Quaternary Benzo[C]Phenanthridine Alkaloids — Biological Activities

QUATERNARY BENZO[C]PHENAI\THRIDINB ALKALOIDS _ BIOLOGICAL

ACTTVITIES

Intracellular Targets of Their Action and Application in Human and Veterinary Medicine

v. ŠnaÁNgKR.VESPALEC*, A.ŠEDo*, J. ULRICHoVÁ, AND J. VICAR Institute of Medical Chemistry,

Palaclql Universiý,

775 I5 Olomouc, Czech Republic, E-mail : vilim@tunw. upol. cz * Inst itute of Analytic al C hemistry, Academy of Sciences of the Czech Republic, Veveří 97, 61 1 42 Brno *Laboratory of Cancer Cell Biolog,t, Institute of Biochemistry and Experimental Oncology,

Charles Universiý, I28 53 Prague, Czech Republic

1.

Introduction

Quarternary benzo[c]phenanthridine alkaloids (QBA) are a small class of compounds commonly isolated from Caprifoliaceae, Fumariaceae, Meliacea, Papaveraceae and Rutaceae plants. QBA belong to the elicitor-inducible secondary metabolites and are considered phytoalexines because of their antifungal and nematocidal activities. Quaternary benzo[c]phenanthÍidine alkaloids whose most studied representatives aÍe sanguinarine (SA), chelerýhrine (CFIE), and fagaronine (FA) (Fig. 1) display a wide spectrum of non-specific biological activities and affect basic molecular targets common to mammalian cells [1]. They are the subject oť sustained practical and research interests because of their pronounced widespread physiological effects [2].

The plants containing SA and CF{E Sanguinaria canaderesls (Papaveraceae, bloodroot, rhizomes contain 4-7oÁ, roots about l.8% alkaloids), Chelidonium majus (Papaveraceae' celandine, roots contain 4.5% alkaloids) and Macleya cordata (Papaveraceae, aerial parts contain about 30Á alkaloids) were utilized in the practice of traditional medicine in North America, Europe and China long before the isolation of the pure alkaloids. SA and CFIE display antimicrobial, anti-inflammatory, adrenolyic, sympatholytic, and local anesthetic effects. The powdered rhizomes and roots of S. canadensis are the active components of the weight gain stimulant for farm animals SANGROVIT@. SANGUINARIA@, the extract from the rhizomes of ,S. canadensis, ffid SANGTIIRITRIN@, QBA fractions fuom M. cordata are used in toothpastes and mouthwashes as antiplaque agents, and the latter is applied as antifungal and anti-inflammatory preparation 245 M.P. Schneider (ed.), Chemical Probes in Biology,245-254. O 2003 Kluwer Academic Publishers. Printed in the Netherlands.

in Russia.

Nevertheless, the

246 molecular basis of SA and CFIE pharmacological activities anďor toxic side effects remain mysterious. In contrast to the beneficial effects of SA/CFIE, the epidemic dropsy syndrome is a well-known toxic effect ascribed to QBA, which is associated with the consumption of edible plant oils contaminated by the oil from Argemone mexicana seeds [3]. The seeds of A. mexicana contun about 0.5yo of QBA [a]. R1

R2

SANGUINARINE

R|+ť=oCHzo, ť+Ra:oCHzo' R5:H

FÍá'^Rtffi,tš^' řl:5i'"-9:ó?'t'-"o'Ť'ilť;t* Figure 1. Structures of quaternary benzo[c]phenanthridine alkaloids

It has been reported that long-term use of oral products containing SANGUINARIA@ appeaÍS to be associated with an increased incidence of leukoplakia of the maxillary uirtiUut. t5l. Furthermore, SA elicited weak positive responses in the Salmonella mutagenicity test after metabolic activation [6] and chelerýhrine induced respirationdeficient mutants in Saccharomyces cereyisieae [7]. However, neither teratogenicity nor increases in preneoplastic or neoplastic lesions have been shown in long-term bioassays with a mixture of SA and CF{E in the rat [6].

FA possesses antileukemic activiry inhibits HIV-1 and HlV-2-reverse transcriptases and DNA topoisomerases I and II [8]. FA is considered a potential antitumor drug. In contrast to SA/CFIE, FA has a different substitution pattern in the ring D.

One of the prerequisites for QBA activity is the presence of an iminium bond. In SA/CHE this bond is susceptible to a nucleophilic attack and consequently plays a key role in the inhibition of SH-proteins. Both alkaloids interconvert between the cationic vs. neutral form (i.e. hydroxide adduct or pseudobase) (Fig. 2) displaýng sort of "Dr Jekyll and Mr Hyde'' duality. R1

RI

R2

+oH

----.-> +

R2

*-an,

Figure 2. Quaternary ioďpseudobase equilibrium in benzo[c]phenanthridine alkaloids.

They penetrate across the cell membrane in the hydrophobic pseudobase form acting as pro-drugs and convert into active cationic form once inside the cell. Unlike SA/CI#, FA, -due to iis different ring substitution pattem, exists at physiological pH as a cation only, is a weak electrophile and its iminium bond is not attacked by nucleophiles. SA/CFE react with

247

biomacromolecule or subcellular structure by covalent bond formation, FA by electrostatic interaction. Both of these interaction modes elicit dissimilar biological

a

response.

In this paper, the following results of our investigation on QBA are presented:

1.

Identification of chemical forms of SA/CF{E that complex with mercapto nucleophiles, and the determination of stability constants that characterize their binding with human serum albumin; 2. Inhibitory effects of SA, CFIE and FA on DPP-IV-like enzymes isolated from human blood plasma and human anďrut glioma cell lines; and 3. QBA role in the genesis of epidemic dropsy syndrome.

SA/CHE that complex with mercapto nucleophileso and the determination of stability constants that characterwe their

2. Identification of chemical forms of

binding with human serum albumin

The red and yellow colors of SA and CFIE, respectively, are ascribed to their quaternary cations [9,10] (Fig. 2). zr-Electron densities on the nitrogen atom in position 5, and on the carbon atom in position 6 in SA, are 1.660 and 0.646, respectively, according to quantum chemistry calculations [11]. Thus, the double bond N(5) : C(6) of these quaternary cations is polar and sensitive to attack by nucleophiles [11]. For hydroxide ions as nucleophiles, a pH dependent equilibrium between the charged quaternary form and the uncharged, so called pseudobase form (Fig. 2) is typical of benzo[c]phenanthridine alkaloids. This equilibrium may be formulďed as a reversible complexing of the heterocyc\ic cation, / and a hydroxide ion, Q* + OIf =- QOH or, more reasonably l7l, as acidobasic

equilibrium

:Fl

Q. + HzO =-

QOH + I{

with an equilib,rium constant

Kn*

pH at which the J [QOH]/[Q.J. heterocyclic cation and pseudobase are present in equal concentrations [11]. The alkanolamine structure given in Fig. 2 has been almost universally adopted because quaternary hydroxides cannot exist U2]. pKp* Constants that characteize equilibria between charged and uncharged forms of SA and CřIE range between 7 anď 9 u3-15]. Thus, at physiological pH7.4, both charged anduncharged forms of the alkaloids exist in In analogy to Bronsted acids, the pKpa denotes the

aqueous solutions and in blood.

reportedly interact with nucleophilic SH- groups of simple organic compounds in a 1:1 ratio [16]. An analogous interaction is suggested with enzymes [16], and with human serum albumin [17] in which the one free SH-group is expected to be the interaction point. The charged (iminium) forms of SA and CFIE have been indicated as the forms interacting chemically with nucleophiles including mercapto ones [16] ; the latter was deduced from static photometric measurements. In contrast, the uncharged

SA and CFm

(pseudobase) form of SA was denoted as the form interacting with human serum albumin in

LI7I.

Our electrophoretic investigation into the type of interaction between SA or CFIE and cysteine or mercaptoethanol evidenced that interaction of these alkaloids with both

mercapto compounds is not a chemical reaction but a complexing. The formed complexes based on non-bonding intermolecular interactions are kinetically labile. Measurements at various pH revealed that only uncharged forms of these alkaloids complex with simple mercapto compounds [18]. Cysteine and mercaptoethanol are electrophoretically uncharged in the pH range 5 - 7.4. Anionic migration of complexes formed by uncharged SA or CFIE

248

and mercaptoethanol at pH 7.4, and at high concentrations of mercaptoethanol in the background electrolýe was observed. This migration evidences that an anion, which may be supplied only from the background electrolýe, participates in the formation of the complex between SA or CF{E and mercaptoethanol. The absence of an analogical effect in experiments with cysteine may be explained by the participation of the negative charge of

cysteine in the complexing. The stability of complexes of SA and CFm with mercaptoethanol and cysteine depends on both buffer cation and buffer anion. Constants corrected for the abundance of the uncharged form of these alkaloids therefore differ markedly depending on the buffer composition. For example, stability of complexes of SA with cysteine was from 8,400 to 18,000 Vmol, stability of complexes of CFm with cysteine was from 21,500 to 30,800 l/mol in the buffers used [18]. The possible structure of complexes of cysteine and mercaptoethanol with uncharged monomeric form of either SA or CHE, which is always present in some equilibrium concentration in water (in aqueous background electrolye), was explored and modeled using the software program HyperChem 2.0. The requirement of minimum total energy was applied. The twisted cysteine molecule, aligned by its carboxylic group to the vicinity of the N(5) - C(6) bond (Fig. 3) was the most probable result for each of the alkaloids. This supports the conclusion from experiments with mercaptoethanol that some participation of the negative charge is necessary for the non-covalent binding of uncharged SA or CF{E with the mercapto group. If cysteine is the ligand, its carboxylic group may supply the necessary negative charge and the resulting complex remains therefore outwardly uncharged. If the negatively charged group is absent from the ligand, e.g., in mercaptoethanol, the necessary negative charge may be supplied only from the solution. In this case, the resulting complex is negative. The simple 1 :1 interaction scheme 116,171, therefore, holds only for mercapto compounds bearing negatively charged groups. Albumins evidently belong to such compounds.

G< ^-':/NH:* R4

Figure 3. Molecular modeling of the non-covalent interaction between cysteine and quatemary benzo[c] phenanthridine alkaloids.

Identical results have been obtained from the investigation of the complexing of SA and CFIE with human Serum ďbumin. Constants corrected for the abundance of the interacting uncharged form of these alkaloids are 332,000+38,400 and297,000+36,000 l/mol for SA and CFm, respectively. The value of stability constant for SA from electrophoretic experiments agrees with the stability constant, K : 385,000 (or log K : 5.59) from static experiments [17].

249

CHB and FA on DPP-IV-like enzymes isolated from human blood plasma and human and rat glioma cell lines

3. Inhibitory effects of SA,

Proteinase inhibitors represent

a very

attractive

and perspective topic in

the

pharmacological research and therapy. As we have shown [9], QBA are potent inhibitors of DPP-IV-like en4rme activity in glioma cells. In fact, the total cellular DPP-N-like enzpe activity represents the sum of hydrolyic activities of several separate molecular species [20] that play a critical role in the regulation of cell proliferation, differentiation,

apoptosis and energy metabolism. Thus, we decided to study the effect of QBA on subseparated DPP-IV-like enzyme activity bearing molecules in order to investigate the complexity of a possible mechanism of the effect of QBA alkaloids. To get a deeper insight into the QBA biological activity, we should focus not only on their "direct" targets in the cell but also on possibly more complex pathways of their action. An example of such an "indirect" QBA effect is the interaction with enzymes involved in a multitude of physiological functions. We have been focusing on heterogeneous DPP-N-like activity bearing enzymes to investigate their inhibition by QBA as one possible pathway that could help to explain the diverse QBA biological activities. Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) was originally believed to be the only membrane-bound enzyme specific for proline as the penultimate residue at the amino-

terminus

of the

polypeptide chain. Many biologically active peptides contain

an

evolutionary conserved proline residue as a proteolysis-processing regulatory element and, therefore, proline-specific proteases could be seen as their important "check-points" [21]. Thus, proteolýic activation and inactivation of such peptides was originally expected to be the main physiological function of DPP-N. Subsequently, three general mechanisms of

DPP-N activity have been postulated: (i) Limited proteolysis, i.e. highly specific processing of biologically active peptides, leading to their functional activation or

inactivation [22]. This mechanism has been shown to play a role in immune and endocrine system regulďions f2l,23f, diabetes mellitus pathogenesis [24] and HIV infection [21]; (ii) Cell-cell, cell-extracellular matrix and cell-virus contacts; DPP-IV was described to be a collagen- and fibronectin-binding protein 1251, a co-receptor for HIV-1 123f, and a homing factor for organ-specific metastasizing of breast tumors [25]; (iii) Signal transduction; DPPIV1CD26 is considered as a co-receptor transmitting specific signals through the plasma membrane [26]. However, other molecules, even structurally non-homologous with the

corresponding enzpe activity, have been identified recently: fibroblast activation protein cr, dipeptidyl peptidase IV-B, N-acetylated cr-linked acidic dipeptidase, quiescent cell proline dipeptidase/dipeptidyl peptidase II, attractin, and dipeptidyl peptidase 8 B0l. Comparing the structure and relatedness of molecules

DPP-N but bearing

associated functionally or structurally to dipeptidyl peptidase IV led to a grouping classified as "DPP-IV activity- andlor structure homologues" (DASH). DASH were shown to participate in a broad ďray of complex processes like cell proliferation and differentiation l2Tl,neoplastic fransformation [28 ] and apoptosis [26].

In our experiment 1291, we found DPP-N-like enzyme activity associated with molecules of about 320 kDa ("low-MW form") in C6 rat glioma cells, human U373 glioma cells, human U87 glioblastoma cells and with molecules of about 570 kDa ("high-MW form") in human U87 cells. In human plasm4 a predominant peak of DPP-N-like activity was found for molecules of about 440 kDA ("intermediate-MW form") and a minor peak of about 600 kDa. Considering MW pH optima and inhibitory parameters of particular DPP-N-like enrqe activity fractions, we assume that the high-MW, intermediate-MW and low-MW

2s0 forms represent attractin [30], DPP-IV1CD26 [31] and DPPS [32], respectively. All three activity fractions were inhibited by the QBA studied. The most potent inhibition was observed in DPP-8, resembling the low-MW form of DPP-IV-like enzyme activity. DPP8 is a ubiquitous soluble non-glycosylated serine protease, localized in the cýoplasmic (nonlysosomal) compartment, acting preferably at neutral pH [32]. Based on the structural similarity with DPP-IV, DPPS was proposed to be involved in T cell activation and

immune function [32]. Functional studies dealing with DPPS have not been published so far and thus, biological impact of its inhibition by QBA remains fully speculative. Attractin was originally described to be an immunoregulatory protein, expressed and secreted by activated T-cells, mediating their costimulation to recall antigen-driven proliferation [33]. Later studies demonstrated its presence in other organ systems, where it is expected to participate in the regulation of pleiotropic phenotypic features including tumor susceptibility, pigmentation and body weight [34]. At least some of these functions may be dependent on attractin enzyme activity. Thus, QBA-mediated attractin inhibition could result in a b'road array of functional effects, depending on the orgaÍl system affected. There is contradictory evidence whether attractin [33] or DPP-N/CD26 I35l represent the main source of serum DPP-IV-like enzyme activity. We observed two fractions of DPP-Nlike enzyme activity in human plasm4 which supports the hypothesis of heterogeneity of DPP-N there. Indeed, it is possible to speculate that their proportion could be dependent on either individual or specific condition. Decreases in serum DPP-N-like enzyme activity were observed to be inversely related to increases in severity of depression in patients exposed to immunochemotherapy [36]. Interestingly, serum DPP-N-like activity is also significantly decreased in patients with food intake disorders. Yet, it is worth mentioning that glucagon-like peptide 1 and 2 are DPP-N substrates and, therefore, QBA mediated inhibition of DPP-IV-like enzyme activity can influence intestinal motility and function as well as to aÍfect the central body weight related effect of both hormones [37]. Additionally, in all glioma cell lines, but not in human plasm4 we have found also a DPPIV-like enzyme activity fraction with parameters (acidic pH optim4 low molecular weight, substrate preference) resembling quiescent cell proline peptidase/dipeptidyl peptidase II (unpublished results). Inhibition of quiescent cell proline peptidase (QPP) is believed to be a trigger of a specific apoptotic pathway in quiescent lymphocýes [38]. Unfortunately, we were unable to detect arry inhibition of the abovementioned DPP-N-like enzyme activity by QBA. This could be due to QBA inactivity below pH 6, i.e. conditions optimal for QPP enrqe activity. Nonetheless, QPP could be speculated as a possible QBA target, at least in

some cell systems. Inhibitors of DPP-IV-like activity bearing molecules are believed to be of significant therapeutic impact in the treďment of HIV infection, diabetes mellitus, and as aÍI immunosupressant in the transplantation surgery and autoimmune diseases, including multiple sclerosis. Even though valuable attempts have been made, there is still a lack of commercially available specific substrates and inhibitors of individual DPP-N-like enzyme activity bearing molecules. On the other hand, from a functional point of view, an inhibitor itself does not need to be ultimately specific for a particular DASH molecule; its ''specificitý' could be provided by cell/immediate environment specific expression paffern of these enzymes.

To conclude, we propose that among numerous others, some of QBA biological effects

could be mediated by their interaction with the heterogeneous group of DASH. Moreover, a specific DASH expression pattern determines the quality of such alkaloid effects, which can be seemingly paradoxical in particular tissues and cell systems.

257 4.

QBA role in the genesis

of epidemic dropsy syndrome

The objective of this study was to investigate whether a long-term oral administration of QBA in feed generates adverse effects in swine. After Sarkar [39] isolated, identified and assessed the toxicity of SA and dihydrosanguinarine (dihydroSA) as the main alkaloids of aÍgemone oil, other investigators have assumed that these alkaloids are responsible for the oral argemone oil toxicity and consequently responsible for the onset of epidemic dropsy. However SA cannot be considered the main toxic principle of argemone oil because the toxicity data obtained with aÍgemone oil on one harrd and with pure SA or with defined fractions of benzo[c]phenanthridine alkaloids on the other hand diÍfer considerably: Sarkar described mortality in young albino rats in less than a week on feed containing 10-25 mg/kg of SA hydrochloride [39]. Oral LDso (rat) of argemone oil was determined as 1.1 ml/kg

[40]. At a 0.5oÁ SA/dihydroSA concentration this corresponds to a 5 mglkg dose for SA/dihydroSA. Other authors report a LD5s in rat for SA of 1658 mglkg [41]. There are no data available on the oral toxicity of dihydroSA; however, this difference is too large to be explained by the toxicity of dihydroSA in argemone oil. The parenteral toxicity of dihydroSA in rats is two andahalftimes lower than that of SA [39]. A 400 mg/kg injection of SA suspended in arachis oil was nontoxic in mice 1421. A daily administration of 3.5 mglkg SA and 10 ml/kg €rrgemone oil (i.e. 46 mýkg SA and dihydroSA) to young and old rats over 225 anď 250 days, respectively' did not induce epidemic dropsy symptoms [42], (i.e. the toxicity was different by one order of magnitude from that reported by Satyavati [a0]. In trials on monkeys it was determined that 2.0 gkg argemone oil (i.e. 10 mg/kg SA and dihydroSA) administered orally ťor 4 weeks induced the development of edema and erýhema along with distinct reddish angiomatous nodules over skin [43,44]. SA only at the daily dose of 1.6 mglkg did not bring about epidemic dropsy [a5]. Only two studies exist of the effects of QBA in swine. Lal et aL la6l in a limited study observed a marked body weight gain after the feeding 5% argemone oil in mustard oil for 3 months. Australian authors studied the tolerarrce of Argemone ochroleuca and A. mexicana seeďs in animal feed |47]. Seeds of the Argemone species (- 20 mg of SA/dihydroSA/other quďernary and dehydrogenated benzo[c]phenanthridine alkaloids in I kg feed) elicited the first signs of intoxication after 3-4 weeks (lower feed intake, lethargy, skin redness, mild diarrhea). At 20Á proportion, the intoxication manifested after 5-7 days and after 2 weeks these animals had to be removed from the experiment. However, toxicity was markedly changed by inadiation of ground seeds with direct sunlight for several days; pigs then to|erated 40Á seeds in the ťeed. Sunlight inadiation of the seed-contaminated feed in the presence of air "destroys" most of the dihydroSA and dihydroCFIE [a7]. This might indicate that these alkaloids rather than SA could be responsible for oral toxicity of A. mexicana. This observation is consistent with Hakim's reference to recognized diminution of argemone oil toxicity by heat, light, and ageingl42l. In our experiment [48], we administered two QBA doses to swine for three months: (i) a low dose (2 mgkg feed) conesponding to the content of QBA recommended for inclusion in commercial feed), atrd (ii) a dose fifty times higher (i.e. 100 mglkg feed), where the onset of adverse reactions could be predicted, based on literature data [3]. We achieved a plasma level of 0.1 1 pglml SA, which is the level comparable with that of a human epidemic dropsy patient [49]. All animals remained in a very good health over the duration of the experiment and did not manifest any signs of toxicity described above. Histological examinations of tissues and hematology did not reveal any toxicological damage. The only significant deviations from the control group clinical chemistry were found for the activity

252

of liver enzymes ALT, AST and GMT. Argemone oil feeding in rats caused an increase in the AST activity in serum [50]; other authors described a significant loss of hepatic ALT/AST activities while the activities were increased in the serum [51]. Dalvi reported an increase in the ALT/AST activity following a single i.p. dose of 10 mg/kg SA in rat 1521. Previously, we found an in vitro itthibition of the ALT/AST activity (IDso 3.4.rc-6 M) in rď liver post-mitochondrial supernatant [16]. Our observation of a decreased ALT/AST in the plasma can be explained as an inhibition of the enzymes by the ca. lO-7 M concentration SA present there, which is a magnitude sufficient to bring about inhibition [16]. The intake of argemone oil (0.5 % of benzo[c]phenanthridine alkaloids) caused a significant stimulation of endogenous lipid peroxidation t531. The serum level of malondialdehyde was found to be significantly increased (172 %) in 10 human (epidemic dropsy patients) following consumption of edible oils adulterated by argemone oil. The malondialdehyde level was found to have positive correlation with the serum SA levels [a9]. we did not observe significant diÍferences between control and experimental groups' i.e. we cannot confirm an increase in lipid peroxidation in the plasma of experimental animals with SA and CFIE levels of 0.11 and 0.024 pglml, respectively (a sanguinarine level comparable with that of an epidemic dropsy patient). 32P-postlabelling assay, we did not detect any SA/CFIE-derived DNA adducts in livers of pigs exposed to the alkaloids [48]. This indicates no genotoxicity of the compounds in

In a

vivo.

In conclusion, orrr experimentď animals, although fed doses of quaternary benzo[c]phenanthridine alkaloids in the range of 5 mg/kg body weight, remained in good health without any symptoms that could be associated to epidemic dropsy. Hence, the results of this study support views of authors who do not consider benzo[c]phenanthridine alkaloids, particularly SA, being responsible for the disease. Attention should rather be directed to other aÍgemone oil components when searching for the cause of epidemic dropsy outbreaks. Acknowledgement Financial support by the Ministry of Education (grant No. MSM 151100003) and Grant Agency of the Czech Republic (grant No. 20310210023) is gratefully acknowledged.

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