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1 Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Strasse 2/015, 97080 Würzburg, Germany
2 Institute of Organic Chemistry, Am Hubland, 97074 Würzburg, Germany
Correspondence
Alicia Ponte-Sucre
aiponte{at}gmail.com
Gerhard Bringmann
bringman{at}chemie.uni-wuerzburg.de
Received July 10, 2009
Accepted September 4, 2009
Naphthylisoquinoline alkaloids equipped with a N,C-hetero-‘biaryl’ axis, and, in particular, simplified synthetic analogues thereof, kill intracellular Leishmania major at concentrations in the low submicromolar range, while being significantly less toxic to their major host cell, the macrophage, at the same concentrations. To further investigate their mechanism of action we evaluated the morphological and ultrastructural changes induced by specific N-arylisoquinolines in L. major, and the correlation of these changes with compound accumulation and disposition by the parasite. After 24 h of treatment with the synthetic arylisoquinolinium salts 3 or 4, dramatic structural changes and cell death were observed. Furthermore, the auto-fluorescent derivative salt 3 accumulates continually in intracellular compartments. Our results thus suggest that the leishmanicidal effect of arylisoquinolinium salts may involve their ability to accumulate and precipitate in intracellular organelles, form a huge vacuole and eventually promote cell lysis.
Abbreviations: IC50, 50 % inhibitory concentration.
Present address: Laboratorio de Fisiología Molecular, Universidad Central de Venezuela, Caracas, Venezuela, c/o Alicia Ponte CCS 81720, PO Box 025323 Miami, Fl 33102-5323, USA. 
These authors contributed equally to this work.
Present address: Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
||Present address: Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093, USA.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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Fortunately, progress in the validation and characterization of drug targets and chemistry of new compounds is raising hope for the future. In fact, different approaches are being used to identify novel pharmacophores against Leishmania, and one scheme has been the analysis of naturally occurring plant-derived compounds, including naphthylisoquinolines, isolated from tropical lianas belonging to the Ancistrocladaceae and Dioncophyllaceae plant families (Bringmann & Pokorny, 1995; Bringmann et al., 1998). These alkaloids are active against Plasmodium, Trypanosoma and Leishmania parasites (François et al., 1996, 1997; Bringmann et al., 2000, 2003a, b).
More recently, a new subclass of these natural products has been discovered, viz. the N,C-coupled naphthylisoquinoline alkaloids, whose representatives are particularly potent anti-infective agents. In fact, previous results demonstrated that some analogues of this novel coupling type, e.g. ancistrocladinium A (1) and B (2) (Bringmann et al., 2006), and their structurally simplified synthetic analogues, e.g. compounds 3 and 4 (Ponte-Sucre et al., 2007, 2009), are effective against intracellular L. major amastigotes in the low submicromolar range. They also demonstrated that the toxicity of these compounds against mammalian cells occurs at higher concentrations than those needed to impair parasite replication inside their host cell (Ponte-Sucre et al., 2007).
One fundamental precondition for the development of a safe drug is low toxicity against mammalian cells (Croft et al., 2006). For this reason, to increase the selectivity of the potent isoquinolinium salts is an important task. Studies on the cellular target and on their metabolic pathway should help us to understand the mode of action of these substrates, and thus help us to (a) improve their structure–activity relationships and (b) define strategies to prevent side effects in an efficient manner.
A drawback in the analysis of the mechanisms of action of classical drugs against Leishmania is that there are no systematic descriptions of the morphological changes induced by toxic concentrations of pentavalent antimonials, polyenic antibiotics like amphotericin B or diamines like pentamidine. Compounds 3 and 4 are the most promising simplified synthetic analogues of the naphthylisoquinoline alkaloids (Ponte-Sucre et al., 2007, 2009). Herein, we investigated the morphological and ultrastructural changes exhibited by Leishmania parasites treated with these arylisoquinolinium salts. In addition, we analysed the uptake and accumulation of these compounds in the parasite. To achieve this last goal we took advantage of the auto-fluorescence of the synthetic derivative salt 3.
Our results strongly suggest that treatment with the N-arylisoquinolinium salts 3 and 4 at concentrations near their IC50 (50 % inhibitory concentration) against Leishmania promastigotes induces morphological alterations in the parasite structure. They varied from discrete changes to total destruction of the parasite, including condensation and distortion of the nucleus, shrinkage of the kinetoplast and the appearance of large cytoplasmic vacuoles. These morphological changes correlated with compound accumulation in intracellular organelles and with a striking decrease of compound levels in culture supernatants. Altogether, these results suggest that the leishmanicidal effect of N-arylisoquinolines initially correlates with their ability to accumulate and precipitate in intracellular organelles and induce cell death.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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Arylisoquinolinium salts 3 and 4.
The synthetic derivatives N-(1'-naphthyl)-6,8-dimethoxy-1,3-dimethylisoquinolinium perchlorate (salt 3) and N-(4'-isopropylphenyl)-6,8-dimethoxy-1,3-dimethylisoquinolinium trifluoroacetate (salt 4) (Fig. 1
) were prepared as described previously (Ponte-Sucre et al., 2007, 2009). For biological studies the compounds were dissolved in DMSO (Sigma-Aldrich) and supplemented either in complete medium or in PBS buffer.
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Determination of L. major structure by electron microscopy. Aliquots (40 µl) of a cell suspension of L. major (2x108 cells ml–1) were incubated in 96-well plates with compound 3 (3 µM), compound 4 (3 µM) or amphotericin B (3 µM), used as a reference compound, for 6 h. The cells were washed three times with PBS and were further fixed by standard procedures using protocols described by Granthon et al. (2007). Cells were dehydrated in increasing concentrations of ethanol and incubated for 1 h in propylene oxide, followed by incubation for 1 h in a 1 : 1 mixture of propylene oxide and Epon (Electron Microscopy Sciences). Specimens were embedded in Epon at 60 °C for 48 h. Post-staining of sections was carried out with 1 % uranyl acetate for 30 min. Photographs were taken with a Zeiss EM10 transmission electron microscope at 100 kV, and scanned images were processed using Adobe Photoshop.
HPLC-MS analysis of L. major culture supernatants. The cell culture supernatants from the experiments to determine changes in cell morphology were collected and vacuum-dried in a desiccator (Lyovac GT 2 E) for 48 h. The vacuum-dried supernatants were extracted by adding 3 ml methanol, filtered with Teflon filters (Phenomenex), and collected in 2 ml HPLC glass vials. The samples were separated by HPLC (Agilent 1100 series) on a Symmetry-C18 column (4.6x250 mm, 5 µm; Waters) with a flow rate of 1.0 ml min–1 and a solvent system consisting of (A) H2O (0.2 % formic acid; Sigma-Aldrich) and (B) ACN (0.2 % formic acid) applying the following gradient: 0 min 10 % B, 30 min 70 % B, 35 min 100 % B, 40 min 100 % B. The analytes were detected and quantified by electrospray ionization-MS (Agilent 1100-SL ion trap). The retention times and the intensities of the resulting peaks were compared to those of reference samples with defined concentrations dissolved in complete medium.
Accumulation of compound 3 in L. major.. Live L. major promastigotes (4x108 cells ml–1) were incubated for 30 min at room temperature in 200 µl PBS supplemented with glucose (5 mM; Sigma-Aldrich). The parasites were then washed with PBS and incubated with compound 3 (3 µM) for 5 and 15 min at room temperature. The parasites were also incubated with N-(3-triethylammoniumpropyl)-4-{6-[4-(diethylamino)phenyl]hexatrienyl}pyridinium dibromide (FM464) (5 µg ml–1, 30 min, room temperature) (Molecular Probes) to label anterior endosomes and the multi-vesicular tube system. In additional experiments the parasites were stained with compound 3 and Lyso Tracker red (Molecular Probes; 1 nM, 5 min, room temperature) to label acidic compartments, or with compound 3 and Mito Tracker red (Molecular Probes; 40 nM, 5 min, 4 °C). Finally, the cells were washed with PBS, mounted in frost line slides and analysed by fluorescent microscopy (with a Zeiss microscope). Images were captured by the Axioscop digital camera at a magnification of x100 and analysed with Adobe Photoshop.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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Although there was a high intrinsic variability in the sizes of the parasites, the total length (body plus flagellum) of the non-treated cells increased from 19.46±0.62 µm to 23.53±0.84 µm (P<0.001) after 24 h of incubation. This is the behaviour normally observed in L. major and Leishmania donovani cultures at their exponential phase (Bandyopadhyay et al., 1991). The size of cells treated with amphotericin B did not change. Direct observation by light microscopy of parasites treated with the isoquinolines 3 or 4 demonstrated that the cells became rounded and lost their motility. The total body and flagellum lengths decreased to 17.67±0.63 (P<0.0001) for compound 3 and to 17.21±0.66 (P<0.0001) for compound 4. The results suggest that compounds 3 and 4 impaired parasite growth (P<0.001), and caused a consistent decrease in the total parasite length, probably as a consequence of the decrease in cell viability; additionally, the cells had the tendency to become ovoid.
Non-treated cells depicted the characteristic promastigote cigar-shaped cell body with a flagellum longer than the cellular body. Promastigotes treated with amphotericin B displayed a morphology either similar to that of the untreated cells, or with a deformation of the anterior part of the body. Treatment with compounds 3 or 4 caused severe changes in the promastigote morphology. A population of elongated cells became ovoid after treatment, whereas another population became extremely slender; cells treated with compounds 3 or 4 had a granular soma with a small nucleus and kinetoplast or were distorted with the appearance of a huge vacuole in the anterior part of the cell, suggesting the accumulation of compounds inside the cell. Of note, amphotericin B toxicity occurs through its binding to sterols in the cell membrane and the formation of aqueous pores (Ramos et al., 1996); intracellular trafficking does not occur for amphotericin B.
Arylisoquinolines 3 and 4 disrupt L. major cell structure
To further analyse the effect of the arylisoquinolinium salts 3 and 4 in L. major we studied their influence on the ultrastructure of the parasites. Untreated promastigotes (Fig. 2a) displayed the typical elongated cell body, including the anterior flagellum and flagellar pocket, a single mitochondrion containing the kinetoplast, and the normal cellular structures. Compounds 3 (Fig. 2b, c) and 4 (Fig. 2d, e) induced remarkable morphological alterations of the parasite structure. The morphological changes were uniform, but varied in intensity from discrete modifications (Fig. 2b) to total disruption of the parasite structure (not shown). Moreover, the treatment with compounds 3 and 4 dramatically affected the cell compartments. In fact, the alteration of the promastigotes to an ovoid shape (Fig. 2c), disturbance and destruction of the nucleus (Fig. 2e), and the appearance of a huge vacuole that tends to occupy the whole cell (Fig. 2c, d), were changes observed frequently. These changes suggest a dramatic distortion of the cell induced by the compounds as a form of cell death.
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Metabolism of N-arylisoquinolines 3 and 4 by L. major
The mechanism of action of compounds 3 and 4 is not known, although some ideas have been put forward. Previous data indicated that N-arylisoquinolines do not seem to be modified by drug-metabolizing enzymes involved in the metabolism of antileishmanial drugs (Bringmann et al., 2006; Ponte-Sucre et al., 2007, 2009). For this reason, measurement of the concentration of these compounds in complex biological samples requires the use of selective analytical techniques. Chromatography can be considered the procedure of choice for the bio-analysis of this class of compounds, as this methodology is characterized by good specificity and accuracy, and it is particularly useful to monitor small drug concentrations. For a more accurate analysis, we decided to quantitatively evaluate the disappearance of the compounds from the culture supernatant.
To analyse the correlation between the observed morphological changes and the handling of compounds by the parasites, L. major promastigotes were treated with compound 3 (3 µM) or compound 4 (3 µM), for 1, 2, 3, 6 or 24 h. The supernatants were collected, centrifuged to eliminate the parasites, desiccated and filtered, and finally analysed by HPLC-MS. The results were compared with those for supernatants obtained from untreated parasites. Standard samples of each compound (without cells present in the system) and of complete medium were run in parallel. The intensity of each analyte at their characteristic retention times [tR (compound 3)=21 min and tR (compound 4)=24 min using HPLC-MS] and the appearance of new peaks were evaluated and compared to the reference samples.
Analysis of the changes in supernatant concentrations of compounds 3 and 4 revealed a significant decrease of both compounds even after only 1 h of incubation with L. major cells (Fig. 3). During this time the compound levels found in the supernatants decreased by approximately 80 % for compound 3 and by 60 % for compound 4, and essentially remained at these levels until the end of the incubation (24 h). Unexpectedly, during this experiment no formation of metabolic products was observed, such as by oxidation or O-demethylation of compounds 3 and 4, as described for other antileishmanial drugs (Berger et al., 1993; Krauth-Siegel et al., 2003), and for C,C-coupled naphthylisoquinoline alkaloids by liver microsomes (Sieber et al., 2006). These results suggest that both compounds accumulate in the parasites and are either permanently bound to the intracellular organelles or metabolized by the parasites to products that were not secreted into the culture supernatant.
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Live promastigotes were treated with compound 3 (3 µM) for various periods of time. After 5 min of incubation, compound 3 could already be detected inside L. major. The accumulation further increased with time and nicely correlated with the radical decrease in concentration of compound 3 observed by HPLC-MS analysis of the culture supernatants from the parasite. This continuous sequestration of compound 3 by intracellular organelles probably disrupts the intracellular organization of the parasite making it difficult to evaluate in which organelle the compound is localized predominantly. In fact, the typical intracellular organelles, nucleus, mitochondria and flagellum were evident in parasites incubated with compound 3 for only 5 min, but are extremely distorted after longer incubations.
In order to determine whether compound 3 initially accumulated in intracellular organelles, we analysed its localization in parasites treated with the compound for only 5, 15 or 30 min, and simultaneously labelled with FM464, Lyso Tracker red or Mito Tracker red. The labelling with compound 3 did not match with that of FM464 (Fig. 4a) or Mito Tracker red (Fig. 4b) indicating that this isoquinoline does not label compartments close to the flagellar pocket, the sole site for exocytosis and endocytosis in the polarized organisms (Dodge et al., 2004), and is not initially incorporated by the kinetoplast from L. major. However, compound 3 was distributed in intracellular organelles that simultaneously showed labelling with Lyso Tracker red (Fig. 4c), a specific marker for acidic compartments. Interestingly, these Leishmania organelles are located at the axial edge of the parasite and either in the middle or towards the anterior end of the cell. Of note, in cells stained with Lyso Tracker red, the lysosomal fluorescence may constitute only a small portion of the total cellular fluorescence, although in Leishmania parasites it has been demonstrated that Lyso Tracker red labels acidocalciosomes (Krauth-Siegel et al., 2003).
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These results suggest that the auto-fluorescent derivative salt 3 accumulates in intracellular acidic compartments, possibly in the acidocalciosomes, which are fundamental for parasite nutrition and survival. No published data are available on the response of acidocalciosomes to stimuli that may induce cell death (Shaha, 2006). For this reason, a detailed investigation on this and on the further metabolic fate of compounds 3 and 4 is currently in progress.
In conclusion, our studies demonstrate that treatment of L. major parasites with isoquinolinium salts like compounds 3 and 4 induces dramatic morphological changes, in particular the appearance of a huge vacuole that eventually may be the cause of cell death. Furthermore, they suggest that specific organelles may constitute the cellular target of the compounds. These findings contribute to the analysis of the mode of action of these promising anti-infective compounds, and should be useful for the design, selection and synthesis of new arylisoquinoline-related substrates with better selectivity.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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References |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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, C., Schaeflein, L., Frank, A., Schultheis, M., Unger, M. & other authors (2009). Structure-activity relationship and studies on the molecular mechanism of leishmanicidal N,C-coupled arylisoquinolinium salts. J Med Chem 52, 626–636.[CrossRef][Medline]
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, Compound 3; 
, compound 4.