Elsevier

Bioorganic Chemistry

Volume 102, September 2020, 104056
Bioorganic Chemistry

Tolnaftate inhibits ergosterol production and impacts cell viability of Leishmania sp.

https://doi.org/10.1016/j.bioorg.2020.104056Get rights and content

Highlights

Abstract

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. The treatment of all forms of leishmaniasis relies on first-line drug, pentavalent antimonial, and in cases of drug failure, the second-line drug amphotericin B has been used. Besides the high toxicity of drugs, parasites can be resistant to antimonial in some areas of the World, making it necessary to perform further studies for the characterization of new antileishmanial agents. Thus, the aim of the present work was to evaluate the leishmanicidal activity of tolnaftate, a selective reversible and non-competitive inhibitor of the fungal enzyme squalene epoxidase, which is involved in the biosynthesis of ergosterol, essential to maintain membrane physiology in fungi as well as trypanosomatids. Tolnaftate eliminated promastigote forms of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum (EC50 ~ 10 μg/mL and SI ~ 20 for all leishmanial species), and intracellular amastigote forms of all studied species (EC50 ~ 23 μg/mL in infections caused by dermatotropic species; and 11.7 μg/mL in infection caused by viscerotropic species) with high selectivity toward parasites [SI ~ 8 in infections caused by dermatotropic species and 17.4 for viscerotropic specie]. Promastigote forms of L. (L.) amazonensis treated with the EC50 of tolnaftate displayed morphological and physiological changes in the mitochondria and cell membrane. Additionally, promastigote forms treated with tolnaftate EC50 reduced the level of ergosterol by 5.6 times in comparison to the control parasites. Altogether, these results suggest that tolnaftate has leishmanicidal activity towards Leishmania sp., is selective, affects the cell membrane and mitochondria of parasites and, moreover, inhibits ergosterol production in L. (L.) amazonensis.

Introduction

Leishmaniasis is an infectious disease caused by parasites of the genus Leishmania and it is transmitted to mammalians through the bite of the insect vector [1]. The infection outcome depends on both the host and parasite interaction as well as the infecting species [2]. Once infected, different clinical forms of leishmaniasis can be identified, ranging from cutaneous lesion to deadly visceral forms. Cutaneous forms can be classified as Localized Cutaneous Leishmaniasis (LCL), that can be caused by different species, such as L. (L.) amazonensis, L. (V.) braziliensis, L. (L.) mexicana, L. (V.) shawi, L. (V.) guyanensis, among other species [3], [4]. Mucocutaneous Leishmaniasis (ML) can be caused by L. (V.) braziliensis and anergic diffuse cutaneous leishmaniasis (ADCL) by L. (L.) amazonensis or L. (L.) mexicana [5]. Otherwise, species such as L. (L.) infantum and L. (L.) donovani infect macrophages from the liver, spleen, lymphoid organs and bone marrow, causing visceral leishmaniasis (VL) [6].

In spite of the diversity of parasite species and clinical forms of leishmaniasis, the treatment is mainly restricted to pentavalent antimonials and amphotericin B. Antimonials have been used since the early 1900s, when cases of American cutaneous leishmaniasis were treated with trivalent antimony. Nowadays pentavalent antimonial is considered the frontline drugs for all the clinical forms of leishmaniasis, although serious side effects, such as hepatotoxicity and cardiotoxicity, limits the use of such drug [7]. Amphotericin B has been used as a second-choice drug and is able to bind to ergosterol, forming complexes that arrange into ion channels, increasing membrane permeability and altering electrolyte balance, that will culminate in parasite death [8]. On the other side, amphotericin B may be a pleiotropic molecule, acting on multiple targets [9]. Although effective, patients treated with this medicine may present fever, rigors, blood pressure changes, hypoxia, renal and gastrointestinal alterations [10], that in fact, limit amphotericin B efficiency. Once formulated to liposomes, amphotericin B (Ambisome®) presents high therapeutic potential and reduced toxicity to patients, however this formulation is expensive and not affordable for the majority of affected countries [11].

Alternative drugs have been employed to overcome the ineffectiveness or side effects of the aforementioned drugs, such as miltefosine and paramomycin. In India, Nepal and Bangladesh [12] miltefosine was used in the treatment of VL due to the high rate of cure and easy administration [13], [14]. After a decade of use, the efficacy decreased and relapses have been reported in such countries [14], [15]. The main side effect of miltefosine is associated to gastrointestinal events, but hepatotoxicity and nephrotoxicity might occur [16]. Paramomycin has also been used, but with variable effectivity in the treatment of human leishmaniasis in India, and in other parts of the World. The main adverse effects include local pain, ototoxicity and hepatic biochemical changes [17], [18]. Altogether, the prevalence of the disease with drastic distinct outcomes, along with the inefficiency and toxicity of the current available drugs, emphasizes the need for more effective and less toxic treatments.

Leishmania parasites present vital biochemical routes that can be explored to find new drugs. In this regard, the sterol biosynthetic pathway can be considered an interesting target to explore and find new leishmanicidal agents. Sterols, more specifically ergosterol, is an important component of Leishmania sp. cell membranes [19]. Notably, the biosynthetic pathway of ergosterol is an interesting specific target for developing new drugs against leishmaniasis, because mammalian cells use cholesterol as the main membrane sterol, while Leishmania synthesize ergosterol and other 24-methyl sterols as important molecules for their growth, differentiation and survival [20]. In fact, it means that drugs targeting specific molecules from this pathway will present high selectivity index toward the parasites [21], [22].

In the present work, the fungicidal drug tolnaftate, that has been employed in the treatment of superficial mycosis, such as tinea cruris, tinea pedis and ringworm, was studied concerning its leishmanicidal properties. This molecule is a thiocarbamate derivative and is a selective, reversible and non-competitive inhibitor of squalene epoxidase, an enzyme involved in the biosynthesis of ergosterol [23]. In this context, it is believed that, once inhibited squalene epoxidase will not be able to convert squalene into 2,3-epoxy derivative, causing ergosterol deficiency as well as cell membrane damage, culminating in parasite death. To the best of our knowledge, this is the first report showing the activity of tolnaftate against parasites belonging to the genus Leishmania. Additionally, the effect of this compound on the parasite morphology, physiology and biochemical parameters was investigated.

Section snippets

Drugs

Tolnaftate (purity ≥ 98%), and amphotericin B (purity ≥ 99%) were purchased from Sigma-Aldrich (USA) and Cristalia Laboratory (Brazil), respectively.

Parasites

Parasites were kindly provided by Prof. Dr. Fernando Tobias Silveira from the cryobank of the “Leishmaniasis Laboratory Prof. Dr. Ralph Laison”, Department of Parasitology, Ministry of Health, Evandro Chagas Institute (Belem, Para – Brazil). They were identified using monoclonal antibodies and isoenzyme electrophoretic profiles at the Leishmaniasis

Effect of tolnaftate on promastigote forms and in host macrophages

Tolnaftate did not display leishmanicidal activity at 24 h of incubation against L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum promastigotes. Conversely, at 48 h leishmanicidal activity was observed and the effect of tolnaftate was similar among all species of tested parasites (EC50 ~ 10 μg/mL), as summarized in Table 1. Amphotericin B, was active at 24 h and its effect was maximized at 48 h on all promastigote species (Table 1).

In respect to the cytotoxicity (Table 1),

Discussion

The scientific basis to study anti-mycotic drugs against trypanosomatids relies on the fact that these parasites are dependent on the formation of ergosterol and other 24-alkyl sterols to constitute and protect biological membranes [32], and thus inhibition of key molecules of this metabolic pathway may generate disturbance in the cell membrane, inducing the death of Leishmania sp. [33]. Therefore, in the present study, the leishmanicidal activity of the antifungal drug tolnaftate, a molecule

Conclusion

Taken together, the results presented herein show that tolnaftate has leishmanicidal activity against both promastigote and amastigote forms of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum, the etiological agents of the most important clinical forms of leishmaniasis such as anergic diffuse, mucosal and visceral leishmaniasis, respectively. Through morphological, physiological, and biochemical studies, it was possible to observe that tolnaftate altered cell membranes of L. (L.)

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors would like to thank the São Paulo Research Foundation (FAPESP) for the support (Grants 2015/17623-6, 2016/00468-0 and 2018/07885-1), HCFMUSP-LIM50 and CNPq scientific research award to JHGL and MDL. Authors also thank Fernanda Thevenard for the English editing.

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