Isoquinoline alkaloids as prolyl oligopeptidase inhibitors Lucie Cahl´ıkov´a, Lucie Hulov´a, Martina Hrabinov´a, Jakub Chlebek, ˇ Anna Hoˇsˇta´ lkov´a, Mark´eta Adamcov´a, Marcela Safratov´ a, Daniel Jun, Lubom´ır Opletal, Miroslav Loˇca´ rek, Kateˇrina Mac´akov´a PII: DOI: Reference:
S0367-326X(15)00094-5 doi: 10.1016/j.fitote.2015.04.004 FITOTE 3161
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
20 February 2015 2 April 2015 3 April 2015
Please cite this article as: Cahl´ıkov´a Lucie, Hulov´a Lucie, Hrabinov´ a Martina, Chlebek ˇ Jakub, Hoˇsˇt´alkov´a Anna, Adamcov´a Mark´eta, Safratov´ a Marcela, Jun Daniel, Opletal Lubom´ır, Loˇc´arek Miroslav, Mac´akov´ a Kateˇrina, Isoquinoline alkaloids as prolyl oligopeptidase inhibitors, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.04.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Isoquinoline alkaloids as prolyl oligopeptidase inhibitors Lucie Cahlíkováa*, Lucie Hulováa, Martina Hrabinováb, Jakub Chlebeka, Anna Hošťálkováa, Markéta Adamcováa, Marcela Šafratováa, Daniel Junb, Lubomír Opletala, Miroslav Ločáreka,
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a
T
Kateřina Macákováa
ADINACO Research Group, Department of Pharmaceutical Botany and Ecology, Faculty of
b
SC
Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defence,
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Třebešská 1575, 500 05 Hradec Králové, Czech Republic
*Corresponding author:
Lucie Cahlíková; Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy,
ED
Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
PT
Tel.: + 420 495 067 311; fax: + 420 495 067 162
AC
CE
E-mail address: [email protected] (L. Cahlíková)
ACCEPTED MANUSCRIPT Abstract Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyses proline-containing peptides at the carboxy terminus of proline residues. It has been associated with
T
schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore
RI P
may have important clinical implications. Thirty-one isoquinoline alkaloids of various structural types, previously isolated in our laboratory, were screened for their ability to inhibit prolyl oligopeptidase. Promising results have been showed by alkaloids californidine (IC50 =
SC
55.6 ± 3.5 µM), dihydrosanquinarine (IC50 = 99.1 ± 7.6 µM), corypalmine (IC50 = 128.0 ±
MA NU
10.5 µM) and N-methyllaurotetanine (IC50 = 135.0 ± 11.7 µM).
Keywords: Isoquinoline alkaloids, prolyl oligopeptidase, californidine, dihydrosanquinarine
Chemical compounds studied in this article
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Argemonine (CID: 442168); Berberine (PubChem CID: 2353); Californidine (PubChem CID: 45266443); Canadine (PubChem CID: 443422); Corypalmine (PubChem CID:
PT
185605); Corynoline (PubChem CID: 177014); Dihydrosanquinarine (PubChem CID:
AC
CE
124069); N-Methyllaurotetanine (PubChem CID: 16573).
ACCEPTED MANUSCRIPT 1. Introduction Prolyl oligopeptidase (POP) is a cytosolic serine peptidase that cleaves peptide bonds at the carboxyl end of proline, and is widely distributed in the organs of the body, including the
T
brain [1,2]. Previous studies indicate that POP activity is involved in key physiological
RI P
functions, such as learning and memory, cell division and differentation, and signaling transduction, as well as in some psychiatric disorders [3]. In recent years, POP has gained
SC
importance as a target for the treatment of schizophrenia (SZ), bipolar affective disorder (BD) and cognitive disturbances, such as those present in Alzheimer´s disease (AD), mainly due to
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its involvement in the metabolism of inositol-1,4,5-P3 (IP3), which is a key molecule in the transduction cascade of neuropeptide signaling. Neuropeptides induce and increase IP3 levels by binding to their receptor in the membrane of the endoplasmatic reticulum and inducing the release of Ca2+, which is believed to play a crucial role in learning and memory [4]. POP has also been involved in the processing of neuropeptide precursors [5]. Moreover, neuroprotective and cognition-enhancing effects of POP inhibitors in experimental animals
ED
have been reported [3,6].
The use of natural products for medical purposes is gaining international popularity.
PT
Medicinal plants are an attractive source for drug research and development as they produce chemically-varying molecules with a wide range of biological activities. Some natural
CE
products are known POP inhibitors in the micromolar range such as the flavonoids baicalin [7] and oroxylin [8], the alkaloid berberine [9], and 6-(8´Z-pentadecenyl)salicylic acid [10].
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Alkaloids are without doubt the most potent therapeutic compounds of natural origin and often have significant pharmacological effects on humans and animals [11]. Isoquinoline alkaloids belong to a large group of nitrogenous compounds with over 400 members. Their biological effects include antimalarial, anti-HIV, antitumor, and antimicrobial activities. They are divided into several different classes including aporphines, protopines, protoberberines, phthalideisoquinolines, spirobenzylisoquinolines
benzophenantheidines, [12].
Some
of
benzylisoquinolines, these
compounds
morphinans possess
and
promising
biological/pharmacological properties for the treatment of important diseases including cancer, AD, and microbial infections. Aporphines, benzylisoquinolines and protoberberines showed a higher cytotoxicity than other structural types of isoquinoline alkaloids. A typical example is berberine with a protoberberine skeleton that showed a remarkable cytotoxicity on a wide range of cancer cell lines (e.g. A549, SK-OV-3, SK-MEL-2 and others) [12]. As mentioned above, it has also been reported to be a potent inhibitor of POP [9], but other
ACCEPTED MANUSCRIPT isoquinoline alkaloids have not been tested until now. On the other hand, a wider range of Amaryllidaceae alkaloids has been tested for POP inhibition activity so far. Important results have been shown by 9-O-demethylgalanthin (IC50 = 150 ± 20 µM) [13] and by further
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Brackenhurst and N. jonquila var. henriquesii, respectively [14].
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Amaryllidaceae alkaloids narwedine and incartine isolated from Narcissus poeticus cv.
The aim of this study was to elucidate the possible POP inhibitory effect of diverse types of
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isoquinoline alkaloids isolated in our laboratory from plant sources.
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2. Results and discussion
In the current study, 31 isoquinoline alkaloids of six structural types: aporphine, benzophenanthridine, benzylisoquinoline, pavinane, protoberberine and protopine, previously isolated in our laboratory, were screened for their ability to inhibit prolyl oligopeptidase (Fig. 1).
Five of the tested compounds showed inhibition activity either stronger than or comparable
ED
with berberine, which has been used as one of the standards (Table 1). The most interesting inhibition activity has been demonstrated by the quaternary pavinane alkaloid californidine
PT
with an IC50 value of 55.6 ± 3.5 μM, isolated from Eschscholtzia californica. It seems that quaternary nitrogen could play an important role in the POP inhibition activity. It is known
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that compounds with quaternary nitrogen are also active AChE inhibitors, such as sanquinarine, chelerythrine [15], berberine [16] and californidine [17]. The quaternary
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nitrogen plays an important role in binding with the enzyme [18]. However, it is reported that these compounds might have problems with crossing the blood-brain barrier (BBB) [19], on the other hand, there is a general view that permeability of the BBB is increased in AD and also that quaternary compounds could cross the BBB [20]. For example, the protoberberine quaternary alkaloid berberine passes through the BBB in rats and is quickly distributed to the thalamus [21]. AD is the most predominant cause of dementia in the elderly, affecting more than 20 million people worldwide and it is estimated that this figure will increase to 114 million by 2040 [22]. Currently, acetylcholinesterase inhibition is the most used therapeutic treatment for the symptoms of AD [20]. In recent studies, some POP inhibitors have been found to be efficacious antidementia drugs [23]. Thus, POP inhibition can represent an important supporting approach in AD treatment, and, therefore, the search for new compounds influencing more therapeutic targets connected with AD is required.
ACCEPTED MANUSCRIPT Recent evidence also pointed to the involvement of POP in cancer and tumor growth. The pattern of POP activity was studied in a large series of human neoplastic tissues. The increased POP activity in Clear Cell Renal Cell Carcinoma (CCRCC), Urothelial Carcinoma
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of the Renal Pelvis (UCRP), Head and Neck Squamous Cell Carcinoma (HNSCC) and
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colorectal adenomatous polyp suggest that this enzyme could be involved also in these malignancies [24].
Further important POP inhibitions have also been shown by the benzophenanthridine alkaloid
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dihydrosanquinarine (IC50 value 99.1 ± 7.6 μM; Fig. 1) isolated from Macleaya cordata [25], two protoberberines corypalmine (IC50 value 128.0 ± 10.5 μM; Fig. 1) and canadine (IC50
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value 152.0 ± 12.5 μM) from Corydalis cava [26], and finally by the aporphine type alkaloid N-methyllaurotetanine (IC50 value 135.0 ± 11.7 μM) from Eschscholtzia californica [17]. All these activities are comparable with those reported and our POP inhibition activity of berberine (IC50 = 145 μM; Table 1) [9].
Table 1. POP inhibition activity of isoquinoline alkaloids
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337.2 ± 23.4 687.0 ± 37.2 55.6 ± 3.5 135.0 ± 11.7 128.0 ± 10.5 152.0 ± 12.5 289.1 ± 47.0 99.1 ± 7.6 392.5 ± 25.0 142.0 ± 21.5 2.75 x 10-3
Results are the means ± standard deviations of three independent replications
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a
Structural type
pavinane pavinane pavinane aporphine protoberberine protoberberine benzophenanthridine benzophenanthridine benzylisoquinoline standard standard
PT
Argemonine Platycerine Californidine iodide N-Methyllaurotetanine Corypalmine Canadine Corynoline Dihydrosanquinarine Reticuline Berberine Z-prolyl-prolinal
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Compound
POP IC50a (µM)
In conclusion, although the mechanism of the POP inhibition of the tested compounds has not been elucidated, the findings of this study indicate the potential of isoquinoline alkaloids as POP inhibitors. Although these inhibitors are not excellent concerning the inhibitory potency compared to known inhibitors (e.g. Z-prolyl-prolinal), these compounds are of interest since they can be used as leads in the development of new potent therapeutic drugs to treat neuropsychiatric disorders. The mechanism of the POP inhibition and prepared semi-synthetic derivatives of the reported active compounds will be studied in our future experiments.
3. Material and methods 3.1. Isoquinoline alkaloids
ACCEPTED MANUSCRIPT (−)-Californidine
iodide,
(−)-eschscholtzine,
(−)-caryachine,
(−)-argemonine,
(−)-O-
methylcaryachine, (-)-O-methylneocaryachine, (+)-reticuline, (+)-salutaridine and (+)-Nmethyllaurotetanine were isolated from aerial parts and roots of Eschscholtzia californica
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[17]. Protopine, allocryptopine, (+)-chelidonine, (+)-homochelidonine and (−)-stylopine were
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isolated from aerial parts and roots of Chelidonium majus [15]. Cryptopine, (+)-bulbocapnine, (+)-canadine, (+)-canadaline, (-)-corycavamine, (±)-corycavidine, (+)-corynoline, (+)corydine,
(−)-corypalmine,
(+)-isocorydine,
(−)-isocorypalmine,
(-)-sinoacutine,
(+)-
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tetrahydropalmatine, (−)-scoulerine and (+)-corydaline were isolated from tubers of Corydalis cava [26]. Dihydrosanguinarine was isolated from a commercial extract of Macleaya cordata
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[25]. (-)-Platycerine was isolated from aerial parts of Argemone platyceras. (+)Thaliktricavine was additionaly isolated from aerial parts of Corydalis cava.
3.2. Isolation of (-)-platycerine
Five kg of aerial parts and roots of Argemone platyceras (Papaveraceae) were extracted with
ED
EtOH (96% (v/v), 2x) at room temperature for 48 h; the combined macerate was filtered and evaporated to dryness under reduced pressure. The crude extract (150 g) was acidified to pH 2
PT
with 2% HCl, defatted with Et2O (4 x 500 mL), and alkalized to pH 9-10 with 10 % Na2CO3. Then, the alkaloids were extracted with CHCl3 (4 x 500 mL). The extract was evaporated
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under vacuum. The extract (10,35 g) was fractionated by CC (Al2O3, ACROS, petrol, gradually enriched with CHCl3). Fractions of 500 mL were collected and monitored by TLC; 9 main fractions were obtained (1-9). Fraction 6 (3.24 g) was treated with 2% HCl, then
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alkalized (Na2CO3) and extracted with Et2O. The residue was crystallized from Et2O. White crystals (500 mg) were obtained and identified as platycerine [27].
3.3. Isolation of (+)-thalictricavine The main procedure is described in reference 27. Preparative TLC (c-hexane: Et2NH, 97:3, 3 times) of fraction A (150 mg) gave (+)-canadine (10 mg) and (+)-corydaline (30 mg). Repeated crystallization of the rest of fraction A from EtOH, followed by preparative TLC (SiO2 F254, Merck, c-hexane: Et2NH 95:5, once) led to the isolation of (+)-thalictricavine (25 mg) [28].
3.4. Prolyl oligopeptidase assay POP was dissolved in phosphate buffered saline (PBS: 0.01 M Na/K phosphate buffer, pH 7.4 containing 137 mM NaCl and 2.7 mM KCl); the specific activity of the enzyme was 0.2
ACCEPTED MANUSCRIPT U/mL. The assays were performed in standard polystyrene 96-well microplates with a flat and clear bottom. Stock solutions of test compounds were prepared in DMSO (10 mM). Dilutions (10-3 M – 10-7 M) were prepared from the stock solution with deionized water; the control was
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performed with the same DMSO concentration. POP substrate, Z-Gly-Pro-p-nitroanilide
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(ZGP-AMC), was dissolved in 40% 1,4-dioxane (10 mM). For each reaction, PBS (170 µL), test compound (5 µL) and POP solution (5 µL) were incubated for 5 min at 37 °C. Then, substrate (20 µL) was added and the microplate was incubated for 30 min at 37 °C. The
SC
formation of p-nitroaniline, directly proportional to the POP activity, was measured spectrophotometrically at 405 nm using a microplate ELISA reader (EL800, Bio-Tek
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Instruments, Inc., Winooski, VT, USA). Inhibition potency of the test compounds was expressed as IC50 value (concentration of inhibitor, which causes 50% POP inhibition). 3.5. Statistical analysis
Calculations were performed using Microsoft Excel software (Redmont, WA, USA) and
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GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego, CA, USA.
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Acknowledgments
This project was supported by grants SVV UK 260 063, Charles University grant Nr.
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17/2012/UNCE and The Grant Agency of the Czech Republic Nr. P303/11/1907. The publication was also co-financed by the European Social Fund and by the state budget of the
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Czech Republic, project No. CZ.1.07/2.3.00/20.0235, the title of the project: TEAB.
References [1] [2] [3] [4]
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García-Horsman JA, Männistö PT, Venäläinen JI. On the role of prolyl oligopeptidase in health and disease. Neuropeptides 2007;41:1-24. Polgár L. The prolyl oligopeptidase family. Cell Mol Life Sci 2002;59:349-62. Toide K, Iwamoto Y, Fujiwara T, Abe H. JTP-4819: a novel prolyl endopeptidase inhibitor with potential as a cognitive enhancer. J Pharmacol Exp Ther 1995;274:1370-78. Komatsu Y. GABAB receptors, monoamine receptors, and postsynaptic inositol triphosphate-induced Ca2+ release are involved in the induction of long-term potentiation at visual cortical inhibitory synapses. J Neurosci 1996;16:6342-52. Wilk S. Prolyl endopeptidase. Life Sci 1983;33:2149-57. Shishido Y, Furushiro M, Tanabe S, Shibata S, Hashimoto S, Yokokura T. Effects of prolyl endopeptidase inhibitors and neuropeptides on delayed neuronal death in rats. Eur J Pharmacol 1999;372:135-42. Tarragó T, Kichik N, Claasen B, Prades R, Teixidó M, Giralt E. Baicalin, a prodrug able to reach the CNS, is a prolyl oligopeptidase inhibitor. Bioorg Med Chem 2008;16: 7516-24.
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Marques MR, Stuker C, Kichik N, Tarragó T, Giralt E, Morel AF, et al. Flavonoids with prolyl oligopeptidase inhibitory activity isolated from Scutellaria racemosa Pers. Fitoterapia 2010;81: 552-6. Tarragó T, Kichik N, Seguí J, Giralt E. The natural product berberine is a human prolyl oligopeptidase inhibitor. ChemMedChem 2007;2:354-9. Lee JH, Lee SY, Lee KS, Jang HJ, Lee KH, Hahn TR, et al. Prolyl endopeptidase inhibitors from the leaves of Gingko biloba. Planta Med 2004;70:1228-30. Rahman A, Choudhary MI. Chemistry and biology of steroidal alkaloids. In The Alkaloids: Chemistry and Biology, ed. G.A. Cordell, Academic Press, San Diego, 1998;50:61-108. Iranshahy M, Quinn RJ, Iranshahi M. Biological active isoquinoline alkaloids with drug-like properties from the genus Corydalis. RSC Adv 2014;4:15900-13. Šafratová M, Novák Z, Kulhánková A, Kuneš J, Hrabinová M, Jun D, et al. Revised NMR data for 9-Odemethylgalanthine: an alkaloid from Zephyranthes robusta (Amaryllidaceae) and its biological activity.Nat Prod Commun 2014;9:787-8. Havlasová J, Šafratová M, Siatka T, Štěpánková Š, Ločárek M, Opletal L, et al. Chemical composition of bioactive alkaloid extracts from some Narcissus species and varieties and their biological activity. Nat Prod Commun 2014;9:1151-5. Cahlíková L, Opletal L, Kurfurst M, Macáková K, Kulhánková A, Hošťálková A. Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Chelidonium majus (Papaveraceae). Nat Prod Commun 2010; 5:1751-54. Huang QQ, Bi JL, Sun QY, Yang FM, Wang YH, Tang GH, et al. Bioactive isoquinoline alkaloids from Corydalis saxicola. Planta Med 2012;78:65-70. Cahlíková L, Macáková K, Kuneš J, Kurfurst M, Opletal L, Cvačka J, et al. Acetylcholinesterase and butyrylcholinesterase nhibitory ompounds from Eschscholzia californica (Papaveraceae).Nat Prod Commun 2010;5:1035-38. Whiteley CG, Daya S. Protein ligand interactions. Part 5:isoquinoline alkaloids as inhibitors of acetylcholinesterase from Electrophorus electricus. J Enzym Inhibit Med Chem 1995;9:285-294. Khorana N, Markmee S, Ingkaninan K, Ruchirawat S, Kitbunnadaj R, Pullagurla MR. Med Chem Res 2009;18:231-41. Zlokovic BV. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 2008;57:178-201. Wang X, Xing D, Wang W, Su H, Tao J, Du L. Pharmacokinetics of berberine in rat thalamus after intravenous administration of Coptidis rhizoma extract. Am J Chin Med 2005;33:935-43. Wimo A, Wimblad B, Stoffler A, Wirth Y, Mobius HJ. The magnitude of dementia occurrence in the world. Alzheimer Dis Assoc Dis 2003;17:63-7. Orhan IE. Current concepts on selected plant secondary metabolites with promising inhibitory effects against enzymes linked to Alzheimer's disease. Curr Med Chem. 2012;19:2252-61. Larrinaga G, Perez I, Blanco L, López JI, Andrés L, Etxezarraga C, et al. Increased prolyl endopeptidase activity in human neoplasia. Regul Peptides 2010;163:102-6. Schramm A, Saxena P, Chlebek J, Cahlíková L, Baburin I, Hering S, et al. Natural products as potential human ether-a-go-go-related gene channel inhibitors – screening of plant derived alkaloids. Planta Med 2014;80:740-6. Chlebek J, Macáková K, Cahlíková L, Kurfurst M, Kuneš J, Opletal L. Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Corydalis cava Schweigger. & Korte (Fumariaceae). Nat Prod Commun 2011;6:607-10. Shamma M, Moniot JL. Novel isoquinoline alkaloid group. Aporphine-pavine dimers. J Am Chem Soc 1974;96:3338-40. Takao N, Iwasa K, Kamigauchi M, Sugiura M. Studies on the alkaloids of papaveraceous plants. XXIX. Conformational analysis of tetrahydroprotoberberines by carbon-13 magnetic resonance spectroscopy. Chem Pharm Bull 1977;25:1426-1435.
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[8]
ACCEPTED MANUSCRIPT Figure 1. Structures of tested isoquinoline alkaloids.
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Figure 2. IC50 calculation curves of californidine iodide, dihydrosanquinarine and corypalmine using ZGP-AMC as substrate.
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Figure 1
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Figure 2
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Graphical Abstract
S0367-326X(15)00094-5 doi: 10.1016/j.fitote.2015.04.004 FITOTE 3161
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
20 February 2015 2 April 2015 3 April 2015
Please cite this article as: Cahl´ıkov´a Lucie, Hulov´a Lucie, Hrabinov´ a Martina, Chlebek ˇ Jakub, Hoˇsˇt´alkov´a Anna, Adamcov´a Mark´eta, Safratov´ a Marcela, Jun Daniel, Opletal Lubom´ır, Loˇc´arek Miroslav, Mac´akov´ a Kateˇrina, Isoquinoline alkaloids as prolyl oligopeptidase inhibitors, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.04.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Isoquinoline alkaloids as prolyl oligopeptidase inhibitors Lucie Cahlíkováa*, Lucie Hulováa, Martina Hrabinováb, Jakub Chlebeka, Anna Hošťálkováa, Markéta Adamcováa, Marcela Šafratováa, Daniel Junb, Lubomír Opletala, Miroslav Ločáreka,
RI P
a
T
Kateřina Macákováa
ADINACO Research Group, Department of Pharmaceutical Botany and Ecology, Faculty of
b
SC
Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defence,
MA NU
Třebešská 1575, 500 05 Hradec Králové, Czech Republic
*Corresponding author:
Lucie Cahlíková; Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy,
ED
Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
PT
Tel.: + 420 495 067 311; fax: + 420 495 067 162
AC
CE
E-mail address: [email protected] (L. Cahlíková)
ACCEPTED MANUSCRIPT Abstract Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyses proline-containing peptides at the carboxy terminus of proline residues. It has been associated with
T
schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore
RI P
may have important clinical implications. Thirty-one isoquinoline alkaloids of various structural types, previously isolated in our laboratory, were screened for their ability to inhibit prolyl oligopeptidase. Promising results have been showed by alkaloids californidine (IC50 =
SC
55.6 ± 3.5 µM), dihydrosanquinarine (IC50 = 99.1 ± 7.6 µM), corypalmine (IC50 = 128.0 ±
MA NU
10.5 µM) and N-methyllaurotetanine (IC50 = 135.0 ± 11.7 µM).
Keywords: Isoquinoline alkaloids, prolyl oligopeptidase, californidine, dihydrosanquinarine
Chemical compounds studied in this article
ED
Argemonine (CID: 442168); Berberine (PubChem CID: 2353); Californidine (PubChem CID: 45266443); Canadine (PubChem CID: 443422); Corypalmine (PubChem CID:
PT
185605); Corynoline (PubChem CID: 177014); Dihydrosanquinarine (PubChem CID:
AC
CE
124069); N-Methyllaurotetanine (PubChem CID: 16573).
ACCEPTED MANUSCRIPT 1. Introduction Prolyl oligopeptidase (POP) is a cytosolic serine peptidase that cleaves peptide bonds at the carboxyl end of proline, and is widely distributed in the organs of the body, including the
T
brain [1,2]. Previous studies indicate that POP activity is involved in key physiological
RI P
functions, such as learning and memory, cell division and differentation, and signaling transduction, as well as in some psychiatric disorders [3]. In recent years, POP has gained
SC
importance as a target for the treatment of schizophrenia (SZ), bipolar affective disorder (BD) and cognitive disturbances, such as those present in Alzheimer´s disease (AD), mainly due to
MA NU
its involvement in the metabolism of inositol-1,4,5-P3 (IP3), which is a key molecule in the transduction cascade of neuropeptide signaling. Neuropeptides induce and increase IP3 levels by binding to their receptor in the membrane of the endoplasmatic reticulum and inducing the release of Ca2+, which is believed to play a crucial role in learning and memory [4]. POP has also been involved in the processing of neuropeptide precursors [5]. Moreover, neuroprotective and cognition-enhancing effects of POP inhibitors in experimental animals
ED
have been reported [3,6].
The use of natural products for medical purposes is gaining international popularity.
PT
Medicinal plants are an attractive source for drug research and development as they produce chemically-varying molecules with a wide range of biological activities. Some natural
CE
products are known POP inhibitors in the micromolar range such as the flavonoids baicalin [7] and oroxylin [8], the alkaloid berberine [9], and 6-(8´Z-pentadecenyl)salicylic acid [10].
AC
Alkaloids are without doubt the most potent therapeutic compounds of natural origin and often have significant pharmacological effects on humans and animals [11]. Isoquinoline alkaloids belong to a large group of nitrogenous compounds with over 400 members. Their biological effects include antimalarial, anti-HIV, antitumor, and antimicrobial activities. They are divided into several different classes including aporphines, protopines, protoberberines, phthalideisoquinolines, spirobenzylisoquinolines
benzophenantheidines, [12].
Some
of
benzylisoquinolines, these
compounds
morphinans possess
and
promising
biological/pharmacological properties for the treatment of important diseases including cancer, AD, and microbial infections. Aporphines, benzylisoquinolines and protoberberines showed a higher cytotoxicity than other structural types of isoquinoline alkaloids. A typical example is berberine with a protoberberine skeleton that showed a remarkable cytotoxicity on a wide range of cancer cell lines (e.g. A549, SK-OV-3, SK-MEL-2 and others) [12]. As mentioned above, it has also been reported to be a potent inhibitor of POP [9], but other
ACCEPTED MANUSCRIPT isoquinoline alkaloids have not been tested until now. On the other hand, a wider range of Amaryllidaceae alkaloids has been tested for POP inhibition activity so far. Important results have been shown by 9-O-demethylgalanthin (IC50 = 150 ± 20 µM) [13] and by further
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Brackenhurst and N. jonquila var. henriquesii, respectively [14].
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Amaryllidaceae alkaloids narwedine and incartine isolated from Narcissus poeticus cv.
The aim of this study was to elucidate the possible POP inhibitory effect of diverse types of
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isoquinoline alkaloids isolated in our laboratory from plant sources.
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2. Results and discussion
In the current study, 31 isoquinoline alkaloids of six structural types: aporphine, benzophenanthridine, benzylisoquinoline, pavinane, protoberberine and protopine, previously isolated in our laboratory, were screened for their ability to inhibit prolyl oligopeptidase (Fig. 1).
Five of the tested compounds showed inhibition activity either stronger than or comparable
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with berberine, which has been used as one of the standards (Table 1). The most interesting inhibition activity has been demonstrated by the quaternary pavinane alkaloid californidine
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with an IC50 value of 55.6 ± 3.5 μM, isolated from Eschscholtzia californica. It seems that quaternary nitrogen could play an important role in the POP inhibition activity. It is known
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that compounds with quaternary nitrogen are also active AChE inhibitors, such as sanquinarine, chelerythrine [15], berberine [16] and californidine [17]. The quaternary
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nitrogen plays an important role in binding with the enzyme [18]. However, it is reported that these compounds might have problems with crossing the blood-brain barrier (BBB) [19], on the other hand, there is a general view that permeability of the BBB is increased in AD and also that quaternary compounds could cross the BBB [20]. For example, the protoberberine quaternary alkaloid berberine passes through the BBB in rats and is quickly distributed to the thalamus [21]. AD is the most predominant cause of dementia in the elderly, affecting more than 20 million people worldwide and it is estimated that this figure will increase to 114 million by 2040 [22]. Currently, acetylcholinesterase inhibition is the most used therapeutic treatment for the symptoms of AD [20]. In recent studies, some POP inhibitors have been found to be efficacious antidementia drugs [23]. Thus, POP inhibition can represent an important supporting approach in AD treatment, and, therefore, the search for new compounds influencing more therapeutic targets connected with AD is required.
ACCEPTED MANUSCRIPT Recent evidence also pointed to the involvement of POP in cancer and tumor growth. The pattern of POP activity was studied in a large series of human neoplastic tissues. The increased POP activity in Clear Cell Renal Cell Carcinoma (CCRCC), Urothelial Carcinoma
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of the Renal Pelvis (UCRP), Head and Neck Squamous Cell Carcinoma (HNSCC) and
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colorectal adenomatous polyp suggest that this enzyme could be involved also in these malignancies [24].
Further important POP inhibitions have also been shown by the benzophenanthridine alkaloid
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dihydrosanquinarine (IC50 value 99.1 ± 7.6 μM; Fig. 1) isolated from Macleaya cordata [25], two protoberberines corypalmine (IC50 value 128.0 ± 10.5 μM; Fig. 1) and canadine (IC50
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value 152.0 ± 12.5 μM) from Corydalis cava [26], and finally by the aporphine type alkaloid N-methyllaurotetanine (IC50 value 135.0 ± 11.7 μM) from Eschscholtzia californica [17]. All these activities are comparable with those reported and our POP inhibition activity of berberine (IC50 = 145 μM; Table 1) [9].
Table 1. POP inhibition activity of isoquinoline alkaloids
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337.2 ± 23.4 687.0 ± 37.2 55.6 ± 3.5 135.0 ± 11.7 128.0 ± 10.5 152.0 ± 12.5 289.1 ± 47.0 99.1 ± 7.6 392.5 ± 25.0 142.0 ± 21.5 2.75 x 10-3
Results are the means ± standard deviations of three independent replications
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a
Structural type
pavinane pavinane pavinane aporphine protoberberine protoberberine benzophenanthridine benzophenanthridine benzylisoquinoline standard standard
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Argemonine Platycerine Californidine iodide N-Methyllaurotetanine Corypalmine Canadine Corynoline Dihydrosanquinarine Reticuline Berberine Z-prolyl-prolinal
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Compound
POP IC50a (µM)
In conclusion, although the mechanism of the POP inhibition of the tested compounds has not been elucidated, the findings of this study indicate the potential of isoquinoline alkaloids as POP inhibitors. Although these inhibitors are not excellent concerning the inhibitory potency compared to known inhibitors (e.g. Z-prolyl-prolinal), these compounds are of interest since they can be used as leads in the development of new potent therapeutic drugs to treat neuropsychiatric disorders. The mechanism of the POP inhibition and prepared semi-synthetic derivatives of the reported active compounds will be studied in our future experiments.
3. Material and methods 3.1. Isoquinoline alkaloids
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iodide,
(−)-eschscholtzine,
(−)-caryachine,
(−)-argemonine,
(−)-O-
methylcaryachine, (-)-O-methylneocaryachine, (+)-reticuline, (+)-salutaridine and (+)-Nmethyllaurotetanine were isolated from aerial parts and roots of Eschscholtzia californica
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[17]. Protopine, allocryptopine, (+)-chelidonine, (+)-homochelidonine and (−)-stylopine were
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isolated from aerial parts and roots of Chelidonium majus [15]. Cryptopine, (+)-bulbocapnine, (+)-canadine, (+)-canadaline, (-)-corycavamine, (±)-corycavidine, (+)-corynoline, (+)corydine,
(−)-corypalmine,
(+)-isocorydine,
(−)-isocorypalmine,
(-)-sinoacutine,
(+)-
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tetrahydropalmatine, (−)-scoulerine and (+)-corydaline were isolated from tubers of Corydalis cava [26]. Dihydrosanguinarine was isolated from a commercial extract of Macleaya cordata
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[25]. (-)-Platycerine was isolated from aerial parts of Argemone platyceras. (+)Thaliktricavine was additionaly isolated from aerial parts of Corydalis cava.
3.2. Isolation of (-)-platycerine
Five kg of aerial parts and roots of Argemone platyceras (Papaveraceae) were extracted with
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EtOH (96% (v/v), 2x) at room temperature for 48 h; the combined macerate was filtered and evaporated to dryness under reduced pressure. The crude extract (150 g) was acidified to pH 2
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with 2% HCl, defatted with Et2O (4 x 500 mL), and alkalized to pH 9-10 with 10 % Na2CO3. Then, the alkaloids were extracted with CHCl3 (4 x 500 mL). The extract was evaporated
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under vacuum. The extract (10,35 g) was fractionated by CC (Al2O3, ACROS, petrol, gradually enriched with CHCl3). Fractions of 500 mL were collected and monitored by TLC; 9 main fractions were obtained (1-9). Fraction 6 (3.24 g) was treated with 2% HCl, then
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alkalized (Na2CO3) and extracted with Et2O. The residue was crystallized from Et2O. White crystals (500 mg) were obtained and identified as platycerine [27].
3.3. Isolation of (+)-thalictricavine The main procedure is described in reference 27. Preparative TLC (c-hexane: Et2NH, 97:3, 3 times) of fraction A (150 mg) gave (+)-canadine (10 mg) and (+)-corydaline (30 mg). Repeated crystallization of the rest of fraction A from EtOH, followed by preparative TLC (SiO2 F254, Merck, c-hexane: Et2NH 95:5, once) led to the isolation of (+)-thalictricavine (25 mg) [28].
3.4. Prolyl oligopeptidase assay POP was dissolved in phosphate buffered saline (PBS: 0.01 M Na/K phosphate buffer, pH 7.4 containing 137 mM NaCl and 2.7 mM KCl); the specific activity of the enzyme was 0.2
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performed with the same DMSO concentration. POP substrate, Z-Gly-Pro-p-nitroanilide
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(ZGP-AMC), was dissolved in 40% 1,4-dioxane (10 mM). For each reaction, PBS (170 µL), test compound (5 µL) and POP solution (5 µL) were incubated for 5 min at 37 °C. Then, substrate (20 µL) was added and the microplate was incubated for 30 min at 37 °C. The
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formation of p-nitroaniline, directly proportional to the POP activity, was measured spectrophotometrically at 405 nm using a microplate ELISA reader (EL800, Bio-Tek
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Instruments, Inc., Winooski, VT, USA). Inhibition potency of the test compounds was expressed as IC50 value (concentration of inhibitor, which causes 50% POP inhibition). 3.5. Statistical analysis
Calculations were performed using Microsoft Excel software (Redmont, WA, USA) and
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GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego, CA, USA.
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Acknowledgments
This project was supported by grants SVV UK 260 063, Charles University grant Nr.
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17/2012/UNCE and The Grant Agency of the Czech Republic Nr. P303/11/1907. The publication was also co-financed by the European Social Fund and by the state budget of the
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Czech Republic, project No. CZ.1.07/2.3.00/20.0235, the title of the project: TEAB.
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ACCEPTED MANUSCRIPT Figure 1. Structures of tested isoquinoline alkaloids.
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Figure 2. IC50 calculation curves of californidine iodide, dihydrosanquinarine and corypalmine using ZGP-AMC as substrate.
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Figure 2
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Graphical Abstract
