Multi-target drugs active against leishmaniasis: A paradigm of drug repurposing

European Journal of Medicinal Chemistry 183 (2019) 111660

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Mini-review

Multi-target drugs active against leishmaniasis: A paradigm of drug repurposing Susana Santos Braga QOPNA & LAQV/REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugal

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 May 2019 Received in revised form 27 August 2019 Accepted 28 August 2019 Available online 29 August 2019

This mini-review focuses on leishmanicidal drugs that were sourced from small molecules previously approved for other diseases. The mechanisms of action of these molecules are herein explored, to probe the origins of their inter-species growth inhibitory activities. It is shown how the transversal action of the azoles e fluconazole, posaconazole and itraconazole e in both fungi and Leishmania is due to the occurrence of the same target, lanosterol 14-a-demethylase, in these two groups of species. In turn, the drugs miltefosine and amphotericin B are presented as truly multi-target agents, acting on small molecules, proteins, genes and even organelles. Steps towards future leishmanicidal drug candidates based on the multi-target strategy and on drug repurposing are also briefly presented. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Leishmanicidal drugs Miltefosine Amphotericin B Fluconazole Itraconazole Posaconazole

1. Introduction 1.1. Leishmaniasis, a global disease Leishmaniasis is an infectious parasitic disease caused by a large variety of parasites e more than twenty e, all belonging to Leishmania genus [1]. It affects humans and a variety of mammals. Having higher incidence rate in countries with warm climate, leishmaniasis is usually classified as a neglected tropical disease [2]. Nevertheless, its present geographical distribution covers roughly 90 countries around the globe and there are records of expansion of its territory due to the climate changes caused by global warming [3]. The transmission of leishmaniasis is done during hematophagy of the female sandflies of the Phlebotomus (Old World) and Lutzomyia (New World) genus [4], and these insects are now expanding their territory due to the increase in temperature. A study based on mathematical modeling of the behaviour of the species Neotoma floridana, N. micropus, Lutzomyia diabolica, and L. anthophora,
xico and Texas, predicts that presently occurring in the north of Me they will broaden their range to central states in the USA and even to the south of Canada, to cause, by 2080, a 70% increase in the risk of exposure to the disease in the North-American population [5]. It

E-mail address: [email protected]. https://doi.org/10.1016/j.ejmech.2019.111660 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

is also believed that globalization contributes to the spreading of leishmaniasis, particularly due to the growing number of international travelers [6] and to the higher significance in international traffic of blood products (anti-leishmanial antibodies screening is not included in the protocols for the control of these products) [7]. 1.2. Clinical forms of leishmaniasis Leishmaniasis starts with the bite of a sand fly carrying the parasite. Then, the parasites spread locally, attacking preferentially macrophagues and other immune competent cells. Following the initial stage of infection, the disease may evolve to different clinical forms, classified as cutaneous, when only skin lesions are observed, mucocutaneous, in which mucosa are also involved besides the skin, and visceral (VL), when the parasite migrates to the bloodstream and affects visceral organs. Cutaneous leishmaniasis (CL), endemic to 42 countries according to the latest statistics [1], is the most common form of the disease, and it is also the most observed form in travelers that get exposed to Leishmania. It causes skin lesions that may take several months or even years to heal and usually leave an atrophic scar [8]. In the Old World (Europe, Asia and the Middle East), CL usually heals by itself and gives the patient protection against subsequent manifestations in the case of re-infection [9]. New World CL (in the Americas), however, is more aggressive and it often requires, along

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with topical treatments, systemic medication (as listed in section 2.1.). Mucocutaneous leishmaniasis (MCL) is known as ‘uta’ in the Andes and endemic in the western slopes of these mountains [10]. The largest number of cases of MCL is found in Bolivia, Peru and Brazil; nevertheless, CL can also be found on another continent altogether, in the country of Ethiopia [10,11]. MCL can be very disfiguring, causing partial or complete destruction of the hard and soft tissues of the nose, palate, and pharynx. Visceral leishmaniasis, called in Hindi ‘kala-azar’, which means black (kala) fever (azar), is endemic to Brazil, East Africa and in South-East Asia. New cases are reported every year and in large numbers, ranging between 50 000 and 90 000. 90% of the new incidences occur in Brazil, Ethiopia, India, Kenya, Somalia, South Sudan and Sudan [11]. VL affects the internal organs, in particular the spleen, the liver, and the bone marrow [8], being characterised by irregular bouts of fever, anemia, and organ enlargement [11]. If left untreated, 95% of the patients will die. These situations make VL responsible for c.a.70000 deaths/year. It is thus mandatory to treat VL, using as first choice Sb(V) salts and AmB or miltefosine as second line treatment [12,13]. Other drugs may also be employed, as listed in the following subsection. 2. Repurposed drugs for the treatment of leishmaniasis 2.1. Drug overview The range of currently available drugs for treating leishmaniasis is relatively small and it includes the pentavalent antimonial salts sodium stibogluconate and meglumine antimonate, amphotericin B, miltefosine, pentamidine, and a family of drugs known as ‘azoles’ e ketoconazole, itraconazole and fluconazole. Notably, 62.5% of these molecules are repurposed (structures represented in Fig. 1). Amphotericin B and the ‘azoles’ are antifungal agents and miltefosine was developed as an antineoplasic agent. But are these activities against various pathologies and pathogens from different regna the result of a multi-target action of a drug? Or is their broad activity range simply the coincidental result of a single-target action on multiple pathogen species? It depends on the drug. A

detailed discussion for each leishmanicidal agent is presented in the following subsections. 2.2. Azole derivatives inhibit biosynthesis of ergosterol Azoles are primarily used as antifungal agents, with fungistatic action against yeasts (e.g. Candida spp) and a fungicide action against molds such as Aspergillus spp [14]. This family of drugs includes both imidazole derivatives, such as ketoconazole, and the triazoles, such as fluconazole and itraconazole. All the drugs have the same molecular target, the enzyme lanosterol 14-a-demethylase (encoded by ERG11 in C. albicans and Criptococcus neoformans and by cyp51A and cyp51B in Aspergillus fumigatus), involved in the biosynthesis of ergosterol, the main cell membrane sterol in yeasts [15e17]. The azoles share a common transport mechanism, being imported into fungal cells not by passive diffusion but by a facilitated diffusion [18]. Once inside the cell, they bind to the iron atom in the heme group located in the active site of ERG11 via an unhindered nitrogen atom in the azole ring [19], blocking in this way the activation of oxygen needed for demethylation of lanosterol. As a result, there is the activation of an alternative biosynthetic route involving another enzyme of the same family (D-5,6-desaturase, encoded by ERG3) and the production of a toxic sterol, 14-a-methyl3,6-diol [20] that causes severe stress on the cell membrane. Ergosterol depletion by action of the azoles also affects the normal functioning of the enzyme Hþ-ATPase existing on the vacuolar membrane, critical for maintaining cellular ion homeostasis via generation of an adequate pH gradient and essential for fungal virulence [21]. Repurposing azoles for the treatment of leishmaniasis (see uses and recommended doses in the Table 1) is mainly based on the fact that Leishmania spp cells also have ergosterol as the main membrane sterol component (note that mammalian cells, in contrast, have cholesterol as the main membrane sterol). This means that, for azoles, the repurposing is not due to a multi-target action but rather by the coincidental existence of the same target, lanosterol 14-a-demethylase [22]. Importantly, sterol 14-a-demethylases have a very rigid active site, unlike many other enzymes of the cytochrome P450 family [23]. This may help explain the

Fig. 1. Chemical structural of the azoles e fluconazole, itraconazole, ketoconazole e, and of the compounds amphotericin B and miltefosine. These compounds are currently marketed drugs for leishmaniasis that were repurposed from other clinical uses.

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Table 1 In vitro activities of the compounds from the azole family that are used against leishmaniasis. The activity of these compounds against lanosterol 14-a-demethylase, the target enzyme, is responsible for the different values of activity against the parasite, as herein demonstrated with L. amazonensis. Compound

Active dose against lanosterol 14-a-demethylase (mM)

a

IC50

at 72h

on L. amazonensis (mM)

promastigotes c

Itraconazole

1.4

0.44

Ketoconazole Fluconazole

1.9 32.7

2.7 d 10.1 e

a b c d e f g h i j

amastigotes 0.08 6.8 e

d

c

Clinical uses and doses b

CL, 200 mg/dayf MCL, 400 mg/dayg CL, 600 mg/day h CL, 400 mg/day i CL, 8 mg/kg/day j

Data was taken from Ref. [25] and converted to micromolar for better comparison. Intracellular amastigotes (in mouse peritoneal macrophages) were used. From Macedo-Silva et al. [27]. From Andrade-Neto et al. [28].
s et al. [29]. From Ginouve From Consigli et al. [30]. From Amato et al. [31]. From Salmanpour et al. [32]. From Emad et al. [33]. From Sousa et al. [34].

transversality of azole growth-inhibitory activity across several species. Azoles have different affinity to lanosterol 14-a-demethylase. This has been correlated with the structure of the non-ligated portion of their molecule [24]. Itraconazole and ketoconazole have better affinity to the enzyme and thus cause inhibition at lower doses; fluconazone has lower affinity and for this the growth inhibition of the parasite requires a higher dose (Table 1). A five-day exposure test with various strains of Leishmania showed that a dose of 10 mg ml1 of fluconazole was needed to achieve the same effect than doses of 1 mg ml1 of itraconazole or ketoconazole. This study is among those with the most comprehensive collection of strains, as it included L. aethiopia, L. donovani, L. major, L. tropica, L. amazonensis, L. Mexicana, L. braziliensis, L. guayanensis, L. panamensis and L. chagasi [25]. Ergosterol depletion by azoles causes several structural changes in the cells of the parasites. In L. Mexicana, the formation of pores in the cells membrane was demonstrated [26]. In L. amazonensis, alterations in the mitochondria, cell deformation and accumulation of lipid bodies are reported [27]. Cell division is also affected, with some cells formed under the treatment with azoles presenting more than two flagella.

2.3. Amphotericin B: pleiotropic properties uncovered Amphotericin B (AmB) is known for several decades to bind to membrane sterols with particular affinity towards ergosterol, forming complexes that arrange into ion channels and increase membrane permeability [35e37]. Ion channel formation, with its consequent electrolyte imbalance, was accepted for many years as the one and only mechanism of death in yeast cells treated with AmB. Recent research, however, has come to challenge the channelbased model of action and to claim that AmB is really a pleiotropic molecule, that is, a molecule that acts on multiple cellular targets. Firstly, an AmB derivative named C35deOAmB and having the particularity of binding ergosterol very strongly while being incapable to form ion channels, has shown substantial in vitro antifungal activity against Candida albicans. Compound C35deOAmB (MIC of 4 mM) was only eightfold less potent than AmB (MIC of 0.25 mM) [35]. These results show that pore formation is not needed to kill yeasts and allow postulating that simply binding ergosterol is enough ensure cell death. Sterols are indispensable for the integrity of yeasts, coordinating membrane heterogeneity to maintain the rigidity and fluidity, and preventing the penetration of water [38].

Secondly, genomic studies on yeasts have shown that AmB has multiple actions on the genomic expression. AmB affects the expression of genes involved in ergosterol synthesis, and it also induces the expression of other genes, associated with cellular stress [39]. Although the role of oxidative damage on the biocidal activity of AmB remains to be elucidated, it is known that the oxidative stress induced by this compound contributes to cell death by apoptosis [40]. In the treatment of human leishmaniasis, AmB is the treatment of first choice for patients with HIV co-infection and also in the case of infections with parasites resistant to Sb(V) salts [41,42]. Being insoluble in water, AmB is administered by perfusion, typically in the form of a lisossome. Similarly to the target in yeasts, ergosterol is recognised as the traditional AmB target in Leishmania parasites. The lethal action of AmB is associated with altered membrane permeability through the formation of non-aqueous and aqueous pores after binding to membrane ergosterol (Fig. 2) [43]. But the effects of ergosterol binding are not limited to pore formation and cell death, as demonstrated by a study on L. Infantum. Ergosterol depletion by AmB in these parasites made them less invasive towards mice [44]. As a downside, depletion of ergosterol and other ergostane-type sterols with strong affinity to bind AmB has also been associated with the development of resistance to this drug [45e47]. In the strain MNYC/BZ/62/M379 of L. mexicana, it was demonstrated that the mechanism of resistance is due to a mutation in the enzyme sterol 14a-demethylase that reprograms the enzyme to produce cholesterol and cholestane-type sterols instead of ergosterol; since these bind less strongly with AmB, the drug's efficacy is hampered [48]. Studies on the influence of AmB exposure on the expression of the genome of Leishmania were also conduted, and these helped confirm that AmB is indeed a pleiotropic drug. Many genes are functionally overexpressed in the presence of AmB, showing its ability to act on more than one target [49]. Highlight was given to the ascorbate peroxidase gene (LdAPx gene) from a Leishmania donovani strain because it is present only in Leishmania, not in humans. The gene was shown to trigger a set of reactions leading to apoptosis-like cell death. LdAPx induces formation of reactive oxygen species and the elevation of the cytosolic Ca2þ level, which leads to depolarisation of the mitochondrial membrane potential and the release of cytochrome c (Cyt c) into the cytosol; then, the redox-active Cyt c activates metacaspases that cause damage to the cell nucleus, fragmenting the DNA and ending in cellular death [49].

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Fig. 2. Schematic representation of the various targets of AmB when in contact with parasitic cells. AmB is able to recognise and bind to ergosterol, leading to the formation of aqueous (herein depicted) and non-aqueous pores. Moreover, AmB leads to the overexpression of various genes, with highlight to LdAPx. This results in increased formation of reactive oxygen species (ROS) and an increase in cytosolic Ca2þ levels, triggering a cascade of events ultimately leading to cell death (see main text for details).

The ability of AmB to bind to cholesterol, although with small affinity, means that it does not interact solely with the Leishmania pathogens but also with the cells of mammal and human hosts, due to the presence of cholesterol and cholestane-like sterols in their membranes. This implies that the patients receiving AmB as a medication for leishmaniasis will experience some degree of cholesterol sequestering at the cellular level. The clinical relevance of AmB-cholesterol binding is that this lipid is essential for the parasite to adhere to target immune cells of the host and invade them. In fact, Leishmania infectivity can be reduced thanks to cholesterol depletion with cyclodextrins [50] or inactivation by AmB [51]. In the case of AmB, cholesterol molecules become immobilised inside the membrane and they can no longer interact with other membrane components, in particular the receptors required for the process of internalisation of the parasite [52]. AmB also has immunomodulatory action, promoting in various human immune cells the release of cytokines [53] and chemokines [54]. The mechanism of this action is not yet fully elucidated, but several studies postulate it to involve activation of toll-like receptors (TLRs) [55], immune “sentinels” that once activated contribute to call other immune cells to the site of the infection. AmB further acts on activated cells of the blood vessel endothelium, stimulating the production of interleukine-1 (IL-1). This mediator causes augment of production of nitric oxide to signal a vasodilating response that plays an important role in the protection against pathogens [56]. 2.4. Miltefosine, the classic multi-target drug Miltefosine, or hexadecylphosphocholine, is a synthetic phospholipid analogue developed originally as an antineoplasic agent. Its chemical structure, with an amphiphillic nature, allows it to be incorporated into the cell plasma membrane, being subsequently distributed to the internal membranes [57]. Its first known target is the enzyme phosphocholine cytidylyltransferase, resulting in inhibition of the biosynthesis of phosphatidylcholine [58]. Maintaining normal amounts of phosphatidylcholine is vital to cell survival, and critically low levels of this phospholipid are known to cause apoptosis [59]. The apoptotic action of miltefosine in cancer cells has also been associated with other targets and molecular pathways:

 In vitro studies with various cell lines demonstrated that miltefosine apoptosis occurs in caspase-3-dependent manner [60,61].  Miltefosine induces an increase in intracellular Ca2þ levels by interfering with the calcium channels of the cell membrane [62,63].  Miltefosine inhibits the enzyme responsible for the biosynthesis of sphingomyelin, leading to the accumulation of its substrate, ceramide, which is pro-apoptotic [64]. Miltefosine is approved for the treatment of human leishmaniasis since 2002 [65]. It is the only available leishmanicidal drug that can be administered orally, being used alone or in combination with other drugs. The mode of action of miltefosine against Leishmania parasites involves an indirect cell death mechanism that is similar, in most aspects, to apoptosis [66e69]. A few of the targets involved in this process have already been identified and they were found to share similarities to those of mammalian cells (Fig. 3). One of these is the family of metacaspases [69], caspase-related cysteine-proteases occurring in organisms that do not have caspases (such as plants, yeast, and protozoan parasites). The enzyme MAP2 (methionine aminopeptidase 2), involved in protein biosynthesis, tissue repair and protein degradation, was also proven to contribute towards the apoptotic-like death of Leishmania parasites treated with miltefosine [70]. Moreover, miltefosine strongly reduces the function of the overall replication machinery of the parasite cells, with RNA synthesis reduced by 96.32%; following, protein synthesis is also reduced and lastly DNA synthesis drops [71]. Miltefosine targets the homeostasis of Ca2þ in Leishmania, similarly to its action on mammal cells. Miltefosine activates a plasma membrane Ca2þ channel that is analogous to the human Ltype VGCC channel [72,73]; this channel is opened by the sphingolipid sphingosine, a mechanism that differs from that of mammal cell Ca2þ channels and, therefore, is a distinctive feature of trypanosomatid channels [73]. In tandem, miltefosine acts directly and rapidly on acidocalcisomes e organelles rich in calcium and phosphate that help regulate osmolarity in the parasite e, alkalinising their interior and causing the fast release of Ca2þ to the intracellular medium [72]. The combined action of miltefosine on these two targets, Ca2þ channels and acidocalcisomes, results in a

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Fig. 3. Schematic representation of the multi-target action of miltefosine. Miltefosine induces cell death by interfering with the various cellular targets of Leishmania spp. represented in the top section of the image. Miltefosine further modulates the activity of host immune cells that are shown in the bottom section. Note that the immunomodulatory activity of miltefosine is not limited to these targets; it can interact with other immune cells (not shown) by mechanisms not yet fully elucidated.

large accumulation of intracellular Ca2þ that also contributes to parasitic cell death. Miltefosine further targets the mitochondrion, leading to the increased production of reactive oxygen species that cause the death of the Leishmania parasites [74]. At least two mitochondrial targets of miltefosine have been identified. These are the enzyme cytochrome c oxidase, which is inhibited by miltefosine in a dosedependent matter [75] and may be linked to apoptosis [76], and the mitochondrial membrane that under the action of miltefosine exhibits a reduction of the hydrogenionic potential [77]. Miltefosine also interacts with the host's immune cells and modulates their response to the parasite. It increases the macrophage production of interferon-g (IFN-g), an immune signalling molecule, as well as the responsiveness of the infected macrophages to IFN-g. This results in a dose-dependent increase in the expression of induced nitric oxide synthase in the macrophages (iNOS2) and in the death of the parasite [78]. Miltefosine also restores the balance between the two types of immune response of Thelper lymphocytes, Th1 and Th2. Th1 responses occur at the stage of infection recognition and they are mostly pro-inflammatory, calling other immune cell to fight the infection while Th2 responses normally occur in a second stage, helping to reduce excessive inflammation. Leishmania parasites induce an excessive Th2 response that allows them to progress with the infection in ‘stealth mode’. By inducing Th1 response and restoring the Th1/Th2

balance [78], miltefosine provides a paramount assistance in the control of the infection progress. Additional immunomodulatory action of miltefosine includes inactivation of mast cells by a process that includes dose-dependent decrease in the production of TNF-a; this was observed both in vitro and in vivo on five human volunteers treated topically with a 6% solution of miltefosine [79]. Such topical action may be helpful in the very early stages (18e24 h) of infection with cutaneous leishmaniasis, in which neutrophils, recruited to the infection site by the TNF-a factor released by the mast cells, are infected and exploited by the parasite for its own reproductory purposes to promote a productive infection [80]. Mast cells themselves may also be involved in leishaniasis exacerbation [81,82], although this correlation is still controversial [83]. In spite of the multiple actions of miltefosine that make it a very robust and powerful medicine, the parasites find ways to survive. Resistence to miltefosine was recently reported in a clinical study conducted in Brazil [84]. Investigation into the genetic background of the involved parasite, L. infantum, allowed to identify a Miltefosine Sensitivity Locus (MLS) in this species and to conclude that patients infected with a strain that lacks this locus have a higher chance to relapse after miltefosine treatment [85]. Although the mechanisms of resistance associated with this genetic mutation are not yet known, the discovery allows screening the patients to identify the genetic background of the parasite and decide on the best medication to employ.

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Fig. 4. Structural representation of a new drug candidates for tuberculosis, SQ109, reported by García-García et al. to also have a very potent inhibitory activity against L. Mexicana amastigotes (IC50 ¼ 11 ± 0.9 nM) [88].

3. Perspectives and future outlook The medication available to treat leishmaniasis, including the repurposed drugs that are highlighted in this work, has many drawbacks and limitations, the most significant being the numerous indesirable reactions due to high toxicity of most of these drugs and the need for parenteral administration (with the exception of miltefosine). Also, treatments require various weeks and often the patient needs to be hospitalised in order to monitor toxic effects, which implies very high costs. Taken together, these factors make therapy inaccessible for many patients or then result in incomplete therapeutic actions, either by lack of follow-up medication or by patient discouragement. This panorama illustrates the strong need to find a broader range of active molecules active against Leishmania, either new or repurposed. Repurposing drugs for leishmaniasis has been used frequently and successfully in the clinical management of this disease. As demonstrated in this review, many of the currently used drugs are repurposed. This practise allows cost-effective and fast way to get new therapeutic alternatives, because the pharmacokinetics and toxicological safety profiles of these molecules are already known, and what lacks is the research to demonstrate the effectiveness of their leishmanicidal action. In the choice of possible candidates for repurposing, research teams seem to have followed the path of the ‘true-and-tried’, choosing antimicrobial and antitumoral drug classes, the two from which the known successful leishmanicidal

medicines were obtained. The recent literature presents a few reports with interesting results for antimicrobials, namely clarithromycin [86], fumagillin [87], and also for a novel antimicrobial with the code name SQ109. Compound SQ109 (Fig. 4) is an orphan drug for the treatment of tuberculosis that is under phase 2 trials. Having been investigated, in parallel, as a leishmanicidal, it featured a very potent action against internalised amastigotes of L. mexicana, with an IC50 value in the nanomolar range [88]. Within antitumorals, sunitinib [89] and tamoxifen [90,91] are reported to have good leishmanicidal activities. Tamoxifen, either alone [90] or in combination with AmB [92] or miltefosine [93], has been pointed out as an adequate candidate for the treatment of the cuteneous leishmaniasis. In the design of new small molecules aiming at being antileishmania drug candidates, one needs to consider not only the effectiveness and low toxicity, but also the low market price, because this disease is most frequent in countries with very limited budgets for healthcare. However, the typical path of discovery and validation of small molecules involves screening millions of compounds that have a high risk of failure, which makes it very expensive and time-consuming. In this regard, pursuing compounds with multi-target activity can be regarded as a promising new technology [94]. Multi-target drugs offer a strong assurance of success. Nevertheless, their discovery does pose some challenges due to the complexity of having to validate action on the various targets. Computational chemistry is very helpful for the initial screening, as it is now possible to use multi-target quantitative structure-analysis relationship analysis to predict the activity of a compound with a single model [95]. While in silico studies are still a bit limited by the number of Leishmania target structures available in the database [96], new models designed for multi-target drug discovery of anti-leishmania drugs are emerging [97] and some of the existing models have already helped to identify and validate active molecules in large families of compounds. Recent examples include xyloguyelline [98], two germacranolide sesquiterpenes and a pseudoguaiacanolide sesquiterpene [99] (Fig. 5).

Fig. 5. Structural representation of a few of drug candidates for leishmaniasis therapy identified by computational methods, either alone or in combination with in vitro screening.

S.S. Braga / European Journal of Medicinal Chemistry 183 (2019) 111660

Xyloguyelline was selected among 142 natural alkaloids extracted from the anonnaceae Anaxagorea dolichocarpa as the most promising drug candidate against L. donovani by a combination of in silico (Random Forest model) studies with in vitro methods, the later involving inhibition tests with a collection of enzymes [98]. The sesquiterpenes (two pseudoguaiacanolides and a germanocrolide) were selected solely based on computational methods that investigated not only the ability to bind strongly to a variety of enzyme targets of Leishmania, but also the physicochemical properties that make molecules good drug candidates, namely partition coefficient (LogP), molar refractivity, polar surface area, hydrogen bonding sites and molecular flexibility (rotatable bonds) [99]. These two examples illustrate well the growing ability to screen and predict the properties of new molecules that will most certainly bring forward a new generation of medicines for Leishmania in the next decades.

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4. Conclusions Leishmaniasis has been a neglected tropical disease (NTD) for many decades, but following the call of attention given to these diseases by the UN in 2009 [94] and the increase in stimuli and funding for research on new drugs for NTDs there was strong response of the international scientific community. In spite of the good progresses achieved, with a growth in the number of new drug candidates and a deeper understanding of the mechanisms of action of the existing drugs, ideal treatment options still remain out of reach. Research efforts must continue, aiming at affordable, safe and effective medications. This short review describes the transversality of targets across pathogens and how the understanding of the action of a pharmacologically active molecule on various targets, within the same pathogen or in pathogens from different species, genres and even regna, can contribute towards the amelioration of patient-adapted treatment and the design of adequate new drugs. Multi-target drugs are a growing trend in pharmacology and in the development of novel therapies, to which the increasing methodologies of in silico, in vitro and combined screening will contribute to accelerate discovery of safer, more active, and patient-compliant drugs. Acknowledgements ~o para a Cie ^ncia The QOPNA research unit acknowledges Fundaça e a Tecnologia (FCT, Portugal), European Union, QREN, European Fund for Regional Development (FEDER), through the programme COMPETE, for general funding (project PEst C-QUI/UI0062/2019; FCOMP-01-0124-FEDER-037296).

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