOHG and BuNOHG

involving: (a) a salt bridge between a glutamate side

chain and the two nonhydroxylated nitrogens of the

N-hydroxyguanidine, and (b) a nonbonded contact

between the hydroxy group and a glycine nitrogen

[17,19] It thus seems that in such a positioning,

chan-ging the N-hydroxyguanidine moiety into a guanidine

moiety does not strongly modify the energy of binding

to iNOS Preliminary data show that this is also true

for nNOS (D Lefe`vre-Groboillot, unpublished data)

The structure–affinity relationship for

alkylguani-dines and N-alkyl-N¢-hydroxyguanialkylguani-dines bearing

non-functionalized linear alkyl chains (ProGua, BuGua,

PentylGua, HexGua, ProNOHG, BuNOHG,

Pentyl-NOHG and HexPentyl-NOHG) showed that the binding

affinity is maximal for the compounds bearing a butyl

chain, i.e BuNOHG and BuGua, with Kd values

around 20 lm (Table 1) Compounds bearing a

n-pentyl chain (PentylNOHG and PentylGua) still bind well to the iNOS active site (Kd around 100 lm) but compounds bearing an n-hexyl chain (HexNOHG and HexGua) interact with iNOS with low affinities (Kd> 150 lm) The crystal structure of the nNOSoxy– BuNOHG complex showed that the butyl chain of BuNOHG interacts with the side chain of a conserved valine residue (V567), a conserved proline (P565) and the amide moiety of a conserved glutamine (Q478) [19] Because BuNOHG has previously been reported

to be similarly efficiently oxidized into NO by both iNOS and nNOS [4,6,9], the binding modes of this compound for the two isoforms is expected to be sim-ilar We measured the Kdfor the binding of BuNOHG

to nNOSoxy and found a somewhat higher value of

100 lm (data not shown), suggesting that the binding

of BuNOHG to iNOS is favoured slightly over its binding to nNOS The Km value for the oxidation of BuNOHG by iNOS and nNOS were also found to fol-low the order iNOS < nNOS [4,6,9] It appears that the hydrophobic contacts such as those observed between the butyl chain and the protein in the nNOSoxy–BuNOHG crystal structure are sufficient to allow compounds BuNOHG and BuGua to bind to the active site of iNOS and nNOS with Kd values in the 20–100 lm range Interestingly, the crystal struc-ture of the nNOSoxy–BuNOHG complex revealed that upon binding of BuNOHG the side chain of residue Q257 has to shift from its position observed in other complexes (including the nNOSoxy–NOHA complex),

in order to accommodate the terminal methyl group

of BuNOHG [19] This is in agreement with the fact that longer compounds such as PentylNOHG or HexNOHG showed lower affinities for the iNOS active site, because their binding may require an important reorganization of the protein environment

The introduction of both an amino function and a carboxylate function on the terminal methyl group of BuGua and BuNOHG in a configuration leading to the natural substrates, l-Arg and NOHA, led to a 10-fold decrease in the observed equilibrium constants (Table 1) The positioning of the a-amino acid moiety

of NOHA or l-Arg analogues appears to be critical for binding to iNOSoxy Indeed, the Kd values for

l-Arg and NOHA were found to be in the 2–4 lm range, in agreement with previously reported data [10,11,13], whereas those for the longer analogues homo-l-Arg and homo-NOHA were found to be in the 10–20 lm range and that for the shorter analogue Nor-l-Arg was found to be > 300 lm This indicates that the alkyl chains of l-Arg (or NOHA) optimally position their guanidine (or N-hydroxyguanidine) and a-amino acid moieties relative to each other in the

Table 3 Apparent association and dissociation rate constants (k onapp

and k offapp) for the binding of guanidines and N-hydroxyguanidines

to iNOS oxy in the presence of 400 l M ImH See Table 1 for the

structure of compounds The rates of spectral transitions (Fig 2)

were fitted vs the postmixing concentrations of the studied

guani-dine or N-hydroxyguaniguani-dine with a linear function, as shown on

Fig 3 koffapp was defined as the y intercept and konapp as the slope.

offapp(s)1)

NOS active site This also indicates that adding one

methylene in the l-Arg chain does not impede efficient

binding, whereas removal of one methylene group is

detrimental for the interaction between the protein and

the substrate The crystal structure of the eNOSoxy–

homo-l-Arg complex (PDB entry 1DM7, C.S Raman

et al 1999) actually showed that homo-l-Arg interacts

with the active site of eNOSoxy in a manner similar to

l-Arg, involving roughly identical positionings of the

guanidine and a-amino acid moieties However, the

longer alkyl chain of homo-l-Arg forms a small bulge

between the two heme propionates in contact with the

heme and the side chain of a conserved valine (V338)

The decrease of the affinity of l-Arg and

homo-NOHA compared with l-Arg and homo-NOHA (Table 1)

could be linked to this unfavourable bulging

confor-mation of the alkyl chain of homo-l-Arg

The simultaneous presence of both the a-amino and

a-carboxylate moieties appears to be necessary because

the NOHA analogue bearing only an a-amino moiety

(NOHAgma) interacted with iNOSoxy with an affinity

(Kd 250 lm) much lower than that found for

BuNOHG, and the NOHA analogue bearing only an

a-carboxylate moiety (NOHGPA) did not interact with

iNOS (Kd> 500 lm) This suggests that the a-amino

and a-carboxylate groups cooperate to provide

favour-able binding enthalpy for the formation of the complex

between NOHA and the protein The crystal structures

of l-Arg or NOHA in NOS active sites actually

showed that the a-amino acid moiety of these

com-pounds interacts with the protein via an H-bond

net-work involving one or two water molecules that links

the a-amino and a-carboxylate moieties one to each

other, and to protein residues [18,19,21]

Finally, six compounds bearing an aryl moiety,

BzNOHG, FPhGua and ClPhGua (Table 1), exhibited

Kd values > 250 lm The crystal structure of the

eNOSoxy–ClPhNOHG complex showed that the phenyl

ring of ClPhNOHG is in close contact with the side

chain of the conserved valine, V338, and with a

pro-pionate of the heme [17] The chlorine atom is also

involved in nonbonded contacts with the conserved

methionine M341 Because ClPhNOHG was previously

reported to be an iNOS-specific substrate [5,6], we also

measured the equilibrium constants for the binding of

these compounds to eNOS and nNOS (data not

shown) ClPhNOHG actually displayed significantly

higher affinities for the two constitutive isoforms than

for iNOS: the Kd values for its binding to nNOSoxy

and eNOSoxywere found to be around 50 and 95 lm,

respectively, whereas that for its binding to iNOSoxywas

found to be close to 350 lm Similar higher affinities

for nNOSoxy compared with iNOSoxy were also observed for TolNOHG, which is also an iNOS specific substrate [5,6], and for FPhNOHG, which is a substrate highly selective for iNOS [5] These results obtained with guanidines or hydroxyguanidines bear-ing an N-aryl moiety recall the well-documented selec-tivity of N-arylamidines for inhibition of nNOS vs iNOS [35]

Relationship between the structure of N-hydroxy-guanidines, their affinity for iNOSoxyand their oxidation by iNOS with formation of NO Previous data showed that a very large number of monosubstituted N-hydroxyguanidines

R-NH-C(¼NOH)-NH2 bearing an alkyl or aryl substituent R, neither too small nor too bulky, led to the detectable production

of NO in the presence of iNOS [4–7,9,29] Formation of

NO from the oxidation of an N-hydroxyguanidine by iNOS is thus not specific to NOHA and can occur with many N-hydroxyguanidines

The rates of NO formation from the oxidation of a great number of N-alkyl- and N-aryl-N¢-hydroxyguan-idines by iNOS were found to be highly dependent

on their structure [4–7,9] However, the kcat values found for NO formation upon iNOS-catalysed oxida-tion of the seven N-hydroxyguanidines menoxida-tioned in Table 2 varied by less than a factor 2, whereas their

Kd values varied by a factor 200 (Table 1) It thus appears that the kcat of NO formation is not simply related to the affinity of the substrate for iNOS For instance, the kcat of NO formation from FPhNOHG oxidation is 83% of that found for NOHA, whereas the Kd of this substrate is 130 times higher than that

of NOHA

As mentioned above and shown in Table 2, very dif-ferent N-hydroxyguanidines leading to similar kcat val-ues (58–100% of that found for NOHA) showed widespread Km⁄ Kdratios (from 1 to  20) This vari-ation may be related to that in koff, as expected by considering the relation Km⁄ Kd¼ 1 + kcat⁄ koff From

a qualitative point of view, this is in agreement with the variation in koffapp for NOHA (0.1 s)1), TFBNOHG (0.5 s)1) and BuNOHG (2 s)1) (Table 3), which is inversely related to that of Km⁄ Kd for these N-hydroxyguanidines (19.3, 8.1 and 3.0 for NOHA, TFBNOHG and BuNOHG, respectively) Rigorous and quantitative correlations could not be done imme-diately, as Km and kcat, Kapp, konapp and koffapp values were measured under different conditions for experi-mental reasons (different temperatures or the presence

of imidazole) However, our data provide a first gen-eral basis to understand the structural factors that are

necessary for guanidines and N-hydroxyguanidines to

efficiently bind to iNOS

Criteria for the formation of NO from the

oxidation of a guanidine by iNOS

Contrary to what is observed for the

N-hydroxyguani-dines, not all the guanidines that bind to iNOS lead to

the production of NO [5–9] For example, all

arylguani-dines assayed to date, among them FPhGua and

TolGua, have failed to lead to any detectable amount

of NO, although their affinity for iNOS is not lower than

that for the corresponding

N-aryl-N¢-hydroxyguani-dines that lead to kcatvalues of formation of NO as high

as 83 and 69% that obtained for NOHA (Table 2)

As in the case of the N-hydroxyguanidines, the kcat

values of NO formation from the oxidation of

guani-dines do not appear to be linked to the affinity of the

compounds for iNOS For example, compound

TFB-Gua led to a kcatvalue of NO formation of 55% that

obtained with l-Arg (Table 2) even though it bound to

iNOS with a Kappvalue 50 times higher (Table 1)

Interestingly, the kcatof production of NO by

oxida-tion of the studied guanidines followed the same order

l-Arg > homo-l-Arg TFBGua > PentylGua > BuGua

as that found for the Km⁄ Kd ratio of the

correspond-ing N-hydroxyguanidines: NOHA > homo-NOHA

TFBNOHG > PentylNOHG > BuNOHG (Table 2)

This suggests that the variations in the kcat values

found for NO formation from the guanidines could

be related to those of the koff of the corresponding

N-hydroxyguanidines Accordingly, the order l-Arg >

TFBGua > BuGua found for the kcatof production of

NO from oxidation of these guanidines corresponds

well to the order NOHA > TFBNOHG > BuNOHG

found for 1⁄ koffapp of the corresponding

N-hydroxygu-anidines (Table 3) These results may suggest that a key

factor for a guanidine to lead to NO formation in the

presence of iNOS could be the ability of the

corres-ponding N-hydroxyguanidine to bind to the active site

without being released before being further oxidized

They could explain why the compound TFBGua is so

far the best non a-amino acid NO precursor upon

oxi-dation by iNOS (Table 2), because the koffapp value of

the corresponding N-hydroxyguanidine TFBNOHG is

particularly low (Table 3) In a more general manner,

our data suggest that changes in the NOS–substrate

complex structure (changes of the substrate structure,

but also mutation or post-translational modification of

the protein) could likely lead to a shift of the activity of

NOS from NO synthesis to N-hydroxyguanidine

syn-thesis Further investigations are currently underway to

test these hypotheses Our results also suggest that the

rational design of guanidines as new NO donors upon

in situ oxidation by NOSs should take into account both thermodynamic and kinetic characteristics of the interaction of the protein not only with the guanidine, but also with the corresponding N-hydroxyguanidine

Experimental procedures

Chemicals and reagents

Paris, France) and l-Arg and homo-l-Arg were from Sigma (Saint-Quentin Fallavies, France) Alkylguanidines were obtained by reaction of the corresponding amine with pyrazole-1-carboxamidine hydrochloride in the presence of diisopropylethylamine following a previously described pro-tocol [8] Arylguanidines were obtained by reaction of the amine with N,N¢-bis(tert-butyloxycarbonyl)pyrazole-1-carb-oxamidine followed by acidic deprotection as previously des-cribed [8] N-Hydroxyguanidines were obtained, as well as small amounts of the corresponding ureas, by the addition of hydroxylamine hydrochloride to intermediate cyanamides in anhydrous ethanol [4–7] Cyanamides were obtained from the amines by addition of BrCN in methanol containing anhydrous sodium acetate [4,5] The physicochemical charac-teristics of N-(n-propyl)-N¢-hydroxyguanidine,

N-(4-fluorophenyl)-N¢-hydroxyguanidine, N-(4-chlorophenyl)-N¢-N-(4-fluorophenyl)-N¢-hydroxyguanidine, N-(4-methylphenyl)-N¢-hydroxyguanidine, N-benzyl-N¢-hydro-xyguanidine, n-butylguanidine, 4,4,4-trifluorobutylguanidine and (4-methyl)phenylguanidine have been published previ-ously [5,6,8] NOHA and homo-NOHA were synthesized as previously reported [29] Other chemicals were from Aldrich (Saint-Quentin Fallavies, France), Sigma or Across (Noisy le Grand, France) unless otherwise indicated and were of the highest purity commercially available

Protein preparation

at its C-terminus was overexpressed in Escherichia coli and

The enzyme concentration was determined from the 444 nm absorbance of its ferrous–CO complex by using an extinction

Assessment of NO formation

using the classical spectrophotometric oxyhemoglobin assay

for NO [38] under conditions described previously [4,5,8].

In some assays, the level of NO formation was measured

by electron paramagnetic resonance spectroscopy following

the formation of the paramagnetic ferrous mononitrosyl

diethyldithiocarbamate complex under previously described

conditions [8,39]

Determination of the dissociation constants for

the complexes between BH4-containing iNOSoxy

and guanidines or N-hydroxyguanidines

Studies were carried out at room temperature in an

UVIKON 942 spectrophotometer (Kontron Biotek), in a

1-cm path length cuvettes (150 lL total volume) Each

dithio-threitol The amplitude of the observed difference spectra

with a linear function The two fits gave dissociation

experiments, indicating that a single binding site model

found, very close to the values reported by others [11–13]

Maximum amplitude of the difference spectrum was

The study of the binding of guanidines and

of 400 lm ImH, a situation that allows the monitoring by

com-plex between the protein and compounds which bind to the

substrate binding site [11,12,14,22,25,36,40–42] The studied

guanidine or N-hydroxyguanidine (dissolved in buffer) was

added stepwise to the sample cuvette, and equivalent

vol-umes of buffer were added to the reference cuvette All

experiments were carried out under conditions where the

concentration of bound ligand was much smaller than the

calcula-tion, the amplitude of the observed difference spectra

N-hydroxy-guanidine concentration [ligand] with a hyperbolic function

[lig-and] with a linear function The two fits always gave

complexes that were less different than the values obtained

from two identical experiments This indicated that a single

binding site model satisfactorily accounted for the observed

spectral changes The maximum amplitude of the difference

signifi-cantly with the structure of the guanidine or

N-hydroxy-guanidine

Rapid kinetic studies of the binding

of guanidines and N-hydroxyguanidines to iNOSoxy

instrument equipped with a rapid-scanning diode array detector (Hi-Tech MG 6000) and following a protocol

400 lm ImH was mixed with a solution of guanidine (or N-hydroxyguanidine) also containing 400 lm ImH Post-mixing heme concentration was 5 lm The reaction was monitored by following the absorbance at 430 and 392 nm Variations of the absorbances at these two wavelengths were fitted with monoexponential functions Observed rate

rate constants measured at the two wavelengths over 5–10 shots

Acknowledgements

The authors thank Sylvie Dijols (UMR 8601 CNRS, Paris) for the synthesis of the guanidines and N-hydroxyguanidines used in this study DL-G thanks Zhi-Qiang Wang, Chin-Chuan Wei and Koustubh Panda (Cleveland Clinic Foundation) for their help with the stopped-flow experiments, and Jeroˆme Santolini (CEA Saclay, France) for his help in the preparation

of proteins and helpful discussions This work was supported by the French Ministry of Research (fellow-ship grant to DL-G), and by National Institutes of Health (grant CA53914 to DJS)

References

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