Introduction
Introduction
Leishmaniasis is an infectious disease caused by a protozoan parasite endemic to tropical
and subtropical countries. This disease primarily affects poor populations and is
estimated to affect 12 million people worldwide [1]. Leishmaniasis consists of four primary clinical syndromes: cutaneous leishmaniasis,
mucocutaneous leishmaniasis (also known as espundia), visceral leishmaniasis (VL;
also known as kala-azar), and diffuse leishmaniasis. Although cutaneous leishmaniasis
may lead to disfigurement, only VL is considered a fatal disease [2]. VL affects approximately 500 000 people worldwide and has a high mortality rate
of 59 000 deaths per year [3]. This disease is caused by the Leishmania (L.) donovani complex in East Africa and the Indian subcontinent, by Leishmania (L.) infantum in Europe, North Africa, and Latin America [4], [5] and by L. (L.) chagasi in Brazil [6]. The incidence of Leishmania spp. and HIV coinfection continues to rise in many countries; coinfection results
in a poor prognosis for VL patients as a consequence of the difficult diagnosis and
reduced responsiveness to treatment [7].
Leishmaniasis, a vector-born disease, is caused by obligate intramacrophage parasites.
Leishmania multiplies in the digestive tract, and the parasites are transmitted to the mammalian
host during blood feeding. Metacyclic promastigotes infect macrophages and transform
into intracellular amastigotes of a non-flagellar spherical form. After multiplication,
the amastigotes lyse the host cells, reinfect other macrophages and then return to
the invertebrated vector during blood feeding [8].
The small number of drugs that are clinically available to treat leishmaniasis are
highly toxic. These drugs are primarily given by injection and could induce resistance
[9]. The pentavalent antimonials sodium stibogluconate and meglumine antimoniate have
been used as first-line drugs for more than 70 years in most countries, despite severe
side effects such as cardiac arrhythmia and acute pancreatitis [10], [11]. Due to increased parasitic resistance, these drugs are nearly obsolete in North
Bihar (India) [12] but can still be used in other countries and Indian states. The antifungal macrolide
amphotericin B, which was first isolated from Streptomyces nodosus, is also a first-line drug with excellent efficacy. However, the primary limiting
factors for amphotericin B use in the clinic are adverse effects including fever,
chills, thrombophlebitis, myocarditis, severe hypokalaemia, renal dysfunction, and
even death. Despite the introduction of the less toxic liposomal formulation of amphotericin
B, Ambisome, therapeutic use is limited in undeveloped countries due to high costs.
In Brazil, the liposomal formulation is provided as a first-line treatment for young
children, HIV‐coinfected patients, and old people and only in the São Paulo state.
Pentamidine is used as a second-line treatment for leishmaniasis; however, it can
cause irreversible insulin-dependent diabetes mellitus and death. In addition to its
severe toxic effects, pentamidine has poor efficacy, resulting in the abandonment
of this treatment in India [13].
The use of “piggy-back” chemotherapy has led to the discovery of the use of the anticancer
drug miltefosine for the treatment of Leishmania spp. parasites. This is the first effective orally dosed drug for VL [14]. Combination therapy for VL has increasingly been advocated as a way to increase
treatment efficacy and tolerance, reduce treatment duration and cost, and limit the
emergence of drug resistance [15]. A phase 2, non-comparative randomised trial performed in India assessed different
combinations of a single dose of liposomal amphotericin B followed by miltefosine
for 7–14 days, resulting in a highly efficacious (more than 95 % of patients cured)
treatment [16]. Despite the efficacy of miltefosine, its adverse effects include severe gastrointestinal
toxicity and significant increases in the levels of serum aspartate aminotransferase,
alanine aminotransferase, and creatinine [17]. In addition, orally dosed miltefosine for the treatment of Indian kala-azar failed
to treat the American species L. (L.) mexicana and L. (V.) braziliensis [18].
Azoles and allopurinol are two of several oral drugs that are weakly active against
Leishmania. These drugs are not useful as a single agent, and case reports record the effective
treatment of immunosuppressed patients when the drugs were used in combination [19]; however, clinical trials to test this combination have not been performed.
The aminoglycoside antibiotic paromomycin has entered clinical studies and displays
antileishmanial activity in India [20], albeit with a significant increase (> 5-fold) in hepatic transaminases [21]. Paromomycin is a potential new candidate; however, treatment failure and relapse
occur when it is used as a monotherapy.
Sitamaquine is an oral 8-aminoquinoline analogue that has shown some efficacy to treat
VL more than 20 years. This drug is being developed by GlaxoSmithKline (GSK), and
marketing for the drug has been delayed [22]. Phase II studies were conducted in Brazil [23] and India [24] with cure rates ranging from 27 % to 87 %; however, there were several cases of
serious adverse renal events.
In addition to specific antileishmanial drugs, other coinfections also need to be
attended. Because the majority of the population affected by leishmaniasis is poor,
a major therapeutic concern is to manage anaemia and malnutrition to ensure the antileishmanial
drugs remain effective. In Brazil, concomitant parasitic infections are frequently
observed in patients from areas endemic with leishmaniasis, resulting in a challenge
when one considers which disease should be treated first. Moreover, the use of a single
drug to treat every Leishmania species is unrealistic. Even in the same country, Leishmania spp. strains vary in terms of sensitivity to antimonial therapy [25]. In practice, no definitive therapeutic protocol is followed in Brazil as a result
of different social realities, patients, nutritional states, and infection by different
Leishmania species and strains.
Differences in Drug Discovery Approaches Using Natural Products
Differences in Drug Discovery Approaches Using Natural Products
The development of simple, reliable, and rapid tests to screen large chemical libraries
for novel antileishmanial agents is a goal for drug discovery groups. In general,
most assays are time consuming and faster methodologies require sophisticated instruments;
however, these are cost prohibitive for most laboratories in developing countries.
Furthermore, the determination of drug activity at the mammalian stage (intracellular
amastigotes) is indispensable and requires intense experimental manipulation over
many days. Finally, the evaluation of new drug candidates requires in vivo assays and also demands time-consuming investigations, ranging from some days to
several months.
Standardisation of drug discovery assays has been a difficult task, especially when
one considers the multidisciplinary research involved in the process. The ineffective
interaction between chemists and biologists is the first major limitation for the
development of new lead compounds. An increasing number of reports describe the antileishmanial
activity of natural products [26], [27], [28], [29], including marine invertebrate metabolites [30]. Few of these compounds have been evaluated for in vitro selectivity or in vivo. In addition, only small amounts of compounds can be isolated from marine animals.
Thus, adequate amounts of compound must be synthesised for the complementary assays.
Only one compound from a library of 5000 to 10 000 compounds has reached the market.
Thus, a major limitation for drug development is the lack of a suitable chemical library.
Increased chemical diversity can be obtained by combinatorial chemistry using natural
compounds as prototypes. Effective interaction between research groups is also essential
for the rapid synthesis of novel selective compounds and would provide a greater chance
for lead development from natural products.
Lack in Consensus on Natural Product Research for the Discovery of Antileishmanial
Agents
Lack in Consensus on Natural Product Research for the Discovery of Antileishmanial
Agents
Oftentimes, when multidisciplinary groups are involved in drug discovery efforts using
natural products, a consensus in methodology and protocols is difficult to obtain.
Some major concerns are presented in the following subsections.
Biological testing of crude extracts or pre-fractionated samples for screening purposes
Natural products must be made into extracts for drug screening purposes. However,
in vitro testing of crude extracts may result in false-negative results. Rapid fractionation
using liquid-liquid partitioning or simple chromatographic techniques, such as solid-phase
extraction, may avoid these problems by enriching for active compounds in specific
fractions and eliminating inactive metabolites. In particular, for marine invertebrate
metabolites, pre-fractionation of crude extracts is an effective way to eliminate
undesirable salts and polar primary metabolites.
What solvents are suitable for microplate assays?
Despite a few hydrophilic secondary metabolites, most natural compounds are solubilised
in an organic solvent before in vitro evaluation. The use of polar solvents to dissolve these compounds results in an inaccurate
concentration, leading to unreliable results. Therefore, test compounds could also
be added directly to the microplate wells and dried in situ before the incubation with parasites. Most 96-well microplates are composed of polystyrene
and do not support organic solvents. Furthermore, a large amount of organic solvents
interfere with spectrophotometer readers. Thus, plates composed of polypropylene may
be used as an alternative. However, in practice, dimethylsulfoxide (DMSO), ethanol
(EtOH), and methanol (MeOH) are the most frequently used solvents, and these do not
interfere with the spectrophotometer. However, the simultaneous dilution of sample
in organic solvents and incubation with parasites is a common practice. Regarding
Leishmania parasites and macrophages, 0.5 % of MeOH or DMSO as a maximum final concentration
is recommended to avoid toxicity and false-positives.
Testing concentrations
Choosing an appropriate compound concentration is important to avoid missing active
compounds and erroneous interpretation of data. For crude and pre-fractionated extracts,
it is important to consider that the active compound(s) might be in a very modest
quantity. For example, if a tested extract is incubated at 10 µg/mL and the active
compound represents only 0.1 % of the crude extract mass, this substance will induce
antiparasitic activity at 0.01 µg/mL, which is, in practice, an unusual event. On
the contrary, if the extract is tested at elevated concentrations (> 300 µg/mL), the
insoluble material on the bottom of the microplate will most likely interfere with
microscopic analysis and the colorimetric or fluorimetric viability tests. An additional
drawback of using elevated compound concentrations is the false selection of poorly
active molecules (50 % effective concentration higher than 100 µg/mL). Based on previous
research, fractionated samples should not be tested at concentrations higher than
300 µg/mL, and the concentration of isolated substances should be limited to 100 µg/mL.
However, structural modification of weakly active compounds may improve efficacy.
Current In Vitro Screening Assays
Current In Vitro Screening Assays
Classical methods
Axenic parasites: The use of axenic promastigotes or amastigotes is a low-cost alternative for screening
large secondary metabolite libraries [18]. Promastigotes are the extracellular form of parasites and live in the gut of the
sand fly insect. The ease of parasite maintenance in culture and assay performance
allows for screening of hundreds of compounds using unsophisticated equipments. Briefly,
promastigotes are counted using a hemocytometer and a light microscope and then incubated
with test compounds in 96-well microplates for 24 to 96 h. Classical methods, such
as direct counting assays using a hemocytometer, are still in use in many laboratories;
however, this technique is time consuming and only tests growth inhibition, not leishmanicidal
activity. The major disadvantages of this assay are the daily time-consuming cell
counting and the potential of inaccurate 50 % effective concentration (EC50) values.
The MTT colourimetric test is a precise and low-cost viability assay [31] that is analysed using a microplate spectrophotometer. This method is based on the
oxidative activity of mitochondria and provides additional information about whether
a compound has leishmanicidal activity. This test can be used to optimise the screening
of large libraries with high reproducibility and accurate determination of EC50 values. A major advantage of this assay is the ease of obtaining EC50 curves, which can be determined after a 24-h incubation with the parasite followed
by a 4-h incubation with the MTT substrate. A major disadvantage of the assay is the
possible oxidation of the MTT substrate by the test compounds, resulting in false-negatives
[32], [33]. The presence of a purple formazan colour is indicative of viable parasites and
is lost upon cell death. A fast plate centrifugation step can be included to extract
the test compound from the media before MTT addition. Care should be taken to avoid
additional errors during plate manipulation. In addition, observation under a light
microscope should be performed prior to the addition of the MTT substrate to evaluate
parasite motility and morphology.
An alternative to the colourimetric MTT assay is the oxidation-reduction indicator
Alamar Blue. This dye has been developed to assess the antileishmanial activity of
drug candidates. After incubation of Alamar Blue with samples (crude extracts, enriched
fractions or pure compounds), promastigote viability is determined at 570 nm in a
microplate reader [34].
The use of axenic amastigotes for compound screening presents some advantages. This
test is performed with a clinically relevant stage of the parasite. In addition, quantification
of drug activity is very feasible. This is achieved with a cell counter [35], evaluating cell viability using an MTT-based method [31], determining ornithine decarboxylase activity [36], or labelling with a fluorescent dye, such as propidium iodide, followed by fluorescence
activated cell sorter (FACS) analysis [37], [38]. However, certain biochemical and immunological markers of the axenic amastigotes
must be confirmed and a high concentration of serum is required in some systems. Differences
in drug sensitivity between axenic L. donovani amastigotes and intracellular amastigotes have been observed [18]. As a general rule, the lack of an effect on macrophages results in free access
of the parasites to drug and consequently, a high number of active compounds are determined.
Because Leishmania is an intracellular pathogen, the use of an intracellular assay to confirm effects
is recommended.
Intracellular assay: Because promastigotes are significantly more susceptible to drug-induced effects
than amastigotes, tests using promastigotes must be considered as preliminary. Use
of an additional intracellular assay will provide the most relevant information about
the compound efficacy [39]. This is also a time-consuming assay, as initial cytotoxicity evaluation of the
test samples is mandatory to avoid the use of high sample concentrations, which could
harm macrophages. Many mammalian cells have been used to host Leishmania amastigotes, including mouse peritoneal macrophages, bone marrow-derived macrophages,
peripheral blood monocyte-derived macrophages, and the tumour monocyte-derived macrophage
THP-1, resulting in very significant variations in drug susceptibility [40]. Data from our lab and from the literature have indicated that mouse peritoneal
macrophages are the most accurate model, providing reproducible results after 96–120 h
and an elevated ratio of infection (> 90 %), which is essential for an accurate analysis.
Amastigotes are appropriate parasites for in vitro macrophage infection. Although promastigotes can be used to infect macrophages as
an alternative, the maintenance of parasites inside the host cell for an adequate
period (96 to 120 h) is not possible with most Leishmania species. This is a result of the effective macrophage elimination of promastigotes
by oxidative mechanisms [41]. Amastigotes are best for in vitro infection, as the penetration into macrophages is “noiseless”. This may be attributed
to an elevated level of phosphatidylserine [42], leading to the expression of anti-inflammatory cytokines, such as TGF-β [43]. Furthermore, amastigotes can resist reactive oxygen species from the host cell
by increasing the expression of protective antioxidant enzymes [44]. Thus, the use of amastigotes is highly recommended for macrophage infections. However,
this is somewhat time consuming since the amastigotes must be isolated from infected
animals. Alternatively, amastigotes may be obtained by in vitro transformation of promastigotes with minimal changes to the growth conditions and
temperature [45]. This method provides effective drug susceptibility assays with persistent infection
in macrophages.
After drug testing (96 to 120 h), slides are fixed with methanol and stained with
Giemsa for microscopic observation (1000 × magnification; oil immersion). Drug efficacy
is determined by scoring the mean reduction in the parasitic index (PI = mean number
of infected cells × mean number of amastigotes per macrophage) out of 500 macrophages.
Other methods
Reporter gene technology: Reporter genes are used to create modified parasites with a readily measurable phenotype
that can be easily distinguished. The use of reporter genes to monitor intracellular
microorganism proliferation has been effectively used for bacteria [46], [47] and other parasites [48], [49]. Such methods produce objective quantitative data, high throughput, and require
minimal manual labour. A variety of reporter genes have been effectively used in biological
screenings, including firefly luciferase, green fluorescent protein (GFP), β-galactosidase, β-lactamase, chloramphenicol acetyltransferase (CAT), and alkaline phosphatase [50]. These reporters facilitate sample screening with high sensitivity.
The firefly luciferase gene is a common reporter gene that catalyses the reaction
of luciferin with adenosine triphosphate (ATP) to generate photons. The biochemical
output can be used to access the transcriptional activity in cells and to measure
the cellular ATP level in a cell viability assay [51]. A recombinant L. (L.) amazonensis that expresses the firefly luciferase gene has been developed [51], allowing the observation of parasitism in real time in live animals. Luciferase
activity, which is derived from a standard curve established with purified amastigotes,
is measured to determine the parasite burden in macrophages or in mouse tissues. The
major disadvantages of this assay are the high cost of the luciferin substrate and
the short half-life of gene expression [52].
Green fluorescent protein (GFP) allows imaging and quantification of parasites using
a fluorimeter [53], [54], a fluorescence microscope, or by fluorescent activated cell sorter (FACS) analysis
[51]. Transgenic L. (L.) donovani expressing GFP has been developed and can be expressed in promastigotes and amastigotes
for approximately one year without drug pressure. Thus, screening of drug candidates
by FACS analysis can be performed [55]. The major disadvantages of this assay are the need for post-translational modifications
and low sensitivity [52].
Colourimetric assays to detect protein expression are also promising techniques for
drug screening with promastigotes and intracellular amastigotes. Promastigotes of
Leishmania that express β-galactosidase [56] can be used for colourimetric detection. However, some of the drawbacks of the β-galactosidase assay include its large size (the monomer is 116 kDa) and the endogenous
expression of this protein by some mammalian cell types, including macrophages, preventing
the use of these cells for drug screening [57]. Buckner and Wilson [58] reported the use of a β-lactamase reporter gene to quantify L. (L.) major and L. (L.) amazonensis amastigotes in macrophages in a 96-well format. The β-lactamase gene was integrated into an rRNA region of the genome, allowing for stable
expression of the enzyme [59], [60]. Previously, Leishmania parasites were quantified and large sample libraries were screened using a spectrophotometer
and a colourimetric nitrocefin reaction [58]. Ideal expression stability was determined by passing the parasites through mice.
However, the use of fluorescent markers or enzymes for the screening of large libraries
has many drawbacks. Typically, transfected parasites do not express a sufficient amount
of fluorescent protein for microplate reader evaluation and thus require the use of
FACS analysis, which decreases the throughput. In addition, the current status of
reporter gene technology is far from ideal conditions. Reporters are not inert and
may affect parasite or host physiology. Furthermore, the use of sophisticated and
expensive equipment, such as a FACS sorter, luminometers, and spectrofluorometers,
limits the use of these tests, especially in developing countries.
Flow cytometry: Flow cytometry is commonly used for diagnostics; however, it is also a useful tool
for screening drug candidates against Leishmania [61]. Leishmania promastigote viability can easily be assessed by SYBR-14, a membrane-permeable nuclear
stain, in combination with propidium iodide (PI), a nucleic acid dye that is unable
to penetrate intact living cells [62]. Alternatively, Leishmania promastigote viability can be quantified by measuring cell division using 5,6-carboxyfluorescein
diacetate succinimidyl ester (CFSE) staining [61]. Transgenic Leishmania that express fluorescent reporter proteins, such as GFP, require a flow cytometry
(FACS)-based method to assess parasite viability [41].
Sample screening can be satisfactorily performed using stained Leishmania promastigotes to infect macrophages in combination with a fluorescent marker, such
as 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF‐Am),
SYTO 17, and PKH2-GL, followed by flow cytometry; however, this method requires drug
incubation periods no longer than 1 day [63]. Though this technique can be used for amphotericin B, it is not suitable for screening
compounds that require a 96- to 120-h incubation time.
Radioactive nucleotides: The incorporation of 3[H] thymidine to assess Leishmania growth has been used since 1977 [64]. The technique was also used for drug discovery to evaluate Leishmania promastigote viability [65]. Despite its high sensitivity, this test has been less popular in recent years because
of the generation of radioactive waste and low throughput.
Acid phosphatase activity: Assays based on the enzymatic hydrolysis of p-nitrophenyl-phosphate are an alternative
method for the spectrophotometric determination of Leishmania promastigote survival [66]. This assay is simple, inexpensive, and highly reproducible, but its use in literature
is very limited.
Therapeutic targets in leishmania and high-throughput screening (HTS): HTS is a well-established process to screen large chemical libraries against biological
targets via the use of automation, miniaturised assays, and large-scale data analysis.
This is widely used by pharmaceutical companies and, to a lesser extent, by academic
researchers, to meet the demand for faster screening of small molecule libraries,
which are based on genomics or combinatorial chemistry [67]. Initially, HTS studies were performed with a library composed of 50 000 to 350 000
compounds in a 96-well microplate format. Currently, most screening projects are performed
with 500 000 to 1 500 000 compounds using 1536-well plates in an ultra-HTS system.
Thus, this assay requires selective and validated biochemical targets as well as a
large number of compounds. However, the majority of targets in an HTS-based lead discovery
using mammalian cells fall into a modest set of target families, such as kinases,
proteases, phosphatises, oxidoreductases, phosphodiesterases, and transferases [68].
Unfortunately, only a small number of potential HTS targets have been validated for
leishmaniasis. These include trypanothione metabolism, cysteine peptidases, sterol
biosynthesis (14-α-demethylase), dihydrofolate reductases, polyamine metabolism (ornithine decarboxylase),
and tubulin [69]. The lack of genetic or chemical validation of novel targets and the difficulty
of obtaining large libraries for the discovery of novel inhibitory compounds limits
the improvement of HTS-based screening against Leishmania. However, a recent study screened 80 000 compounds using Leishmania GDP-mannose phosphorylase as a readout, resulting in twenty selective nontoxic candidates.
These compounds were also active in vitro against intracellular amastigotes [70].
Complementary assays – the selectivity index (SI)
For proper drug evaluation, it is good practice to evaluate in vitro cytotoxicity in mammalian cells. The correlation between in vitro cytotoxicity and antiparasitic activity (EC50 against mammalian cells/EC50 against Leishmania) is given by the selectivity index (SI). The EC50 value for test compounds is determined by treating one or a panel of mammalian cells
with a serial dilution of the compound. A candidate compound must have an SI higher
than 1, otherwise the compound is more toxic in mammalian cells than in Leishmania. In particular, for antileishmanial assays, the use of macrophages is recommended
in order to determine the highest drug concentrations for the intracellular assay.
One should consider that SI is a prediction of in vitro toxicity, and a large panel of cells must be tested to assess in vitro efficacy of the candidate compound.
Marine Organisms as a Source of Antileishmanial Compounds
Marine Organisms as a Source of Antileishmanial Compounds
During the past 60 years, marine organisms have been investigated as a source of structurally
unique and biologically active secondary metabolites [71]. The discovery of the first antiparasitic marine natural product in the late 1980s
gained the attention of several research groups around the world, and marine secondary
metabolites are now being evaluated as drug leads for treatment of neglected diseases,
such as malaria, Chagas disease, and leishmaniasis. Currently, there are numerous
academic initiatives to discover antiparasitic marine natural products from specific
organisms, such as cyanobacteria, or in specific geographical regions, such as in
Central America, New Zealand, and South America. Other research programs have also
significantly contributed to the discovery of antileishmanial marine natural products.
The findings of these groups are comprehensively summarised and discussed in the following
sections.
Peptides ([Figs. 1] and [2])
Fig. 1 Structures of antileishmanial modified peptides 1–9.
Fig. 2 Structures of antileishmanial modified peptides 10–16.
The marine cyanobacterium Lyngbya majuscula is an outstanding source of biologically active and structurally unique secondary
metabolites. A strain of L. majuscula isolated from mangrove roots yielded a crude extract that displayed strong in vitro antileishmanial activity against L. (L.) donovani amastigotes. Chromatographic separation of this crude extract yielded the N-methylated linear lipopeptide, almiramide A–C (1–3). Biological evaluation of these three peptides showed that almiramide B (2) and C (3) display strong in vitro antiparasitic activity against L. (L.) donovani amastigotes (EC50 = 2.4 and 1.9 µM for 2 and 3, respectively) and weak cytotoxicity in mammalian Vero cells (52.3 and 33.1 µM for
2 and 3, respectively). Almiramide A (1) was inactive up to 13.5 µM [72].
Dragonamide A (4), E (5), and herbamide B (6) were also isolated from Lyngbya majuscula and displayed comparable in vitro activity against L. (L.) donovani (axenic amastigotes) (EC50 = 6.5, 5.1, and 5.9 µM, respectively) [73].
Viridamide A (7) isolated from Oscillatoria nigro-viridis displayed antiparasitic activity against L. (L.) mexicana amastigotes as assessed by a DNA fluorescence method. In addition, viridamide A (7) showed antileishmanial activity with an EC50 of 1.5 µM [74].
Bioassay-guided fractionation of organic extracts from the cyanobacteria Schizothrix sp. led to the isolation of a highly functionalised linear peptide, gallinamide A.
Gallinamide A (8) was tested against Leishmania (L.) donovani promastigotes and displayed antiparasitic activity at EC50 9.3 µM. It also displayed moderate cytotoxicity in Vero cells (EC50 = 10.4 µM) and in vitro cytotoxicity in NCI-H460 human lung tumour and neuro-2a mouse neuroblastoma cell
lines up to 16.9 µM [75].
Kahalalide F (9) is a cyclic depsipeptide derived from the Hawaiian herbivorous marine mollusc Elysia rufescens. Kahalalide F (9) was tested for its activity and plausible mode of action against Leishmania promastigotes (L. [L.] donovani and L. [L.] pifanoi) and amastigotes (L. [L.] pifanoi). In general, amastigotes were more resistant than promastigotes. Kahalalide F inhibited
L. (L.) donovani and L. (L.) pifanoi at concentrations of 6.13 µM and 8.31 µM, respectively, and the amastigotes of L. (L.) pifanoi at 29.53 µM. Compound 9 also showed cytotoxic activity against peritoneal macrophages (10.23 µM) and bovine
aortic endothelial cells (25.8 µM) [76].
Ciliatamides A–C (10–12) isolated from the deep sea sponge Aaptos ciliata were evaluated for antileishmanial activity against L. (L.) major promastigotes using a fluorometric assay. Ciliatamides A (10) and B (11) showed 50 % growth inhibition at 10.0 µg/mL, but ciliatamide C (12) was not active [77].
Valinomycin (13) is a cyclic depsipeptide isolated from Streptomyces sp. strains found in the Mediterranean sponges Axinella polypoides and Aplysina aerophoba. Valinomycin (13) displayed antiparasitic activity against L. (L.) major promastigotes (EC50 < 0.11 µM) and also exhibited cytotoxicity against 293T kidney epithelial cells (EC50 = 11.24 µM) and J774.1 macrophages (EC50 < 0.10 µM) [78].
The cyanobacterium Symploca sp. from Papua New Guinea yielded symplocamide A (14), which displayed activity against another protozoan parasite, Plasmodium falciparum; however, no activity against L. (L.) donovani promastigotes was observed up to 9.5 µM. A citrulline residue and a N,O-diMe-Br-Tyr moiety are unique structural features of symplocamide A [79].
Oscillatoria sp., a cyanobacterium from the Portobelo National Marine Park, yielded venturamide
A (15) and B (16). Modified peptides 15 and 16 exhibited mild activity against L. (L.) donovani amastigotes, both with an EC50 of 19.0 µM [80].
The glycoprotein pachymatismin was isolated from the sponge Pachymatisma johnstonii as a cytotoxic agent that was active against promastigotes of various Leishmania species and amastigotes of L. (L.) mexicana. Pachymatismin induced changes in Leishmania spp. cell shape, inhibited phospholipase A2 activity and decreased the invasion capacity.
Every promastigote species that was tested was inhibited by pachymatismin with an
EC50 value between 0.6 and 2.5 µg/mL. Pachymatismin was also active against L. (L.) mexicana promastigotes between 1.0 and 1.2 µg/mL. In an attempt to evaluate the therapeutic
potential of this glycoprotein, the effects on the amastigote stage of Leishmania spp. were studied. In acidic pH, antileishmanial activity was obtained against axenic
amastigotes at the same EC50 value [81].
Terpenoids ([Figs. 3], [4] and [5])
Fig. 3 Structures of antileishmanial terpenes 17–26.
Fig. 4 Structures of antileishmanial terpenes 27–34.
Fig. 5 Structures of antileishmanial terpenes 35–37 and antileishmanial alkaloids 38–53.
A series of terpenoids (17–29) was isolated from the sponges Spongia sp. and Ircinia sp. at the Turkish coastline of the Aegean Sea. Most of these terpenoids inhibited
L. (L.) donovani (axenic amastigotes) growth. Among these compounds, 11β-acetoxyspongi-12-en-16-one (28) had the most potent antileishmanial activity against L. (L.) donovani with an EC50 value of 0.75 µg/mL; this value is comparable to that of miltefosine (EC50 = 0.20 µg/mL). Unfortunately, however, the same compound showed almost identical
cytotoxicity toward mammalian cells, indicating a lack of selectivity. Furospongin-1
(18) and 4-hydroxy-3-octaprenylbenzoic acid (23) also displayed antileishmanial activity with EC50 values of 4.80 and 5.60 µg/mL, respectively. The remaining active compounds had moderate
EC50 values ranging from 9.6 µg/mL to 18.9 µg/mL. Compounds 17 and 18 are structurally very similar, with the exception that 18 contains an additional isoprene unit. The length of the isoprene chain appears to
have no impact on the inhibition of L. (L.) donovani axenic amastigotes in culture. The remaining compounds had either low or no cytotoxic
potential against mammalian cells (EC50 > 90 µg/mL) [82].
Sesquiterpene (S)-(+)-curcuphenol (30), which was isolated from the Jamaican sponge Myrmekioderma styx, showed activity against several human cancer cell lines, in vitro antimicrobial activity, and antimalarial and antileishmanial activity against L. (L.) donovani promastigotes with an EC50 of 11.0 µM, which is similar to the EC50 of pentamidine and amphotericin B (EC50 = 4.7 and 1.2 µM, respectively) [83].
Holothurins A (31) and B (32) were isolated from the sea cucumber Actinopyga lecanora. Compound 31 showed moderate antiparasitic activity in vitro and in vivo against L. (L.) donovani. At 100.0 µg/mL, holothurin A induced 73.2 ± 6.8 % and 66 ± 6 % inhibition in promastigotes
and intracellular amastigotes, respectively. Holothurin A also displayed 44.6 ± 10.6 %
inhibition of L. (L.) donovani at a dose of 100 mg · kg−1 · day−1 after a 5-day treatment of infected hamsters. Compound 32 inhibited promastigotes growth 82.5 ± 11.6 % and 47.3 ± 6.5 % at 100.0 and 50.0 µg/mL,
respectively. Holothurin B 32 also inhibited intracellular amastigotes 57.6 ± 8.4 % and 78 ± 10 % at 50.0 and 100.0 µg/mL,
respectively. In L. donovani-infected hamsters, holothurin B had a better activity profile (71.5 ± 12.8 %; p < 0.005)
than holothurin A, after a 5-day treatment at 100 mg · kg−1 · day−1 [84].
Pandaroside G (33) and its methyl ester pandaroside G (34) were isolated from the Caribbean sponge Pandaros acanthifolium. The glycosides 33 and 34 inhibited L. (L.) donovani (axenic amastigotes) growth with an EC50 of 1.3 and 0.051 µM, respectively. Compounds 33 and 34 appeared to be highly toxic against L6 cells, a primary cell line derived from mammalian
(rat) skeletal myoblasts (EC50 = 5.4 and 0.22 µM, respectively). Thus, 33 and 34 may be viable antiparasitic lead compounds [85].
The meroterpenoid isoakaterpin (35) was isolated from the Brazilian marine sponge Callyspongia sp. as an inhibitor of Leishmania tarentolae adenine phosphoribosyl transferase (L-APRT). This enzyme participates in the Leishmania spp. purine salvage pathway. Isoakaterpin (35) inhibited Leishmania tarentolae APRT with an EC50 of 1.05 µM, and it is the most potent known inhibitor of this enzyme [86].
The unique terpenoid euplotin C (36) was isolated from the marine ciliate Euplotes crassus. This is a relatively abundant compound, suggesting a possible ecological role in
E. crassus. Euplotin C (36) displayed antiparasitic activity to both promastigotes of L. (L.) major and L. (L.) infantum with EC50 values of 4.6 and 8.1 µg/mL, respectively. Leishmanicidal activity was also observed
in the euplotin-producing cells of E. crassus when cultured with promastigotes; this activity increased in a time-dependent manner
during a 6-h incubation. Euplotin C induced nonsignificant cytotoxicity (EC50 > 200 µg/mL) against the J774 cell line [87].
Ilimaquinone (37), isolated from the marine sponge Smenospongia sp., inhibited the L. (L.) mexicana promastigote growth. In addition, it induced promastigote morphological and ultrastructural
changes. Comparison of the effects of ilimaquinone, ketoconazole, terbinafine, and
dermaseptin on L. (L.) mexicana promastigotes showed that ilimaquinone activity was equivalent to that of terbinafine.
Both compounds showed 100-fold less antiparasitic activity than ketoconazole. Dermaseptin
has been shown to bind the surface of parasites and alter the lipid bilayer. Ilimaquinone
(37) inhibited L. (L.) mexicana promastigote growth in a dose-dependent manner, killing 100 % of parasites at 24 µM.
The ilimaquinone EC50 was 5.6 µM, whereas the EC50 for terbinafine was 8.5 µM. Finally, the dermaseptin EC50 against promastigote growth was 3 µM. Ketoconazole completely lysed L. (L.) mexicana promastigotes at 0.06 µM [88].
Alkaloids ([Figs. 5] and [6])
Fig. 6 Structures of antileishmanial bromopyrrole alkaloids 54–65.
Staurosporine (38), a well-known inhibitor of protein kinase C and platelet aggregation, was isolated
from Streptomyces sp. strain 11 (GU214750) found in an unidentified Mediterranean sponge. Staurosporine
was active against L. (L.) major promastigotes with an EC50 of 5.30 µM; however, it showed general cytotoxicity against 293T kidney epithelial
cells (EC50 = 1.30 µM) and J774.1 macrophages (EC50 < 0.13 µM) [78].
The indole alkaloid tryptophol (39), isolated from the marine sponges Spongia sp. and Ircinia sp. collected from the Turkish coastline of the Aegean Sea, was evaluated for in vitro activity against the mammalian stage of L. (L.) donovani (axenic amastigotes). This compound had an EC50 of 9.60 µg/mL and a low level of mammalian cell cytotoxicity (EC50 = 63.46 µg/mL) [80].
The alkaloid araguspongin C (40), isolated from the marine sponge Haliclona exigua, moderately inhibited promastigote and intracellular amastigote growth at 100 µg/mL.
In addition, it had weak in vivo antileishmanial activity without inducing cytotoxic effects up to a concentration
of 100 µg/mL [89], [90].
Three tricyclic guanidine alkaloids, 1,8a;8b,3a-didehydro-8b-hydroxyptilocaulin (41), 1,8a;8b,3a-didehydro-8ahydroxyptilocaulin (42), and mirabilin B (43), were identified from the marine sponge Monanchora unguifera. Mirabilin B (43) was tested against L. (L.) donovani promastigotes and amastigotes, and presented an EC50 value of 17 µg/mL [91].
Bioassay-guided fractionation of extracts from the sponge Neopetrosia sp. yielded renieramycin A (44), which inhibited the viability of a recombinant L. (L.) amazonensis promastigote that expresses the enhanced green fluorescent protein La/egfp with an EC50 of 0.2 µg/mL. Renieramycin A (44) was also cytotoxic against P388 murine leukaemia cells at a 10-fold higher concentration
(EC50 = 2.2 µg/mL) [92].
A series of ascididemin and pyridoacridine alkaloid derivatives have been tested against
extracellular and intracellular L. (L.) donovani. Compound 45 (unnamed) displayed the most potent extracellular antiparasitic activity using promastigotes
(EC50 = 0.78 µg/mL). Since the majority of the derivatives evaluated displayed significant
in vitro cytotoxicity against RAW 264.7 cells, it was difficult to evaluate their activity
against L. (L.) donovani amastigotes. Derivative 46 was the strongest inhibitor of intracellular L. (L.) donovani axenic amastigote viability (EC50 = 35.0 µg/mL) [93]. Isoaaptamine (47), which was isolated from an Aaptos sponge, is structurally related to 46 and displayed potent antileishmanial activity against L. (L.) donovani promastigotes (EC50 = 0.7 µg/mL). Isoaaptamine was more active than both pentamidine (EC50 = 1.6 µg/mL) and amphotericin B (EC50 = 1.1 µg/mL), drugs which are currently used for leishmaniasis treatment. Moreover,
isoaaptamine had no cytotoxicity against the Vero TC50 cell line [94]. Thus, the structure scaffolds of both 46 and 47 deserve further investigation for the development of antileishmanial agents.
The common Indo-Pacific sponge Acanthostrongylophora sp. is a rich source of bioactive manzamine-related alkaloids. This class of alkaloids
is known to display a number of potent biological activities, including cytotoxic,
insecticidal, antibacterial, anti-inflammatory, anti-infective, and antiparasitic
activities [95], [96], [97]. Several of such alkaloids displayed potent antiparasitic activity against L. (L.) donovani [95], [96], [97], [98], including manzamine A (48), (+)-8-hydroxymanzamine A (49), manzamine Y (50), manzamine E (51), 6-hydroxymanzamine E (52), and manzamine F (53). In tests using L. (L.) donovani promastigotes, manzamine A (48) displayed the most potent activity against viability ([Table 1]) [95], [97]. However, manzamine alkaloids also induce significant cytotoxicity in mammalian
cells. Compounds 51 and 53 did not induce cytotoxicity at a concentration of 4.76 µg/mL [95], [98].
Table 1 Antiparasitic activity of manzamine derivatives [95], [98].
|
Manzamine derivative
|
Leishmania (L.) donovani*
|
Cytotoxicity (Vero cells)
|
|
|
EC50 (µg/mL)
|
EC90 (µg/mL)
|
EC50 (µg/mL)
|
|
48
|
0.9
|
1.8
|
1.2
|
|
49
|
6.2
|
11
|
1.1
|
|
50
|
1.6
|
8.0
|
3.9
|
|
51
|
3.8
|
6.8
|
n. c.
|
|
52
|
2.5
|
4.3
|
4.3
|
|
53
|
4.2
|
7.0
|
n. c.
|
|
* Promastigotes; n. c. = no cytotoxicity
|
The effects of bromopyrrole alkaloids 54–65, which are obtained from marine sponges belonging to the Axinella and Agelas genera, were evaluated in vitro against L. (L.) donovani (axenic amastigotes) and against L6 cells. The majority of alkaloids induced L. (L.) donovani growth inhibition, particularly longamide B (60) (EC50 = 3.85 µg/mL) and dibromopalau'amine (63) (EC50 value 1.09 µg/mL). The activity profiles of these compounds are quite remarkable;
the effective concentration ranges on the same order of potency as the control compound,
miltefosine (EC50 = 0.21 µg/mL). The other bromopyrrole alkaloids were much less active than 63 and 60, and dispacamide B (54), bromoaldisin (59), and longamide A (61) were completely inactive. When tested against mammalian (L6) cells, only dibromopalau'amine
(63) and longamide B (60) induced toxicity (EC50 = 4.46 and 9.94 µg/mL, respectively) [99].
Polyketides ([Fig. 7])
Fig. 7 Structures of antileishmanial polyketides 66–79, antileishmanial xanthones 80–82, purine 83 and antileishmanial sterols 84–90.
The effects of pseudopyronines A (66) and B (67), which were isolated from fermentation extracts of Pseudomonas fluorescens, on the growth of L. (L.) donovani were evaluated as well as their cytotoxicity against the mammalian L6 and P388 cell
lines. Pyrones (66) and (67) exhibited antiparasitic activity against L. (L.) donovani axenic amastigotes (EC50 = 2.63 and 1.38 µg/mL, respectively) and induced cytotoxicity in P388 leukaemia cells
(EC50 = 4.7 and 5.4 µg/mL, respectively) at a lower concentration than against primary
mammalian L6 cells (EC50 = 23.2 and 17.9 µg/mL, respectively) [100].
Polyketides 68–72 isolated from the sponge Plakortis angulospiculatus from Brazil killed 100 % of L. (L.) chagasi promastigotes at a maximal concentration of 25 µg/mL. These effects of polyketides
68–72 were dose-dependent with an EC50 value in the range of 1.9–8.5 µg/mL. Plakortide P (69) had the most effective antileishmanial activity (EC50 = 1.9 µg/mL). Compounds (68–72) were also submitted to an intracellular amastigote assay and demonstrated significant
anti-amastigote and leishmanicidal activity (EC50 = 0.50–3.40 µg/mL). Each compound (68–72) tested also induced mammalian cytotoxicity in a dose-dependent manner (EC50 = 16.6–31.6 µg/mL) [101].
The investigation of the antiparasitic effects of polyketides 68–72 was based on previous studies of the antiparasitic activity against L. (L.) mexicana reported for very similar polyketides, which were also isolated from Plakortis angulospiculatus. Among the polyketides 73–76, which are members of the plakortin family, (3S,6R,8S)-4,6-diethyl-3,6-epidioxy-8-methyldodeca-4-enoic
acid (73) displayed the most potent activity (EC50 = 0.29 µg/mL) and disrupted the L. (L.) mexicana promastigote cell membrane after 24-h treatment at a concentration of 1 µg/mL. A
significant decrease in parasite mobility was noted within 30 minutes after drug addition.
Compound 74 (EC50 = 1.0 µg/mL) was less effective, whereas the polyketides 75 and 76 showed similar activity but at higher doses (EC50 = 1.86 and 2.7 µg/mL, respectively) [102].
In addition, the antiparasitic polyketides gracilioethers A–C (77–79) have been isolated from the sponge Agelas gracilis. Gracilioether B (78) inhibited 68 % of L. major promastigote growth at 10 µg/mL; however, no such activity was reported for compounds
77 and 79 [103].
Xanthones ([Fig. 7])
The marine-derived fungus Chaetomium sp. yielded chaetoxanthones A–C (80–82) and these compounds were tested against L. (L.) donovani intracellular amastigotes. Chaetoxanthone B (81) was active at an EC50 of 3.4 µg/mL and displayed no cytotoxicity against L6-cells (EC50 > 90 µg/mL) or tumour cell lines (mean EC50 > 10 µg/mL). Chaetoxanthone C (82) was moderately active with an EC50 value of 3.1 µg/mL, and cytotoxicity against L6 cells was not observed (EC50 = 46.7 µg/mL) [104].
Agelasine D (83), isolated from the marine sponge Agelas sp., was tested against L. (L.) infantum amastigotes and for toxic effects against MRC-5 fibroblast cells. Compound (83) displayed weak in vitro activity against L. (L.) infantum with an EC50 value of 1.5 µg/mL and induced toxic effects against MRC-5 cells (EC50 = 6.7 µg/mL) [105].
Isolated from the sponge Petromica ciocalyptoides, the steroid halistanol A (84) inhibited adenine phosphoribosyl transferase isolated from L. tarentolae (L-APRT) in a dose-dependent manner; 92 % inhibition was observed at a concentration
of 25 µg/mL (EC50 of 2.87 µg/mL). However, a desulphated derivative of 84 did not inhibit L-APRT. Halistanol A (84) was also tested on the promastigotes of L. (L.) chagasi but did not show any antiparasitic activity [106].
24-Ethyl-cholest-5α-7-en-3-β-ol (85) isolated from Agelas oroides inhibited L. (L.) donovani amastigotes with an EC50 of 29.5 µg/mL and induced cytotoxicity with an EC50 higher than 90 µg/mL in L6 cells [107]. Norselic acids A–E (86–90), isolated from the sponge Crella sp. from Antarctica, were also active against the Leishmania parasite. Each norselic acid was active against Leishmania sp. promastigotes at the following concentrations: 2.5 µM for 86; 2.4 µM for 87; 2.6 µM for 88; 2.0 µM for 89 and 3.6 µM for 90 [108].
Conclusion
Conclusion
There is a pressing need for inexpensive, rapid, and reproducible techniques to screen
candidate compounds for the treatment of leishmaniasis. Without access to large libraries
of compounds and without the collaborative work of multidisciplinary research groups,
novel antileishmanial drugs are difficult to develop. Due to these factors, as well
as the poor population that this disease affects, it is clear that big pharmaceutical
companies will not include neglected parasitic diseases in their research programs.
Therefore, major drug discovery efforts must come from academic research institutions.
Secondary metabolites isolated from both marine macro- and microorganisms are usually
structurally complex and display potent biological activities. Since the pioneering
discovery of the antileishmanial compound ilimaquinone (37) in 1997 [88] and of Plakortis polyketides in 1998 [102], several marine natural products have shown to inhibit several Leishmania strains. Considering that leishmaniasis is one of the major public health problems
in developing countries, the discovery of these marine metabolites is very relevant
to the search for novel antileishmanial chemotherapy.
Although modified peptides that were isolated from marine sources are very potent
against Leishmania parasites, these compounds are not suitable as scaffolds for the development of drug
leads because peptide synthesis is expensive and the active peptides are usually toxic
and easily hydrolysed. A possible alternative to circumvent these limitations is the
use of peptide mimetics to enhance compound bioavailability and stability under hydrolytic
conditions. Of note is the discovery of the marine polyheteroaromatic alkaloid saponins
and sterols as active cytotoxic agents against Leishmania spp. However, saponins are usually hemolytic. Thus, their use as a model for the
development of antileishmanial drugs is very limited. On the other hand, several synthetic
approaches have been developed for the production of polyheteroaromatic compounds
and steroid derivatives. Therefore, it is possible to investigate the mode of action
of such compounds in order to discover additional drug leads or novel cellular death
routes in Leishmania sp. Finally, fermentation technologies are being used to discover antileishmanial
agents from marine-derived microbial strains.
Undoubtedly, marine organisms are a very promising source of novel antileishmanial
compounds, which can be further developed as drug prototypes for leishmaniasis treatment.
Acknowledgements
Acknowledgements
The authors thank the FAPESP BIOTA/BIOprospecTA funding program (grant 05/60175-2)
for financial support to prepare this review.
