Beyond topoisomerase inhibition: Antitumor 1,4-naphthoquinones as potential inhibitors of human monoamine oxidase

Article abstract of DOI:10.1111/cbdd.12255

Monoamine oxidase (MAO) action has been involved in the regulation of neurotransmitters levels, cell signaling, cellular growth, and differentiation as well as in the balance of the intracellular polyamine levels. Although so far obscure, MAO inhibitors are believed to have some effect on tumors progression. 1,4-naphthoquinone (1,4-NQ) has been pointed out as a potential pharmacophore for inhibition of both MAO and DNA topoisomerase activities, this latter associated with antitumor activity. Herein, we demonstrated that certain antitumor 1,4-NQs, including spermidine-1,4-NQ, lapachol, and nor-lapachol display inhibitory activity on human MAO-A and MAO-B. Kinetic studies indicated that these compounds are reversible and competitive MAO inhibitors, being the enzyme selectivity greatly affected by substitutions on 1,4-NQ ring. Molecular docking studies suggested that the most potent MAO inhibitors are capable to bind to the MAO active site in close proximity of flavin moiety. Furthermore, ability to inhibit both MAO-A and MAO-B can be potentialized by the formation of hydrogen bonds between these compounds and FAD and/or the residues in the active site. Although spermidine-1,4-NQs exhibit antitumor action primarily by inhibiting topoisomerase via DNA intercalation, our findings suggest that their effect on MAO activity should be taken into account when their application in cancer therapy is considered. Antitumor spermidine-1,4-naphthoquinones are reversible and competitive MAO inhibitors. Spermidine-1,4-NQs that act as potent inhibitors of MAO are capable of binding to FAD moiety. The application of spermidine-1,4-naphthoquinones in cancer therapy should consider their effect on MAO activity.

Full text of DOI:10.1111/cbdd.12255

Chem Biol Drug Des 2014; 83: 401–410
Research Article
Beyond Topoisomerase Inhibition: Antitumor
1,4-Naphthoquinones as Potential Inhibitors of
Human Monoamine Oxidase
Eduardo Coelho-Cerqueira1, Paulo A. Netz2,
Vanessa P. do Canto2, Angelo C. Pinto3
and Cristian Follmer1,*
Monoamine oxidases (MAO) [amine: oxygen oxidoreduc-
tase (deamination) (flavin containing) EC 1.4.3.4.] catalyze
the major route of inactivation of neurotransmitters, such
as dopamine, epinephrine, and norepinephrine by promot-
ing the oxidation of amines to the corresponding alde-
hydes, with the generation of hydrogen peroxide (1). Two
MAO isoforms have been identified, MAO-A and MAO-B,
which have a sequence identity of approximately 72% and
differ from each other in relation to substrate and inhibitor
specificity. MAO-A acts preferentially on 5-hydroxytrypta-
mine, whereas MAO-B is more active on arylalkylamines
such as benzylamine, preventing exogenous amines from
acting as false neurotransmitters (1,2). Additionally, MAO-B
participates in the oxidative metabolism of exogenous
amines such as the neurotoxin 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP), which is converted to MPP+
that causes Parkinson-like symptoms in human and other
primates (2,3).
1Department of Physical Chemistry, Institute of Chemistry,
Federal University of Rio de Janeiro, Rio de Janeiro
21941-909, Brazil
2Institute of Chemistry, Federal University of Rio Grande
do Sul, Porto Alegre, 91501-970, Brazil
3Department of Organic Chemistry, Institute of Chemistry,
Federal University of Rio de Janeiro, Rio de Janeiro
21941-909, Brazil
*Corresponding author: Cristian Follmer, follmer@iq.ufrj.br
Monoamine oxidase (MAO) action has been involved in
the regulation of neurotransmitters levels, cell signal-
ing, cellular growth, and differentiation as well as in
the balance of the intracellular polyamine levels.
Although so far obscure, MAO inhibitors are believed
to have some effect on tumors progression. 1,4-naph-
thoquinone (1,4-NQ) has been pointed out as a poten-
tial pharmacophore for inhibition of both MAO and
DNA topoisomerase activities, this latter associated
with antitumor activity. Herein, we demonstrated that
certain antitumor 1,4-NQs, including spermidine-
1,4-NQ, lapachol, and nor-lapachol display inhibitory
activity on human MAO-A and MAO-B. Kinetic studies
indicated that these compounds are reversible and
competitive MAO inhibitors, being the enzyme selectiv-
ity greatly affected by substitutions on 1,4-NQ ring.
Molecular docking studies suggested that the most
potent MAO inhibitors are capable to bind to the MAO
active site in close proximity of flavin moiety. Further-
more, ability to inhibit both MAO-A and MAO-B can be
potentialized by the formation of hydrogen bonds
between these compounds and FAD and/or the resi-
dues in the active site. Although spermidine-1,4-NQs
exhibit antitumor action primarily by inhibiting topo-
isomerase via DNA intercalation, our findings suggest
that their effect on MAO activity should be taken into
account when their application in cancer therapy is
considered.
The activity of both MAO isoforms is augmented with
aging, leading to an increase in the side product hydrogen
peroxide, accompanied by a reduction in the levels of cer-
tain neurotransmitters (1,4). While the level of MAO-B
increases 4- to 5-fold during aging and was associated
with neurodegenerative disorders (4), MAO-A level was
significantly enhanced in hearts of aged rats and was
associated with cardiac cellular degeneration (5). In addi-
tion, MAO activity is enhanced in certain tumor cells (6,7).
In this context, extensive efforts have been made to
develop efficient and safe MAO inhibitors, notably for Alz-
heimer disease and Parkinson’s disease therapy (inhibition
of MAO-B isoform) and for depression illness (inhibition of
MAO-A) (1).
Several evidences have pointed out that MAO is a promis-
ing target for cancer therapy: (i) A significant and selective
MAO-B activity is enhanced in malignant tumors in central
nervous system (6,7). In astrocytomas, the degree of
tumor malignancy has been positively correlated with the
amine oxidase activity (8). (ii) Isatin, an endogenous MAO-
B inhibitor, is capable to induce apoptosis or necrosis in
human neuroblastoma cells (9) due to an intracellular
accumulation of monoamines and increase in the produc-
tion of reactive oxygen species (ROS) (10). (iii) In prostate
cancer, MAO-A inhibitors such as clorgyline might restore
Key words: cancer, flavin, lapachol, monoamine oxidase,
naphthoquinones, spermidine
Received 25 July 2013, revised 24 September 2013 and
accepted for publication 22 October 2013
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12255
401

Cerqueira et al.
differentiation and hence reverse the aggressive character-
istic of high-grade cancer (11). Therefore, MAO-A and
MAO-B might represent an important target for new thera-
peutic strategies for different types of tumors.
MAO activity. In light of these findings, we have discussed
the role of MAO activity in cancer growth as well as the
importance of MAO inhibition in antitumor therapies.
Recently, it has been shown that 1,4-naphthoquinone
(1,4-NQ) is a potential scaffold for designing reversible
MAO inhibitors in which the selectivity may be easily
altered by changes in the substituents on the naphthoqui-
none ring (12,13). Furthermore, certain 1,4-NQs display
inhibitory properties on important biological targets, includ-
ing DNA topoisomerase activity (14), amyloid b-peptide
and a-synuclein fibrillization (15,16), and Hsp90 activity
(17). Naphthoquinones such as lapachol (2-hydroxy-3-pre-
nyl-1,4-naphthoquinone) and nor-lapachol [2-hydroxy-3-(2-
methylpropenyl)-1,4-naphthoquinone] were described to
have antitumor action against several cancer cells (14,18).
Recently, the antitumor activity of 2-spermidine-3-R-1,4-
naphthoquinones was demonstrated for cancer cell lines
such as human promyelocytic leukemia, lung cancer, Bur-
kitt lymphoma, and mouse breast tumor (18). Studies of
the antitumor properties and mechanisms of action of qui-
none derivatives have shown that they can act as topo-
isomerase inhibitors via DNA intercalation (14). These
molecules display a 1,4-NQ scaffold, which is associated
with both DNA topoisomerase and MAO inhibition, conju-
gated with a polyamine such as spermidine, which is sub-
strate for MAO and other amine oxidases. Interestingly,
the level of polyamines such as N1-(3-aminopropyl)-
1,4-butanediamine (spermidine, d) is increased in tumors
as compared to normal tissues, and their intracellular
accumulation can induce apoptosis in different cell lines
(19,20). Analogs of natural polyamines might compete with
naturally occurring polyamines for critical cellular binding
sites (18). In this context, the conjugation of spermidine
analogs with a cytotoxic compound was used to increase
in their chemotherapeutic activity by either facilitating their
entry into tumor cells (via polyamine uptake systems) or
increasing their selectivity to DNA (18).
Experimental
Synthesis of spermidine-1,4-NQs
Spermidine-1,4-NQs a1, b1, and c1 were synthesized and
characterized as described by Cunha et al. (21). The syn-
thesis steps are indicated in Figure 1, in which one can
verify that a1 and a2 compounds are structurally related to
lapachol (a), whereas the c1 and c2 compounds are
related to nor-lapachol (c). The b1 and b2 compounds are
related to lawsone (b), that is, they display a hydrogen at
the 3-position in the 1,4-NQ ring. The steps of the synthe-
sis were as follows: (i) the methylation of lapachol (a) and
nor-lapachol (c) with dimethylsulphate in acetone and
potassium carbonate to yield a0 and c0, and synthesis of
methoxylawsone (b0) from the sodium salt of 1,2-naphtho-
quinone-4-sulfonic acid; (ii) preparation of the protected
derivative of spermidine d0 in a four-step synthesis; (iii)
nucleophilic displacement of the methoxyquinones a0, b0,
and c0 with compound d0 (Figure 1). To remove the pro-
tecting group BOC, a solution of TFA (0.20 ml; 2.6 mmol)
in CH2Cl2 (5 mL) was slowly added to a solution of b1
(62.7 mg; 0.13 mmol) in methanol (20 mL) at 0 °C. After
10 min, the reaction was taken to room temperature and
kept under stirring for 4 h until total consumption of the
starting material. The solvent was removed under reduced
pressure; addition of 10% KHCO3 (10 mL) was followed
by extraction with CH2Cl2 (3 9 20 mL). The organic phase
was dried with anhydrous Na2SO4 and concentrated
under reduced pressure to give the free amine b2
(50.3 mg, 99%), as a red oil that was purified by flash
chromatography (ethyl acetate/methanol, 6:4). [Rf = 0.2
(CH2Cl2/methanol/triethylamine 10:89:1)]. Infrared (film)
mmax (cmÀ1): 3353, 3063, 3003, 2973, 2805, 1708,
1606,1509, 721, 698. 1H NMR (200 MHz, CDCl3): d 1.59
(m, 6H), 2.42 (t, J = 6.2 Hz, 2H), 2.50 (t, J = 6.2 Hz, 2H),
3.11 (m, 4H), 3.53 (s, 2H), 5.28 (brs, 1H), 5.68 (s, 1H),
5.99 (s, 1H), 7.30 (m, 5H), 7.61 (brt, J = 7.5, 1H), 7.73
(brt, J = 7.5, 1H), 8.05 (d, J = 7.5, 1H) and 8.11 (d,
J = 7.5 Hz, 1H). 13C NMR (50 MHz, CDCl3): d 24.6, 25.9,
26.1, 28.5, 34.0, 42.4, 52.4, 53.1, 58.9, 126.2, 129.0,
131.9, 134.7, 139.2, 149.2, 183.1, 184.3. MS found:
392.2359 [b2+1]+; calculated for [C24H29N3O2]+ 392.2338.
In this work, we synthesized and evaluated the inhibitory
activity on human MAO-A and MAO-B of antitumor
polyamine-1,4-NQs containing
a
spermidine analog
(benzyl-spermidine) conjugated to 1,4-NQ, lapachol, or
nor-lapachol. The molecular mechanisms lying behind the
inhibition of MAO isoforms were investigated using molec-
ular modeling tools. These antitumor compounds inhibited
MAO isoforms by a competitive mechanism in which the
enzyme selectivity was greatly influenced by substitutions
on 1,4-NQ ring. In addition, molecular docking results sug-
gested that spermidine-1,4-NQs that act as potent inhibi-
tors of MAO are capable of binding to the catalytic site of
MAO in close proximity of flavin moiety. Although previous
studies have already pointed out that 1,4-NQ represents
an important scaffold for the development of both MAO
and DNA topoisomerase inhibitors, it was the first time that
1,4-NQs with potential antitumor activity against several
cancer cell lines were evaluated in respect to inhibition of
Similar procedure was followed for the syntheses of com-
pounds a2 and c2. Product a2 was purified as above using
ethyl acetate/methanol 6:4 as eluent and obtained as
brown reddish oil (57.9 mg, 97%). [Rf = 0.2 (CH2Cl/meth-
anol/triethylamine 10:89:1)]. Infrared (film) mmax (cmÀ1):
3347, 3063, 3027, 2930, 2863, 1712, 1602, 1570, 1515,
721, 698. 1H NMR (200 MHz, CDCl3): d 1.60 (m, 4H),
1.68 (brt, 3H), 2.42 (t, J = 6.4 Hz,2H), 2.48 (t, J = 6.4 Hz,
2H), 3.15 (m, 2H), 3.40 (m, 4H), 3.52 (s, 2H), 5.06 (m,
1H), 5.25 (m, 1H), 5.67 (m, 1H), 7.56 (brt, J = 7.5 Hz, 1H),
402
Chem Biol Drug Des 2014; 83: 401–410

MAO Inhibition by Antitumor 1,4-Naphthoquinones
O
O
R
NH
2
H N
NH
HO
2
(d) spermidine
R = CH CH=C(CH
2
) (a) lapachol
3 2
R = H ( b) lawsone
R = CH=C(CH
)
(c) nor-lapachol
3 2
O
O
O
R
R
O
O
+
4
3
O
NH
N
NH
2
4
3
O
NH
N
NH
O
O
d
0
a
R = CH CH=C(CH ) ,
2
3 2
0
b
R = H,
0
a
R = CH CH=C(CH ) ,
2
b
3 2
1
c
0
R = CH=C(CH ) ,
3 2
R = H,
1
c
1
R = CH=C(CH ) ,
3 2
N-BOC removal
O
R
4
3
H N
N
NH
2
O
a
R = CH CH=C(CH ) ,
2
3 2
2
b
R = H,
2
Figure 1: Steps of the synthesis of
spermidine-1,4-NQs.
c
2
R = CH=C(CH ) ,
3 2
7.67 (brt, J = 7.5 Hz, 1H), 7.99 (dd, J = 7.5 Hz, 1H), 8.09
(dd, J = 7.5 Hz, 1H). 13C NMR (50 MHz, CDCl3): d 18.2,
23.8, 24.3, 25.8, 26.6, 29.2, 39.3, 45.0, 52.7, 53.4, 58.9,
115.4, 123.2, 126.2, 127.2, 128.7, 130.5, 132.0, 133.6,
134.4, 139.4, 145.7, 183.1. MS found: 460.2943 [a2+1]+;
calculated for [C29H37N3O2]+ 460.2964. Product c2 was
purified as above using ethyl acetate/methanol, 6:4 as elu-
ent as red oil (55.5 mg, 96%) [Rf = 0.2 (CH2Cl2/methanol/
triethylamine 10:89:1)]. Infrared (film) mmax (cmÀ1): 3354–
3347, 3063, 3027, 2972, 2806, 1712, 1602, 1515, 1170,
721, 698. 1H NMR (200 MHz, CDCl3): d 1.46 (m, 5H);
1,61 (m, 4H); 1,92 (s, 3H); 2,42 (m, 4H, J = 6.3); 2.61 (t,
2H, J = 6.3 Hz); 4,60 (t, 2H, J = 6.3); 3.50 (s, 2H); 5.34
(br s, H); 5.91 (t, 1H); 6.10 (s, 1H); 7,54 (m, 5H); 7,63 (dt,
2H, J = 7.5); 8,06 (dd, 2H, J = 7.5). 13C NMR (50 MHz,
CDCl3): d 20.3, 24.5, 25.6, 26.9, 28.0, 39.9, 44.4, 39.9,
53.5, 59.0, 113.5, 118.0, 126.1, 126.5, 127.3, 128.5,
129.1, 130.6, 132.0, 133.8, 134.7, 135.3, 138.7, 139.5,
144.8, 183.1, 183.5. MS found: 446.2837 [c2+1]+; calcu-
lated for [C28H35N3O2]+ 446.2807.
Carlsbad, CA, USA) in the presence of hydrogen peroxide
(generated by MAO activity) and horseradish peroxidase
(HRP) (22). These assays were carried out in a 96-well
microplate. The fluorescence intensity was measured by
a fluorescence microplate reader in a Cary Eclipse Fluo-
rimeter (Agilent Technologies, Santa Clara, CA, USA) with
excitation at 571 nm and emission at 585 nm. The effect
of the inhibitors on the fluorescence emission of resorufin
at 585 nm was previously evaluated, and a correction
was performed when necessary. The reaction mixture
(final volume of 200 lL) contained 5 lg/mL MAO-A or
MAO-B in a 50 mM sodium phosphate buffer pH 7.4,
1 mM p-tyramine (substrate for MAO-A) or 1 mM benzyl-
amine (substrate for MAO-B), 1 U/mL HRP, and 200 lM
AR. This mixture was incubated, in the presence or
absence of the inhibitors, at 37 °C for 45 min using a
Thermomixer equipment (Eppendorf, Hamburg, Germany).
The enzyme plus the inhibitors were incubated at 37 °C
for 20 min prior to the addition of the substrates, HRP
and AR. The inhibitors were dissolved in 100% DMSO,
and equivalent concentrations of DMSO alone (1–2%)
were used as controls. Clorgyline (MAO-A inhibitor) or
pargyline (MAO-B inhibitor) at 5 lM concentration was
used as positive control for inhibition. To verify whether
the inhibitors affect the enzymatic assay by either inhibit-
ing the AR oxidation by HRP or by scavenging the hydro-
gen peroxide that was generated, hydrogen peroxide was
preincubated in the presence or absence of the inhibitors,
and 1 U/mL HRP, 1 mM substrate, and 200 lM AR were
MAO assay
Microsomes from baculovirus-infected insect cells that
express recombinant human MAO-A and MAO-B were
purchased from Sigma-Aldrich Co (St Louis, MO, USA).
The activities of MAO-A and MAO-B were evaluated by a
fluorometric assay that measures the amount of resorufin
produced from Amplex Redꢀ (AR) (Life Technologies,
Chem Biol Drug Des 2014; 83: 401–410
403

Cerqueira et al.
added to the reaction mixture. This mixture was incu-
bated at 37 °C for 45 min, and the resorufin content was
determined by fluorescence, as described above. The
enzymatic activity was expressed as nanomols of hydro-
gen peroxide produced per milligram of enzyme per min-
ute at pH 7.4 and 37 °C. We varied the amount of
hydrogen peroxide used (0.4–3.2 nmols) to create a cali-
bration curve.
dimeric form of the human MAO-B, with each chain con-
taining FAD and benzylhydrazine. For docking purposes,
only the co-ordinates of chain A and FAD were considered
as starting point for the construction of the receptor
structure.
Molecular modeling: grid and docking
The spermidine derivatives are bulky ligands and are able
to fit only roughly in the binding site as displayed in the ori-
ginal PDB receptor structures. The structures obtained
from the Protein Data Bank depend strongly on the experi-
mental conditions at which they were determined. It is rea-
sonable, thus, to relax the receptor’s structures before the
docking and to obtain an adequate ensemble of confor-
mations, where the receptor is able, in principle, to interact
with ligands of several sizes. Therefore, we choose an
ensemble docking method similar to the Relaxed Complex
Scheme (33–35) in order to sample adequate conforma-
tions, suitable to interact with those ligands. We carried
out 50 ns long molecular dynamics simulations of the Holo
form [bound with FAD and menadione (13)] of MAO-A and
MAO-B. At each 5 nseconds, snapshots were extracted
and the protein and FAD co-ordinates used as receptor
for docking purposes. The best scores for each ligand in
each snapshot were recorded, and we could therefore
obtain a dynamical evaluation of the docking poses, which
can be correlated with receptor’s structural changes. The
molecular dynamics (MD) simulations for the generation of
the trajectory were carried out with the GROMACS pack-
age (36) using GROMOS 53A6 force field (37), in the NPT
ensemble, using PME electrostatic calculations. Explicit
SPC (simple point charge) water molecules located in a
simulation box was used. Snapshots were generated
using programs into Gromacs package, and the structures
of the receptors were prepared using AutoDock tools. The
receptor structures in each snapshot were fitted to the ini-
tial structure, using the FAD moiety as reference. The
ensemble docking was carried out, with help of home-
Kinetic parameters
The enzymatic kinetic parameters Michaelis–Menten con-
stant (Km) and maximum reaction rate (Vmax) were deter-
mined by nonlinear fitting using the NLfit tool and the Hill
function in OriginLab 8.0 software (OriginLab Co, North-
ampton, MA, USA). The inhibitory constant Ki was deter-
mined by the relation: Km’/Km = 1 + [I]/Ki, where Km′ and
Km are the Michaelis–Menten constant in the presence
and the absence of inhibitor, respectively, and [I] is the
concentration of the inhibitor. The mechanism of inhibition
was confirmed by the Lineweaver–Burk curves (double
reciprocal plot) by plotting the inverse of initial velocity (1/
V) as a function of the inverse of the substrate concentra-
tion (1/S) (23).
Molecular modeling: ligand and receptor building
The structures of menadione, lapachol, nor-lapachol, and
the spermidine-1,4-naphthoquinones (a2, b2, and c2)
were built with GaussView (24) and optimized with RHF/
6-31G (d,p) using GAUSSIAN 09 (25). Considering that
the spermidine derivatives have many potential proton-
ation sites, the pKA of each amine group and the
protonation pattern at physiological conditions were cal-
culated with MarvinSketch [Marvin 5.12.3 (Evaluation
Mode), 2013, ChemAxon (www.chemaxon.com/marvin/
sketch/index.php)], and the resulting protonated struc-
tures were re-optimized with AM1 method using GAUSS-
IAN 09. The final structures were converted to the mol2
format. Using AutoDock Tools (26), the non-polar hydro-
gen atoms were merged to the heavy atoms. Gasteiger–
Marsili partial charges are assigned as described (27).
The final structures were written as pdbqt files for further
use in AutoDock (28,29) and AutoDockVina (30)
dockings.
made scripts, with AutoDock Vina, using
a grid of
23 9 23 9 23 Angstroms centered in the active site.
All dockings using AutoDock Vina were carried out in
triplicate.
For dockings using the AutoDock program, the receptor
structures were chosen according to AutoDock Vina
results, selecting the snapshots that provided the major
discrimination between ligands, 30 nseconds for MAO-A
and 20 nseconds for MAO-B. A grid box was built with a
resolution of 0.375 Angstrons and 61 9 61 9 61 points
and constituted a region surrounding the interaction site
close to FAD, inside the enzymes. The docking was car-
ried out by AutoDock 4.2 (38) with a Lamarckian Algorithm
(Genetic Algorithm combined with a local search). The fol-
lowing parameters were chosen: 100 GA runs, population
size of 50, number of evaluations 50 000 000, number of
generations 27 000, maximum number of top individuals
of 1, gene mutation rate of 0.02, and a crossover rate of
The MAO-A receptor structure was built as a modification
of a structure obtained from the Protein Data Bank (PDB:
2Z5X) (31). This structure includes a monomeric form of
MAO-A interacting non-covalently with FAD and the
reversible inhibitor harmine (HRM, 7-methoxy-1-methyl-
9H-beta-carboline). The co-ordinates of harmine and
crystallization water molecules were deleted, and only the
co-ordinates of the protein and FAD were used as starting
point for the construction of the receptor structures, as
detailed below. The MAO-B receptor structure was built
as a modification of the structure obtained from the Pro-
tein Data Bank (PDB: 2VRL) (32). This structure contains a
404
Chem Biol Drug Des 2014; 83: 401–410

MAO Inhibition by Antitumor 1,4-Naphthoquinones
0.8. The AutoDock 4.2 default values were used for the
remaining docking parameters, except for the step-size
Analyzing the data in Table 1, one can observe that all
spermidine-1,4-NQs were capable to inhibit MAO iso-
forms with a poor selectivity, exhibiting Ki values in the
range of 12–63 lM. The most potent inhibitor of MAO-B
was b2 with a Ki = 12 lM and a 2-fold selectivity for this
isoform. For MAO-A, a2 exhibited the lowest Ki value
(22 lM), being the most selective among the spermidine-
1,4-NQs. The effect of the inhibitors (50 lM) on the
parameters Km and Vmax for MAO-A and MAO-B is
shown in Table 2. The Km values for MAO-A and MAO-B
in the absence of the inhibitors were 370 Æ 68 lM
ꢀ
parameters that were chosen to be 0.2 A (translation) and
5.0 degrees (quaternion and torsion). The docked confor-
mations were clustered according to their geometrical sim-
ꢀ
ilarity (rms of 2.0 A) and docked energy. The interactions
between the ligand and protein residues were analyzed
with AutoDockTools (26).
Results and Discussion
(Vmax = 36 Æ 4 nmol/min.mg)
and
497 Æ 203 lM
Kinetic features of MAO inhibition
(Vmax = 19 Æ 4 nmol/min.mg), respectively. An increasing
of Km without changes in Vmax was observed for all inhib-
itors, which indicates a competitive mechanism. The most
remarkable alteration for Km was observed for the com-
pounds exhibiting the lowest Ki values, that is, a2 for
MAO-A and nor-lapachol and b2 for MAO-B. The minor
MAO inhibitors, lapachol, a2, and c2 for MAO-B and c2
for MAO-A, did not produce significant effects on Km at a
concentration of 50 lM (Table 2). Collectively, our data
suggest that antitumor 1,4-NQs are competitive MAO
inhibitors in which the enzyme selectivity is remarkably
dependent on the substitutions on 1,4-NQ ring. While
1,4-NQ inhibits MAO-A by a non-competitive mechanism
(13), its derivatives with substitutions on either 2- or
3-position of 1,4-NQ ring behave exclusively as
competitive inhibitors. Molecular modeling data suggest
that 1,4-NQ interacts with MAO-A residues located in the
inner portion of the enzyme instead of those in the cata-
lytic site, which may explain the observed non-competi-
tive mechanism (13).
By using a very sensitive fluorescent method to measure
MAO activity, the inhibitory effect of a series of antitumor
1,4-NQs (spermidine-1,4-NQs, lapachol, and nor-lapachol)
on MAO activity was investigated. It is important to note
that these 1,4-NQs did not interfere in the reaction of for-
mation of resorufin from AR in the presence of hydrogen
peroxide and HRP, similarly to described for 1,4-NQ and
menadione (13). In addition, these compounds neither
interfered in resorufin fluorescence (e.g., by quenching
the fluorescence) nor act as scavenger of hydrogen per-
oxide that was produced during the reaction (data not
shown).
Table 1 shows the inhibitory constant Ki and the enzyme
selectivity (KiMAO-A/KiMAO-B) for MAO inhibition by spermi-
dine-1,4-NQs (a2, b2, and c2), lapachol, and nor-lapachol.
The mechanisms of inhibition as well as the Ki values were
determined through a nonlinear regression of Michaelis–
Menten curves as illustrated in Figure 2. Both lapachol
and nor-lapachol were less effective MAO inhibitors than
either 1,4-NQ or menadione, this latter being the most
potent and selective 1,4-NQ described so far with
Ki = 0.4 lM and 65-fold selectivity for MAO-B (13). Lapa-
chol inhibited MAO-B with a Ki value of 59 lM, but it did
not have any effect on MAO-A at a concentration of
100 lM. On the other hand, nor-lapachol inhibited both
isoforms with similar Ki values: 35 and 18 lM for MAO-A
and MAO-B, respectively. Therefore, nor-lapachol, with a
poor selectivity for MAO-B, was less potent and selective
than menadione or 1,4-NQ for this isoform.
Two important features must be taken into account for the
development of new MAO inhibitors: the reversibility and
the selectivity. The reversibility and non-selectivity might
represent, in certain cases, an advantage in comparison
with irreversible and selective inhibitors, even though
reversible inhibitors are often less potent than irreversible
inhibitors. Similar to other 1,4-NQs, spermidine-1,4-NQs,
lapachol, and nor-lapachol behave as reversible MAO
inhibitors. The reversibility is an important feature for inhibi-
tors targeting MAO-A. As tyramine is metabolized by
MAO-A in the intestine, the irreversible inhibitors of MAO-A
might produce a side-effect known as ‘cheese reaction’
that is characterized by an elevation of the sympathetic
cardiovascular activity via the release of norepinephrine
(39). Because the intestine contains a low MAO-B content,
the administration of MAO-B inhibitors does not promote
the cheese reaction unless the inhibitor is administered in
Table 1: Kinetic data of the inhibition of MAO by 1,4-NQs.
Results are shown as mean Æ standard deviation for three experi-
#
ments.*Ref (7.); no inhibition at up 100 lM
Inhibitory constant Ki (lM)
MAO-A
MAO-B
KiMAO-A/KiMAO-B
a
dose high enough to inhibit MAO-A. Furthermore,
spermidine-1,4-NQs and nor-lapachol were in general
poorly selective inhibitors. In contrast with menadione that
exhibits 65-fold selectivity to MAO-B, the most selective
inhibitors of MAO-B among the antitumor 1,4-NQs, i.e.,
nor-lapachol and b2, exhibited ~ 2-fold selectivity for this
isoform. Similar selectivity for MAO-A was observed for a2,
the most potent inhibitor among antitumor 1,4-NQs
1,4-NQ*
Menadione*
Lapachol#
Nor-lapachol
a2
7.7
26
1.5
0.4
5.1
65
–
1.9
0.5
2.3
1.1
–
59 Æ 9
18 Æ 8
40 Æ 6
12 Æ 3
58 Æ 7
35 Æ 6
22 Æ 3
28 Æ 5
63 Æ 12
b2
c2
Chem Biol Drug Des 2014; 83: 401–410
405

Cerqueira et al.
A
B
C
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
D
E
F
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
Figure 2: Kinetics of the inhibition of human MAO by antitumor spermidine-1,4-NQs. The effect of varying concentrations of the inhibitors
a2, b2, and c2 on MAO-A (A, B, and C, respectively) and on MAO-B (D, E, and F, respectively) was analyzed by nonlinear fitting.
Benzylamine and p-tyramine were used as substrates for MAO-B and MAO-A, respectively.
Docking of antitumor 1,4-NQs with MAO isoforms
To understand the structural features associated with the
inhibitory properties of lapachol, nor-lapachol, and spermi-
dine-1,4-NQs on MAO isoforms, molecular docking stud-
ies were carried out. As described in the Experimental
section, the Ensemble Docking method, using AutoDock
Vina, was carried out taking receptor’s structures from
snapshots obtained by MD simulations. For docking of all
ligands into MAO-A or MAO-B, we have chosen the
receptor structures from MD with favorable docking poses
that correspond to the snapshots at t = 30 nseconds and
t = 20 nseconds for the MAO-A and MAO-B structures,
respectively. AutoDock Vina was used as the first
approach to evaluate the interaction of antitumor 1,4-NQs
with MAO isoforms because this program is much faster
than Auto-Dock (28,29). Further, AutoDock was applied in
a new docking series for the refinement of the results
obtained using Autodock Vina. As discussed in the Experi-
mental section, with the AutoDock program, a whole set
of conformations is generated, which are arranged in clus-
ters considering the geometrical similarity and ranked
according to the interaction energy. Therefore, in our stud-
ies, the results were analyzed taking into account not only
enzyme–inhibitor interaction energy, but also the orienta-
tion of the 1,4-NQ ring in relation to the FAD moiety, the
proximity of the inhibitor to the FAD, and the number of
hydrogen bonds (HB) between the ligand and the residues
of the active site (Table 3).
Table 2: Kinetic parameters (Km and Vmax) for MAO-A and MAO-
B in the presence of 50 lM of lapachol, nor-lapachol, or spermi-
dine-1,4-NQs. Results are shown as mean Æ standard deviation
for three experiments. *Mean Æ SD for six experiments
MAO-A
MAO-B
Vmax
Vmax
(nmol/min.
mg)
(nmol.min.
mg)
Km (lM)
Km (lM)
Control*
Lapachol
Nor-
lapachol
a2
b2
370 Æ 68
–
759 Æ 54
36 Æ 4
–
35 Æ 1
497 Æ 203
749 Æ 107
1208 Æ 162
19 Æ 4
13 Æ 2
17 Æ 2
789 Æ 64
758 Æ 92
490 Æ 60
32 Æ 2
36 Æ 2
41 Æ 1
650 Æ 127
1587 Æ 298
587 Æ 63
20 Æ 1
24 Æ 2
22 Æ 1
c2
(Table 1). The non-selectivity in the MAO inhibition is an
important feature when the goal is the oxidation of amines
that are substrates of both MAO isoforms. For instance,
the administration of non-selective MAO inhibitors might
result in an increase in dopamine level that is not observed
for many selective inhibitors (40). Dopamine is metabolized
by both MAO isoforms: by MAO-A intraneuronally and by
MAO-A/MAO-B in glial and astrocyte cells (1). Therefore,
the selective inhibition of an isoform can be counterbal-
anced by an increase in the activity of the other isoform. In
this context, the poor selectivity displayed by antitumor
spermidine-1,4-NQs and nor-lapachol might be an inter-
esting feature if the goal is degradation of amines that are
metabolized by both MAO isoforms.
The most stable conformations (clusters of most negative
interaction energy) generated by docking lapachol or
nor-lapachol with MAO isoforms are shown in Figure 3.
406
Chem Biol Drug Des 2014; 83: 401–410

MAO Inhibition by Antitumor 1,4-Naphthoquinones
Table 3: Docking of antitumor 1,4-NQs into active site of MAO isoforms
MAO-A
MAO-B
Energy
Orientation
Energy
Orientation
(kcal/mol)
1,4-NQ/FAD
FAD
HB
(kcal/mol)
1,4-NQ/FAD
FAD
HB
a2
b2
À9.44
À9.85
À9.33
À7.08
À7.00
Perpendicular
Parallel
Perpendicular
Perpendicular
Perpendicular
HB
3
2
1
No
No
À8.08
À8.15
À7.32
À7.78
À7.39
Parallel
Parallel
Parallel
–
Close
HB
Close
Far
No
3
1
2
2
Close
Close
Close
Close
c2
Lapachol
Nor-lapachol
Perpendicular
Close
A
B
Tyr188
Cys172
Tyr398
FAD
Cys192
Tyr398
Gln206
Gly434
Ser433
Gly434
nor-lapachol
Tyr435
lapachol
Trp432
Cys172
Ser433
Tyr435
Tyr188
C
D
Flavin
Figure 3: Docking of MAO
Flavin
inhibitors into the active site of
MAO isoforms. The MAO-B isoform
is shown in the presence of
lapachol (A), nor-lapachol (B). For
comparison, the interaction of
1,4-NQ (C) and menadione (D) with
FAD into the MAO-B active site
was illustrated; (E) Stabilizing effect
due to the formation of an
Tyr398
Tyr398
Tyr435
Tyr435
‘aromatic sandwich’ between the
reversible MAO-A inhibitor harmine
with the tyrosine residues Tyr407/
444 in MAO-A as well as for the
reversible and irreversible MAO-B
inhibitors, isatin and selegiline,
respectively, with Tyr398/435 in
MAO-B. The illustrations were built
using the program SwissPDB
Viewer 4.0.4.
E
Tyr444
Tyr435
Tyr407
Tyr398
Tyr435
Tyr398
Selegiline
IsaƟn
Harmine
The kinetic studies have indicated that lapachol is a less
potent MAO-B inhibitor than nor-lapachol, with Ki values
of 59 and 18 lM, respectively. The Table 3 shows that the
energy of interaction of lapachol or nor-lapachol with
MAO-B was nearly similar and both inhibitors display two
HBs with the residues Cys172 and Tyr398 in the active
site of MAO-B (Table S1). However, in the lowest energy
conformation, lapachol in MAO-B active site is not close
enough to form effective interactions with the flavin moiety,
whereas nor-lapachol displays interaction with FAD (Fig-
ure 3A and B). Moreover, a higher proximity between nor-
lapachol and the residues Tyr398 and Tyr435 of MAO-B
was observed. These differences could explain the greater
inhibitory activity of nor-lapachol in comparison with lapa-
chol. Our previous studies on the interaction of 1,4-NQ or
menadione with MAO-B revealed that the naphthoquinone
ring is positioned between Tyr435 and Tyr398 residues in
MAO-B, perpendicularly to the re-face of flavin moiety of
Chem Biol Drug Des 2014; 83: 401–410
407

Cerqueira et al.
FAD cofactor, which allows the phenolic side chains to
form an ‘aromatic sandwich’ structure (Figure 3C and D)
(13). Both 1,4-NQ and menadione interact with flavin
through a HB. Similar stabilizing effect was described for
the reversible MAO-A inhibitor harmine with the tyrosine
residues Tyr407/444 in MAO-A, and for the MAO-B inhibi-
tors isatin (reversible) and selegiline (irreversible) with
Tyr398/435 in MAO-B (Figure 3E). Crystallographic data
suggest that both inhibitors and substrates must pass
between these two tyrosines to interact with the flavin
group, making this ‘aromatic sandwich’ an important fea-
ture for enzyme functionality (40). Overall, these findings
suggest that the close proximity of nor-lapachol and the
FAD moiety as well as the orientation of the 1,4-NQ ring of
the inhibitor and its location into the Tyr435/Tyr398 aro-
matic sandwich might contribute to the inhibitory proper-
ties against MAO-B.
MAO-A differently than observed in MAO-B. For MAO-A,
the 1,4-NQ ring is oriented perpendicularly to the flavin
ring instead of parallel as found in MAO-B. Moreover, the
interaction of a2 with MAO-A displays more HBs than with
MAO-B.
Overall, the docking results suggested that spermidine-
1,4-NQs that act as potent inhibitors of MAO are capable
of binding to the catalytic cavity of MAO in close proximity
of flavin moiety. Furthermore, the formation of HBs
between these compounds and FAD and/or the residues
in the active site might explain their ability to inhibit both
MAO-A and MAO-B. Interestingly, the most potent inhibi-
tors of MAO-A and MAO-B among the spermidine-1,4-
NQs, a2, and b2, respectively, are the only capable of
forming HB with the flavin besides a number of HBs with
residues in the catalytic site. Considering that b2 is ori-
ented parallel to the flavin in MAO-B, whereas a2 is per-
pendicular to the flavin in MAO-A, we can speculate that
the 1,4-NQ ring orientation in relation to the flavin does
not play a critical role for MAO inhibition by spermidine-
1,4-NQs.
Lapachol did not inhibit significantly MAO-A at a concen-
tration of up to 100 lM, whereas nor-lapachol exhibited a
Ki of 35 lM. The AutoDock results have indicated that the
energy of interaction of lapachol or nor-lapachol with
MAO-B is more favorable (more negative) than observed
for MAO-A. In the lowest energy cluster, lapachol and nor-
lapachol interact with MAO-B with energy of À7.78 and
À7.39 kcal/mol, respectively (Table 3). For MAO-A with
either lapachol or nor-lapachol, the lowest energies of
interaction were À7.08 and À7.00 kcal/mol, respectively.
Furthermore, lapachol and nor-lapachol did not display
any HB with the residues in MAO-A active site (Table S1).
Although both inhibitors are located close to the FAD moi-
ety of MAO-A and their 1,4-NQ rings are oriented perpen-
dicularly to FAD as observed for nor-lapachol with MAO-B,
the lower energy of interaction with the absence of HBs
might explain the less pronounced inhibitory properties of
these molecules against MAO-A.
Topoisomerase and MAO as targets for cancer
therapy
The inhibition of DNA topoisomerases is an important
strategy in cancer therapy due to the role of these
enzymes in the modulation of the topological state of DNA
and in apoptosis (41). In vitro cytotoxic studies of spermi-
dine-1,4-NQs against diverse cancer cell lines demon-
strated that these polyamine-conjugates displayed
a
higher activity than lapachol, nor-lapachol or lawsone,
according to the activity of these compounds on topo-
isomerase inhibition. Spermidine-1,4-NQs a2, b2, and c2
are capable to induce DNA fragmentation in HL-60 cells at
the dose of 25 lM, while only partial inhibition of topoisom-
erase-2-alpha was observed for lapachol at 200 lM (18).
Importantly, no effect of the R-side chain on the activity of
the spermidine-1,4-NQs conjugates was observed, in con-
trast with that verified for the inhibitory activity on MAO.
These data suggest that the cytotoxic activity of these
compounds on cancer cells, at least in in vitro studies,
cannot be associated with their inhibitory activity on MAO,
even though the action on MAO might influence their antit-
umoral properties.
The analysis of the interaction of MAO-B with a2, b2, or
c2, in the lowest energy conformation, indicated that the
1,4-NQ ring is positioned close to the flavin moiety of FAD
cofactor in a parallel orientation. In this case, the energies
of interaction were À8.08, À8.15, and À7.32 kcal/mol for
a2, b2, and c2, respectively. Importantly, b2 was the only
antitumor 1,4-NQ that forms a HB with FAD into the active
site of MAO-B. b2 builds three HBs, with FAD, Leu171,
and Tyr398, whereas c2 only one, with Cys172, and a2
neither (Table S2). These data might explain the fact of b2
was the most potent MAO-B inhibitor among the spermi-
dine derivatives with a Ki of 12 lM (Table 3).
Ornithine decarboxylase, an enzyme involved in polyamine
biosynthesis, is enhanced in tumor cells, which results in
an increase pool of polyamines such as spermine and
spermidine that are necessary for cell growth (42). The
degradation of these amines by polyamine oxidase and
MAO action is accompanied by an elevation of the pro-
duction of cytotoxic metabolites, such as hydrogen perox-
ide and aldehydes (43). Therefore, MAO plays an
important function not only in the process of cell growth
and differentiation but also in the development and growth
regulation of tumors. In this context, MAO inhibitors might
For MAO-A, the energies of interaction with a2, b2, and c2,
in the lowest energy conformation, were À9.44, À9.85,
and À9.33 kcal/mol, respectively. In this case, a2 builds
three HBs, with FAD, Gly67, and Gln215 in active site and
was the strongest MAO-A inhibitor with a Ki of 22 lM. On
the other hand, b2 and c2 form, respectively, two and one
HB with Ile180. Analyzing the a2 docked in the active site
of MAO-A, one can verify that this ligand is positioned in
408
Chem Biol Drug Des 2014; 83: 401–410

MAO Inhibition by Antitumor 1,4-Naphthoquinones
interfere in these processes by controlling the formation of
hydrogen peroxide and aldehydes from MAO action
(43,44).
cardiac monoamine oxidase A in rats. Am J Physiol
Heart Circ Physiol;284:1460–1467.
ꢁ
6. Marcozzi G., Befani O., Mondovı B. (1998) Type B
monoamine oxidase activity in human brain malignant
tumors. Cancer Biochem Biophys;16:287–294.
7. Gabilondo A.M., Hostalot C., Garibi J.M., Meana J.J.,
Callado L.F. (2008) Monoamine oxidase B activity is
increased in human gliomas. Neurochem Int;52:230–
234.
Conclusions
Spermidine-1,4-NQs, lapachol, and nor-lapachol behave
as reversible and competitive MAO inhibitors in which
the enzyme selectivity and the potency of the inhibitor
might be greatly influenced by substitutions on different
positions of the 1,4-NQ ring. These data reinforce the
idea that 1,4-NQ is a potential pharmacophore for the
development of MAO inhibitors and, more importantly,
suggest that certain antitumor 1,4-NQs might block
MAO activity. Although spermidine-1,4-NQs exhibit anti-
tumor action primarily by inhibiting topoisomerase via
DNA intercalation, our findings suggest that their effect
on MAO activity should be taken into account when
their application in cancer therapy is considered.
Whether the inhibitory activity on MAO of spermidine-
1,4-NQs, lapachol, and nor-lapachol contributes posi-
tively or negatively to the tumor growth is a question for
further investigations.
ꢁ
8. Mondovı B., Riccio P., Riccio A., Marcozzi G. (1983)
Amine oxidase activity in malignant human brain
tumors. In: Bachrach U., Kaye A., Chayen R., editors.
Advances in Polyamine Research 4. New York: Raven
Press.
9. Igosheva N., Lorz C., O’Conner E., Glover V., Mehmet
H. (2005) Isatin, an endogenous monoamine oxidase
inhibitor, triggers a dose- and time-dependent switch
from apoptosis to necrosis in human neuroblastoma
cells. Neurochem Int;47:216–224.
10. Toninello A., Salvi M., Pietrangeli P., Mondovi B. (2004)
Biogenic amines and apoptosis: minireview article.
Amino Acids;26:339–343.
11. Zhao H., Flamand V., Peehl D.M. (2009) Anti-onco-
genic and pro-differentiation effects of clorgyline, a
monoamine oxidase A inhibitor, on high grade prostate
cancer cells. BMC Med Genomics;2:55–69.
Acknowledgments
12. Khalil A.A., Steyn S., Castagnoli N. (2000) Isolation
and characterization of a monoamine oxidase inhibitor
from tobacco leaves. Chem Res Toxicol;13:31–35.
13. Coelho-Cerqueira E., Netz P.A., Diniz C., Petry do
Canto V., Follmer C. (2011) Molecular insights into
human monoamine oxidase (MAO) inhibition by 1,4-
naphthoquinone: evidences for menadione (vitamin K3)
acting as a competitive and reversible inhibitor of
MAO. Bioorg Med Chem;19:7416–7424.
~
This work was supported by Souza Cruz Co., Fundacßao
ꢁ
de Amparo a Pesquisa do Estado do Rio de Janeiro
(FAPERJ), Conselho Nacional de Desenvolvimento
ꢂ
ꢂ
Cientıfico e Tecnologico (CNPq) and International Founda-
tion for Science (IFS). P.A.N and V.P.C are grateful to the
National Supercomputing Center – CESUP/UFRGS.
14. Balassiano I.T., De Paulo S.A., Henriques Silva N.,
Cabral M.C., da Gloria da Costa Carvalho M. (2005)
Demonstration of the lapachol as a potential drug
for reducing cancer metastasis. Oncol Rep;13:329–
333.
References
1. Youdim M.B., Bakhle Y.S. (2006) Monoamine oxidase:
isoforms and inhibitors in Parkinson’s disease and
depressive illness. Br J Pharmacol;147:287–296.
2. Javitch J.A., D’Amato R.J., Strittmatter S.M., Snyder
S.H. (1985) Parkinsonism-inducing neurotoxin, N-
methyl-4-phenyl-1,2,3,6 -tetrahydropyridine: uptake of
the metabolite N-methyl-4-phenylpyridine by dopamine
neurons explains selective toxicity. Proc Nat Acad Sci
USA;82:2173–2177.
3. Langston J.W., Langston E.B., Irwin I. (1984) MPTP-
induced parkinsonism in human and non-human pri-
mates - clinical and experimental aspects. Acta Neurol
Scand Suppl;100:49–54.
ꢂ
ꢂ
ꢂ
ꢂ
15. Bermejo-Bescos P., Martın-Aragon S., Jimenez-Aliaga
K.L., Ortega A., Molina M.T., Buxaderas E., Orellana
ꢂ
€
G., Csaky A.G. (2010) In vitro antiamyloidogenic prop-
erties of 1,4-naphthoquinones. Biochem Biophys Res
Commun;400:169–174.
16. Silva F.L., Coelho-Cerqueira E., Freitas M.S., Goncal-
ß
ves D.L., Costa L.T., Follmer C. (2013) Vitamins K
interact with N-terminus a-synuclein and modulate the
protein fibrillization in vitro. Exploring the interaction
between quinones and a-synuclein. Neurochem
Int;62:103–112.
4. Fowler J.S., Logan J., Volkow N.D., Wang G.J., Mac-
Gregor R.R., Ding Y.S. (2002) Monoamine oxidase:
radiotracer development and human studies. Meth-
ods;27:263–277.
17. Hadden M.K., Hill S.A., Davenport J., Matts R.L.,
Blagg B.S. (2009) Synthesis and evaluation of Hsp90
inhibitors that contain 1,4-naphthoquinone scaffold.
Bioorg Med Chem;17:634–640.
5. Maurel A., Hernandez C., Kunduzova O., Bompart G.,
18. Esteves-Souza A., Lucio K.A., Cunha A.S., Cunha
Pinto A., Silva Lima E.L., Camara C.A., Vargas M.D.,
Gattass C.R. (2008) Antitumoral activity of new
ꢂ
Cambon C., Parini A., Frances B. (2003) Age-depen-
dent increase in hydrogen peroxide production by
Chem Biol Drug Des 2014; 83: 401–410
409

Cerqueira et al.
polyamine-naphthoquinone
Rep;20:225–231.
conjugates.
Oncol
ing receptor flexibility: the relaxed complex system.
J Am Chem Soc;124:5632–5633.
ꢁ
19. Toninello A., Salvi M., Mondovı B. (2004) Interaction of
biologically active amines with mitochondria and their
role in the mitochondrial- mediated pathway of apopto-
sis. Curr Med Chem;11:2349–2374.
34. Lin J.H., Perryman A.L., Schames J.R., McCammon
J.A. (2003) The relaxed complex method: accommo-
dating receptor flexibility for drug design with and
improved scoring scheme. Biopolymers;68:47–62.
35. Sinko W., Lindert S., McCammon J.A. (2013) Account-
ing for receptor flexibility and enhanced sampling
methods in computer-aided drug design. Chem Biol
Drug Des;81:41–49.
36. Lindahl E., Hess B., van der Spoel D. (2001) GRO-
MACS 3.0: a package for molecular simulation and
trajectory analysis. J Mol Model;7:306–317.
37. Oostenbrink C., Villa A., Mark A.E., van Gunsteren
W.F. (2004) A biomolecular force field based on the
free enthalpy of hydration and solvation: the GROMOS
force-field parameter sets 53A5 and 53A6. J Comput
Chem;25:1656–1676.
20. Agostinelli E., Arancia G.Dalla., Vedova L., Belli F.,
Marra M., Salvi M., Toninello A. (2004) The biological
functions of polyamine oxidation products by amine
oxidases: perspectives of clinical applications. Amino
Acids;27:347–358.
21. Cunha A.S., Lima E.L.S., Pinto A.C., Esteves-Souza
A., Echevarria A., Camara C.C., Vargas M.D., Torres
J.C. (2006) Synthesis of novel naphthoquinone-spermi-
dine conjugates and their effect on DNA topoisomeras-
es I and II-a. J Braz Chem Soc;17:439–442.
22. Guang H., Du G. (2006) High-throughput screening for
monoamine oxidase-A and monoamine oxidase-B
inhibitors using one-step fluorescence assay. Acta
Pharmacol Sinica;27:760–766.
23. Dixon M. (1953) The determination of enzyme inhibitor
constants. Biochem J;55:170–171.
24. Dennington R., Keith T., Millam J. (2007) GaussView,
Version 4.1. Shawnee Mission, KS: Semichem Inc.
25. Frisch M.J., Trucks G.W., Schlegel H.B., Scuseria
G.E., Robb M.A., Cheeseman J.R., Scalmani G. et al.
(2009) Gaussian 09, Revision A.1. Wallingford, CT:
Gaussian, Inc.
26. Sanner M.F. (1999) Python: a programming language
for software integration and development. J Mol Graph
Model;17:57–61.
27. Gasteiger J., Marsili M. (1980) Iterative partial equaliza-
tion of orbital electronegativity – a rapid access to
atomic charges. Tetrahedron;36:3219–3228.
28. Morris G.M., Goodsell D.S., Halliday R.S., Huey R.,
Hart W.E., Belew R.K., Olson A.J. (1998) Automated
docking using Lamarckian Genetic Algorithm and an
38. Morris G.M., Huey R., Lindstrom W., Sanner M.F., Be-
lew R.K., Goodsell D.S., Olson A.J. (2009) Autodock4
and AutoDockTools4: automated docking with
selective receptor flexibility. J Comput Chem;16:2785–
2791.
€
€
39. Da Prada M., Zurcher G., Wuthrich I., Haefely W.E.
(1988) On tyramine, food, beverages and the reversible
MAO inhibitor moclobemide. J Neural Transm Sup-
pl;26:31–56.
40. Nagatsu T., Sawada M. (2006) Molecular mechanism
of the relation of monoamine oxidase B and its inhibi-
tors to Parkinson’s disease: possible implications of
glial cells. J Neural Transm Suppl;71:53–65.
41. Chikamori K., Grozav A.G., Kozuki T., Grabowski D.,
Ganapathi R., Ganapathi M.K. (2010) DNA topoisom-
erase II enzymes as molecular targets for cancer
chemotherapy. Curr Cancer Drug Targets;10:758–
771.
42. Russel D.H. (1980) Ornithine decarboxylase as
a
empirical binding free-energy function.
J
Comput
biological and pharmacological tool. Pharmacol-
Chem;19:1639–1662.
ogy;20:117–121.
ꢁ
29. Huey R., Morris G.M., Olson A.J., Goodsell D.S. (2007)
A semiempirical free energy force field with charge-
based desolvation. J Comput Chem;28:1145–1152.
30. Trott O., Olson A.J.J. (2010) Auto-DockVina: improving
the speed and accuracy of docking with a new scoring
function, efficient optimization, and multithreading.
Comput Chem;31:455–461.
43. Pietrangeli P., Mondovı B. (2004) Amine oxidases and
tumors. Neurotoxicology;25:317–324.
44. Maslinski C., Bieganski T., Fogel W.A., Kitler M.A.
(1985) Diamine oxidase in developing tissues. In:
ꢂ
Mondovı B., editor. Structure and Functions of Amine
Oxidases. Boca Raton, FL: CRC Press; p. 153–166.
31. Son S.Y., Ma J., Kondou Y., Yoshimura M., Yamashita
E., Tsukihara T. (2008) Structure of human monoamine
oxidase A at 2.2-A resolution: the control of opening
the entry for substrates/inhibitors. Proc Natl Acad Sci
USA;105:5739–5744.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
32. Binda C., Wang J., Li M., Hubalek F., Mattevi A.,
Edmondson D.E. (2008) Structural and mechanistic
studies of arylalkylhydrazine inhibition of human
monoamine oxidases A and B. Biochemistry;47:5616–
5625.
Table S1. Interaction of lapachol and nor-lapachol with
the FAD/residues into active site of MAO in the lowest
energy conformation
Table S2. Interactions of anti-tumor spermidine-1,4-NQs
with the FAD/residues into active site of MAO isoforms in
the lowest energy conformation
33. Lin J.H., Perryman A.L., Schames J.R., McCammon
J.A. (2002) Computational drug design accommodat-
410
Chem Biol Drug Des 2014; 83: 401–410