Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors

Phytochemistry 86 (2013) 8–20

Contents lists available at SciVerse ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors Carolina S. Passos a,⇑, Claudia A. Simões-Pires b, Alessandra Nurisso b, Tatiane C. Soldi a, Lucilia Kato c, Cecilia M.A. de Oliveira c, Emiret O. de Faria c, Laurence Marcourt b, Carmem Gottfried d, Pierre-Alain Carrupt b, Amélia T. Henriques a,⇑ a Laboratório de Farmacognosia, Departamento de Produção de Matéria-Prima, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, UFRGS, 91610-00 Porto Alegre, RS, Brazil b School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CH-1211 Geneva, Switzerland c Instituto de Química, Universidade Federal de Goiás, UFG, 74001-970 Goiânia, GO, Brazil d Laboratório de Plasticidade Neuroglial, Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, 90035-000 Porto Alegre, RS, Brazil

a r t i c l e

i n f o

Article history: Received 9 August 2012 Received in revised form 29 October 2012 Available online 19 December 2012 Keywords: Psychotria suterella P. laciniata P. leiocarpa P. umbellata P. myriantha P. prunifolia Rubiaceae Monoterpene indole alkaloids Quaternary-b-carboline alkaloids Acetylcholinesterase Butyrylcholinesterase Monoamine oxidases Molecular docking

a b s t r a c t Thirteen Psychotria alkaloids were evaluated regarding their interactions with acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidases A and B (MAO-A and MAO-B), which are enzymatic targets related with neurodegenerative diseases. Two quaternary b-carboline alkaloids, prunifoleine and 14-oxoprunifoleine, inhibited AChE, BChE and MAO-A with IC50 values corresponding to 10 and 3.39 lM for AChE, 100 and 11 lM for BChE, and 7.41 and 6.92 lM for MAO-A, respectively. Both compounds seem to behave as noncompetitive AChE inhibitors and time-dependent MAO-A inhibitors. In addition, the monoterpene indole alkaloids (MIAs) angustine, vallesiachotamine lactone, E-vallesiachotamine and Z-vallesiachotamine inhibited BChE and MAO-A with IC50 values ranging from 3.47 to 14 lM for BChE inhibition and from 0.85 to 2.14 lM for MAO-A inhibition. Among the tested MIAs, angustine is able to inhibit MAO-A in a reversible and competitive way while the three vallesiachotamine-like alkaloids display a time-dependent inhibition on this target. Docking calculations were performed in order to understand the binding mode between the most active ligands and the selected targets. Taken together, our findings established molecular details of AChE, BChE and MAO-A inhibition by quaternary b-carboline alkaloids and MIAs from Psychotria, suggesting these secondary metabolites are scaffolds for the development of multifunctional compounds against neurodegeneration. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Neurodegenerative disorders, such as Parkinson disease (PD) and Alzheimer disease (AD), are the result of multiple pathophysiological pathways contributing to the neurodegenerative cascade. For this reason, the search for compounds able to interact with CNS biotargets, such as cholinesterases (AChE and BChE) and monoamine oxidases (MAO-A and MAO-B), is considered an attractive goal in drug design (Novaroli et al., 2005; Geldenhuys et al., 2011; Youdim and Buccafusco, 2005). Multipotent ligands able to simultaneously inhibit cholinesterases, as well as MAOs, have been already developed in the context of a multi-target-directed ligand ⇑ Corresponding authors. Tel.: +55 51 33085417; fax: +55 51 33085243 (A.T. Henriques) E-mail addresses: [email protected] (C.S. Passos), amelia@ farmacia.ufrgs.br (A.T. Henriques). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.11.015

approach (MTDL), leading to the identification of HLA20, M30 and ladostigil, a brain selective molecule acting on different targets on the CNS which is in clinical trials for the treatment of AD (Geldenhuys et al., 2011). Despite the great amount of studies related to design and synthesis of multi-target compounds, the identification of multifunctional scaffolds in the natural products field is still scarce. It is possible to highlight the alkaloid berberine as a lead structure for AChE, BChE and amyloid b (Ab) inhibition (Jiang et al., 2011), together with other activities mainly related with AD. Considering that the alterations in the monoaminergic transmission are related to PD and to behavioral disturbances in patients with AD, monoamine oxidases (MAOs, EC 1.4.3.4) may be considered as targets for the treatment of these multifactorial diseases (Youdim and Bakhle, 2006; Youdim et al., 2006). Monoamine oxidases are classified as MAO-A and MAO-B according to their substrate and inhibitor affinities, and are responsible for the oxidative deamination of a range of monoamines, including

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

5-hydroxytryptamine (5-HT), histamine, catecholamines (dopamine, noradrenaline and adrenaline), as well as xenobiotic amines, such as tyramine, benzylamine and b-phenylethylamine (Youdim et al., 2006). MAO-A selective inhibitors are used in the treatment of anxiety and depression, while the selective irreversible MAO-B inhibitors, L-deprenyl and rasagiline, are clinically employed in the therapy of PD by increasing brain dopamine levels (Youdim and Bakhle, 2006; Youdim et al., 2006). It has been demonstrated that MAO-A inhibition can also affect brain dopamine levels. Thus, a dual reversible MAO-A/B inhibition or a brain selective irreversible inhibition could be an interesting feature for PD treatment, overcoming the cardiovascular effects caused by tyramine, known as cheese reaction (Youdim and Bakhle, 2006). Acetylcholinesterase (AChE, EC 3.1.1.7) inhibitors are largely employed for the symptomatic treatment of AD by increasing the brain levels of acetylcholine (ACh). Consequently, they promote improvements in cognition in patients suffering from this condition. Besides the AChE, butyrylcholinesterase (BChE, EC 3.1.1.8) is also responsible for inactivating ACh in the brain tissue (Francis et al., 1999). Moreover, in AD, AChE is lost by up 85% in specific brain regions, whereas BChE levels rise with disease progression, mainly in the hippocampus and temporal cortex, suggesting that the ACh degradation could occur in a greater extent via BChE catalysis (Lane et al., 2006; Greig et al., 2005; Fernández-Bachiller et al., 2012). Despite this fact, selective AChE inhibition is still considered an important feature in the search for new drugs against AD, since BChE inhibition leads to unwanted cholinergic peripheral side effects (Francis et al., 1999). Brain selective AChE/BChE inhibitors, possessing less peripheral activities, could represent a better alternative for AD treatment than highly selective AChE inhibitors (Greig et al., 2005). Alkaloids from neotropical Psychotria species (subg. Heteropsychotria) have been subjected to in vitro and in vivo biological studies involving CNS activities. Psychollatine, the major alkaloid found in Psychotria umbellata leaves, displayed mild analgesic activity, and anxiolytic (7.5 and 15 mg/kg), antidepressive (3 and 7.5 mg/ kg) and amnesic effects (100 mg/kg) in mice models. Its effects seem to be related with the modulation of opioid (Both et al., 2002), glutamatergic NMDA (Both et al., 2006) and serotonergic 5-HT2A/C systems (Both et al., 2005). Strictosidinic acid seems to act on 5-HT and DA systems in rat striatum (Farias et al., 2010) and hippocampus (Farias et al., 2012), affecting the monoamines metabolism in this brain area. Recently, fractions and alkaloids from Psychotria suterella and Psychotria laciniata were evaluated on MAO-A and MAO-B from rat brain mitochondria. In these experiments, RP-MPLC fractions containing mixtures of vallesichotamine-like alkaloids were able to inhibit MAO-A and MAO-B with IC50 values ranging from 0.57 to 68 lg/mL (Passos et al., 2012). On the other hand, the monoterpene indole alkaloids lyaloside (6) and strictosamide (7) (Fig. 1) inhibited both enzymes in higher concentrations: MAO-A IC50 of 118 and 133 lM for 6 and 7, respectively; and MAO-B IC50 of 724 and 646 lM for 6 and 7, respectively. Therefore, considering the central activities described for Psychotria alkaloids as well as the multiple mechanisms which are related to these actions, the present study aims at (i) evaluating the effects of 13 Psychotria alkaloids on AChE, BChE, recombinant human MAO-A and MAO-B; (ii) retrieving structural information about such interactions by molecular docking. 2. Results and discussion 2.1. Chemistry The ethanol extracts of P. laciniata Vell. leaves were partitioned by acid-basic extraction affording the P. laciniata alkaloid fraction (LAE). LAE was further subjected to RP-MPLC to yield five major

9

fractions, which were tested on AChE, BChE, MAO-A and MAO-B (Supplementary data, Table S1). The RP-MPLC fractions LAE-F4 and LAE-F5 were able to inhibit BChE (71.42% and 79.03%, respectively) and MAO-A (94.94 and 101.69%, respectively) at 40 lg/mL and, therefore, they were subjected to separation by preparative HPLC, affording angustine (1), vallesiachotamine lactone (2), and E/Z-vallesiachotamine (3 and 4). LAE-F2, which is also able to inhibit BChE (48.41%) and MAO-A (95.23%), was subjected to column chromatography on Sephadex LH-20 in MeOH, affording pauridianthoside (5). Alkaloids 1–4, which derive from strictosidine, were previously described in Psychotria and Palicourea species, the last one being a Rubiaceae genus closely related to Psychotria (Solis et al., 1993; Paul et al., 2003; Vencato et al., 2006). Regarding to alkaloid 5, it seems to be the first time that this lyaloside derivative is found in Psychotria. This compound and its epimer at C-21 (isopauridianthoside) were first described in Pauridiantha lyallii (Levesque et al., 1977, 1983). The other eight alkaloids investigated in this study regarding their activities on cholinesterases and monoamine oxidases were previously isolated from different Psychotria species, with their identity and purity confirmed by NMR or MS analyses as described in Section 4. The detailed spectral data of alkaloids 1–13 are given in Supplementary data. 2.2. Screening on AChE, BChE, MAO-A and MAO-B In a second step, 13 Psychotria alkaloids (1–5; lyaloside 6; strictosamide 7; vincosamide 8; brachycerine 9; psychollatine 10; strictosidinic acid 11; prunifoleine 12; and 14-oxoprunifoleine 13) (Fig. 1) were evaluated at 10 and 100 lM on AChE, BChE, MAO-A and MAO-B assays. From these experiments, it was observed that compounds 12 and 13 were able to strongly inhibit AChE, BChE and MAO-A in a concentration dependent-way (Supplementary data, Table S2). Moreover, alkaloids 1–4 showed inhibition on BChE and MAO-A higher than 80% at 100 lM. However, the inhibition of AChE by 1–4 was much lower (<65%) at the same concentration (Supplementary data, Table S2). Based on these results, compounds 1–4, 12 and 13 were selected for complementary experiments. 2.3. AChE and BChE inhibition studies The AChE and BChE inhibitory potencies of alkaloids 1–4, 12 and 13 are shown in Table 1. The results demonstrate that 12 and 13 are non-selective inhibitors for both cholinesterases, exhibiting selectivity indexes (SI) of 0.99 and 0.5 when their pIC50 values are compared. The IC50 values displayed by 12 and 13 on AChE (10 and 3.39 lM, respectively) are close to that observed for galanthamine hydrobromide at the same test conditions (2.14 lM) (Table 1). For BChE, 12 and 13 showed IC50 of 100 and 11 lM, respectively. These AChE/BChE inhibitions are in agreement with the data reported in literature for other quaternary b-carbolines (bCs), such as nostocarboline iodide (AChE IC50 = 5.3 lM; BChE IC50 = 13 lM), deschloro-nostocarboline iodide (2-methylnorharmane iodide; BChE IC50 = 11 lM) (Becher et al., 2009, 2005), infractopicrin and 10-hydroxy-infractopicrin (bovine erythrocytes AChE IC50 = 9.72 and 13 lM) (Geissler et al., 2010), respectively. Additionally, some other synthetic quaternary b-carbolines possessing the harmane (14) scaffold are also able to inhibit AChE and BChE displaying similar IC50 values (Schott et al., 2006; Torres et al., 2012). The presence of alkyl groups at N-4 seems to be a requirement for cholinesterase inhibition, since this pattern is observed for 12 and 13 and for the other cited compounds. Moreover, AChE-selectivity is attributed to alkyl groups as substituents at the indole nitrogen (Geissler et al., 2010) and their absence in 12 and 13 as well as in nostocarboline could explain the lack of

10

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

Fig. 1. Chemical structures of the Psychotria alkaloids investigated in this study: angustine (1), vallesiachotamine lactone (2), E-vallesiachotamine (3), Z-vallesiachotamine (4), pauridianthoside (5), lyaloside (6), strictosamide (7), vincosamide (8), brachycerine (9), psychollatine (10), strictosidinic acid (11), prunifoleine (12), and 14oxoprunefoleine (13). The chemical structures of harmane (14) and clorgyline (15), respectively a reversible and an irreversible MAO-A inhibitor, are also shown in this figure.

AChE-selectivity exhibited by these compounds. Contrary to what was observed for the quaternary alkaloids, 1–3 were not able to inhibit more than 50% of AChE at concentrations of 10 or 100 lM (Supplementary data, Table S2). Despite the 63% of inhibition on AChE displayed by 4 at 100 lM, this value is lower than the corresponding values of 12 and 13 (90% and 89%, respectively). Additionally, 12 and 13 also inhibited AChE in more than 50% at 10 lM. However, 1–4 inhibited BChE showing IC50 values (Table 1) in a similar range as 13. It is important to highlight that alkaloids

12 and 13 are bCs, while 1–4 are THbCs, suggesting that the aromatic bC system is not essential for BChE inhibition. 2.4. AChE kinetic studies The inhibition mode of prunifoleine (12) and 14-oxoprunifoleine (13) on AChE was further investigated in order to determine their Ki values. Lineweaver–Burk plots were constructed by measuring the initial catalytic rates of AChE in the presence of

11

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20 Table 1 The IC50 (pIC50) values for the inhibition of AChE, BChE, MAO-A and MAO-B by Psychotria alkaloids. IC50 lM (pIC50)a AChE

BChE

1 2 3 4 5 6 7 12 13

>100 (<4) >100 (<4) >100 (<4) >10 (<5) >100 (<4) >100 (<4) >100 (<4) 10 (4.99 ± 0.26) 3.39 (5.47 ± 0.25)

3.47 (5.46 ± 0.19) 14 (4.85 ± 0.07) 7.08 (5.15 ± 0.43) 9.77 (5.01 ± 0.00) >100 (<4) 23 (4.63 ± 0.11) >100 (<4) 100 (4.00 ± 0.51) 11 (4.97 ± 0.10)

Tacrine Galanthamine Clorgyline Harmane Pargyline

0.07 (7.14 ± 0.11) 2.14 (5.67 ± 0.03) N.T. N.T. N.T.

0.008 (8.10 ± 0.06) N.T. N.T. N.T. N.T.

S.I.b

MAO-A

MAO-B

S.I.b

138 (3.86 ± 0.12) 34 (4.47 ± 0.38) 120 (3.92 ± 0.06) 126 (3.90 ± 0.04) 316 (3.50 ± 0.21) >100 (<4) >100 (<4) 41 (4.39 ± 0.01) 81 (4.09 ± 0.01)

2.10 1.59 1.75 2.17 1.23

0.99 0.50

1.10 (5.96 ± 0.04) 0.87 (6.06 ± 0.01) 2.14 (5.67 ± 0.02) 0.85 (6.07 ± 0.01) 19 (4.73 ± 0.06) 182 (3.74 ± 0.25) 141 (3.85 ± 0.15) 7.41 (5.13 ± 0.06) 6.92 (5.16 ± 0.04) N.T. N.T. 0.004 (8.42 ± 0.14) 0.13 (6.87 ± 0.06) 4.07 (5.39 ± 0.03)

N.T. N.T. 11 (4.96 ± 0.01) N.T. 0.13 (6.87 ± 0.04)

()0.96

0.74 1.07

3.46 1.48

N.T.: not tested. a The pIC50 values, which represent the negative logarithms of the molar concentrations of inhibitor required to decrease the enzymatic activities by 50%, are expressed in parenthesis as mean ± SD of 2–4 replicates. The IC50 values, expressed in lM, were determined by the formula IC50 = antilog pIC50  106. b The selectivity indexes (S.I.) correspond to DpIC50: pIC50 AChE  pIC50 BChE and pIC50 MAO-A  pIC50 MAO-B.

Fig. 2. Lineweaver–Burk plots of AChE activity over a range of substrate concentrations (0.05–1.2 mM) for prunifoleine (12, 2A) (6.75 and 13.5 lM) and 14-oxoprunifoleine (13, 2B) (7.25 and 14.5 lM). Ki values estimated from a replot of the slopes of the Lineweaver–Burk plots versus inhibitor concentrations were 15.78 and 8.93 lM for 12 and 13, respectively.

two different concentrations of 12 and 13. As shown in Fig. 2, the Lineweaver–Burk plots constructed for AChE inhibition are linear

and the intercept at x-axis suggests a noncompetitive inhibition, in line with the data reported for nostocarboline (Becher et al.,

12

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

2009) and for two synthetic b-carbolinium salts derived from harmane (Torres et al., 2012). The Ki values were estimated by plotting the slope of the individual Lineweaver–Burk plots versus the inhibitor concentrations. For prunifoleine (12) the estimated Ki was 15.78 lM, while for 14-oxoprunifoleine (13), the Ki was 8.93 lM. 2.5. MAO-A and MAO-B inhibition studies The MAO-A and MAO-B inhibitory potencies of 1–4, 12 and 13 are given in Table 1. The results showed that all tested compounds possess a degree of selectivity for MAO-A, with SI values between 0.74 and 2.17. Moreover, these alkaloids displayed MAO-A IC50 values in the lM range, exhibiting concentrations varying from 0.85 to 7.41 lM, being about 10-fold less potent than harmane (14) (MAO-A IC50 = 0.13 lM) and 1000-fold less potent than harmine (MAO-A IC50 = 0.002 lM) (Samoylenko et al., 2010). The MAO-A IC50 for 6 and 7 were also determined (Table 1) in order to compare the results with those described for the enzymes obtained from rat brain mitochondria (Passos et al., 2012). From these experiments, it was verified that lyaloside (6) and strictosamide (7) seem to have the same potency on human (Table 1) and rat brain MAO-A (rat brain MAO-A IC50 = 118 lM and 133 lM) (Passos et al., 2012), respectively. The alkaloid pauridianthoside (5), which has not been previously evaluated on rat brain MAO-A, inhibited the human enzyme with IC50 = 19 lM. This inhibition, about 10-fold more potent than that observed for 6, suggests that the oxygen substituent at C14 could contribute to the ligand binding in MAO-A. Considering the tested alkaloids, vallesiachotamine lactone (2) and prunifoleine (12) were the most potent inhibitors of MAO-B with IC50 values of 34 and 41 lM, respectively. 2.6. Reversibility of MAO-A inhibition MAO inhibitors can be classified as reversible or irreversible, according to their interaction with the target protein. MAO irreversible inhibitors have been extensively used as clinical drugs. However, this type of inhibition may induce cardiovascular toxic effects, mainly provoked by the inhibition of the peripheral MAO-A located in gut, liver and endothelium (Youdim and Bakhle, 2006; Youdim et al., 2006). Additionally, the slow and variable enzyme recovery following the withdrawal of irreversible inhibitors is a disadvantage in clinical use, since the turnover rate for MAO biosynthesis in the human brain seems to require about 40 days

(Fowler et al., 1994). On the other hand, competitive reversible inhibitors have less influence in the enzyme recovery after withdrawal, and MAO-A reversible inhibitors such as moclobemide are devoid of cardiovascular effects, since the ingested tyramine is able to displace the inhibitor from the MAO active site and be metabolized in the normal way by peripheral enzyme in gut and liver (Youdim and Bakhle, 2006). In order to clarify if 1–4, 12 and 13 act as reversible or irreversible inhibitors, the time-dependence of MAO-A inhibition was evaluated according to method described by Legoabe et al. (2012). Harmane (14) and clorgyline (15) (Fig. 1) were used as controls for reversible (non time-dependent) and irreversible (timedependent) inhibition, respectively. In these assays, the enzymatic activity, when the compounds are preincubated with MAO-A for various periods of time (0, 15, 30 and 60 min), remains unchanged regardless of the time period for which the inhibitor is incubated with the enzyme. By contrast, irreversible inhibitors affect the enzyme activity in a time-dependent way after preincubation (Legoabe et al., 2012). Psychotria alkaloids 1–4, 12 and 13 were preincubated for different time periods (0, 15, 30, 60 min) with MAO-A at concentrations corresponding to twofold their measured IC50. After addition of the kynuramine substrate and after a new period of incubation, the residual enzyme activities were measured and bar graphs were constructed. One-way ANOVA followed by post hoc analysis by Tukey’s test (P < 0.05 was considered significant) were used for evaluating inhibition. The results observed for time-dependence experiments are shown in Fig. 3. By graphical analysis, it is possible to observe that harmane (14) and clorgyline (15) clearly behave as non timedependent and time-dependent inhibitors, respectively, over the time period (0–60 min), and at the evaluated concentrations. Regarding the tested alkaloids, only angustine (1) seems to display a non time-dependent MAO-A inhibition, suggesting a reversible mechanism. On the other hand, alkaloids 2–4, 12 and 13 displayed a time-dependent MAO-A inhibition, possessing an intermediate behavior between harmane and clorgyline. These results suggest that these compounds can act as irreversible or slowly-reversible inhibitors. 2.7. MAO-A kinetic studies Kinetic studies were performed in order to provide additional evidence for the reversible interaction of angustine (1) with

Fig. 3. Time-dependent inhibition of hMAO-A by angustine (1), vallesiachotamine lactone (2), E-vallesiachotamine (3), Z-vallesiachotamine (4), prunifoleine (12) and 14oxoprunifoleine (13). The evaluated compounds were preincubated for different periods of time (0, 15, 30 and 60 min) with the target enzyme in concentrations corresponding to twofold their calculated IC50 values for hMAO-A inhibition. After addition of kynuramine, in concentration corresponding to its Km, and consequent dilution of the inhibitors solutions to concentrations equal their IC50 values, the hMAO-A activity was determined. Results are expressed as mean ± SD of triplicate determinations. Statistical significance was determined by one-way ANOVA followed by Tukey’s test; a: significant difference from t = 0 min (P < 0.05).

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

13

(Supplementary data, Table S4). Additionally, it was found that 5 water molecules located in the active site of hMAO-A crystals (Son et al., 2008; Reniers et al., 2011; Legoabe et al., 2012) were essential for stabilizing the complexes as also reported in literature. Thus, such water molecules were retained in the docking studies.

Fig. 4. Lineweaver–Burk plots of the human MAO-A activity over a range of substrate concentrations (22–220 lM) for angustine (1). The Ki value estimated from replots of the slopes of the Lineweaver–Burk plots versus inhibitor concentrations was 0.50 lM.

MAO-A. The kinetic parameters Km (Michaelis–Menten constant) and Vmax (maximal velocity) were determined by means of Lineweaver–Burk (LB) plots over a substrate range varying from 22 to 220 lM. The MAO-A catalytic rates were measured in the absence of inhibitor, and presence of three different concentrations of 1 (0.5; 1; and 2 lM). The graphical analysis of the LB plots suggests competitive inhibition, characterized by linear plots and intersection at y-axis (Fig. 4). Additionally, the Ki value (0.50 lM) for MAO-A inhibition by 1 was estimated from replots of the slopes of the LB plots versus inhibitor concentrations, as performed for AChE. These kinetics data support that 1 is a reversible MAO-A inhibitor. 2.8. Docking studies Docking studies were carried out in order to understand the interactions of compounds 12 and 13 with AChE, BChE and MAOA, and of compounds 1–4 with BChE and MAO-A, according to the most relevant results found for these biotargets in the enzymatic assays. Structural information about AChE from Torpedo californica (TcAChE) in complex with galanthamine (PDB ID 1DX6) (Greenblatt et al., 1999), the human BChE (hBChE) in complex with butyrate (PDB ID 1P0I) (Nicolet et al., 2003) and the human MAO-A (hMAO-A) in complex with harmine (PDB ID 2Z5X) (Son et al., 2008) were retrieved from the Protein Data Bank (http:// www.rcsb.org/). TcAChE was chosen for this study because no reliable information about the electric eel enzyme, previously used in the enzymatic tests, was available. Nevertheless, TcAChE shares more than 60% of sequence identity with EeAChE. The human BChE was chosen for the same reason: no equine serum BChE structure could be found in the Protein Data Bank. It should be noticed that this protein has more than 85% sequence identity with the equine serum BChE. In all cases, the selected proteins were co-crystallized with ligands: the presence of such compounds in the pockets induces the side-chains to adopt conformations that can better accommodate ligands during docking. Before docking Psychotria alkaloids, the docking methodology was set up and validated by re-docking co-crystallized ligands in the selected proteins. Docking calculations were performed by the GOLD software v.5 (CCDC, Cambridge, UK) using the Molecular Lipophilicity Potential as hydrophobic descriptor (Nurisso et al., 2012), since all targets possess a Lipophilic Index higher than 10 (Supplementary data, Table S3). The best ranked solutions for the re-docked ligands presented RMSD values lower than 2.0 Å with respect to the X-ray ligands

2.8.1. Docking of 12 and 13 on AChE and BChE The best ranked solutions observed for both prunifoleine (12) and 14-oxoprunifoleine (13) in AChE (Fig. 5) were taken into account for structural inspections. Both alkaloids displayed a similar orientation in the enzyme active site, being the endocyclic oxygen of both ligands involved in a hydrogen bond with the hydroxyl group of Tyr-121 from the peripheral anionic binding site (PAS). A hydrophobic niche present in the cholinesterase binding site, characterized by Phe-290, Phe-330 and Phe-331 residues, accommodated 12 and 13 through van der Waals contacts. Moreover, a stacking interaction between Trp-84 and the aromatic ring of these compounds was also observed. The amino acids involved in these interactions have been previously described for Cortinarius infractus quaternary b-carbolines (Geissler et al., 2010) and for two synthetic b-carbolinium salts derived from harmane (14) (Torres et al., 2012) in TcAChE. The orientation of 12 and 13 in the BChE active site was different from what was observed in AChE (Fig. 5). Indeed, the active site of hBChE presents structural differences when compared to the TcAChE one. Although a peripheral anionic binding site, similar to that described for TcAChE, had been previously described for human BChE, site-direct mutagenesis and photo-affinity labeling studies showed that its location and the response upon ligand binding differ significantly from those of AChE (Nicolet et al., 2003; Masson et al., 1997; Nachon et al., 2006). Moreover, in BChE active site, an Ala residue replaces the Phe-330 from AChE, and the size of the binding pocket differs, being more voluminous than in AChE (Nicolet et al., 2003). For prunifoleine (12) a stacking interaction with Trp-82 (which corresponds to Trp-84 in TcAChE) was observed. Ala-328, Phe-329 and Tyr-332, with their hydrophobic side-chains, further stabilize the complex through hydrophobic interactions with the aromatic carbon atoms characterizing the alkaloid scaffold. For 13, molecular modeling suggested a diverse binding mode. The best ranked solution, according to the GoldScore (55.75), showed polar contacts between the nitrogen of the indole group and Ser-198 and His-438 side-chains. Hydrophobic interactions were also retrieved, involving the carbon atoms of the ligand and Trp-82, Trp-231, Leu-286 and Phe-329 side-chains. The recent findings of Torres et al. (2012) concerning the binding of b-carbolinium salts on both cholinesterases are in line with the present data: different ligand orientations in the two cholinesterase enzymes were observed. Moreover, a noncompetitive inhibition mode was found, supported by structural data retrieved by docking. Due to the high similarity to these compounds, it is supposed that 12 and 13 could also bind to the enzymes without displacing their natural substrates (Torres et al., 2012). 2.8.2. Docking of 1–4 on BChE Considering the interesting IC50 values displayed by 1–4 on BChE (10 lM), molecular modeling studies were carried out in order to obtain structural information on the complexes. The vallesiachotamine-like alkaloids (2–4) bind to the BChE active site maintaining the same orientation of their scaffold: the indole ring faces to the Ser-198 and His-438 residues, establishing hydrogen bonds whereas Trp-82, Trp-231, Leu-286, Phe-329 and Ile-442 residues contribute to complexes stabilization through van der Waals contacts with the carbon atoms characterizing the ring system (Supplementary data, Fig. 1SB–D). On the other hand, angustine (1) binds to BChE in a different way (Supplementary data,

14

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

Fig. 5. Docking of prunifoleine (12) and 14-oxoprunifoleine (13) in AChE (green; PDB ID: 1DX6) and BChE (cyan; PDB ID: 1P0I). In 5A are shown the best ranked solutions obtained from docking of 12 in AChE (orange; 12 docked in AChE; hydrogen bond between the endocyclic oxygen of 12 and the hydroxyl group of Tyr-121) and BChE (magenta; 12 docked in BChE). In 5B are shown the best ranked solutions obtained from docking of 13 in AChE (orange; 13 docked in AChE; hydrogen bond between the endocyclic oxygen of 13 and the hydroxyl group of TYR-121) and BChE (magenta; 13 docked in BChE; polar contacts between the nitrogen of the indole group and Ser-198 and His-438 side-chains). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Figs. 1SB–D and 1SA), mainly stabilized by hydrophobic interactions involving Trp-82, Trp-231, Leu-286 and Phe-329 and its aromatic moieties. The steric hindrance due to the introduction of substituents on the pyridine ring of 2–4 may be the cause of this different binding mode. Again, considering the work from Torres et al. (2012), it can be supposed that there is an analog mode of action, characterized by noncompetitive inhibition (Torres et al., 2012). 2.8.3. Docking of 1–4, 12 and 13 on MAO-A Compounds 1–4, 12 and 13 were docked in the MAO-A catalytic site in order to structurally understand the interactions with this biotarget together with their inhibitory profiles.

Docking indicated that both indole and pyridine groups of angustine (1), through their nitrogen atoms, can establish hydrogen bonds with structural water molecules WAT-746 and WAT805, respectively, acting as bridges between the ligand and the protein. Additionally, the pyridine ring is also involved in p stacking interactions with the Tyr-407 aromatic ring, whereas Tyr-69, Phe-208, Ile-335 and Leu-337 residues establish van der Waals contacts with the carbons of the alkaloid cycle (Fig. 6A). Interactions between MAO-A and 1 are similar to those observed for harmine in the co-crystal (Supplementary data, Fig. 1SC) (Son et al., 2008). As an analogy, a reversible and competitive inhibitory profile of compound 1 towards MAO-A can be expected, as demonstrated by the experimental data of the present study.

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

15

Fig. 6. Docking of alkaloids 1–4 in MAO-A (PDB ID: 2Z5X). MAO-A is represented in grey and FAD in yellow. (A) Docking of angustine (1) in MAO-A (hydrogen bonds between the indole nitrogen of 1 and WAT-746; and the pyridine nitrogen of 1 and WAT-805); (B) Docking of 2 (cyan), 3 (green) and 4 (orange) in MAO-A active site (hydrogen bonds: indole nitrogen of 2–4 with WAT-746; aldehyde oxygen of 3 with WAT-710; aldehyde oxygen of 4 with Gln-215 side-chain; lactone oxygen of 2 with Tyr-444 side chain and WAT-718). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The other monoterpene indole compounds 2, 3 and 4 bind in a similar way the MAO-A pocket (Fig. 6B). As for compound 1, their scaffolds are accommodated through hydrophobic interactions with the apolar side-chains of the amino acids characterizing the binding site. A conserved hydrogen bond between the nitrogen of the indole ring and WAT-746 characterizes the binding of these alkaloids. Other polar contacts can be observed in 3 and 4, between the oxygen of the aldehyde and WAT-710 and Gln-215 side chain, respectively. In 2, the oxygen of the lactone group acts as a polar bridge between the hydroxyl group of the Tyr-444 side-chain

and WAT-718. The carboxymethyl moieties characterizing the MIAs 2, 3 and 4 are close to the FAD cofactor. This group could be subjected to a nucleophilic attack by the N5 of FAD, creating a covalent bond between the enzyme and the ligands. This reminds the irriversible inibitory mechanism of action of acetylsalicylic acid towards the COX enzyme (Marnett, 2002). The hypothesis could justify the apparent irreversible behavior (time-dependent inhibition) of such compounds towards MAO-A (Fig. 3). Finally, prunifoleine (12) and 14-oxoprunifoleine (13) were docked in human MAO-A binding pocket (Fig. 7). Both quaternary

16

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

Fig. 7. Docking of prunifoleine (12) and 14-oxoprunifoleine (13) in MAO-A (PDB ID: 2Z5X). MAO-A is represented in grey and FAD in yellow. (A) Prunifoleine (12) docked in MAO-A (hydrogen bond between the indole nitrogen of 12 and the carboxyl group of the main chain of Phe-208); and (B) 14-oxoprunifoleine (13) docked in MAO-A (hydrogen bond between the indole nitrogen of 13 and the carboxyl group of the main chain of Phe-208, and between the keto-oxygen and WAT-746). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

b-carboline systems are buried inside the hydrophobic niche created by Tyr-407, Tyr-444, Tyr-69, Phe-352, Ile-335 and Ile-325 residues. A hydrogen bond between the nitrogen from both indole moieties with the carboxyl group of the main chain of Phe-208 can also be observed, whereas the exclusive keto-oxygen in 13 creates polar contacts with WAT-746. It should be noticed that the vi-

nyl group, present in both 12 and 13 structures, is faced to the FAD cofactor. By analogy with the propargyl-containing MAO-A inhibitors, such as clorgyline, able to inactivate the enzyme by covalent interactions with the N5 of FAD isoalloxazine (MA et al., 2004), 12 and 13 could also establish such covalent interactions via their vinyl groups, which can be considered the irreversible-enzyme

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

inactivating moieties. Taken together, the docking and the experimental results observed in the enzymatic assays suggest MAO-A irreversible inhibition for alkaloids 12 and 13. 3. Concluding remarks The present study shows that monoterpene indole alkaloids (MIAs) and quaternary b-carboline alkaloids obtained from Brazilian Psychotria species act as cholinesterases and monoamine oxidases inhibitors. The quaternary b-carbolines prunifoleine (12) and 14-oxoprunifoleine (13) found in Psychotria prunifolia were able to inhibit both cholinesterases (AChE and BChE) and MAO-A. Regarding AChE and MAO-A assays, compounds 12 and 13 showed a similar profile on these targets, characterized by noncompetitive AChE inhibition with IC50 values of 10 lM (12) and 3.39 lM (13), and time-dependent MAO-A inhibition with IC50 values in the same range verified for AChE. The time-dependence of MAO-A inhibition suggests an irreversible mechanism, which is supported by the docking simulations showing the possibility of covalent interactions between the N5 from FAD and the vinyl groups of 12 and 13. Concerning the BChE inhibition, alkaloids 12 and 13 displayed IC50 values of 100 and 11 lM, respectively, suggesting that 13 is approximately 10-fold more potent than 12 on this target. The docking simulations support the differences observed in the IC50 values, showing that 13 may establish polar contacts between the nitrogen of the indole group and the side-chains of Ser-198 and His-438 from the active site. The MIAs angustine (1), vallesiachotamine lactone (2) and E/Zvallesiachotamine (3,4) were able to inhibit BChE and MAO-A. Compounds 1–4 inhibited BChE with IC50 values ranging from 3.47 to 14 lM. The docking simulations added information about the binding mode of 1–4 on BChE, showing that 2–4 seem to establish similar interactions with this target whereas 1 binds to the enzyme in a different orientation. Regarding the MAO-A inhibition, alkaloids 1–4 inhibited MAO-A displaying IC50 values ranging from 0.85 to 2.14 lM. The kinetics and time-dependent studies showed that angustine (1) clearly behaves as a reversible and competitive inhibitor, whereas 2–4 displayed a time-dependent behavior, suggesting irreversible inhibition. The experimental data are in agreement with the docking simulations, which suggest that angustine (1) bind to MAO-A in a similar way than harmine, reinforcing the hypothesis of reversible and competitive inhibition. On the other hand, the binding mode of 2–4 on MAO-A active site suggest the possibility of covalent interactions between the N5 from FAD and the carboxymethyl moieties of these alkaloids. In conclusion, the Psycohtria alkaloids evaluated in the present study could be new scaffolds for the development of multifunctional cholinesterases and MAOs inhibitors. The six alkaloids showing activity on the selected targets may be divided into two different classes: monoterpene indole alkaloids (MIAs) (1–4) and quaternary b-carbolines (12, 13). All the tested substances were able to interact with one or more target presenting different mechanisms of inhibition. 4. Experimental section 4.1. General NMR spectra were recorded in d6-DMSO (Dr. Glaser, AG, Basel, Switzerland) on a Varian Inova 500 MHz spectrometer (Palo Alto, CA, USA). The instrument was controlled using a Varian V NMR software installed on a Sun SPARCstation (Santa Clara, CA, USA). The chemical shifts are given in d (ppm) with residual d6-DMSO as internal reference. The coupling constants are given in Hz.

17

UHPLC/HR-TOF-MS analyses were performed on a MicromassLCT Premier Time of Flight mass spectrometer from Waters (Milford, MA, USA) with an electrospray (ESI) interface coupled with an Acquity UPLC system from Waters. Detection was performed in positive ion mode in the range m/z 100–1000 in centered mode with a scan time of 0.2 s and an interscan delay of 0.3 s for polarity switching. ESI conditions were capillary voltage 2800 V, cone voltage 40 V, source temperature 120 °C, desolvation temperature 250 °C, cone gas flow 20 L/h, and desolvation gas flow 800 L/h. For internal calibration, a solution of leucine/enkephalin from Sigma–Aldrich (Steinheim, Germany) at 5 lg/mL was infused through the lockmass probe at a flow rate of 5 lL/min, using a second Shimadzu LC-10ADvp LC pump (Duisburg, Germany). The separation was performed on a 150 2.1 mm i.d., 1.7 lm particle size, Acquity BEH C18 UPLC column (Waters) in the gradient mode at a flow rate of 0.3 mL/min with the following solvent system: (A) 0.1% HCO2H in H2O, (B) 0.1% HCO2H in CH3CN; in 7.0 min. The temperature was set at 30 °C and the injected volume was 5 lL. Reversed-phase medium pressure liquid chromatography (RP-MPLC) was carried out on a BUCHI system equipped with a B-688 BUCHI chromatography pump, a B-687 BUCHI gradient former, and a BUCHI borosilicate column filled out with RP-18 LiChrospherÒ (Merck, Darmstadt, Germany). Separation by preparative HPLC was performed in a system composed by pumps Shimadzu LC-8A, a system controler Shimadzu SCL-10A, and a UV–Vis detector Shimadzu SPD-10A, using a Waters XBridgeTM prep C18 Column (250  10 mm I.D.; 5 lM particle size, Part Number 186003256). Sephadex LH-20 was used for column chromatography (CC). Fractions were concentrated on a R-114 BUCHI rotavapor equipped with a V-710 BUCHI vacuum pump. The spectrophotometric readings for AChE and BChE assays were performed on a PowerWavex spectrophotometer (Bio-Tek Instruments, Winooski, Vermont, USA). The fluorescence readings for MAO-A and MAO-B experiments were carried out on a FLx 800 microplate fluorescent reader (FLx 800, Bio-Tek Instruments, Winooski, Vermont, USA).

4.2. Materials Acetylcholinesterase from Electrophorus electricus (electric eel AChE), butyrylcholinesterase from equine serum (BChE), acetylthiocholine iodide (ATCI), S-butyrylthiocholine iodide (BTCI), 5,50 dithiobis(2-nitrobenzoic acid) (DTNB), galanthamine hydrochloride, tacrine hydrochloride, kynuramine dihydrobromide, pargyline hydrochloride, clorgyline hydrochloride, dimethyl sulfoxide (DMSO), human albumin, and acetonitrile HPLC grade were purchased from Sigma–Aldrich Chemical (St. Louis, MO, USA). 4Hydroxyquinoline (4-OH), potassium phosphate salts, sodium phosphate salts, potassium chloride and sodium hydroxide came from Fluka (Buchs, CH). Human MAO-A and MAO-B supersomes were acquired from BD Gentest (Woburn, MA). All remaining chemicals used were of analytical grade and were purchased from Sigma–Aldrich. The monoterpene indole alkaloids lyaloside (6) and strictosamide (7) were isolated from P. suterella and P. laciniata (de Santos et al., 2001; Passos et al., 2012). Vincosamide (8) was obtained from Psychotria leiocarpa (Henriques et al., 2004), brachycerine (9) from P. brachyceras (Kerber et al., 2001), psychollatine (10) from P. umbellata (Kerber et al., 2008), and strictosidinic acid (11) from Psychotria myriantha (Simões-Pires et al., 2006). The quaternary b-carboline alkaloids, named prunifoleine (12) and 14-oxoprunifoleine (13) were isolated from P. prunifolia (Faria et al., 2010). Before the enzymatic experiments, all alkaloids had their purity assessed by UHPLC/HR-TOF-MS, and alkaloids 1–7 had their purity further assessed by NMR spectroscopy given the same results as for the MS method (Supplementary data).

18

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

4.3. Plant Material Leaves of P. laciniata Vell. (Rubiaceae) were collected from the Atlantic forest of Cocal do Sul (Santa Catarina State, Brazil S26°800 ; W48°950 ; ICN 182552). The material was identified by Dr. Sergio L. Bordignon (Fundação La Salle, RS, Brazil) and the voucher specimens have been deposited in the herbarium of Departamento de Botânica from Universidade Federal do Rio Grande do Sul (UFRGS). 4.4. Extraction and isolation The dried leaves of P. laciniata (410 g) were extracted with EtOH (5 x 5 L) at room temperature. The solvent was removed under vacuum and the resulting dark green syrups were dissolved in 1 N HCl (0.6 L) and exhaustively extracted with CH2Cl2, in order to remove non-polar constituents. Subsequently, the acid extracts were alkalinized with 25% NH4OH (pH 9–10) and partitioned with CH2Cl2, resulting in the P. laciniata alkaloid fraction (LAE; 0.98 g, 0.24%, w/w). LAE was fractionated by reversed phase medium pressure liquid chromatography (RP-MPLC) with a H2O–CH3CN gradient step, affording five fractions: LAE-F1–5. LAE-F4 and LAE-F5 were submitted to preparative HPLC with isocratic elution (H2O:CH3CN, 40:60, v/v), rate of 5 mL/min, and detection at 280 nm, affording the known compounds angustine (1, 1.1 mg), vallesiachotamine lactone (2, 1.7 mg), E-vallesiachotamine (3, 5.5 mg) and Z-vallesiachotamine (4, 5.8 mg), respectively. LAE-F2 was subjected to Sephadex LH-20 CC (MeOH), affording the known compound pauridianthoside (5, 3.4 mg). The detailed spectroscopic data for alkaloids 1–5 are provided in Supplementary data. 4.5. Enzymatic assays 4.5.1. AChE and BChE inhibition The AChE and BChE inhibitory properties of Psychotria alkaloids were assessed using the Ellman’s method (Ellman et al., 1961) modified by Di Giovanni et al. (2008), employing tacrine and galanthamine as reference compounds. For the AChE assay, wells were filled with Ellman’s reagent (158 lL, 0.15 mM final concentration of 5,50 -dithiobis-(2-nitrobenzoic acid) in 0.1 M phosphate buffer pH 7.4), acetylthiocholine iodide (ATCI) solution (20 lL) in demineralised water (final concentration equal to the Km value; Km = 0.33 mM), and test compound solutions in DMSO (2 lL). For controls, test compound solutions were replaced by the corresponding volume of DMSO or tacrine and galanthamine solutions in concentrations corresponding to their IC50 values (50% of inhibition) and 100-fold their IC50 (100% of inhibition). The enzymatic reaction was initiated by the addition of an electric eel AChE solution (20 lL, 1 U.I./mL in 0.1 M phosphate buffer pH 7.4, containing human serum albumin at 1 mg/mL). The BChE assay was carried out in a similar way to the AChE experiments, except by using the substrate (S-butyrylthiocholine iodide (BTCI); Km = 0.30 mM) and the enzyme (serum horse BChE 1 U.I./mL in 0.1 M phosphate buffer pH 7.4, containing human serum albumin at 1 mg/mL). Assays were performed at room temperature with a PowerWavex microplate spectrophotometer (BioTek Instrument, Winooski, Vermont, USA) following the rate of increase in the absorbance at 412 nm during 6 min (intervals of 30 s between readings). Enzyme activities were first determined for one or two concentrations of the evaluated alkaloids and controls: 104 and 105 M. Subsequently, the IC50 values were evaluated for at least 5 different concentrations for the alkaloids displaying inhibition higher than 50% at 104 M. The inhibitory activities were expressed as percentage of inhibition at 104 and 105 M. The percentage of inhibition was calculated relative to a control sample, for which the AChE and BChE activities were assessed under identical conditions but in the

absence of test compounds (considered to be 100%). Data are means of n = 3. The inhibitory potencies expressed as IC50 values (pIC50), represent the concentrations of inhibitor required to decrease AChE and BChE activity by 50% and were calculated by curve fitting according to classical sigmoidal dose–response equation. All IC50 values were determined in 2–4 replicates from at least six compound concentrations. 4.5.2. Kinetic analysis of the AChE inhibition Reciprocal plots of 1/V versus 1/[S] were constructed at different concentrations of the substrate ATCI (0.05 to 1.20 mM) for prunifoleine (12) and 14-oxoprunifoleine (13) in order to estimate their inhibition constants (Ki). Experiments were performed under the same conditions previously described at two different concentrations of each inhibitor: 0, 6.75 and 13.5 lM for 12; and 0, 7.25 and 14.5 lM for 13. Reactions were performed in triplicate. Progress curves were characterized by a linear steady-state turnover of the substrate and values of a linear regression were fitted according to Lineweaver–Burk replots using GraphPad Prism 5.0 software. 4.5.3. MAO-A and MAO-B inhibition Monoamine oxidase inhibition assays were carried out with fluorescence based method (end-point lecture) using kynuramine as non-selective substrate of MAO-A and MAO-B as previously described by Novaroli et al. (2006). Briefly, reactions were performed in black, flat-bottomed polystyrene 96-well microtiter plates (FluoroNunc/LumiNund, MaxiSorpTM surface, NUNC, Roskild, Denmark) containing potassium phosphate buffer (158 lL), an aqueous stock solution of kynuramine 0.5 mM (final kynuramine concentration corresponding to 50 lM), and DMSO inhibitor solution. This assay mixture was incubated at 37 °C, and then diluted human recombinant MAO-A and MAO-B were delivered to obtain final protein concentrations of 0.009 mg/mL and 0.015 mg/mL, respectively. Incubation was carried out at 37 °C, and the reactions were stopped by addition of 25 lL of a 6 M NaOH solution. Formation of 4-hydroxyquinoline was quantified with a 96-well microplate fluorescent reader (FLx 800, BioTek Instruments, Inc. Winoosli, U.S.A.) at excitation/emission wavelengths of 310/ 400 nm (20 nm slit width for excitation, 30 nm slit width for emission). Data analysis was performed with GraphPad Prism 5.0 software. The degree of inhibition IC50 (pIC50) was assessed by a sigmoidal dose–response curve using duplicate values. 4.5.4. Time-dependent studies of MAO-A To determine whether the inhibition of MAO-A by Psychotria alkaloids could be reversible or irreversible, the time-dependence of inhibition of angustine (1), vallesiachotamine lactone (2), Eand Z-vallesiachotamines (3, 4), prunifoleine (12), and 14-oxoprunifoleine (13) were evaluated according to Legoabe et al. (2012). The selected alkaloids were allowed to preincubate with human MAO-A (final protein concentration of 0.03 mg/mL) for different periods of time (0, 15, 30, 60 min) at 37 °C in potassium phosphate buffer (0.1 M, pH 7.4, made isotonic with KCl). For this purpose, the alkaloid concentrations were equal to twofold their measured IC50 values for MAO-A. Subsequently, the reactions were diluted twofold by the addition of kynuramine to yield a final enzyme concentration of 0.015 mg/mL and alkaloid concentrations corresponding to their IC50. These assays were also performed in 96-well microplates, being the final volume in each well equal to 200 lL and the final kynuramine substrate concentration to 50 lM. The reactions were incubated at 37 °C for a further 30 min period and terminated with the addition of NaOH 6 M (25 lL). The rates of the MAO produced 4-hydroxyquinoline were measured and calculated as described above. All measurements were carried out in triplicate and are expressed as mean ± SD.

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

4.5.5. Lineweaver–Burk plots Lineweaver–Burk plots were constructed in order to evaluate the mode of MAO-A inhibition by compound 1. Alkaloid 1 was evaluated in 3 different concentrations and the substrate, kynuramine, in four concentrations ranging from 22 to 220 lM. Recombinant human MAO-A was employed at a final concentration corresponding to 0.009 mg/mL. The initial catalytic rates were measured considering the reaction product, 4-OH (lM). Linear Regression analysis was performed according described for AChE (Section 4.5.2).

4.6. Molecular modeling studies 4.6.1. Protein preparation The crystallographic structures of Torpedo californica AChE in complex with galanthamine (TcAChE; PBD ID: 1DX6; Resolution = 2.30 Å) (Greenblatt et al., 1999), human BChE in complex with butyrate (hBChE; PDB ID: 1P0I; Resolution = 2.00 Å) (Nicolet et al., 2003) and human MAO-A in complex with harmine (hMAO-A; PDB ID: 2Z5X; Resolution = 2.20 Å) (Son et al., 2008) were retrieved from the Protein Data Bank (PBD; http:// www.rcsb.org/pdb/) (Berman et al., 2000). Protein structures were prepared before docking simulations by using the Biopolymer module implemented in Sybyl X-1.3 (Tripos Inc., St. Louis, MO).

4.6.2. Ligand preparation The starting geometries of the ligands (Fig. 1) were built in Sybyl X-1.3. Bond and atom types were carefully checked. Gasteiger– Marsili partial charges were calculated for each ligand, successively optimized by using the Tripos force field (Clark et al., 1989) with 1000 cycles of conjugate gradient algorithm and convergence criterion was 0.01 kcal/mol.

4.6.3. Docking procedure The docking simulations were carried out using the program GOLD (Verdonk et al., 2003) (CCDC, Cambridge, UK) version 5.0 with the MLP filter approach (Nurisso et al., 2012). The interaction sphere in the docking was delimited by a 6 Å radius from the cocrystallized ligand for all the selected targets (AChE, BChE and MAO-A). No water molecules were retained in AChE and BChE pockets. On the other hand, five water molecules located in the active site were considered in the MAO-A model (Reniers et al., 2011). For each ligand, 100 docking solutions were generated by using 100 000 GOLD Genetic Algorithm iterations (Preset option). Docking poses were evaluated and ranked according to the GoldScore scoring functions. Before starting the simulations with the alkaloids, the co-crystalized ligands from the respective PDB were re-docked into the respective catalytic site in order to validate the methodology. In this case, RMSD values between the docking solutions and the crystallized ligand of reference were calculated.

Acknowledgments C.S.P. acknowledges the fellowships from CNPq/Brazil and CAPES/Brazil (for financial support during internship at the University of Geneva). T.C.S. is recipient of a fellowship from CNPq (BIC/ UFRGS). This work was supported by CNPq, Grant #472287/ 2009-5. Authors also acknowledge Professors Jean-Luc Wolfender and Muriel Cuendet from the University of Geneva for making NMR and MS analyses available.

19

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2012. 11.015. References Becher, P.G., Baumann, H.I., Gademann, K., Jüttner, F., 2009. The cyanobacterial alkaloid nostocarboline: an inhibitor of acetylcholinesterase and trypsin. J. Appl. Phycol. 21, 103–110. Becher, P.G., Beuchat, J., Gademann, K., Jüttner, F., 2005. Nostocarboline: isolation and synthesis of a new cholinesterase inhibitor for Nostoc 78-12A. J. Nat. Prod. 68, 1793–1795. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N., Bourne, P.E., 2000. The protein data bank. Nucleic Acids Res. 28, 9750–9755. Both, F.L., Meneghini, L., Kerber, V.A., Henriques, A.T., Elisabetsky, E., 2005. Psychopharmacological profile of the alkaloid psychollatine as a 5HT2A/C serotonin modulator. J. Nat. Prod. 68, 374–380. Both, F.L., Meneghini, L., Kerber, V.A., Henriques, A.T., Elisabetsky, E., 2006. Role of glutamate and dopamine receptors in the psychopharmacological profile of the indole alkaloid psychollatine. J. Nat. Prod. 69, 342–345. Both, F.L., Kerber, V.A., Henriques, A.T., Elisabetsky, E., 2002. Analgesic properties of umbellatine from Psychotria umbellata. Pharm. Biol. 40, 336–341. Clark, M., Cramer III, R.D., Van Opdenbosch, N., 1989. Validation of the general purpose Tripos 5.2 force field. J. Comput. Chem. 10, 982–1012. de Santos, L.V., Fett-Neto, A.G., Kerber, V.A., Elisabetsky, E., Quirion, J.C., Henriques, A.T., 2001. Indole monoterpene alkaloids from leaves of Psychotria suterella Müll. Arg. (Rubiaceae). Biochem. Syst. Ecol. 29, 1185–1187. Di Giovanni, S., Borloz, A., Urbain, A., Marston, A., Hostettmann, K., Carrupt, P.A., Reist, M., 2008. In vitro screening assays to identify natural or synthetic acetylcholinesterase inhibitors: thin layer chromatography versus microplate methods. Eur. J. Pharm. Sci. 33, 109–119. Ellman, G.L., Courtney, D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Faria, E.O., Kato, L., de Oliveira, C.M.A., Carvalho, B.G., Silva, C.C., Sales, L.S., Schuquel, I.T., Silveira-Lacerda, E.P., Delprete, P.G., 2010. Quaternary b-carboline alkaloids from Psychotria prunifolia (Kunth) Steyerm. Phytochem. Lett. 3, 113–116. Farias, F.M., Passos, C.S., Arbo, M.D., Barros, D.M., Gottfried, C., Steffen, V.M., Henriques, A.T., 2012. Strictosidinic acid, isolated from Psychotria myriantha Mull. Arg. (Rubiaceae), decreases serotonin levels in rat hippocampus. Fitoterapia 83, 1138–1143. Farias, F.M., Passos, C.S., Arbo, M.D., Zuanazzi, J.A.S., Steffen, V.M., Henriques, A.T., 2010. Monoamine levels in rat striatum after acute intraperitoneal injection of strictosidinic acid isolated from Psychotria myriantha Mull. Arg. (Rubiaceae). Phytomedicine 17, 289–291. Fernández-Bachiller, M.I., Pérez, C., Monjas, L., Rademann, J., Rodríguez-Franco, M.I., 2012. New tacrine-4-oxo-4H-chromene hybrids as multifunctional agents for the treatment of Alzheimer’s disease, with cholinergic antioxidant, and bamyloid-reducing properties. J. Med. Chem. 55, 1303–1317. Fowler, J.S., Volkow, N.D., Logan, J., Wang, G.J., MacGregor, R.R., Schlyer, D., Wolf, A.P., Pappas, N., Alexoff, D., Shea, C., Dorflinger, E., Kruchowy, L., Yoo, K., Fazzini, E., Patlak, C., 1994. Slow recovery of human brain MAO B after L-deprenyl (seleginine) withdrawal. Synapse 18, 86–93. Francis, P.T., Palmer, A.M., Snape, M., Wilcock, G.K., 1999. The cholinergic hypothesis of Alzheimer’s disease: a review of progress. J. Neurol. Neurosurg. Psychiatry 66, 137–147. Geissler, T., Brandt, W., Porzel, A., Schlenzig, D., Kehlen, A., Wessjohann, L., Arnold, N., 2010. Acetylcholinesterase inhibitors from the toadstool Cortinarius infractus. Bioorg. Med. Chem. 18, 2173–2177. Geldenhuys, W.J., Youdim, M.B.H., Carroll, R.T., Van der Schyf, C.J., 2011. The emergence of designed multiple ligands for neurodegenerative diseases. Prog. Neurobiol. 94, 347–359. Greenblatt, H.M., Kryger, G., Lewis, T., Silman, I., Sussman, J.L., 1999. Structure of acetylcholinesterase complexed with ()-galanthamine at 2.3 Å resolution. FEBS Lett. 463, 321–326. Greig, N.H., Utsuki, T., Ingram, D.K., Wang, Y., Pepeu, G., Scali, C., Yu, Q.S., Mamczarz, J., Holloway, H.W., Giordano, T., Chen, D.M., Furukawa, K., Sambamurti, K., Brassi, A., Lahiri, D.K., 2005. Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer b-amyloid peptide in rodent. Proc. Natl. Acad. Sci. U.S.A. 102, 17213–17218. Henriques, A.T., Lopes, S.O., Paranhos, J.T., Gregianini, T.S., von Poser, G.L., Fett-Neto, A.G., Schripsema, J., 2004. N, b-D-Glucopyranosyl vincosamide, a light regulated indole alkaloid from the shoots of Psychotria leiocarpa. Phytochemistry 65, 449– 454. Jiang, H., Wang, X., Huang, L., Luo, Z., Su, T., Ding, K., Li, X., 2011. Benzenediol– berberine hybrids: multifunctional agents for Alzheimer’s disease. Bioorg. Med. Chem. 19, 7228–7235. Kerber, V.A., Gregianini, T.S., Paranhos, J.T., Schwambach, J., Farias, F., Fett, J.P., FettNeto, A.G., Zuanazzi, J.A.S., Quirion, J.C., Elisabetsky, E., Henriques, A.T., 2001. Brachycerine, a novel monoterpene indole alkaloid from Psychotria brachyceras. J. Nat. Prod. 64, 677–679.

20

C.S. Passos et al. / Phytochemistry 86 (2013) 8–20

Kerber, V.A., Passos, C.S., Verli, H., Fett-Neto, A.G., Quirion, J.P., Henriques, A.T., 2008. Psychollatine, a glucosidic monoterpene indole alkaloid from Psychotria umbellata. J. Nat. Prod. 71, 697–700. Lane, R.M., Potkin, S.G., Enz, A., 2006. Targeting acetylcholinesterase and butyrylcholinesterase in dementia. Int. J. Neuropsychopharmacol. 9, 101–124. Legoabe, L.J., Petzer, A., Petzer, J.P., 2012. Inhibition of monoamine oxidase by selected C6-substituted chromone derivatives. Eur. J. Med. Chem. 49, 343–353. Levesque, J., Jacquesy, R., Merienne, C., 1983. Un gluco-alcaloide de configuration inhabituelle isole de Pauridiantha lyallii: l’isopauridianthoside. J. Nat. Prod. 46, 619–625. Levesque, J., Pousset, J.L., Cave, A., 1977. Pauridianthoside, a new gluco-alkaloid isolated form Pauridiantha lyallii. Fitoterapia 48, 5–8. Ma, J., Yoshimura, M., Yamashita, E., Nakagawa, A., Ito, A., Tsukihara, T., 2004. Structure of rat monoamine oxidase A and its specific recognitions for substrates and inhibitors. J. Mol. Biol. 338, 103–114. Marnett, L.J., 2002. Recent developments in cyclooxygenase inhibition. Prostaglandins Other Lipid Mediat. 68–69, 153–164. Masson, P., Legrand, P., Bartels, C.F., Froment, M.T., Schopfer, L.M., Lockridge, O., 1997. Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase. Biochemistry 36, 2266–2277. Nachon, F., Ehret-Sabatier, L., Loew, D., Colas, C., van Dorsselaer, A., Goeldner, M., 2006. Trp82 and Tyr332 are involved in two quaternary ammonium binding domains of human butyrylcholinesterase as revealed by photoaffinity labeling with [3H]DDF. Biochemistry 37, 10507–10513. Nicolet, Y., Lockridge, O., Masson, P., Fontecilla-Camps, J.C., Nachon, F., 2003. Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products. J. Biol. Chem. 278, 41141–41147. Novaroli, L., Daina, A., Bertolini, F., Di Giovanni, S., Bravo, J., Reist, M., Carrupt, P.A., 2005. Identification of novel multifunctional compounds for the treatment of some aging related neurodegenerative diseases. Chimia 59, 315–320. Novaroli, L., Daina, A., Favre, E., Bravo, J., Carotti, A., Leonetti, F., Catto, M., Carrupt, P.A., Reist, M., 2006. Impact of species-dependent differences on screening, design and development of MAO-B inhibitors. J. Med. Chem. 49, 6264–6272. Nurisso, A., Bravo, J., Carrupt, P.A., Daina, A., 2012. Molecular docking using the molecular lipophilicity potential as hydrophobic descriptor: impact on GOLD docking performance. J. Chem. Inf. Model. 52, 1319–1327. Passos, C.S., Soldi, T.C., Abib, R.T., Apel, M.A., Simões-Pires, C., Marcourt, L., Gottfried, C., Henriques, A.T., 2012. Monoamine oxidase inhibition by monoterpene indole alkaloids and fractions obtained from Psychotria suterella and Psychotria

laciniata. J. Enzyme Inhib. Med. Chem.. http://dx.doi.org/10.3109/ 14756366.2012.666536. Paul, J.H.A., Maxwell, A.R., Reynolds, W.F., 2003. Novel bis(monoterpenoid) indole alkaloids from Psychotria bahiensis. J. Nat. Prod. 66, 752–754. Reniers, J., Robert, S., Frederick, R., Masereel, B., Vincent, S., Wouters, J., 2011. Synthesis and evaluation of b-carboline derivatives as potential monoamine oxidase inhibitors. Bioorg. Med. Chem. 19, 134–144. Samoylenko, V., Rahman, M.M., Tekwani, B.L., Tripathi, L.M., Wang, Y.H., Khan, S.I., Khan, I.A., Miller, L.S., Joshi, V.C., Muhammad, I., 2010. Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative constituents for the activities relevant to neurodegenerative disorders and Parkinson’s disease. J. Ethnopharmacol. 127, 357–367. Schott, Y., Decker, M., Rommelspacher, H., Lehmann, J., 2006. 6-Hydroxy- and 6methoxy-b-carbolines as acetyl- and butyrylcholinesterase inhibitors. Bioorg. Med. Chem. Lett. 16, 5840–5843. Simões-Pires, C.A., Farias, F.M., Marston, A., Queiroz, E.F., Chaves, C.G., Henriques, A.T., Hostettmann, K., 2006. Indole monoterpenes with antichemotactic activity from Psychotria myriantha: chemotaxonomic significance. Nat. Prod. Commun. 1, 1101–1106. Solis, P.N., Wright, C.W., Gupta, M.P., Phillipson, J.D., 1993. Alkaloids form Cephaelis dichroa. Phytochemistry 33, 1117–1119. Son, S.Y., Ma, J., Kondou, Y., Yoshimura, M., Yamashita, E., Tsukihara, T., 2008. Structure of human monoamine oxidase A at 2.2-Å resolution: the control of opening the entry for substrates/inhibitors. Proc. Natl. Acad. Sci. U.S.A. 105, 5739–5744. Torres, J.M., Lira, A.F., Silva, D.R., Guzzo, L.M., Sant’Anna, C.M.R., Kümmerle, A.E., Rumjanek, V.M., 2012. Structural insights into cholinesterases inhibition by harmane b-carbolinium derivatives: a kinetics – molecular modeling approach. Phytochemistry 81, 24–30. Vencato, I., da Silva, F.M., de Oliveira, C.M.A., Kato, L., Tanaka, C.M.A., da Silva, C.C., Sabino, J.R., 2006. Vallesiachotamine. Acta Crystallogr. E 62, 0429–0431. Verdonk, M.L., Cole, J.C., Hartshorn, M.J., Murray, C.W., Taylor, R.D., 2003. Improved protein–ligand docking using GOLD. Proteins Struct. Funct. Genet. 52, 609–623. Youdim, M.B.H., Bakhle, Y.S., 2006. Monoamine oxidase: isoforms and inhibitors in Parkinson’s disease. Br. J. Pharmacol. 147, S287–S296. Youdim, M.B.H., Buccafusco, J., 2005. Multi-functional drugs for various CNS targets in the treatment of neurodegenerative diseases. Trends Pharmacol. Sci. 26, 27– 35. Youdim, M.B.H., Edmondson, D., Tipton, K., 2006. The therapeutic potential of monoamine oxidase inhibitors. Nat. Rev. Neurosci. 7, 295–309.