When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guana-benz, 2-2-benzofuranyl-2-imidazoline and idazoxan was competitive wit
Trang 1purified monoamine oxidase A
Tadeusz Z E Jones1, Laura Giurato2, Salvatore Guccione2and Rona R Ramsay1
1 Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, UK
2 Dipartimento di Scienze Farmaceutiche, Facolta` di Farmacia, Universita` degli Studi di Catania, Ed 2 Citta` Universitaria, Catania, Italy
In 1984, Bousquet et al [1] found that clonidine
pro-duced a pharmacologic response, independent of
a-adrenoceptors, at high-affinity imidazoline-binding
sites These binding sites were later classified into three
different subtypes, and some progress was made in
determining their identity and function [2,3] Ligands
to the type 2 binding sites (I2BS) [4] also inhibit
monoamine oxidase (MAO) but with lower affinity [5]
Some MAO substrates and inhibitors bind to I2BS [6]
Harmane, a b-carboline formed from tryptamine, has
nanomolar affinity both for I2BS and for MAO, and
mimics the hypotensive effect of clonidine [7]
How-ever, a careful study using b-carboline derivatives
showed no correlation between MAO-A inhibition and
binding to either high- or low-affinity I2BS [8]
Both MAO and I2BS are located on the outer mit-ochondrial membrane [9] and show coexpression dur-ing the agdur-ing process in humans [10] The protein labeled by I2BS ligands has a similar molecular weight and peptide sequence to MAO [6,11] Transfection of yeast cells with MAO led to the expression of imidazo-line-binding sites not previously observed, although the correlation between the number of binding sites and MAO activity was poor [6,12] However, a decrease in the number of I2BS in suicide victims was not accom-panied by a decrease in MAO-B [13] Furthermore, MAO-A knockout mice did not lose the high-affinity imidazoline binding, although a peptide ( 60 kDa) was no longer labeled by the covalent imidazoline ligand [14] At the protein level, photoaffinity labeling
Keywords
docking; I2binding sites; imidazoline;
kinetics; monoamine oxidase
Correspondence
R R Ramsay, Centre for Biomolecular
Sciences, University of St Andrews, North
Haugh, St Andrews, Fife KY16 9ST, UK
Fax: +44 1334 463400
Tel: +44 1334 463411
E-mail: rrr@st-and.ac.uk
(Received 26 September 2006, revised 15
January 2007, accepted 17 January 2007)
doi:10.1111/j.1742-4658.2007.05704.x
The two forms of monoamine oxidase, monoamine oxidase A and mono-amine oxidase B, have been associated with imidazoline-binding sites (type 2) Imidazoline ligands saturate the imidazoline-binding sites at nanomolar concentrations, but inhibit monoamine oxidase activity only at micromolar concentrations, suggesting two different binding sites [Ozaita A, Olmos G, Boronat MA, Lizcano JM, Unzeta M & Garcı´a-Sevilla JA (1997) Br J Pharmacol 121, 901–912] When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guana-benz, 2-(2-benzofuranyl)-2-imidazoline and idazoxan was competitive with kynuramine as substrate, giving Ki values of 3 lm, 26 lm and 125 lm, respectively Titration of monoamine oxidase A with imidazoline ligands induced spectral changes that were used to measure the binding affinities for guanabenz (19.3 ± 3.9 lm) and 2-(2-benzofuranyl)-2-imidazoline (49 ± 8 lm) Only one type of binding site was detected Agmatine, a putative endogenous ligand for some imidazoline sites, reduced monoamine oxidase A under anaerobic conditions, indicating that it binds close to the flavin in the active site Flexible docking studies revealed multiple orienta-tions within the large active site, including orientaorienta-tions close to the flavin that would allow oxidation of agmatine
Abbreviations
2-BFI, 2-(2-benzofuranyl)-2-imidazoline; I 2 BS, imidazoline-binding site (type 2); MAO, monoamine oxidase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
Trang 2of the imidazoline site on MAO-B identified a labeled
peptide containing amino acids 149–222 of the MAO-B
sequence [12] The crystal structure of MAO-B shows
that some of the residues in this peptide lie between
the entrance cavity and the active site [15] On the
other hand, inhibition of MAO with a
mechanism-based irreversible inhibitor did not prevent binding of
the I2 ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI),
suggesting that the I2BS was not the active site of
MAO [16]
Imidazolines reversibly inhibit MAO, but with IC50
or Ki values in the micromolar range [5,6,17–20]
Examples of the literature values for MAO inhibition
measured in membranous samples with the ligands
used in this study are included in Table 1 It has been
proposed that I2BS ligands bind both to the MAO
act-ive site, thereby inhibiting the enzyme, and to a second
site with substantially higher affinity present on a
sub-population of MAO-B enzymes [21] Recently, some
imidazole derivatives were reported to have high I2BS
affinity but negligible MAO inhibitory activity [22],
reinforcing a separate identity of the I2BS from the
MAO active site
The kinetics of MAO inhibition by imidazolines
have been described as noncompetitive [6,20],
noncom-petitive and mixed [17], or comnoncom-petitive for MAO-A
inhibition and mixed for MAO-B inhibition [5] This
study examines binding of the imidazoline ligands to
purified enzyme of a single subtype (MAO-A) to
characterize their active site binding and the resulting
inhibition The use of purified enzyme simplifies the
system for kinetics and also allows observation of the
spectrum of the flavin cofactor in MAO-A The subtle
changes in the flavin spectrum on ligand binding in the
active site [23,24] are concentration-dependent and
saturable, permitting calculation of the dissociation constant for binding to the active site of MAO-A To complement these two methods, flexible docking [25] was used to visualize the interactions between imidazo-lines and the MAO-A active site
Results
Kinetics of inhibition Guanabenz, 2-BFI and idazoxan all act as inhibitors
of MAO-A, but no inhibition was seen below 0.1 lm (Fig 1) Reported KD values for I2BS vary consider-ably [5,6,18,19,26], but all indicate much more than 50% occupancy of the I2BS at 0.1 lm There was also
no activation of MAO-A by any ligand, either with kynuramine (Fig 1) or with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (not shown) as a sub-strate, in contrast to the activation by imidazolines of benzylamine oxidation catalyzed by bovine plasma semicarbazide-sensitive amine oxidase [27] Allosteric activation of semicarbazide-sensitive amine oxidase by imidazolines was seen only with selected substrates, so both kynuramine and MPTP were tested for MAO-A
As some MAO-A inhibitors (such as the b-carbolines [28], also known to bind to both I2BS and MAO-A [8]) are time-dependent, the time dependence of inhibition was investigated For 2-BFI, the inhibition immediately upon mixing was 40 ± 3% (n¼ 3) When 2-BFI was incubated with the enzyme in the cuvette before addi-tion of substrate, the inhibiaddi-tion was 37 ± 5% (n ¼ 4) after either 1 min or 5 min of preincubation A similar lack of any time-dependent increase in inhibition was found for the other two inhibitors Thus, there is no tighter, time-dependent binding that might account for the discrepancy between the reported affinities for the
I2BS and the MAO-A active site
Table 1 Binding and inhibition constants for I2ligands with MAO-A.
The K i values for purified human MAO-A were determined as
des-cribed in Experimental procedures The KDvalues for human MAO-A
were calculated from the absorbance change at 500 nm induced by
ligand binding (from two experiments for guanabenz and one for
2-BFI and idazoxan, each with 14–18 points) The literature values for
inhibition of MAO-A were measured in membranes from rat liver.
ND, not determined.
Ligand
Ki
(l M )
Ki (l M )
KD (l M )
IC50 (l M ) [5]
IC50 (l M ) [9]
Idazoxan 165 ± 30 125 ± 40 > 100 280 220
Guanabenz Idazoxan
–9
100 80 60 40 20 0
Log ([I],M)
2-BFI
Fig 1 Inhibition of MAO-A by guanabenz (solid line), 2-BFI (dotted line), and idazoxan (dashed line) The activity of MAO-A was deter-mined with 0.5 m M kynuramine (4 · K m ) The unit for the inhibitor concentration is molÆL)1.
Trang 3When MAO-A was assayed with kynuramine at
0.5 mm (4· Km), the IC50 values obtained were 6 lm
for guanabenz, 48 lm for 2-BFI, and over 500 lm for
idazoxan, giving a good range of affinity suitable for
the binding studies These values are consistent with
the IC50 values in the literature for membrane-bound
enzyme (Table 1) The type of inhibition and the
inhi-bitory constants (Ki) were determined by varying both
substrate and inhibitor (see Experimental procedures)
The Lineweaver–Burk plot used for illustration in
Fig 2 shows that the inhibition was competitive with
the amine substrate The Ki values are given in
Table 1 There are only small differences between the
values obtained with the two substrates, despite the
different steady-state level of oxidized enzyme available
for inhibitor binding with kynuramine and MPTP [29]
Difference spectra and binding curves
When ligands bind to the active site of MAO-A, the
environment of the flavin is altered, resulting in
spec-tral changes [24] The I2BS ligands caused similar,
sat-urable changes in the MAO-A spectrum (shown for
guanabenz in Fig 3), suggesting that these I2BS
lig-ands might be expected to bind in the active site in the
same way as other reversible MAO-A inhibitors In
the experiment in Fig 3, the extinction coefficient
for guanabenz from the change at 500 nm was
1913 m)1Æcm)1, and the dissociation constant
calcula-ted directly from the change in absorbance of MAO-A
after addition of guanabenz fitted to a rectangular
hyperbola was 20.3 ± 1.3 lm The mean KD value for guanabenz and the values for 2-BFI and idazoxan are given in Table 1
Evidence for the proximity of these ligands to the flavin in MAO-A comes from the influence of the lig-and on the redox properties of the flavin Like other inhibitors of MAO-A [23,30], 2-BFI strongly increased the amount of anionic flavosemiquinone observed at
412 nm during reduction of the flavin in MAO-A by dithionite (Fig 4A) In contrast, agmatine [(4-amino-butyl)guanidine], the endogenous compound that binds
to some imidazoline sites [31,32], is a substrate for MAO-A Figure 4B shows the reduction of anaerobic MAO-A by agmatine without addition of chemical
Fig 2 Inhibition of MAO-A by guanabenz is competitive
Kynuram-ine was varied (0.1–0.9 m M ) to determine the apparent K m in the
absence (solid squares) and presence (other symbols) of inhibitor at
the concentrations indicated Secondary plots of the apparent Km
against the inhibitor concentration were used to determine the K i
values given in Table 1 The inhibition is shown as Lineweaver–
Burk plots, prepared by plotting the reciprocal data in CRICKETGRAPH ,
to illustrate the unchanged V max
0.02
A
B
0.02
–0.02
–0.03
0.03
Wavelength (nm)
0.01
0.01
–0.01 0
0
[Guanabenz] µM
Fig 3 Spectral changes on ligand binding to MAO-A (A) Selected difference spectra (the spectrum for MAO-A alone subtracted from the spectrum for MAO-A + inhibitor) are shown for guanabenz (17 l M MAO-A with 8, 16, 32 and 72 l M guanabenz) (B) The satu-rable changes at 500 nm indicate the amount of enzyme with ligand bound The line fitted to the data is a rectangular hyperbola.
Trang 4reductant As with all substrates, no semiquinone is
observed The transfer of electrons from the amine to
the flavin requires a catalytic interaction, so the
nitro-genous group of agmatine must be within 3 A˚ of N5 of
the isoalloxazine ring of the flavin Agmatine, however,
binds poorly to MAO-A, giving only 50% inhibition of
the oxidation of 0.3 mm kynuramine at 1 mm
Docking of imidazolines to the structure of the
MAO-A active site
Using qxp⁄ flo software [33], three imidazoline
com-pounds were docked into the human MAO-A structure
(Protein Data Bank code 2BXS, 3.15 A˚ [25]) The
cov-alently bound clorgyline present in the structure of
MAO-A was removed Hydrogens were added to the FAD to reflect the oxidized state of unligated enzyme Residues lining the site (listed in Experimental proce-dures) were allowed to move to give flexibility to the binding site The initial energy-minimized orientations for guanabenz, 2-BFI and idazoxan placed the com-pounds at the entrance of the active site cavity near amino acid residues found in the peptide photolabeled with an I2BS ligand This would be sufficient to block access of substrate and so inhibit the enzyme How-ever, this position is too far from the flavin for the reaction that takes place with agmatine Alternative, higher-energy orientations placed the ligands near the flavin in MAO-A, similar to the position previously found for linezolid, an oxazolidinone inhibitor of MAO-A [24], and as expected for ligands that alter the spectrum of the enzyme
In order to compare the conformation of all three ligands near the flavin in MAO-A, the docking was repeated with the compounds bound through a zero-order bond to C4a of the flavin molecule The results obtained under this condition are shown in Fig 5 The imidazoline groups of 2-BFI and idazoxan may form a hydrophobic interaction with Tyr407, which is about 3.30 A˚ away The flexibility of guanabenz allowed sev-eral orientations equally close to the flavin in two binding modes In one mode, its aromatic ring occu-pies the same area as the aromatic rings of 2-BFI and idazoxan (Fig 5A,B) in the MAO-A active site area outlined by residues Tyr69, Tyr197, Phe208, Tyr407, Phe352, Tyr444, and the isoalloxazine ring Guanabenz also assumes an alternative binding mode with respect
to the other ligands (Fig 5C), with the guanidinium group bent away from the tyrosines This alternative binding mode may indicate an enhanced probability of
Fig 4 Inhibitor and substrate imidazolines have opposite effects
on stabilization of the anionic semiquinone (A) During reduction of
MAO-A (17 l M ) by dithionite, the presence of 2-BFI (2.5 m M )
increases the yield of red anionic semiquinone at 414 nm (B)
Agm-atine reduces MAO-A (18 l M ) without the addition of dithionite, but
no semiquinone peak at 414 nm is seen Additions are 0.12, 0.16,
0.20, 0.40, 0.50 and 1.0 m M agmatine over 4 h.
B
FAD
CYS 406
TYR 69
TYR 407
GLU 216
TYR 444 TYR 197 ASN 181
PHE 352
PHE 208
C
FAD
CYS 406
TYR 69
TYR 407
GLU 216
TYR 444 TYR 197
ASN 181
PHE 352
PHE 208
A
CYS 406
TYR 197 TYR 444
TYR 407
FAD
TYR 69 PHE 352
PHE 208
GLU 216
ASN 181
Fig 5 Binding of the stereoisomers of 2-BFI (A) and idazoxan (B), and of guanabenz (C) to MAO-A The active site of MAO-A (2BXS) was prepared as described in Experimental procedures Residues allowed flexibility are shown in purple, and the flavin is orange The R forms of the ligands are green and the S forms are yellow For 2-BFI, the oxygen atoms in the heterocycle are marked by a circle.
Trang 5good interactions with the enzyme, and so might
explain the better Ki value of guanabenz (Table 1)
Interestingly, the result of docking a positively charged
(protonated) guanabenz was the same as for the
un-charged molecule At the pH of the assay and
physio-logically, most of the guanabenz would be in the
protonated, positively charged form
Docking of 2-BFI and idazoxan was performed for
both enantiomers The orientations for the R (green)
and S (yellow) forms of 2-BFI and idazoxan in
Fig 5A,B show relatively similar localization for the
imidazole ring, with the rest of the molecule easily
accommodated in the relatively large cavity of MAO-A
Figure 6 shows that the optimum position of neutral
agmatine in MAO-A is consistent with the reduction
observed experimentally, as the primary amino group
is between the two tyrosines, with the amino nitrogen
almost close enough to act as a substrate (mean
dis-tance in the three best orientations was 4 A˚)
All the three possible forms of agmatine were docked
into MAO-A: neutral, protonated, and diprotonated
Although the three forms will be in equilibrium, the
pKavalue of 8.93 for the amino group [34] means that
the diprotonated form will be the most abundant at the
assay pH of 7.2 With a pKa of 12.48, the guanidinium
group is positively charged (protonated) in most
envi-ronments but, because of the conjugation between the
double bond and the nitrogen lone pairs, the positive
charge is delocalized on the three nitrogens [35] On the
basis of titrations in octanol, it has been suggested that compounds such as guanidine retain basicity in low-dielectric medium to a greater extent than does a simple amine [36] The guanidinium group is also able to form multiple hydrogen bonds [34] The conformational searches, carried out with macromodel [37], highlighted the fact that the diprotonated form is the most stable () 1325 kJÆmol)1), followed by the mono-protonated form () 1070 kJÆmol)1) and by the neutral form () 705 kJÆmol)1) Despite the different charges, the free energies derived from the docking results were very close one another, with a maximum difference of about 6 kJÆmol)1 between the neutral form (best) and the diprotonated one The slightly better value for the neutral form could be explained by analysis of the ener-getic contributions: the negative hydrophobic term is lower for the more lipophilic neutral structure, and for the protonated forms the positive term for the polar desolvation is higher, making the total interaction energy less favorable This insight is useful because the poor inhibition of MAO-A by agmatine (IC50 of
1 mm at 0.3 mm kynuramine) makes further studies impractical
To investigate whether the guanidinium or amino group would approach closer to the flavin in a less flexible molecule, the serum protease inhibitor 4-amino-benzamidine was docked to MAO-A The same com-ments as were made for agmatine apply to the charges
on this structure As with agmatine, either orientation was possible When the amino group was deprotonated
in either agmatine or 4-aminobenzamidine, it was more likely to lie between the two tyrosines close to the flavin than the guanidinium group, but again, energy differ-ences were small
Discussion
The association of MAO with I2BS was established by the appearance of an I2BS in yeast expressing human MAO-A or MAO-B [6], but high-affinity, specific bind-ing of [3H]idazoxan was not altered in MAO-A knock-out mice, although covalent labeling of one peptide was lost [14] The irreversible inhibition of MOA
in vivo has been shown to reduce the density of imida-zoline-binding sites in rat brain [38,39] Here we have used kinetic and spectral techniques to characterize the binding of imidazolines to the active site of purified MAO-A The representative I2BS ligands used are lin-ear competitive inhibitors of purified MAO-A, in agreement with the data of Ozaita et al (1997) for membrane-bound MAO-A and MAO-B [5] Spectral changes and the alteration of the redox properties of the flavin (Figs 2 and 3) confirm that I2BS ligands bind
FAD
CYS 406
PHE 352
PHE 208
TYR 69
TYR 407 TYR 444
ASN 181 TYR 197
GLU 216
Fig 6 Agmatine in the active site of MAO-A Binding of the
neut-ral form of agmatine (green) close to the flavin in MAO-A is
stabil-ized by the hydrogen bonds with Glu216, Tyr444, Tyr197, and
Asn181 Colours are as in Fig 5.
Trang 6at the active site Typical changes in the flavin
spec-trum are seen on binding of I2BS ligands, and the KD
values calculated from the saturable decreases in
absorbance at 500 nm were slightly higher than the
kinetic Ki values (Table 1) The spectral change
requires close association of the ligand with the flavin,
whereas competitive inhibition requires only blocking
of access to the active site The multiple orientations
seen for guanabenz in the docking study included some
that are less likely to influence the flavin and the
adja-cent tyrosine rings directly in the way seen for 2-BFI
This may be an explanation for the higher KDthan Ki
with guanabenz
The reduction of MAO-A by agmatine (Fig 4B)
establishes that the ligand can bind near the flavin,
because proximity is required for electron transfer
However, the very poor inhibition of kynuramine
oxi-dation by agmatine (50% inhibition at 1 mm in the
presence of 0.3 mm kynuramine) and its very slow
reduction of anaerobic MAO-A indicate that MAO-A
is not a pathway for agmatine metabolism The
copper-containing amine oxidases are more likely to contribute
to the oxidation of this biologically active amine [35]
Inhibition of MAO-A by I2BS ligands is shown here
to require over 1000-fold higher concentrations than
that reported in the literature for saturation of I2BS
[6] Binding to the active site of solubilized MAO-A
has been measured directly (Fig 3); this revealed only
one site with the same low affinity as found for both
MAO-A and MAO-B in membranous preparations [5]
These differences add to the evidence that the I2BS is
distinct from the active site of MAO
The flexible docking models (Fig 5) revealed that all
three I2BS ligands were easily accommodated in the
hydrophobic region surrounded by residues Tyr69,
Tyr197, Phe208, Tyr407, Phe352, Tyr444, and the
iso-alloxazine ring, with rather small differences in free
energy that did not reflect the differences in KDvalues
The optimal orientation for guanabenz in MAO-A has
the closest nitrogen atom less than 5 A˚ from N5 of the
isoalloxazine ring This orientation and the alternative
binding modes found may explain why it is the best of
the inhibitors examined here
Given that all three ligands tested had similar
ener-gies despite their well-separated Ki values, no
conclu-sions can be drawn from the similar energies and
orientations for the R and S stereoisomers of both
2-BFI and idazoxan As only the racemic mixture was
available, discrimination of the stereoisomers by
MAO-A was not tested experimentally The I2BS does
show discrimination between the (+) and (–) forms of
idazoxan, with the (–) isomer more potent on
periph-eral I2sites and the (+) isomer more potent on central
I2BS sites [40,41] Further experimental data on both MAO and the I2BS stereospecificities would be useful Interestingly, the lowest-energy orientation for each
of the three ligands in the large active site of MAO-A put them in the middle of the active site pocket, adja-cent to residues in the peptide identified by photoaffin-ity labeling [12] This suggests that the conclusion that this peptide forms part of the I2BS location rather than just the active site must be taken with caution
In conclusion, this study has characterized the bind-ing to MAO-A of three commonly used I2BS ligands, and had provided evidence for only one binding site adjacent to the flavin in this isolated preparation All three show three orders of magnitude lower affinity for the active site than reported values for I2BS It seems clear that the active site of MAO is not the site to which
I2BS ligands bind with nanomolar affinity However, imidazolines do bind at the active site of MAO-A with dissociation constants in the 10–200 lm range, causing spectral and redox changes as seen for other active site inhibitors The modeling studies revealed multiple ori-entations within the hydrophobic active site that did not differ much with the protonation state and provi-ded a possible explanation for the covalent labeling of
an active site peptide in MAO by an imidazoline ligand
Experimental procedures
Materials
MOA-A (human liver form) expressed in Saccharomyces cerevisiae [42] was purified and stored at) 20 C in a solu-tion of 50 mm potassium phosphate (pH 7.2), 0.8% n-octyl-b-d-glucopyranoside (Melford Laboratories Ltd, Ipswich, UK), 1.5 mm dithiothreitol, 0.5 mm d-amphetamine, and 50% glycerol The specific activity was 1 lmolÆmin)1Æ(mg protein))1 with kynuramine as the substrate Only one major peak was seen in the mass spectrum, and the purity was greater than 95% as determined by silver-stained SDS gel electrophoresis Before use, dithiothreitol, d-amphetam-ine and glycerol were removed by gel filtration in a spin col-umn of G-50 Sephadex equilibrated with 50 mm potassium phosphate (pH 7.2) containing detergent (0.05% Brij) [30] 2-BFI was purchased from Tocris (Bristol, UK), and all other chemicals were obtained from Sigma-Aldrich Co Ltd (Poole, UK)
MAO-A assays and spectral titrations
Initial rates of oxidation were measured spectrophotometri-cally at 30C in 50 mm potassium phosphate (pH 7.2) con-taining 0.05% Triton X-100 Ki values were determined from three to five separate experiments, using a substrate
Trang 7range of 0.1–0.9 mm kynuramine or 0.025–0.5 mm MPTP
at six different inhibitor concentrations over a 10-fold
range The formation of the product was followed
spectro-photometrically at 314 nm in assays with kynuramine [43]
and at 343 nm with MPTP [44] The kinetic constants were
obtained by direct fit for each group of six substrate
con-centrations in duplicate in the Shimadzu kinetics program
of the UV-2101PC spectrophotometer (Shimadzu UK Ltd.,
Milton Keynes, UK) The linear secondary plots were fitted
in Excel
Spectra were recorded approximately 5 min after each
addition in a Shimadzu UV-2101PC spectrophotometer in
an anaerobic cuvette containing MAO-A at about 10 lm in
50 mm potassium phosphate (pH 7.2) containing 0.05%
Brij The change in absorbance at 500 nm was used to
determine the binding parameters, fitting the data to a
rect-angular hyperbola in enzfitter
Modeling
The molecular modeling studies were carried on an
SGI-OCTANE R12000 workstation operating under irix 6.5+
(Silicon Graphics Inc., Sunnyvale, CA, USA) The ligand
structures were built through the building tool of the
soft-ware macromodel version 8.1 [37] (Schrodinger, Inc.,
Port-land OR, USA; http://www.schrodinger.com) Aqueous
conformational analyses by macromodel were performed
using the Monte Carlo Multiple Minimum (MCMM)
Search protocol with the Merck Molecular Force Field
(MMFFs) Prior to submitting the ligands to the search
protocol, a minimization was carried out using the MMFFs
as implemented in macromodel Default options were used
with the Polak–Ribiere Conjugate Gradient scheme, until a
gradient of 0.001 kJÆA˚)1 mol was reached To search the
conformational space, 5000 Monte Carlo steps were
per-formed on each starting conformation An energy cut-off of
20.0 kJÆmol)1, high enough to map the conformational
space, including the bioactive conformation, was applied to
the search results The lowest-energy conformer of each
ligand was exported to Protein Data Bank format to be
read by the docking software
Docking studies were performed using the qxp⁄ flo
soft-ware using the qxp mcdock+ module (1000 cycles) qxp is
the molecular mechanics module in flo+ (version
April03), a molecular design program (Thistlesoft,
Cole-brook, CT, USA) [33]
The enzyme structure used for the calculations was the
human MAO-A (Protein Data Bank code 2BXS, 3.15 A˚
[25]) The covalently bound clorgyline present in the
struc-ture of MAO-A was removed Hydrogens were added to
the FAD to reflect the oxidized state of unligated enzyme
To give flexibility to the binding site, residues lining the site
were allowed to move These residues were: Cys406 (to
which the flavin is covalently bound), Tyr69, Tyr444,
Tyr407, Phe208, Phe352, Tyr197, Asn181, and Glu216 To
speed the calculations, the enzyme was cut to 12 A˚ around the active site before each docking The docking procedure was tested using clorgyline as a control for comparison with the original structure When the covalent bond was present, the optimum position was in the same area as in the crystal structure With oxidized FAD and a zero-order bond to the flavin, binding was similar but not exactly superimposable
In the absence of any bond, the clorgyline molecule moved away from the flavin
Acknowledgements
T Z E Jones and R R Ramsay thank AstraZeneca for support Dr Colin McMartin (Thistlesoft, Cole-brook, CT, USA) is gratefully acknowledged for the software flo+ (version April03) We thank Dr Robert
A Scherrer (BIOpK, White Bear Lake, MN, USA) for helpful discussion and suggestions on the physicochem-ical properties of the guanidine derivatives The mode-ling work is part of Laura Giurato’s PhD thesis, to be presented at the Universita` degli Studi di Catania
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