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Introduction
Monoamine oxidase (MAO) is responsible for oxidative deamination of endogenous and xenobiotic amines located at the
outer membrane of mitochondria in neuron and non-neuronal cells. Two isoforms of MAO have been identified and
designated as MAO-A and MAO-B[1], coded by similar but distinct genes, having different substrate preference and inhibitor
specificity[2]. Experimentally, MAO-A preferentially oxidizes serotonin (5-hydroxytryptamine, 5HT), noradrenaline and
adrenaline, and is inhibited by low concentrations of clorgyline. In contrast, MAO-B preferentially oxidizes dopamine,
b-phenylethylamine (PEA), benzylamine and is inhibited by low concentrations of deprenyl or
pargyline[3].
Changes of MAO activity are involved in some central and peripheral nervous system diseases. Extra high MAO-B
activity in the brain appears in neurological degenerations involving Alzheimer¡¯s disease, Huntington¡¯s disease, some forms
of Parkinson¡¯s disease and normal
aging[4]. Abnormal MAO-A activity is implicated in depression,
anxietyand psychiatric
disorders[5]. Clinically, MAO inhibitors have been found to alleviate symptoms or slow deterioration of these diseases,
which impels the necessity of discovering more MAO inhibitors (MAO-I).
The establishment of high-throughput screening (HTS) assays is an essential element in the drug discovery pro-cesses.
Based on the general reaction
RCH2NR1R2+
H2O+O2 ¡ª¡ú RCHO +
NHR1R2 +
H2O2, available assays to measure MAO
activity can be summarized either by determining the rate of product formation or substrates depleted in an MAO-catalyzed
reaction: (1) the direct measurement of oxygen consumption by polarographic
detection[6]; (2) the detection of oxidized
monoamine products by spectrophotometry or radiometric
assay[7,8]; (3) liquid chromatography with tandem mass
spectrometry measuring MAO activity has been recently applied to HTS, but it has less specificity and limited
convenience[9]; (4) the assessment of co-product hydrogen peroxide
directly[10] or indirectly, a continuous fluorescence assay with a sensitive,
stable H2O2 probe, N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). Because of its convenience and continuity, this
one-step method was considered more suitable to detect the activity of enzymes whose co-product is hydrogen peroxide.
However, there has been no establishment detecting MAO-A and MAO-B activity by this method until now. HTS protocol
discovering MAO-A-inhibitor (MAO-A-I) and MAO-B-inhibitor (MAO-B-I) applying this assay has not yet been developed.
In the current experiment, we detected both MAO-A and MAO-B activity by this fluorescence probe based method with
evaluations of its sensitivity, stability and specificity. The reliability of this assay was valued by comparing MAO-A and
MAO-B dynamic parameters with those obtained by traditional assays. In regard to enzyme source facilitations, we purified
rat brain mitochondria by graded extractions to obtain higher specific activity. Reaction conditions including enzyme
sources, substrates concentrations, incubation volume and reaction time in 384-well format were optimized to make
MAO-A-I and MAO-B-I screening protocols with
convenience, sensitivity, and low consumption. Moreover, precision parameters
were calculated to ensure the confidence of a developed system, intending to provide convenient and robust protocols in
MAO-A-I and MAO-B-I screening procedures.
Materials and methods
Materials and animals Horseradish peroxidase (HRP), hydrogen peroxide, benzylamine, serotonin, Triton X-100, bovine
serum albumin, Cu-Zn superoxide dismutase (Cu-Zn SOD), clorgyline, and deprenyl were purchased from Sigma (St Louis,
MO, USA). Amplex Red was from Molecular Probes (Eugene, OR, USA). Other high-graded chemical reagents were
commercially available.
Adult male Wistar rats (280-300 g) were obtained from the Chinese Academy of Medical Sciences, Experimental Animal
Center.
Enzyme preparations Different enzyme preparation conditions were conducted to obtain a high specific activity.
Mitochondria were isolated according to our developed assay with
modifications[11]. In brief, crude mitochondria were obtained
by centrifuging rat brain homogenates at
2000×g for 10 min in MSETB buffer (210 mmol/L mannitol, 70
mmol/L sucrose, 0.5 mmol/L ethylenediamine tetra-acetate, 10
mmol/L Tris-HCl and 0.2% bovine serum albumin, pH 7.4). Sonicated crude
mitochondria were gained by sonicating crude mitochondria suspension at 20 Hz twice for 20 s with an interval of 10 s and further
centrifuging at 16 000×g for 10 min. Washed mitochondria were obtained by resuspending the crude mitochondria in 1:40
(w/v) SET buffer (280 mmol/L sucrose, 0.5 mmol/L EDTA and 10 mmol/L Tris-HCl pH 7.4) and further centrifuged at 16
000×g for 8 min. Mitochondria were then subsequently thrice frozen at -20 ºC and thawed at 4 ºC. Further purifications were conducted
by solubilizing mitochondria in 0.1% Triton X-100 to reduce homogenate interferences.
To further assess MAO-A and MAO-B quantifications in each extraction step, Western blotting assay was carried out as
previously described. Equivalent amounts of total proteins were separated by PAGE/SDS. The primary antibody
concentration for MAO-A is 1:2000 (Santa Cruz Biotechnology, Santa Cruz, USA ), MAO-B 1:1000 (Santa Cruz Biotechnology, Santa
Cruz, USA), and anti-actin antibody 1:2000 (Santa Cruz Biotechnology, Santa Cruz, USA). Immunoreactive deposits were
detected using horseradish peroxidase-conjugated secondary antibodies and enzyme-linked chemiluminescence according
to manufacturer¡¯s instructions (ECL, Pierce, Rockford, IL, USA)
Suspension of each purification step was stored at -40 ºC in aliquots until required for experiment within 48 h. Specific
activity of MAO-A or MAO-B and inhibition properties of clorgyline to MAO-A or deprenyl to MAO-B were examined by
fluorescence assay. Protein quantification was evaluated by Lowry¡¯s
assay[12].
MAO activity measurement The experiment was performed by optimization at 37 ºC (pH 7.4) in 384-well microplate
(Greiner, Bio-One, Germany). The reaction mixture contained total enzyme protein at the final concentration of 0.2 mg/mL, and
0.02 mmol/L serotonin as a substrate for MAO-A, 0.01 mmol/L benzylamine as a substrate for MAO-B, with 10
U/mL HRP, 2.5 µmol/L Amplex Red and 40 U/ml Cu-Zn SOD previously supplemented. Amplex Red was oxidized into
resorufin (Figure 1) in the reaction, directly detected at 560± 10 nm at excitation and 590±10 nm at emission. As a blank control,
an identical vehicle solution for samples was added in place of enzyme solution. The
Km value of serotonin to MAO-A and
that of benzylamine to MAO-B were determined by adding graded concentrations of each substrate to the reaction system.
At the same time, time and concentration dependent manners of the standard solution were obtained by applying series of
hydrogen peroxide concentrations in Figure 6C.
Evaluations Different reagent combinations were
conducted with and without substrates or enzyme incubated in the
reaction system with the fluorescence density demonstrated in Figure 5. Fluorescence in the MAO-A and MAO-B catalyzed
reaction at different time points was detected with the time-dependent manner.
HTS protocol HTS procedures were performed on
conditions of optimized reaction volume
as 50 µL, in which contained optimized concentrations of enzyme, substrates, and sample. Incubation time of MAO-A catalyzed reaction was 60 min and
that of MAO-B catalyzed reaction was 45 min. In primary screening, the samples were examined on their inhibitory potency
on MAO-A and MAO-B activity. During the protocol, clorgyline and deprenyl were positive controls in MAO-A inhibitor
screening and MAO-B inhibitor screening, respectively, with PBS buffer as the negative control. In further screening,
compounds were serially
diluted into graded concentrations and the inhibition was measured with
IC50.
Data processing Inhibition of samples on MAO activity was represented by the percentage of enzyme activity relative
to that of the negative control. Data processing and validation were undertaken according to our standard laboratory data
processing technology. Precision parameters involving coefficient of variation (CV), signal to background value (S/B), and
Z¡¯ factor were calculated according to Zhang¡¯s
method[13].
Results
Enzyme preparations Specific activities of MAO-A and MAO-B in different preparation steps are shown in Table 1.
Taking intra-day evaluation for instance, MAO-A had its highest activity as 52.91±7.54
nmol·min-1·mg protein-1
in sonicated crude mitochondria, while exhibiting a decreasing tendency in the following steps. In contrast, MAO-B demonstrated
an increase of activity in the five preparation steps, reaching its peak of 102.34±11.21
nmol·min-1·mg protein-1
at the Triton X-100 treated step.
Interestingly, MAO-A and MAO-B immunoblotting evaluations were consistent with performances of specific activity.
Inference could be obtained from this coincidence that further extraction increased enzyme content ratio in samples instead
of demolishing their structure and activity, inversely confirming enhancements on enzyme specific activity in the
purifications (Figure 2).
For further evaluation, sensitivities of MAO-A and MAO-B from each step to their specific inhibitors were examined with
the tendency curves. MAO-A had the highest sensitivity against clorgyline in the sonicated crude mitochondria step, while
demonstrating decreasing tendency in the last three steps. MAO-B demonstrated an increasing sensitivity trend in the five
successive enzyme extraction steps. Tendency demonstrated was in accordance with the results shown in Table 1, which
demonstrated further different enzyme extraction conditions in MAO-A and MAO-B preparations (Figure 3).
MAO parameters Dynamic parameters of MAO-A and MAO-B from the determined extraction steps were examined with
graded concentrations of their substrates added (Figure 4), the
Km value of serotonin to MAO-A was 1.66 µmol/L and
Vmaxwas 14.66
nmol·min-1·mg protein-1
in MAO-A catalyzed reaction. In the MAO-B catalyzed reaction, the
Km value of benzylamine to MAO-B was 0.80 µmol/L, and
Vmax was 14.81
nmol·min-1·mg
protein-1.
Evaluations From Figure 5, group VI, which had Amplex Red, HRP, MAO-B, benzylamine co-incubated together in the
reaction, demonstrated the highest fluorescence, while group V
having Amplex Red, HRP, MAO-A and serotonin together,
displayed the second highest fluorescence. How-ever, other groups in the absence of even one reagent demonstrated a low
fluorescence, which implied the specificity of this applied assay. Interestingly, group
VII, containing Amplex Red, HRP, MAO-A and benzylamine, and
group VIII, containing Amplex Red, HRP, MAO-B and serotonin, both had a lower
fluorescence also suggesting the sensitivity of this fluorescence assay.
The fluorescence was also detected at successive time points in MAO-catalyzed reaction with graded
concentrations of substrates. In Figure 6A, the reaction with 0.02
mmol/L serotonin as its optimum concentration substrate showed a fluorescence peak at 60 min, while in Figure
6B the
reaction with 0.01 mmol/L benzylamine as the optimum concentration substrate showed a fluorescence peak at 45 min. In
either an MAO-A or MAO-B catalyzed reaction, there existed a stable tendency after the peak in time-dependent curve.
HTS experiment In optimum conditions, compounds in our library were tested for their inhibitory profile on MAO
activity, with a final concentration of 10
µg/mL in the primary screening process. Results showed that in an MAO-A HTS
system 0.6% compounds had relative inhibition higher than 70.1%, and 1.0% compounds had a relative inhibition on
MAO-B higher than 70.0%. In further screening processes, there was one compound exerting specific inhibitory potency against
MAO-A activity in a concentration-dependent manner, with its
IC50 as 0.36 µmol/L. Meanwhile, three compounds had
specific inhibitory potency against MAO-B activity with their
IC50 as 0.13, 0.19, and 0.13 µmol/L.
In the present experiment, the applied method was subjected to validation from several batches of quality control samples,
with precision presented in Table 1. The inter-day CV was 14.2 and intra-day CV was 5.3 in the applied MAO-A extraction
procedure, while in the MAO-B extraction procedure inter-day precision was 10.9 and 4.8 for the intra-day precision. The
IC50 value of clorgyline to MAO-A was 2.99 nmol/L, and that to deprenyl was 7.04 nmol/L, in accordance with those detected by
the traditional assays[13,14]. The
S/Bvalue of the two HTS systems were higher than 3. The Z¡¯ factor for MAO-A-I HTS
performance was 0.71±0.03 and
0.75±0.03 for the MAO-B-I HTS system.
Discussion
Abnormal MAO changes are involved in neurological diseases, however, most of the available MAO inhibitors have
undesirable side effects[16]. This means that more MAO inhibitors need to be discovered. By using robotic automation
system to test numbers of compounds against novel biological targets, high-throughput screening (HTS) dramatically
accelerates the pace of drug discovery with large amounts of drug candidates efficiently screened out. How-ever, until now,
no HTS assay for MAO inhibitors has been developed by appropriately convenient, sensitive and efficient methods.
In retrospect, several conventional methods detecting MAO activity are available but most of them are not suitable for
rapid and continuous screen protocols for large amounts of samples. For example, the direct measurement of oxygen
consumption needs a very rigid condition
control[4]. Other methods such as spectrophotometry or liquid
chromatography[17,18] used for analyzing aldehydes formed in the reaction still can not be conveniently utilized in HTS because of low specificity
and limited sensitivity. Low sensitivity of an analytical method inevitably requires high concentrations of reaction
substances and long incubation periods, resulting in significantly high consumable costs and low throughputs. The radiometric
approach using 14C-labeled substrates is more specific and sensitive, but handling of radioactive material or isolation of
products by extraction is much more troublesome and costly. Therefore, HTS processes for MAO inhibitors require an MAO
detection assay with high specificity, sensitivity, and convenience.
Because MAO acting on its substrates also generates co-product hydrogen peroxide generally independent to the
substrate, spectrometric assays designed to directly detect hydrogen peroxide production have been applied to estimate
MAO activity or enzyme classification in recent years. However, because direct detection on absorption of hydrogen
peroxide is often interfered by crude tissue homogenates at the wavelength of 230 nm, it is not suitable to use HTS protocol
with its low sensitivity. Indirect measurement on hydrogen peroxide production based on the HRP-coupled reaction system
has only ever been conducted with a hydrogen peroxide-sensitive probe, Amplex Red in human
leukocytes[19]. But there have been no studies on the feasibility of detecting fluorescent MAO activity with Amplex Red. HTS protocols applying this
assay have not been developed, either.
Attempts to apply this assay to MAO-I HTS platform have been carried out in the current experiment, with sensitivity,
specificity and stability evaluations. The low fluorescence background of Amplex Red, homogenates and the individual
substrate indicated the high sensitivity of this assay. The fluorescence demonstration in Figure
5 also confirmed specific substrates to their specific MAO subtypes. The sensitivity of the protocol was also proven by the
concentration-dependence effect and time-dependence manner shown in Figure 6, in which the peak of MAO-A catalyzed reaction at 60 min and
MAO-B catalyzed reaction at 45 min was shown in the present reaction system. Interestingly, it could also be noticed that a
stable platform after the reaction peak existed in time-dependence curve, together with time-dependence manner with
hydrogen peroxide as the standard solution demonstrating the stability of this applied fluorescence assay.
Considering that the production of hydrogen peroxide was also formed by other oxidative enzymes present in enzyme
preparations, Cu-Zn SOD was previously added in the reaction system, preventing the auto-oxidation of Amplex Red that
interfered with the quantitative assessment of low rates of hydrogen peroxide production in the MAO-catalyzed reaction.
Even with this procedure, the applied assay was still a one-step method with specificity and sensitivity, enabling a
convenient platform for this HTS system.
For facilitation, rat brain tissues were chosen as enzyme preparation sources. Regarding the fact that endogenous MAO
inhibitors may cause under-determination in the
activity evaluation[20], we tried to perform different enzyme extraction protocols to get a high enzyme quality. By
optimi-zation, the optimum enzyme preparation conditions were determined by examining the specific activity of enzyme and
sensitivity to their specific inhibitors at different extraction steps. As shown in Table 1, MAO-B had its highest activity in the
Triton X-100 treated step of the present assay, in which the purification of MAO-B was enhanced or endogenous MAO
inhibitors may be washed out, also reversely confirming the existence of endogenous MAO inhibitors. However, MAO-A
had very low activity in the last three steps indicating its activity loss in the further extraction procedures, which may not only
be a result of its low content in the rat brain but also its comparative vulnerability as outlined in a previous
study[21]. From the consistence of MAO-A and MAO-B demonstrations in immuno-blotting evaluations with performances of specific activity
measurement, inference could be deduced that further extraction increased enzyme content ratio in samples, which confirmed
the enhancement of enzyme specific activity in purifications. Figure 3 illustrates the sensitivity of MAO against their
inhibitors, in which MAO-A had its highest sensitivity against clorgyline in the sonicated crude mitochondria step, while
displaying a decreasing tendency in the last three steps. MAO-B had an increasing sensitivity trend in the five successive
extraction steps further confirming the sensitivity of this assay.
Dynamic parameters of MAO-A and MAO-B were measured to evaluate the reliability of this applied assay. Results
demonstrated that the Km value of serotonin to MAO-A was 1.66 µmol/L, while that of benzylamine to MAO-B was 0.80
µmol/L and the IC50 value of clorgyline to MAO-A was
2.99 nmol/L, while that of deprenyl to MAO-B was 7.04
nmol/L. These matched those obtained by the traditional
assays[14,15].
During the development of the HTS model, other optimizations were also
conducted involving concentrations of reaction
substances such as enzyme, substrates, samples and the incubation volume minimization to 50 µL with obtained data
reproducible. The Z¡¯ factors were calculated to evaluate and validate the quality of the overall assay. Results
revealed that the Z¡¯ factor was both higher than 0.7 in the MAO-A-I and
MAO-B-I HTS performances, indicating high quality and
reproducibility of the established HTS protocols.
Taken together, the fluorescence probe based and HRP-coupled fluorescence assay in MAO detection was evaluated as
sensitive, reliable, and convenient in the present paper. It can not only be used for MAO activity assay and MAO parameters
profile but is suitable for HTS processes. The established HTS models applying this one-step fluorescence were robust for
discovering MAO-A and MAO-B inhibitors. This was achieved with high efficiency, reproducibility, and low consumption
of HTS characteri-zations.
With the development of recombinant technology, commercial enzymes as recombinant proteins will be applied in this
HTS protocol in our future studies for further standardizations and lead-compound evaluations. This applied
assay could prospectively be extended to detect other biological compounds or enzymes that also require further investigation.
References
1 Abell CW, Kwan SW. Molecular characterization of monoamine oxidases A and B. Prog Nucleic Acid Res Mol Biol 2001; 65: 129-56.
2 Nakagawasai O, Arai Y, Satoh SE, Satoh N, Neda M, Hozumi M,
et al. Monoamine oxidase and head-twitch response in mice. Neurotoxicology
2004; 25: 223-32.
3 Sandler M. My fifty years (almost) of monoamine oxidase. Neuro-toxicology 2004; 25: 5-10.
4 Rehman HU, Masson EA. Neuroendocrinology of ageing. Age Ageing 2001; 30: 279-87.
5 Jean CS. Cloning, after cloning, knock-out mice, and
physiological functions of MAO A and B. Neurotoxicology 2004; 25: 21-30.
6 Averill-Bates DA, Agostinelli E, Przybytkowski E, Mateescu MA, Mondovi B. Cytotoxicity and kinetic analysis of purified bovine serum
amine oxidase in the presence of spermine in Chinese hamster ovary cells. Arch Biochem Biophys 1993; 300: 75-9.
7 Zhou JJ, Zhong B, Silverman RB. Direct continuous
fluorometric assay for monoamine oxidase B. Anal Biochem 1996; 234: 9-12.
8 Yoshimi K, Kozuka M, Sakai J, Iizawa T, Shimizu Y, Kaneko I,
et al. Novel monoamine oxidase inhibitors,
3-(2-aminoethoxy)-1,2-benzisoxazole derivatives, and their differential reversibility. Jpn J Pharmacol 2002; 88: 174-82.
9 Yan ZY, Caldwell GW, Zhao BY, Reitz AB. A
high-throughput monoamine oxidase inhibition assay using liquid chromatography with
tandem mass spectrometry. Rapid Commun. Mass Spectrom 2004; 18: 834-40.
10 Stevanato R, Vianello F, Rogo A. Thermodynamic analysis of the oxidative deamination of polyamines by bovine serum amine oxidase.
Arch Biochem Biophys 1995; 324: 374-8.
11 Zhang, HX, Du GH, Zhang JT. Ischemic pre-conditioning preserves brain mitochondrial functions during the middle cerebral artery
occlusion in rat. Neurol Res 2003; 25: 471-6.
12 Lowry OH, Rosebrougy NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-71.
13 Zhang JH, Chuang TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening
assays. J Biomol Screen 1999; 4: 67-73.
14 Saura J, Kettler R, Prada MD, Richards JG. Quantitative enzyme radioautography with 3H-Ro 41-1049 and 3H-Ro 19-6327
in vitro: localization and abundance of MAO-A and MAO-B in rat CNS, peripheral organs, and human brain. J Neurosci 1992; 12: 1977-99.
15 Hirata M, Kagawa S, Yoshimoto M, Ohmomo Y. Synthesis and characterization of radioiodinated MD-230254: a new ligand for potential
imaging of monoamine oxidase B activity by single photon emission computed tomography. Chem Phram Bull 2002; 50: 609-14.
16 Yamada M, Yasuhara H. Pharmacology of MAO inhibitors: safety and future. Neurotoxicology 2004; 25: 215-21.
17 Riederer P, Danielczyk W, Grunblatt E. Monoamine oxidase-B
inhibition in Alzheimer's disease. Neurotoxiology 2004; 25: 271-7.
18 Markianos M, Panas M, Kalfakis N, Vassilopoulos D. Platelet monoamine oxidase activity in subjects tested for Huntington's disease gene
mutation. J Neural Transm 2004; 111: 475-83.
19 Mohanty JG, Jaffe JS, Schulman ES, Raible DG. A highly sensitive fluorescent micro-assay of
H2O2 release from activated human
leukocytes using a dihydroxyphenoxazine derivative. J Immunol Methods 1997; 202: 133-41.
20 Medvedev AE, Glover V. Tribulin and endogenous MAO-inhibitory regulation
in vivo. Neurotoxiology 2004; 25: 185-92.
21 Matsukawa M, Hirai T, Karita S, Akizawa T, Hou HP, Yoshioka M,
et al. A screening system of prodrugs selective for MAO-A or
MAO-B. Neurotoxicology 2004; 25: 293-302.
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