In vitro cultures for testing anti-leishmanial compounds

Anti-leishmanial drugs are tested in vitro, using both the intracellular amastigotes and the free-swimming promastigotes. For culturing Leishmania parasites from cutaneous leishmaniasis patients, parasites are recovered from a needle biopsy sample of the skin of an infected person, and after purification, parasites are put in culture either in blood agar slants or in enriched media (Ref Reference Paun22). For collecting specimens for culture for the parasites causing the visceral form of the disease, bone marrow, spleen, liver, lymph node or other tissue biopsies are taken and put in culture either in blood agar slants or in enriched media. After incubation, parasites are retrieved (Ref Reference Sundar and Rai23).

The promastigotes divide rapidly in vitro in the logarithmic phase which transforms into stationary phases from late log phase promastigotes and are highly infective (Ref Reference Arjmand, Madrakian, Khalili, Najafi Dastnaee, Zamani and Akbari24). Amastigotes obtained from human biopsies or infected animals or macrophages infected in vitro (Ref Reference Sudhandiran and Shaha25) are used for experiments. Axenic amastigotes, a type of amastigote commonly employed in in vitro research, are generated from promastigotes cultivated at 37°C and pH 5.0, which replicates the conditions of a phagolysosome of the host macrophage that the parasite uses as the survival niche (Ref Reference Chanmol, Jariyapan, Somboon, Bates and Bates26).

Typically, three methods are employed for in vitro experiments with amastigotes. First, mammalian macrophage cultures are infected with promastigotes of Leishmania spp. These parasites will transform into amastigotes between 3 and 5 days (depending on the species of the parasite). The parasites are then collected from the macrophages by differential centrifugation (30 g for macrophages and 700 g for the parasites). The second technique is the use of axenic amastigotes in vitro, generated by the method stated in the earlier paragraph. The third possible way is to collect amastigotes from skin lesions or liver biopsies and use them for in vitro testing.

In vivo testing for anti-leishmanial compounds

For in vivo animal testing, mice or hamsters are the most common models while dog and non-human primates are used less frequently (Ref Reference Loría-Cervera and Andrade-Narváez27). Several Leishmania species like Leishmania major (L. major), Leishmania mexicana (L. Mexicana) and Leishmania amazonensis (L. amazonensis) are used to generate CL (Ref Reference Loría-Cervera and Andrade-Narváez27) by injection in the foot pad of the animal. Sensitive BALB/c mice get extensive lesions that lead to the progression of the illness and serve as models for the study of CL (Ref Reference Loría-Cervera and Andrade-Narváez27).

For generating VL, mice are challenged intravenously with late log or stationary phase promastigotes of Leishmania infantum (L. infantum) or Leishmania donovani (L. donovani). Two to 4 weeks after injection, amastigotes can be recovered from the spleen and liver (Ref Reference Shivahare, Vishwakarma, Parmar, Yadav, Haq, Srivastava and Kar28). The most suitable model for visceralizing Leishmania infections is the Syrian golden hamster (Mesocricetus auratus) (Ref Reference Shivahare, Vishwakarma, Parmar, Yadav, Haq, Srivastava and Kar28). A canine model and the Asian rhesus macaques (Macaca mulatta) are quite susceptible to the Leishmania infection and are used as disease models (Ref Reference Loría-Cervera and Andrade-Narváez27).

Artemisinin

Artemisinin, a sesquiterpene lactone, and its derivatives have been widely used for malaria. Based on references to the use of the herb Artemisia annua in Chinese traditional medicine, artemisinin was discovered by Tu Youyou in 1972 (Ref Reference Wang, Xu, Wong, Liao, Jiang and Tu43). Through a collaborative effort, Chinese scientists prepared dihydroartemisinin, artemether and artesunate generically known as ‘artemisinins’ from Artemisia annua that were used as anti-malarial medications (Ref Reference Wang, Xu, Wong, Liao, Jiang and Tu43). WHO recommends artemisinin-based combination therapies as the second line of treatment for P. vivax that are resistant to chloroquine and the first line of treatment for uncomplicated P. falciparum malaria (Ref Reference Kong and Tan44). As a remedy for malaria in many endemic areas, artemisinin derivatives such as artesunate and artether are used solo or in combination (Ref Reference Chen and Hsiang45, Reference Krishna, Bustamante, Haynes and Staines46). Artemisinin is a tetracyclic 1,2,4-trioxane containing an endoperoxide bridge (C–O–O–C), the key pharmacophore of the drug. (Figure 4). Notably, it is not only malaria that artemisinin is used for, but it is also prescribed for phylogenetically unrelated parasitic infections of public health importance like schistosomiasis (Ref Reference Issa, Warsame, Mahamat, Saleh, Boulotigam, Djimrassengar and Menard47) or other helminthic diseases caused by nematodes, trematodes, cestodes and others (Ref Reference Salas-Coronas, Vázquez-Villegas, Villarejo-Ordóñez, Sánchez-Sánchez, Espada-Chavarría, Soriano-Pérez and Cabezas-Fernández48–Reference Bergquist and Elmorshedy50). Therefore, this drug has a wide range of efficacy against parasites which makes it suitable for repurposing.

Figure 4.

In vivo tested plant-based possible anti-leishmanials that could target mitochondria. Artemisinin, chloroquine, quercetin, coumarin and R1–R5 quinolone are shown.

In an in vitro and an ex-vivo study for VL, apoptotic death of L. donovani promastigotes and intracellular amastigotes was induced by artemisinin with IC₅₀ values of 160 and 22 μM, respectively (Ref Reference Sen, Bandyopadhyay, Dutta, Mandal, Ganguly, Saha and Chatterjee51). A loss of mitochondrial potential occurred before initiation of the death process (Ref Reference Sen, Bandyopadhyay, Dutta, Mandal, Ganguly, Saha and Chatterjee51). Other studies confirmed similar observations in in vitro and ex-vivo studies showing leishmanicidal activity against L. donovani with 50 % inhibitory concentration of 14.63 ± 1.49 μg ml⁻¹ and 7.3 ± 1.85 μg ml⁻¹, respectively, against the promastigotes and intracellular amastigotes. Reactive oxygen species (ROS) and nitric oxide NO were shown to precipitate mitochondrial changes leading to parasite death by apoptosis (Ref Reference Islamuddin, Chouhan, Tyagi, Abdin, Sahal and Afrin52). The leishmanicidal activity shown in in vitro and ex-vivo experiments was supported by in vivo studies in a model of leishmaniasis in infected BALB/c mice where the extracts were 90% effective in eliminating parasites (Ref Reference Islamuddin, Chouhan, Tyagi, Abdin, Sahal and Afrin52). Artemisinin incorporated in liposomes with increased availability demonstrated a noteworthy anti-leishmanial efficacy both in vivo in L. donovani-infected BALB/c mice and ex vivo in murine macrophages (Ref Reference Want, Islammudin, Chouhan, Ozbak, Hemeg, Chattopadhyay and Afrin53, Reference Aderibigbe54). In the BALB/c mice, the efficacy was excellent with percentage inhibition of the parasite load of 82.4% ± 3.8% in the liver and 77.6% ± 5.5% in the spleen (Ref Reference Aderibigbe54). Already tested in humans for safety (Ref Reference Li, Guo, Fu, Jian and Wang55), artemisinin and its derivatives or the combinations could be considered as potential anti-leishmanials.

For CL, intracellular amastigotes of L. amazonensis, prevalent in the Brazilian Amazon region and capable of causing CL and MCL, were eliminated when exposed to artemisinin, artemether, artesunate and dihydroartemisinin (Ref Reference Machín, Nápoles, Gille and Monzote56). The in vitro studies were supported by in vivo observations of CL in BALB/c mice created by L. amazonensis where artemisinin and artesunate could reduce parasite load and lesion size (Ref 56). Artesunate, a more stable artemisinin derivative effective in eliminating L. amazonensis parasites, also reduced lesion size and associated inflammation (Ref Reference Gugliandolo, Palma, Peritore, Siracusa, D’Amico, Fusco and Crupi57). L. major, another species of Leishmania parasite causing CL was also eliminated from skin lesions (Ref Reference Ghaffarifar, Esavand Heydari, Dalimi, Hassan, Delavari and Mikaeiloo58). The efficacy shown in vivo generates possibilities for artemisinin and its derivatives as possible drugs for CL as well.

Mechanistically, artemisinin inhibits complexes I–III and II–III of the Leishmania ETC in vitro rapidly increasing ROS, thus disrupting mitochondrial function (Ref Reference De Sarkar, Sarkar, Sarkar, Dighal, Chakrabarti, Staniek and Chatterjee59). The endoperoxide 1,2,4-trioxane ring in artemisinin is responsible for the action of artemisinin against malaria (Ref Reference Wang, Huang, Li, Fan, Long, Li and Zhou60). Structurally, the unusual peroxide bridge of artemisinin in the presence of reducing agents such as Fe²⁺, haem and Cu²⁺ becomes unstable by cleavage, a cause of the generation of carbon and oxygen-centred free radicals (Ref Reference Geroldinger, Tonner, Hettegger, Bacher, Monzote, Walter and Gille61). The suppression of ETC is a major source of ROS (Ref Reference Staniek, Gille, Kozlov and Nohl62) especially superoxide. The resulting mitochondrial malfunction affects the energy needs of Leishmania parasites, with ATP depletion causing an energy catastrophe that ultimately results in death (Ref Reference De Sarkar, Sarkar, Sarkar, Dighal, Chakrabarti, Staniek and Chatterjee59). Data on mitochondrial ΔΨm in the Leishmania parasite post artemisinin treatment corroborates observations of the collapse of the ΔΨm in malaria parasites (Ref Reference Antoine, Fisher, Amewu, O’Neill, Ward and Biagini63). The respiratory control rate which is a measure of functioning of the mitochondrial ETC, is reduced by 30% when dihydroartemisinin is added to L. braziliensis cultures, suggesting a mitochondria-targeted action (Ref Reference Grazzia, Boaventura, Garcia, Gadelha and Miguel64). The above studies give a clear indication that the primary site of artemisinin action in the parasite is the mitochondria and with its anti-leishmanial efficacy could be considered for therapy against leishmaniasis.

Chloroquine

Encouraged by the scaffold of quinine, chloroquine, an alkaloid, was first synthesized by chemists in 1934 in Germany (Ref Reference Zhou and Yue65, Reference Frosch, Schmitt, Bringmann, Kiefer and Popp66). It is an analog of quinoline with an aliphatic amino side chain at the 4-position and a chloro group at the 7-position (Ref Reference Frosch, Schmitt, Bringmann, Kiefer and Popp66)(Figure 4). Chloroquine was the drug of choice for malaria till new antimalarials like artemisinin, mefloquine and pyrimethamine were developed. Since then, chloroquine and its derivatives have been repurposed for HIV, systemic lupus erythematosus, rheumatoid arthritis and several other diseases (Ref Reference Carmo, Silva, Machado, Fontes, Pavan, Leite and Da Silva67, Reference Pandey, Sharma, Pandey, Alam, Shaquiquzzaman and Akhter68) and are being tested for anti-tumor therapies based on their action on the autophagic activities of the cell (Ref Reference Ferreira, Soares, Barreto-Junior, Nascimento, Freire-de-Lima, Delorenzi and Pinto-da-Silva69). The distribution of its range of action prompted scientists to test chloroquine and its derivatives for anti-leishmanial efficacy.

Quinolinyl β-enaminone derivatives showed leishmanicidal activity against L. donovani by impairing the mitochondrial ETC complex and inducing ROS-mediated programmed cell death with 84% clearance of parasites in infected mice and hamsters (Ref Reference Rani, Khanikar, Dutta, Katiyar, Qamar, Seth and Kar70). The addition of small heterocyclic rings on the pendent nitrogen of the 4-amino-7-cholroquinoline with replacement of the 7-chloro group with a trifluoromethyl group showed 81-99% inhibition at 10 μM concentration of promastigotes in in vitro cultures. The majority of the analogs demonstrated in vitro anti-amastigote action (IC₅₀ = 5.3–9.6 μM). The substances that demonstrated anti-amastigote activity in vitro were then examined in vivo on a hamster model of VL generated by L. donovani. At 27 days after infection, the 2,6-trichlorophenylthiazolidin-4-one molecule outperformed others, showing around 80% elimination of the Leishmania parasite in treated hamsters (Ref Reference Singh, Kashif, Srivastava and Manna71). One benefit of these compounds is that they are well-tolerated by humans and the thiazolidin/thiazinan-4-ones are biologically advantageous scaffolds (Ref Reference Verma and Saraf72). Other studies reported the identification of two 4-amino-7-chloroquinoline compounds with adamantane or benzothiophene moieties being highly effective as anti-leishmanial candidates in a mouse model of VL using L. infantum. Parasite clearance from the liver was seen at 95% and 99% for doses of 50 and 100 mg/kg, respectively (Ref Reference Konstantinović, Videnović, Orsini, Bogojević, D’Alessandro, Scaccabarozzi and Šolaja73). Interestingly, Sitamaquin, an 8-aminoquinoline analogue, demonstrated superior anti-leishmanial effectiveness in vivo (Ref Reference Loiseau, Cojean and Schrével74). This orally administered 8-amino-quinoline showed efficacy in various animal models of leishmaniasis and encouraging efficacy against various species of Leishmania was found in preliminary clinical studies in Kenya and Brazil (Ref Reference Jha, Sundar, Thakur, Felton, Sabin and Horton75). Nitroimidazo-oxazole compound DNDI-VL-2098 was previously identified as a favourable candidate but was found to cause organ toxicity in animal studies of longer duration and hence was not taken further (Ref Reference Gupta, Yardley, Vishwakarma, Shivahare, Sharma, Launay and Puri76). However, its oxazine derivative DNDI-0690 was recognized as a promising candidate for a Phase I trial for VL (Ref Reference Van Bocxlaer, McArthur, Harris, Alavijeh, Braillard, Mowbray and Croft77) and also for CL (Ref Reference Wijnant, Croft, de la Flor, Alavijeh, Yardley, Braillard and Van Bocxlaer78). The added advantage of the above compound is its efficacy against CL in addition to VL. In 2022, the evaluation of the safety and pharmacokinetic properties of DNDI-0690 was completed. The drug was advanced for consideration for efficacy testing in leishmaniasis patients (Ref Reference Antinarelli, Dias, Souza, Lima, Gameiro, da Silva and Coimbra79). Therefore, the above compounds or their modified versions offer possibilities for treatment of VL.

The 4,7-dichloroquinoline linked together with diamines, diaminealkynes and diaminedialkynes, a series of 4-amino-7-chloroquinoline derivatives showed strong anti-leishmanial activity against L. chagasi, L. braziliensis, L. major and L. amazonensis all causing CL (Ref Reference Carmo, Silva, Machado, Fontes, Pavan, Leite and Da Silva67). These studies again suggested that modification at the 7-position is an attractive strategy for the development of anti-leishmanial compounds. Synthesized 4-aminoquinoline derivatives through conjugation of sulfonamide, hydrazide and hydrazine residues were effective in killing L. amazonensis promastigotes and intracellular amastigotes (Ref Reference Herrera, Llanes, Álvarez, Degracia, Restrepo, Rivera and Fernández80) showing their activity against CL. A chloroquine analog tested on L. panamensis causing tegumentary leishmaniasis showed good efficacy in an in vivo model of BALB/c mice but no mitochondrial interference was checked (Ref Reference Carvalho, Luque-Ortega, Manzano, Castanys, Rivas and Gamarro81).

Mechanistically, aminoquinolines particularly target mitochondrial complex enzymes to disrupt the electron transport system in the mitochondria of protozoan parasites like Plasmodium, Leishmania, etc. (Ref Reference Carvalho, Luque-Ortega, López-Martín, Castanys, Rivas and Gamarro82). Sitamaquin inhibits complex II of the respiratory chain of L. donovani promastigotes (Ref Reference Wang, Sheaff and Hussaini83) where oxidative stress is generated because the drug targets succinate dehydrogenase of complex II. Tafenoquine, an antiplasmodial 8-aminoquinoline, targets Leishmania respiratory Complex III and induces apoptosis (Ref Reference Carvalho, Luque-Ortega, López-Martín, Castanys, Rivas and Gamarro82). Exposure to 7-chloro-4-quinolinylhydrazone derivative to Leishmania induces a loss of mitochondrial potential and generates ROS leading to cell death (Ref Reference Herrera, Llanes, Álvarez, Degracia, Restrepo, Rivera and Fernández80). Chloroquine derivatives are also important disruptors of mitochondrial function through ATP inhibition because any interference with ETC would disrupt ATP (Ref Reference Wang, Sheaff and Hussaini83).

Coumarins

Coumarins are a naturally occurring class of phenolic compounds of the benzopyrone family first isolated from the plant named Dipteryx odorata Willd. by Vogel in 1820 (Ref Reference Küpeli Akkol, Genç, Karpuz, Sobarzo-Sánchez and Capasso84). Coumarins have a wide variety of structures with coumarin 7 as the privileged scaffold (Ref Reference Stefanachi, Leonetti, Pisani, Catto and Carotti85). They are aromatic organic chemical compounds that could be described as having a benzene molecule with two adjacent hydrogen atoms replaced by an unsaturated lactone ring forming a 6-membered heterocycle that shares two carbons with the benzene ring (Figure 4). This bicyclic heterocycle is capable of performing interactions with various biological targets (Ref Reference Carvalho, Luque-Ortega, López-Martín, Castanys, Rivas and Gamarro82) and show a vast range of biological properties like anticoagulant, anticancer, antiviral, antitrypanosomal, antioxidant and also anti-leishmanial activities. Their biological activity as antimicrobials makes them attractive candidates for the development of antibacterial and antiprotozoal medicines. Coumarin derivatives are known to interfere with mitochondrial function and lately, several coumarins have been developed with mitochondrial target function that needs to be tested as anti-leishmanial agents (Ref Reference Ortega-Forte, Rovira, Gandioso, Bonelli, Bosch, Ruiz and Marchán86). Coumarins have been used to treat prostate cancer, renal cell carcinoma, leukemia (Ref Reference Küpeli Akkol, Genç, Karpuz, Sobarzo-Sánchez and Capasso84) and used as anticoagulants (Ref Reference Flores-Morales, Villasana-Ruíz, Garza-Veloz, González-Delgado and Martinez-Fierro87).

Studies conducted in silico, in vitro and in vivo demonstrate that coumarins are suitable candidates for assessment as anti-leishmanial medications (Ref Reference Mandlik, Patil, Bopanna, Basu and Singh88). Five coumarins selected based on in silico studies were tested against CL. One derivative C2(6-Amino-3-(1,3-benzodioxol-5-yl)-2H-chromen-2-one) was used as nanoliposomal formulation against L. major promastigotes and significant anti-leishmanial activity was observed. There was a loss of ΔΨm after treatment with the compound in promastigotes. Continuing further with the study, testing in in vivo conditions showed a reduction in CL lesion sizes formed by L. major in BALB/c mice (Ref Reference Mandlik, Patil, Bopanna, Basu and Singh88). Mammea A/BB, a coumarin isolated from leaves of Calophyllum brasiliense showed anti-leishmanial activity on both promastigotes and amastigotes of L. amazonensis with mitochondrial effects. Good in vivo activity was visible in the reduced size of skin lesions in the footpads of BALB/c mice (Ref Reference Cardoso, De Mello, Lera, Brenzan, Cortez, Donatti and Lonardoni89, Reference Brenzan, Nakamura, Prado Dias Filho, Ueda-Nakamura, Young and Aparício Garcia Cortez90). Treatment with Mammea A/BB induced changes in the mitochondria of both promastigotes and amastigotes in terms of mitochondrial swelling with reduced density of the mitochondrial matrix and extra vesicles visible inside the mitochondrion, indicating damage and significant change in this organelle (Ref Reference Brenzan, Nakamura, Prado Dias Filho, Ueda-Nakamura, Young and Aparício Garcia Cortez90, Reference Tiuman, Brenzan, Ueda-Nakamura, Filho, Cortez and Nakamura91). The details of any effect on the ETC are not known. Another coumarin derivative, Mammea A/BA showed IC₅₀ of 85.8 and 36.9 μM for epimastigotes and trypomastigotes, respectively, of Trypanosoma cruzi a related parasite where a significant increase in ROS with decrease ΔΨm was observed (Ref Reference Rodríguez-Hernández, Martínez, Reyes-Chilpa and Espinoza92). Subcutaneous treatment with (+)-3-(1′-dimethylallyl)-decursinol and (-)-heliettin, two coumarins extracted from Helietta apiculata Benth reduced size of cutaneous lesions generated in BALB/c mice with L. amazonensis by 95.6% and 98.6%, respectively (Ref Reference Ferreira, de Arias, Yaluff, de Bilbao, Nakayama, Torres and Fournet93). Osthole, a natural coumarin displayed dose-dependent leishmanicidal activity against intracellular amastigotes with IC₅₀ value of 14.95 μg/ml. CL lesions generated in BALB/c mice with osthole resulted in declined lesion progression in comparison to suitable controls (Ref Reference Kermani, Sajjadi, Hejazi, Arjmand, Saberi and Eskandarian94). Earlier, aurapten, a 7-geranyloxy-coumarin was shown to have anti-leishmanial activity (Ref Reference Napolitano, Silva, Ellena, Rodrigues, Almeida, Vieira and Thiemann95). A review of natural and synthetic coumarins with anti-leishmanial efficacy details several important experiments and discusses the possibility of obtaining novel coumarin-based inhibitors (Ref Reference Gonçalves, Spillere, das Neves, Kagami, von Poser, Canto and Eifler-Lima96). To identify the ETC complex component that coumarins affect, further study is required but coumarins could be considered as potential anti-leishmanials.

Quinolones

Simple 3 carboxy quinolones were first described by J.R. Price and L.J. Drummond in 1949 (Ref Reference Bisacchi97). Quinoline alkaloids are secondary metabolites found in plants and are also produced by some bacterial species. Quinoline alkaloids have a wide range of biological activities including activity against cancer, malaria, inflammation and viruses (Ref Reference Yun, Yoon, Park and Park98, Reference Shang, Morris-Natschke, Liu, Guo, Xu, Goto and Lee99). Numerous quinolones like quinine, quinidine, cinchonine and cinchonidine have been obtained from natural sources since quinine was first extracted from the bark of the Cinchona plant in 1811 (Ref Reference Heeb, Fletcher, Chhabra, Diggle, Williams and Cámara100). Some of these have excellent medicinal properties and served as lead structures that provided the basis for synthetic quinolones as useful drugs (Ref Reference Heeb, Fletcher, Chhabra, Diggle, Williams and Cámara100). A few of these are very effective medications and were the model compounds from which synthetic quinolones were derived (Ref Reference Heeb, Fletcher, Chhabra, Diggle, Williams and Cámara100). Isolated from plant or microbial sources, several 4-quinolone alkaloids showed antimicrobial activities. The core structure is bicyclic (Figure 4; Ref Reference Pham, Ziora and Blaskovich101). Endochin-like quinolones (ELQs) show promise as novel antiparasitic drugs, they are analogs of ubiquinone that can act as competitive inhibitors of cytochrome bc1 by binding to the Qi ubiquinone binding site (Ref Reference McConnell, Bruzual, Pou, Winter, Dodean, Smilkstein and Doggett102, Reference Ortiz, Forquer, Boitz, Soysa, Elya, Fulwiler and Landfear103).

Studies show the efficacy of quinolones against CL caused by L. amazonensis and L. venezuelensis in infected BALB/c mice (Ref Reference Coimbra, Antinarelli, Silva, Souza, Meinel, Rocha and da Silva104). Other quinoline alkaloids isolated from the Galipea longiflora Krause D and B act against L. braziliensis and L. donovani promastigotes (Ref Reference Calla-Magariños, Quispe, Giménez, Freysdottir, Troye-Blomberg and Fernández105). N-methyl-8-methoxyflindersin, isolated from the leaves of Raputia heptaphylla also known as 7-methoxy-2,2-dimethyl-2H,5H,6H-pyran[3,2-c]quinolin-5-one, shows anti-parasitic activity against Leishmania promastigotes and amastigotes (Ref Reference Torres Suarez, Granados-Falla, Robledo, Murillo, Upegui and Delgado106). In silico tools used to identify synthetic quinoline alkaloids with similar structure to that of 7-methoxy-2,2-dimethyl-2H,5H,6H-pyran[3,2-c]quinolin-5-one tested for their in vitro antiparasitic activity against Leishmania (Viannia) panamensis, and in vivo therapeutic response in hamsters with experimental cutaneous leishmaniasis (CL) was reported (Ref Reference Torres Suarez, Granados-Falla, Robledo, Murillo, Upegui and Delgado106). Endochin like quinolones deplete ATP in promastigotes and amastigotes but at a slow pace (Ref Reference McConnell, Bruzual, Pou, Winter, Dodean, Smilkstein and Doggett102). Buparvaquone is a hydroxynaphthoquinone that is also related in structure to ubiquinone and is another potential inhibitor of cytochrome bc1 (Ref Reference McConnell, Bruzual, Pou, Winter, Dodean, Smilkstein and Doggett102). This compound has demonstrated efficacy against L. donovani in vitro and in a BALB/c model of visceral leishmaniasis (Ref Reference Coy Barrera, Coy Barrera, Granados Falla, Delgado Murcia and Cuca Suarez107). Buparvaquone and its phosphate prodrugs (BPQ-3-PHOS and 3-POM-BPQ) were tested against several species of Leishmania (L. major, L. amazonensis, L. aethiopica, L. mexicana and L. panamensis) in vitro. BPQ, BPQ-3-PHOS and 3-POM-BPQ demonstrated low IC₅₀ values against promastigotes and amastigotes of all Leishmania species tested (Ref Reference Croft, Hogg, Gutteridge, Hudson and Randall108). Buparvaquone is a potent inhibitor of electron transport and robustly depletes ATP at submicromolar concentrations within an hour in vitro resulting in amastigote death. Buparvaquone’s toxicity may stem from its capacity to enhance reactive oxygen species (ROS) production, possibly as a result of its inhibition of cytochrome bc1 activity (Ref Reference Menna-Barreto and de Castro41). Buparvaquone significantly eliminates L. donovani parasites in mice models of leishmaniasis (Ref Reference Coy Barrera, Coy Barrera, Granados Falla, Delgado Murcia and Cuca Suarez107, Reference Luque-Ortega, Rivero-Lezcano, Croft and Rivas109). In an in vivo experiment, buparvaquone loaded with lipid nanoparticles with enhanced solubility demonstrates a 80% reduction in the livers’ burden of L. infatum (Ref Reference Smith, Serrano, Mauger, Bolás-Fernández, Dea-Ayuela and Lalatsa110, Reference Monteiro, Löbenberg, Barbosa, de Araujo, Sato, Kanashiro and Bou Chacra111). In a different study, lipid nanoparticles coated with buparvaquone demonstrated 100% clearance of parasites in an in vivo model, whereas pure buparvaquone demonstrated only 50% efficiency in this regard (Ref Reference Thapa, Mondal, Riikonen, Rantanen, Näkki, Nissinen and Lehto112). Cytochrome bc1 is thus a promising target for novel anti-leishmanial drugs, and further improvements on the buparvaquone scaffold are warranted for the development of enhanced therapeutics.

Quinolinyl β-enaminone derivatives showed anti-leishmanial activity through disruption of the mitochondrial ETC complex and induction of PCD after significant ROS production (Ref Reference Frosch, Schmitt, Bringmann, Kiefer and Popp66). Of the several compounds reported, the disruption of complex II by compound 3D ((Z)-1-(4-chlorophenyl)-3-(quinolin-8-ylamino)prop-2-en-1-one) was followed by oxidative burst, intracellular Ca²⁺ accumulation and activation of programmed cell death. In silico studies with compound 3D showed that mitochondrial complex II (SDH) or complex III (CCR) of L. donovani, modelled using the SWISS-Model server had more affinity towards SDH over CCR, which invariably correlates with its higher anti-leishmanial efficacy of 3D. This compound showed superior efficacy in vivo as well in infected BALB/c mice and hamsters (Ref Reference Rani, Khanikar, Dutta, Katiyar, Qamar, Seth and Kar70) indicating this as a potential compound that can be tested for therapeutic purposes.

Quercetin

Quercetin is a polyphenolic flavonoid with antioxidant characteristics found in a wide range of foods we consume, including citrus fruits and green leafy vegetables (Ref Reference Qi, Xiong and Long113). It was first isolated in 1854 (Ref Reference Ghareeb, Zayan, Shari, Sayed and Osredkar114). In essence, quercetin is a pentahydroxyflavone with five hydroxy groups positioned at positions 3, 3, 3’, 4, 5 and 7 (Figure 4) and is known to possess anti-inflammatory, anti-proliferative, anti-oxidative, anti-diabetic, anti-carcinogenic and anti-viral properties. Several in vivo and in vitro studies demonstrate the role of quercetin in various diseases and a detailed review is available (Ref Reference Deepika and Maurya115). The potential of quercetin as an anti-leishmanial agent was described in an early review (Ref Reference Sarkar, Mandal, Sinha, Mukhopadhyay, Das and Basu116). Following the release of the review, in vitro and in vivo investigations provided evidence supporting quercetin‘s potential as an anti-leishmanial agent. L. amazonensis and L. (V.) braziliensis, the causative agents of CL, L. donovani responsible for VL are sensitive to quercetin in vitro (Ref Reference Sousa-Batista, Poletto, Philipon, Guterres, Pohlmann and Bergmann117–Reference Cataneo, Tomiotto-Pellissier, Miranda-Sapla, Assolini, Panis, Kian and Pavanelli119) and mitochondrial disruption is observed in both L. amazonensis and L. (V.) braziliensis along with ROS production (Ref Reference Cataneo, Tomiotto-Pellissier, Miranda-Sapla, Assolini, Panis, Kian and Pavanelli119, Reference Fonseca-Silva, Inacio, Canto-Cavalheiro and Almeida-Amaral120). In vivo studies in a murine model of CL created by L. amazonensis, a reduction in lesion size was observed (Ref Reference Muzitano, Falcão, Cruz, Bergonzi, Bilia, Vincieri and Costa121). Effective against both L. (V) brazilensis promastigotes and intracellular amastigotes, quercetin showed anti-amastigote IC₅₀ (21 ± 2.5 μM) and anti-promastigote IC₅₀ (25 ± 0.7 μM) activities with a selectivity index of 22 (Ref Reference Dos Santos, Da Silva, Brito, Inácio, Ventura, Mendes and Da Silva122). Quercetin was also effective as an oral drug in hamsters with CL (Ref Reference Dos Santos, Da Silva, Brito, Inácio, Ventura, Mendes and Da Silva122, Reference Ribeiro-Romão, Moreira, Osorio, Cysne-Finkelstein, Gomes-Silva, Valverde and Pinto123). Earlier studies with CL induced in BALB/c mice using L. amazonensis showed a reduction in lesion size with quercetin (Ref Reference Muzitano, Falcão, Cruz, Bergonzi, Bilia, Vincieri and Costa121). Five weeks after inoculation, CL generated in BALB/c mice using L. major showed a dramatic reduction in lesion size following oral quercetin exposure for a sequential 28 days, (Ref Reference Dos Santos, Da Silva, Brito, Inácio, Ventura, Mendes and Da Silva122). The anti-leishmanial impact of CL made with L. amazonensis in BALB/c mice was enhanced by the encapsulation of quercetin in lipid nanocapsules of poly(ϵ-caprolactone) (Ref Reference Sousa-Batista, Poletto, Philipon, Guterres, Pohlmann and Bergmann117). Quercetin may be taken into consideration for additional development given its effectiveness as an anti-leishmanial agent, as demonstrated by investigations both in vitro and in vivo.

Quercetin exerts its anti-leishmanial effect on L. amazonensis promastigotes through the disruption of mitochondria and generation of ROS (Ref Reference Fonseca-Silva, Inacio, Canto-Cavalheiro and Almeida-Amaral120). In mammalian cells, quercetin shows redox interaction with the mitochondrial ETC affecting its membrane potential and reducing ATP production. A review detailing interactions of quercetin with the ETC is available (Ref Reference Carrillo-Garmendia, Madrigal-Perez and Regalado-Gonzalez124). Studies with Leishmania mitochondria need to be explored further to assess the effect of quercetin on mitochondria.

In vitro tested plant compounds that affect the mitochondria

The following compounds have not been tested in higher animal models but are tested in vitro for efficacy with different Leishmania species and their effect on mitochondria has been analyzed. These could provide clues to taking up compounds for further studies for the development of anti-leishmanial agents. In L. amazonensis promastigotes, phytol-rich hexane fraction from the leaves of Lacistema pubescens induces depolarization of the mitochondria with an increase in ROS levels (Ref Reference da Silva, Antinarelli, Ribeiro, Coimbra and Scio125). Xanthohumol and resveratrol are naturally occurring constituents of the human diet. Resveratrol is formed as a phytoalexin in plants to defend against parasitic and fungal infections while xanthohumol belongs to the group of polyphenols. When tested with Leishmania parasites, both exerted anti-leishmanial action but in contrast to Resveratrol, xanthohumol strongly inhibited oxygen consumption in Leishmania tarantolae promastigotes. This was due to the inhibition of the mitochondrial electron transfer complex II/III by xanthohumol, which was less pronounced with Resveratrol (Ref Reference Monzote, Lackova, Staniek, Steinbauer, Pichler, Jäger and Gille126). Essential oil from Chenopodium ambrosioides L. and caryophyllene oxide, a compound derived from the essential oil was able to inhibit the electron transport chain of L. tarantolae promastigotes depriving the parasite of energy sources (Ref Reference Monzote, Lackova, Staniek, Steinbauer, Pichler, Jäger and Gille126). Iron can generate toxic ROS via the Fenton reaction. Studies to establish the influence of iron on the anti-leishmanial action of artemisinin in vitro showed that in Sneider’s insect medium, artemisinin caused a greater redox imbalance as compared to M199, a culture media where many experiments on Leishmania parasites grown in vitro are done (Ref Reference Dighal, De Sarkar, Gille and Chatterjee127). Therefore, the media type should be taken into account while analyzing the Leishmania in vitro tests. Several other compounds tested for anti-leishmanial effects through disruption of mitochondria primarily tested in vitro are described in a recent review (Ref Reference Lazarin-Bidóia, Garcia, Ueda-Nakamura, Silva and Nakamura128). Berberine chloride is an alkaloid that shows anti-leishmanial effects where death is preceded by an upsurge in the generation of ROS along with a depletion of ATP. In addition, mitochondrial superoxide, depolarization of the mitochondrial membrane potential, dose-dependent inhibition of mitochondrial complexes I-III and II-III collectively suggest interference with mitochondria in L. donovani (Ref Reference De Sarkar, Sarkar, Sarkar, Dighal, Staniek, Gille and Chatterjee129). Plumbagin, a naphthoquinone derivative results in the depolarization of the mitochondrial membrane, and depletion in ATP levels in the promastigotes of L. donovani inducing apoptosis-like death (Ref Reference Awasthi, Kathuria, Pant, Kumari and Mitra130). Essential oil from Chenopodium ambrosioides, an aromatic herb, kills the parasite by causing a breakdown of mitochondrial membrane potential and a modification of redox indexes (Ref Reference Zinovkin and Zamyatnin35). Formulations of saponins and hydrazones cause changes in the ATP levels and induce depolarization of membrane potential with ROS increase (Ref Reference Upegui Zapata, Echeverri, Quiñones, Torres, Nacher, Rivas and Robledo131). Ethanolic extract of Croton blanchetianus Ball is capable of causing depolarization of the mitochondria in Leishmania amazonensis promastigotes leading to death (Ref Reference Pereira, Vasconcelos, Braz, InÁcio, Estevam, Correa and Scher132). Phloroglucinol derivatives from Hypericum species induce mitochondrial dysfunction like hyperpolarization and ROS generation in promastigotes of L. amazonensis promastigotes (Ref Reference Dagnino, Mesquita, Dorneles, Teixeira, de Barros, Vidal Cana-Capatinta and Romão133). A naturally occurring plant flavone Apigenin, shows anti-leishmanicidal action against L. amazonensis promastigotes through induction of swelling of the parasite mitochondria resulting in a loss of mitochondrial membrane potential with accompanying ROS increase (Ref Reference Fonseca-Silva, Canto-Cavalheiro, Menna-Barreto and Almeida-Amaral134). Melatonin, a neurohormone found in animals, plants and microbes used in humans as medication changes calcium homeostasis of the mitochondrion of L. infantum, with the opening of the mitochondrial permeability transition pore, loss of ATP and mitochondrial potential loss (Ref Reference Elmahallawy, Jiménez-Aranda, Martínez, Rodriguez-Granger, Navarro-Alarcón, Gutiérrez-Fernández and Agil135). Two plant-based compounds, piperine and phenylamide eliminate Leishmania promastigotes and amastigotes through induction of mitochondrial swelling and a loss of mitochondrial membrane potential (Ref Reference Ferreira, Soares, Barreto-Junior, Nascimento, Freire-de-Lima, Delorenzi and Pinto-da-Silva69). Caffeic acid shows anti-leishmanial activity against promastigotes and amastigotes of Leishmania amazonensis by the increase of oxidant activities (Ref Reference Bortoleti, Tomiotto-Pellissier, Gonçalves, Miranda-Sapla, Assolini, Carloto and Pavanelli136). In mammalian cancer cells a variety of caffeic acid derivatives targeted to mitochondria have been developed (Ref Reference Bastidas, Araya-Valdés, Cortés, Jara and Catalán36). These should be tested in the Leishmania parasites both in vitro and in vivo. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a natural polyphenol found in the rhizomes of Curcuma spp. and is a well-known diferuloylmethane. Curcumin is a potent antioxidant agent with anti-inflammatory and anti-tumor properties. In several species of Leishmania, curcumin causes mitochondrial membrane depolarization along with hyperproduction of ROS (Ref Reference Das, Roy, Dutta and Majumder137). In cancer cells, curcumin was shown to cause mitochondrial disruption leading to ROS production (Ref Reference Jung, Lee, Park, Moon, Cho, Choe and Lee138).