Human African Trypanosomiasis, Chagas Disease and Leishmaniases

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Review An Overview on Target-Based Drug Design against Kinetoplastid Protozoan Infections: Human African Trypanosomiasis, Chagas Disease and Leishmaniases

Violeta Kourbeli 1, Eleni Chontzopoulou 2, Kalliopi Moschovou 2, Dimitrios Pavlos 2, Thomas Mavromoustakos 2 and Ioannis P. Papanastasiou 1,*

1 Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimioupoli-Zografou, 157 84 Athens, Greece; [email protected] 2 Department of Organic Chemistry, Faculty of Chemistry, National and Kapodistrian University of Athens, Panepistimioupoli-Zografou, 157 71 Athens, Greece; [email protected] (E.C.); [email protected] (K.M.); [email protected] (D.P.); [email protected] (T.M.) * Correspondence: [email protected]

Abstract: The protozoan diseases Human African Trypanosomiasis (HAT), Chagas disease (CD), and leishmaniases span worldwide and therefore their impact is a universal concern. The present regimen against kinetoplastid protozoan infections is poor and insufﬁcient. Target-based design expands the horizon of drug design and development and offers novel chemical entities and potential drug candidates to the therapeutic arsenal against the aforementioned neglected diseases. In this review, we report the most promising targets of the main kinetoplastid parasites, as well as their Citation: Kourbeli, V.; Chontzopoulou, E.; Moschovou, K.; corresponding inhibitors. This overview is part of the Special Issue, entitled “Advances of Medicinal Pavlos, D.; Mavromoustakos, T.; Chemistry against Kinetoplastid Protozoa (Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.) Papanastasiou, I.P.. An Overview on Infections: Drug Design, Synthesis and Pharmacology”. Target-Based Drug Design against Kinetoplastid Protozoan Infections: Keywords: kinetoplastid protozoan diseases; Human African Trypanosomiasis; Chagas disease; Human African Trypanosomiasis, leishmaniases; target-based drug design Chagas Disease and Leishmaniases. Molecules 2021, 26, 4629. https:// doi.org/10.3390/molecules26154629 1. Introduction Academic Editor: Giulio Rastelli The protozoan diseases Human African Trypanosomiasis (HAT), Chagas disease (CD), and leishmaniases are infectious diseases, which belong to Neglected Tropical Diseases Received: 29 June 2021 (NTDs), according to World Health Organization (WHO). These kinetoplastid protozoan Accepted: 27 July 2021 Published: 30 July 2021 infections not only affect low- and middle-income countries (LMICs) like other NTDs, but they span worldwide, because of globalization caused by human and capital translocation.

Publisher’s Note: MDPI stays neutral More than 20% of the world’s population lives in an area where NTDs are endemic and with regard to jurisdictional claims in over 1 billion people are affected by these diseases each year [1]. Thus, vector and host published maps and institutional affil- transportation and new outbreaks happen all around the world. In the last 70 years, public iations. health policy has focused on the eradication of infectious microbes using powerful medical weaponry. Antibiotics and vaccines provoked a bold confidence that the end of infectious diseases was ahead. The devastating COVID-19 pandemic has proven that this grandiose optimism was premature. The evolutionary history of pathogens and human infectious diseases was unpredictably accelerated by the human transformation of global ecosystems Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and the construction of massive transportation networks connecting human societies This article is an open access article worldwide. Emerging infectious diseases are a new threat against human dominance, distributed under the terms and which might prove more fragile than ever before in history [2]. conditions of the Creative Commons The kinetoplastid diseases, sleeping sickness (HAT), Chagas disease (CD), and leish- Attribution (CC BY) license (https:// maniases, as well as all NTDs, suffer from impoverished funding and insufficient alignment creativecommons.org/licenses/by/ among relevant partners to achieve overall goals, based on a strategic plan [3]. The treat- 4.0/). ment of these diseases and overall reduction in burden is a multidisciplinary endeavor that

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involves changes to health management policies, coordinated public health interventions, and the improvement of health disparities. In 2012, WHO released the first bold roadmap towards 2020, against NTDs. However, glaring gaps remain from these achievements because of failing approaches [4]. A second 2021–2030 road map for NTDs, guided again by WHO, provides an accelerated and ambitious coordination between public organizations and stakeholders towards the prevention, control, elimination, and eradication of these infections [5]. A community of partners, known as Uniting to Combat NTDs, has endorsed and supported the vision of both WHO roadmaps and has updated the London Declaration on Neglected Tropical Diseases [6]. Public–private consortia are forms of cooperation between public authorities and private businesses that carry out funding streams covering the full range of activities required for drug discovery against NTDs [7]. The Drugs for Ne- glected Disease Initiative (DNDi) is the most successful model of the product development partnership (PDP), which is implicated in drug innovation involving research institutions, pharmaceutical companies, government agencies and international organizations [8,9]. An open-source model example is the Tres Cantos Open Lab Foundation (TCOLF), which allows academics to use GlaxoSmithKline’s R&D facilities in Spain. This GSK-TCOLF partnership has fostered a lot of scientific exchanges, research grants, and follow-up en- gagement [10]. The Novartis Institute for Tropical Disease (NITD), in cooperation with the Singapore Economic Development Board, have also been involved in a multidisciplinary fight against NTDs [11]. The Wellcome Trust, the Bill and Melinda Gates Foundation, and the NIH also contribute to the research and development against NTDs [12]. The current drugs used against NTDs are suboptimal. The available regimen for the treatment of kinetoplastid diseases is far from satisfactory, with many drugs currently in use dating from early in the 20th century, being essentially poisons, for example, arsenic used to treat sleeping sickness and antimonials for leishmaniasis. The approved therapeutical arsenal presents substantial restrictions, serious adverse side effects, and excessive toxicity, as well as limited efficacy and increasing resistance [13]. Most of the currently used drugs probably have multiple mechanisms of action, as they address to various parasite targets. There is a need for the drug discovery of completely new classes of therapeutic agents, with reduced host toxicity and improved administration profile [14]. The challenge of a typical small-molecule drug discovery follows three pathways [15]: 1. Large-scale cell-based (phenotypic) screening approach. The phenotypic screening identifies drug candidates that are active against the whole cell, without any knowl- edge of the mechanism of action or a specific cellular or molecular target. 2. Target-based screening approach. There are a few validated targets against the parasitic diseases because of insufficient understanding of the detailed biology of the pathogens. 3. Compound repurposing. Marketed drugs are being tested for redirecting or reprofiling, which has become a recent strategy in medicinal chemistry and drug discovery [16]. In this review, we highlight the challenges associated with target-based drug discovery for the kinetoplastid diseases, Human African Trypanosomiasis (HAT), Chagas disease (CD), and leishmaniases.

2. Kinetoplastid Parasites and Related Diseases 2.1. The Life Cycles of Trypanosomids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. share a common, but distinct from other protozoan parasites, mitochondrial DNA, the kinetoplast, which defines their class (kinetoplastida) of phylogenetic order. Kinetoplastida trypanosomatidae protozoans have divergent evolutionary patterns from the eukaryotic mammal lineages, a fact which is reflected in their distinct biology, as shown in Figure1. T. brucei, T. cruzi, and Leishmania spp. are eukaryotic parasites that exhibit a developmental stage within the gut of insect vectors and tissues of vertebrate hosts. During the vertebrate infective stages, the above protozoans alter the differential expression of virulence genes, modifying their biological and antigenic properties to subvert the host protective immune responses and establish a persistent infection. One of the characteristics Molecules 2021, 26, 4629 3 of 34

their class (kinetoplastida) of phylogenetic order. Kinetoplastida trypanosomatidae protozoans Molecules 2021, 26, 4629 have divergent evolutionary patterns from the eukaryotic mammal lineages, a fact which 3 of 41 is reflected in their distinct biology, as shown in Figure 1. T. brucei, T. cruzi, and Leishmania spp. are eukaryotic parasites that exhibit a developmental stage within the gut of insect vectors and tissues of vertebrate hosts. During the vertebrate infective stages, the above protozoans alter the differential expression of virulence genes, modifying their biological of these protozoansand is antigenic their evasion propertiesmechanisms to subvert the host from protective host immune immunity, responses which and establish results a in disease chronification [17].persistent infection. One of the characteristics of these protozoans is their evasion mechanisms from host immunity, which results in disease chronification [17].

FigureFigure 1. The 1. Thelife cycle life of cycleT. brucei of, T.T. cruzi brucei, and ,LeishmaniaT. cruzi spp., and (a) LeishmaniaT. brucei is transmittedspp. ( aby) tsetseT. brucei flies tois humans transmitted or by tsetse livestock animals. The parasites proliferate as procyclic forms, which transform into epimastigotes and then into infectious flies to humans or livestock animals. The parasites proliferate as procyclic forms, which transform into epimastigotes and then into infectious metacyclic trypomastigotes, in the tsetse fly. These metacyclic trypomastigotes infect mammals, where they differentiate into bloodstream “slender” forms that circulate in the bloodstream, skin, adipose tissue or the brain. The slender forms differentiate into bloodstream “stumpy” (nonreplicative) forms which can be transmitted to the insect vector when it takes a blood meal. (b) T. cruzi: Initially, an infected triatome insect vector (known as “kissing bug”) takes a blood meal and releases trypomastigotes near the site of bite injury. Trypomastigotes enter the host cells’ intact mucosal membranes (conjunctiva) or through the bite injury. Then, inside the host, the trypomastigotes infiltrate cells near the site of injury and transform to amastigotes. Subsequently, amastigotes multiply in binary fission, and they differentiate into trypomastigotes where they are released into the blood circulation (bloodstream trypomastigotes). The vectors become infected by feeding on human or animal blood that contains circulating parasites. Trypomastigotes transform into epimastigotes in the vector’s midgut where they multiply. Finally, they transform into infective metacyclic trypomastigotes and they migrate into the rectum of vector. (c) Leishmania spp. are transmitted in the saliva of the sandfly vector to humans and domesticated dogs. In the sandfly vector, the leishmanial species replicate as promastigotes and differentiate into metacyclic promastigotes. Then, the parasites are injected into the skin of the vertebrate host, and invade mononuclear cells, mainly macrophages, where the parasites differentiate to replicative amastigote forms within the parasitophorous vacuole. Upon replication, the amastigotes lyse the cells and infect other macrophages, which can be ingested by female sandflies during bloodmeal. Molecules 2021, 26, 4629 4 of 41

2.2. Human African Trypanosomiasis HAT is characterized as a tropical disease, since it mostly affects the population of West/Central Africa (95% of the cases) and East Africa (5% of the cases) [18]. It is also considered as one of the “Neglected Diseases”, given that the drugs that are prescribed for its treatment have not been improved since their initial circulation (1960), due to the limited market share [19]. In 1995, about 25,000 cases were detected with HAT disease, 300,000 undetected cases were estimated, and 60 million people were estimated to be at risk of HAT infection. In the ensuing years, the mortality rates for HAT have decreased substantially and fewer than 1000 cases were found in 2019 [20]. The disease has been characterized as endemic and consequently, the need for the development of a vaccine is fundamental. However, vaccination will not bring a radical solution to the HAT issue, since the parasites’ antigenic variation will possibly make the vaccine ineffective, and the disease will continue to infect thousands of people per year. In conclusion, the development of improved drugs, presenting high bioavailability and low toxicity, is crucial for the battle against HAT [14]. The main subspecies of T. brucei parasites using humans as a host are Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, and Trypanosoma brucei. The first two are detected in West/Central Africa and East Africa, respectively, while the last is rarely spotted with the population of these regions. The parasite is transmitted to humans and domestic animals through the bite of infected tsetse flies and causes the endemic HAT disease [21]. People diagnosed with HAT suffer from fever, headaches, weakness, pain in the joints, lymphadenopathy, and stiffness. The infected population may not present these symptoms immediately, but only when the parasite has penetrated the blood–brain barrier (BBB). At this point, the parasite affects many human biological functions. The name of the disease, i.e., sleeping sickness, is attributed to its primary symptom, sleeping disorder. The central nervous system (CNS) is penetrated, causing a variety of different neuronal misfunctions, such as deep sensory disturbances, abnormal tone and mobility, ataxia, psychiatric disorders, seizures which slowly lead to coma, and finally, death. The duration of the disease is differentiated from T.b. gambiense to T.b. rhodesiense. The progression of the disease happens during two distinguishable stages, defined by the parasite’s penetration to the BBB. Before the parasite enters the CNS, which is considered as the first stage of the disease or the hemato-lymphatic stage, patients suffer from vague symptoms linked to a variety of diseases, such as fever, pruritus, arthralgia, enlarged lymph nodes, fatigue, and headaches, making the disease difficult to recognize. After the entrance of the parasite in CNS, there is the second or meningo-encephalitic stage, where the symptoms get more intense and specific. Finally, the presence of severe symptoms, including neuropsychiatric conditions, ultimately lead to coma and death [22]. Hence, HAT’s diagnosis and treatment should be performed during the expression of the first symptoms in patients. However, as it has been already mentioned, the first stage of HAT includes common symptoms that are also linked to many other diseases, rendering HAT’s diagnosis difficult to perform. Consequently, HAT’s diagnosis includes the operation of laboratorial tests to people populating these regions, who present “suspicious” symptoms that could reveal the possibility of infection. The confirmation of the results derives from a three-step testing procedure. First, blood samples are taken from patients and they are screened, following the card agglutination test for trypanosomiasis’ protocol. Patients who are positive to the presence of the parasite undergo further tests to identify trypanosomes in blood and lymph node aspirate samples. The third step is identifying the stage of the disease in patients. This is the most challenging part of the procedure, since mild symptoms overlap between the two stages. Herein, samples of cerebrospinal fluid are taken from patients and cell cultures reveal the stage of their disease. Recently, rapid diagnostic tests (RDTs) have been also developed in order to detect the presence of the same native antigen as the one used in the test process described above. Rapid tests were developed in 2016 and 2017 by two different companies and they Molecules 2021, 26, 4629 5 of 41

presented high accuracy (99%), important speciﬁcity, and stability in various temperatures. Thus, RDTs are currently advantageous, offering accurate, sensitive results and they are strongly recommended for use to all primary medical facilities [23].

2.3. Chagas Disease Trypanosoma cruzi is a hemoflagellate protozoan, transmitted mainly by blood-sucking triatomine bugs and it causes human Chagas disease (CD), also known as American trypanosomiasis [24]. It was identified in the first decade of the 20th century and it still remains a major problem for public health [25]. More specifically, Chagas disease is a neglected tropical illness from Latin America that spread through immigration and became a global issue [25]. An estimated 6 to 7 million people worldwide are infected with T. cruzi and 75 million people are at risk of infection [26]. CD was discovered by Brazilian researcher Dr. Carlos Chagas in 1909 [27]. Chagas disease can cause a sudden illness after 1–2 weeks (acute phase), or it may appear 10–20 years after initial infection (chronic phase). The acute phase of CD that can last a few weeks or months (4–8 weeks), is often asymptomatic. Acute phase symptoms include fever, fatigue, rash, body aches, eyelid swelling, headache, loss of appetite, nausea, diarrhea or vomiting, swollen glands, enlargement of the liver or spleen, and Romana sign (also known as chagoma: unilateral palpebral oedema when the conjunctiva is the portal of entry). Chronic phase symptoms may include irregular heartbeat, heart failure, sudden cardiac arrest, difficulty swallowing due to enlarged esophagus, stomach pain, or constipation due to enlarged colon. In both acute and chronic phases, the disease can impact multiple organ systems, leading to cardiopathy or megaviscera [25]. There are two mechanisms of Chagas infection transmission, primary (or main) and secondary [28]. Primary mechanisms include transmission by vectors (triatomines), by blood transfusion, orally (contaminated food), and across the placenta or through the birth canal [29,30]. Secondary mechanisms of transmission are: handling infected animals, organ transplantation, sexually, criminally induced infection by inoculation or orally, and laboratory accidents [31].

2.4. Leishmaniases The Leishmaniases are a group of infectious diseases caused by protozoan parasites from more than twenty Leishmania species. The insect vector is a female phlebotomine sand- ﬂy, which transmits the parasite to mammalian hosts (humans, canines). The parasites that cause leishmaniasis are present in 98 countries around the world and more than 1 billion people live in areas endemic for leishmaniasis and is at risk of infection [29]. Leishmaniasis is widespread on all continents except Australia and Antarctica and according to WHO, 1.3 million of new cases occur annually [30]. There are three main forms of the disease: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), also known as kala-azar, and mucocuntaneous leishmaniasis (MCL). The clinical features vary depending on the parasite’s characteristics and on the genetic aspects of the host that determine the effectiveness of the immune response [32]. Dogs are considered main reservoirs of the parasite, although rodents, wild rabbits, hares, and cats can also play a relevant role as reservoir host [29]. Cutaneous leishmaniasis is the most prevalent clinical form of leishmaniasis worldwide, and 90% of all CL cases occur in only seven countries: Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia, and Syria. It usually produces ulcers on the exposed parts of the body, such as the face, arms and legs [33]. Psychiatric disorder is associated with patients with extensive lesions that are formed due to cutaneous leishmaniasis. These patients were found to have a low quality of life with symptoms of depression, high anxiety, and low body image satisfaction [34]. CL is the most common form, but VL is responsible for the majority of deaths from leishmaniasis. In visceral leishmaniasis (caused by L. donovani or L. chagasi), the parasite systematically attacks and spreads throughout the body, infecting the body’s macrophage cells, which then carry the parasite to the spleen, lymph nodes, liver, and bone marrow [35]. Of the 200,000 to 400,000 new cases of VL worldwide, more than Molecules 2021, 26, 4629 6 of 41

90% occur in six countries: India, Bangladesh, Sudan, South Sudan, Ethiopia, and Brazil. MCL is clearly distinguishable from other cutaneous leishmaniases by its chronic, latent, and metastatic behavior. In mucocutaneous leishmaniasis, the lesions can lead to the partial or total destruction of the mucous membranes of the nose, mouth, and throat cavities and surrounding tissues [33].

3. Current Regimen for Kinetoplastid Diseases 3.1. HAT Trypanocidals Nowadays, the treatment of HAT disease includes the following five drug options: pentamidine, suramin, melarsoprol, eflornithine (administrated as monotherapies), and nifurtimox– eflornithine combination therapy (NECT) [36]. Pentamidine is considered as the first line treatment for early-stage HAT caused by T.b. gambiense, while T.b. rhodiense is treated with suramin. Pentamidine, an aromatic diamine, was first introduced in 1940s and is considered a broad-spectrum drug, targeting parasitic worms, protozoa and fungi [36]. Apart from early- stage HAT, it has been also administrated for the treatment of leishmaniasis. Pentamidine presents relative toxicity and patients need to be monitored during therapy. This drug acts by preventing the synthesis of trypanosomal proteins, nucleic acids, phospholipids, and folate and its mechanism of action includes the inhibition of the enzymes participating in the polyamine synthesis and the RNA polymerase activity [37]. Suramin is one of the first successful drugs of early medicinal chemistry and it is administrated for first-stage HAT caused by T.b. rhondesiense. This compound, first introduced in 1917, is considered a broad-spectrum drug, presenting very promising results against other trypanosomes. Its administration for HAT disease involves five weekly intravenous injections of 20 mg/kg. Suramin’s mechanism of action towards HAT has not been established yet, even though it has been an extensively used drug for the past century. There is some evidence that suramin inhibits glycolytic enzymes of T. brucei that have high isoelectric points (>9), because of the presence of basic aminoacids in high ratio. The question that has been addressed but not answered yet is whether the drug penetrates the membrane and acts on the glycolytic enzymes inside the glycosomes or whether it binds firstly to the cytosolic glycolytic enzymes and then enters the glycosomes, where these enzymes reside. Apart from the inhibition of glycolytic enzymes, suramin could also prevent sleeping sickness by inhibiting glycerophosphate oxidase, serine oligopepti- dase, and the RNA-editing ligase of the trypanosome’s kinetoplast, although the drug’s penetration to the mitochondrial membrane still remains unclear [38]. Eflornithine, available in the market since 2001, has replaced melarsoprol, as it is much safer to consume. Melarsoprol presented high toxicity that leads to reactive encephalopa- thy [39,40] followed by death. However, eflornithine requires huge doses of administration (56 infusions during 14 days in 14 liters of sterile saline) and consequently, daily visits of patients in health facilities and hospitals. This is a very difficult issue to resolve, since the accessibility to medical care facilities is considered as a privilege of big cities, in the regions that mostly suffer from HAT. Habitants of rural areas do not have easy access to hospitals; thus, the toxic but easily administrated melarsoprol is still used for HAT treatment. Moreover, apart from the difficulty in administration, eflornithine presents a high risk of resistance to patients, resulting in the possibility of untreated second-stage HAT. The concept of combining therapies started to gain ground, as a way of optimizing drugs’ efficacy and lowering the resistance to therapy [41]. Various clinical studies have been performed using a combination of efthornithine, melarsoprol, and nifurtimox (drug administrated for the treatment of Chagas disease). All the studies including the administration of melarsoprol resulted in a high level of toxicity, while promising results were revealed for the combination of eflornithine and nifurtimox (NECT therapy) concerning the treatment of second-stage HAT caused by Trypanosoma brucei gambiense. In order to eliminate eflornithine’s difficulty in administration, WHO designed a medical kit, consist- ing of the drugs in an accessible form, and trained the health workers to implement the Molecules 2021, 26, 4629 7 of 41

aforementioned drug therapy [42]. The NECT therapy does not present the same results for infection caused by Trypanosoma brucei rhodesiense, thus melarsoprol remains the only efﬁcient drug for this disease.

3.2. Antichagastic Drugs Treatment with antitrypanosomal drugs is indicated for acute and congenital CD, chronic disease in infants, and reactivated infections [18]. Benznidazole (BZN) and nifurtimox (NFX) have been the only two nitroheterocyclic available drugs for CD’s treatment since the 1960s [25,28,43]. These drugs are effective in the acute phase, whereas in adults with chronic infection, clinical trials have shown limitations, and both drugs cause several toxic effects leading to restrict their use [28,43]. The mechanism that results in the activation of nitroheterocyclic drugs in trypanosomes is achieved through the involvement of the nitroreductase enzyme (NTR). The resistance of BZN and NFX occurs through the reduced activity of NTR [18]. Benznidazole prevents the multiplication of a parasite, covalently modifying macromolecules. Nifurtimox generates nitro radical anions and becomes reactive in the presence of oxygen [19]. Nifurtimox is no longer used as a therapeutical agent of CD in Brazil due to the fact that it causes neurological disorders or psychiatric episodes as side effects [20,21]. Many patients ﬁnd out that they have contracted of Chagas disease years after the infection. Therefore, there is an urgent need to design novel, effective, and non- toxic drugs.

3.3. Antileishmanial Drugs The chemotherapy currently used for the treatment of leishmaniases include a small spectrum of drugs, that are used as monotherapy or in combination. Provided that there is no vaccine available for the prevention of leishmaniases, the control of the disease depends mainly on these drugs. The therapeutic scheme depends on the type of the disease (cutaneous or visceral leishmaniasis), the parasite species, the geographic location, and probable underlying diseases of the patient. These drugs are in general repurposed from other indi- cations and there are severe limitations concerning their use, which indicates the need of new regimen options. The significant toxicity, low efficacy, and increasing drug resistance are some of the most important problems of the current drugs. The high cost and poor availability of drugs, along with the long duration of therapy and need of hospitalization, are also major concerns, especially for countries in which leishmaniasis is endemic [13,44]. Pentavalent antimonials are the oldest category of antileishmanial drugs. The use of antimony for the treatment of leishmaniasis has been known for several centuries and the first pentavalent antimony compound was synthesized and proved effective against VL in 1925. Today, there are two pentavalent antimonials that are used as first-line antileishmanial drugs: sodium stibogluconate and meglumine antimonate. These drugs are inhibiting enzymes of the glycolytic pathway and fatty acid oxidation in Leishmania parasites. [44] While pentavalent antimonials are used as first-line drugs, their efficacy varies significantly depending on the country, and especially in India, where high rates of resistance are observed [45]. Amphotericin B is a polyene antibiotic which has been used as an antileishmanial drug for several decades. Amphotericin B increases the membrane permeability of Leishmania spp., by binding to ergosterol of the parasite membrane [43]. This drug is used as a second- line drug for VL, and even though it exhibits major side effects such as nephrotoxicity [44], it is recommended from WHO as first-line therapy in South Asia, where the cure rate of antimonial drugs is low. AmBisome®, a liposomal form of amphotericin B, is very effective and lacks of the side effects of the conventional amphotericin B, but its cost is unaffordable, especially for some endemic countries [46]. Paromomycin is another aminoglycoside antibiotic, which has been used as an antileishmanial agent since the 1980s and is currently administrated as a second-line drug, but it is not used as a single agent, because of the risk of developing drug resistance [44,46]. Molecules 2021, 26, 4629 8 of 41

Miltefosine is an alkylphospholipid derivative, approved for the treatment of leishmaniasis in 2002. It was firstly developed as an antineoplastic agent but showed activity against Leishmania spp., and it is the only administrated oral treatment until now. Mil- tefosine modulates parasite membrane receptors and changes the structure of the cell membrane by inhibiting the metabolism of phospholipids. It also affects various intracellular pathways leading to regulated cell death [47]. It has significant toxicity and teratogenic properties and there are several restrictions on its use [44,45]. Combination therapy with at least two drugs is proposed to increase the therapeutic efficacy and avoid drug resistance. The use of a combined dosage form also aims to shorten the duration of therapy and reduce the drug doses as well as to eliminate the toxicity and increase the compliance of patients. Moreover, this therapeutical approach is cost effective for public health systems. There are several forms that are used, such as sodium stibogluconate with paromycin and amphotericin or paromycin in combination with miltefosine. There are also some repurposed drugs, e.g., pentoxyfilline, allopurinol, and various azoles that are used in combination dosage forms, along with antileishmanial drugs [44,46]. The current regimen against kinetoplastid diseases is depicted in Table1. Molecules 2021, 26, 4629 8 of 34

hospitalization, are also major concerns, especially for countries in which leishmaniasis is endemic [13,44]. Molecules 2021, 26, 4629 8 of 34 Pentavalent antimonials are the oldest category of antileishmanial drugs. The use of antimony for the treatment of leishmaniasis has been known for several centuries and the first pentavalent antimony compound was synthesized and proved effective against VL hospitalization, are also major concerns, especially for countries in which leishmaniasis is in 1925. Today, there are two pentavalent antimonials that are used as first-line antileish- endemic [13,44]. manialPentavalent drugs: sodium antimonials stibogluconate are the oldest and catego megluminery of antileishmanial antimonate. drugs. These The drugs use areof inhib- itingantimony enzymes for the of treatmentthe glycolytic of leishmaniasis pathway andhas been fatty known acid oxidation for several incenturies Leishmania and the parasites. [44]first While pentavalent pentavalent antimony antimonials compound are was used synthesized as first-line and drugs,proved their effective efficacy against varies VL signif- icantlyin 1925. depending Today, there on are the two country, pentavalent and antimonialsespecially in that India, are used where as first-line high rates antileish- of resistance aremanial observed drugs: [45]. sodium stibogluconate and meglumine antimonate. These drugs are inhib- itingAmphotericin enzymes of the B glycolytic is a polyene pathway antibiotic and fatty which acid oxidationhas been in used Leishmania as an parasites.antileishmanial drug[44] Whilefor several pentavalent decades. antimonials Amphotericin are used B as increases first-line drugs, the membrane their efficacy permeability varies signif- of Leish- maniaicantly spp., depending by binding on the to country, ergosterol and of especially the parasi in teIndia, membrane where high [43]. rates This of drugresistance is used as a are observed [45]. second-line drug for VL, and even though it exhibits major side effects such as nephrotox- Amphotericin B is a polyene antibiotic which has been used as an antileishmanial icitydrug [44], for severalit is recommended decades. Amphotericin from WHO B increases as first-line the therapymembrane in permeabilitySouth Asia, ofwhere Leish- the cure ® ratemania of spp.,antimonial by binding drugs to ergosterolis low. AmBisome of the parasi, tea liposomalmembrane [43].form This of amphotericin drug is used as B, a is very effectivesecond-line and drug lacks for of VL, the and side even effects though of it theexhibits conventional major side effectsamphotericin such as nephrotox- B, but its cost is unaffordable,icity [44], it is recommendedespecially for from some WHO endemic as first-line countries therapy [46]. in South Paromomycin Asia, where is the another cure ami- noglycosiderate of antimonial antibiotic, drugs whichis low. AmBisomehas been used®, a liposomal as an antileishmanial form of amphotericin agent B, since is very the 1980s andeffective is currently and lacks administrated of the side effects as a ofsecond-line the conventional drug, butamphotericin it is not usedB, but as its a costsingle is agent, becauseunaffordable, of the especiallyrisk of developing for some endemic drug resistance countries [44,46].[46]. Paromomycin is another ami- noglycosideMiltefosine antibiotic, is an alkylphospholipidwhich has been used derivative, as an antileishmanial approved agent for the since treatment the 1980s of leish- and is currently administrated as a second-line drug, but it is not used as a single agent, maniasis in 2002. It was firstly developed as an antineoplastic agent but showed activity because of the risk of developing drug resistance [44,46]. againstMiltefosine Leishmania is anspp., alkylphospholipid and it is the only derivative, administrated approved oral for treatment the treatment until of now. leish- Miltefo- sinemaniasis modulates in 2002. parasite It was firstly membrane developed receptors as an antineoplastic and changes agent the structure but showed of activitythe cell mem- braneagainst by Leishmania inhibiting spp., the and metabolism it is the only of administratedphospholipids. oral It treatment also affects until various now. Miltefo- intracellular pathwayssine modulates leading parasite to regulated membrane cell receptors death [47]and . changesIt has significant the structure toxicity of the celland mem- teratogenic propertiesbrane by inhibiting and there the are metabolism several restrictions of phospholipids. on its useIt also [44,45]. affects Combination various intracellular therapy with atpathways least two leading drugs to is regulated proposed cell to death increase [47]. Itthe has therapeutic significant toxicityefficacy and and teratogenic avoid drug resistance.properties The and use there of aare combined several restrictions dosage form on its also use aims [44,45]. to Combinationshorten the durationtherapy with of therapy andat leastreduce two the drugs drug is dosesproposed as well to increase as to eliminate the therapeutic the toxicity efficacy and and increase avoid drugthe compliance resistance. The use of a combined dosage form also aims to shorten the duration of therapy of patients. Moreover, this therapeutical approach is cost effective for public health sys- and reduce the drug doses as well as to eliminate the toxicity and increase the compliance tems.of patients. There Moreover,are several this forms therapeutical that are used,approa suchch is ascost sodium effective stibogluconate for public health with sys- paromy- cintems. and There amphotericin are several or forms paromycin that are used,in combination such as sodium with stibogluconate miltefosine. withThere paromy- are also some repurposedcin and amphotericin drugs, e.g., or paromycin pentoxyfilline, in combination allopurinol, with andmiltefosine. various There azoles are alsothat someare used in Molecules 2021, 26, 4629 combinationrepurposed drugs,dosage e.g., forms, pentoxyfilline, along with allopurinol, antileishmanial and various drugs azoles [44,46]. that are used in 9 of 41 combinationThe current dosage regimen forms, againstalong with kinetoplastid antileishmanial diseases drugs is [44,46]. depicted in Table 1. The current regimen against kinetoplastid diseases is depicted in Table 1. Table 1.Table Current 1. Current drugs for drugs kine fortoplastid kinetoplastid diseases. diseases. Table 1. Current drugs for kinetoplastid diseases. DrugDrug Structure Structure Comments Comments [14,45] [14 ,45] Efficacy [45] Efﬁcacy [45] Drug Structure Comments [14,45] Efficacy [45] Chagas disease Chagas diseaseChagas disease • • p.o.p.o. • 60–85% in• BenznidazoleBenznidazoleBenznidazole • p.o. • 60–85% in 60–85% in acute disease. Ineffective in • • • Tolerability,Tolerability,Tolerability, toxicity, tox- tox- incompliance.acute disease.acute disease. the chronic stage. icity,icity, incompliance. incompliance. IneffectiveIneffective in in the chronicthe chronic stage. stage. •• p.o. • NifurtimoxNifurtimoxNifurtimox • p.o.p.o. Effective• Effective • Effective against acute disease. •• Tolerability, toxicity, incompliance. • Tolerability,Tolerability, tox- tox- againstagainst acute acute Molecules 2021, 26, 4629 icity, incompliance. disease. 9 of 34 icity, incompliance. disease. Human African Trypanosomiasis (HAT) Human AfricanHuman AfricanTrypanosomiasis Trypanosomiasis (HAT) (HAT) Suramin • i.v. • Effective against hemolymphatic stage of Suramin • i.v. • Effective • Toxicity. Rhodesian trypanosomiasis. • Toxicity. against hemolymphatic stage of Rho- desian trypanosomiasis.

Pentamidine • i.m. • Effective • Toxicity. against hemolymphatic stage of Gam- bian trypanosomiasis only. Melasoprol • i.v. • Effective • Suitable for sec- against menin- ond-stage disease. goencephalic • High toxicity. stage of both • High levels of forms of the treatment failure in disease. It is some regions. used only for Rhodesian trypanosomiasis. Eflornithine • i.v. • Most effective • Suitable for sec- against menin- ond-stage disease. goencephalic • High cost. stage of Gam- • Not efficacious bian trypano- against T.b. somiasis. Ef- rhodesiense. fective in mon- NECT Eflornithine-Nifurtimox combination • Suitable for sec- otherapy or in ond-stage disease. combination • Reduced cost and with nifur- duration compared timox. to monotherapy. Leishmaniases Sodium • i.v. or i.m. • 35–95% de- stibogluconate • First-line treat- pending on ment. country, re- • High resistance in sistance in In- some regions of In- dia. dia. • Toxicity. Meglumine an- • i.v. or i.m. • 35–95% de- timonate • First-line treat- pending on ment. country. • High resistance in some regions of In- dia. • Toxicity.

Molecules 2021, 26, 4629 9 of 34

Suramin • i.v. • Effective Molecules 2021, 26, 4629 • 9 of 34 Toxicity. against hemolymphatic stage of Rho- Suramin • i.v. • desianEffective trypa- • Molecules 2021, 26, 4629 Toxicity. nosomiasis.against hemo-9 of 34 lymphatic stage of Rho- desian trypa- • • Suramin i.v. nosomiasis.Effective Pentamidine • i.m.Toxicity. • Effectiveagainst hemo- Molecules 2021, 26, 4629 9 of 34 • Toxicity. againstlymphatic hemo- lymphaticstage of Rho- Suramin • i.v. • Effective stagedesian of trypa- Gam- Pentamidine • Toxicity.• i.m. against hemo-• bianEffectivenosomiasis. trypano- Molecules 2021, 26, 4629 10 of 41 • Toxicity. lymphatic somiasisagainst hemo- only. • stage of Rho-• lymphatic Melasoprol i.v. desian trypa- Effective • Suitable for sec-nosomiasis. againststage of menin- Gam- Table 1. Cont. ond-stage disease. goencephalicbian trypano- Pentamidine • i.m. • Effective • High toxicity. stagesomiasis of both only. • Toxicity. against hemo- DrugMelasoprol Structure• Highi.v. Comments levels of [14,45]• formsEffective of the Efficacy [45] lymphatic Pentamidine Pentamidine • • i.m.• i.m. treatmentSuitable for failure• sec- Effective in disease.against menin- It• is Effective against hemolymphatic stage of • • Toxicity. against hemo- stage of Gam- Toxicity.someond-stage regions. disease. usedgoencephalic only for Gambian trypanosomiasis only. lymphatic bian trypano- • High toxicity. Rhodesianstage of both stage of Gam- somiasis only. • High levels of bian trypano- trypanosomia-forms of the Melasoprol • • i.v. • • Effective against meningoencephalic Melasoprol treatmenti.v. failuresomiasis in only. sis.disease.Effective It is • •Suitable for second-stage disease. stage of both forms of the disease. It is Melasoprol • i.v.• Suitable for• sec- Effective• against menin- Eflornithine • • Highi.v.some toxicity. regions. Mostused onlyeffective forused only for Rhodesian Suitable• ond-stage for sec- disease.against menin-goencephalic • ond-stage HighSuitable levelsdisease. of for treatment sec-goencephalic failure in againstRhodesian some menin- trypanosomiasis. • High toxicity. stage of both • Highregions. toxicity.ond-stage disease.stage of both goencephalictrypanosomia- • High levels of forms of the • High• levelsHigh of cost. forms of the stagesis. of Gam- • treatmenttreatment failure in failuredisease. in It is disease. It is EflornithineEflornithine • i.v. Noti.v. efficacious • bianMost trypano- effective• Most effective against • some regions. used only for • Suitablesome for regions. second-stage disease. used only formeningoencephalic stage of Gambian • againstSuitable T.b. for sec-Rhodesian somiasis.against menin- Ef- High cost. Rhodesian trypanosomiasis. Effective in • rhodesienseond-stage .disease. trypanosomia- fectivegoencephalic in mon- Not efficacious against T.b. rhodesiensetrypanosomia-. monotherapy or in combination with NECT Eflornithine-Nifurtimox combination • SuitableHigh cost. for sec-sis. otherapystage of Gam- or in • i.v. • Most effective sis. nifurtimox. NECTEflornithine Eflornithine-Nifurtimox combination • • Suitableond-stageNot forefficacious second-stage disease. disease. combinationbian trypano- • Suitable• for sec- against menin-• Eflornithine • • ReducedReducedagainsti.v. cost T.b. andcost duration and comparedwithsomiasis.Most to nifur-effective Ef- ond-stage• Suitable disease. for sec-goencephalic against menin- • Highmonotherapy. cost.durationrhodesiense compared. stage of Gam- timox.fective in mon- • ond-stage disease. goencephalic NECT Eflornithine-Nifurtimox combination Not• efficacioustoSuitable monotherapy. for sec-bian trypano- otherapy or in • Leishmaniasesagainst T.b.ond-stageHigh cost. disease.somiasis. Ef- combinationstage of Gam- Leishmaniases rhodesiense• . fective in mon- • • Not efficacious • bian trypano-• Sodium stibogluconateSodium NECT Eflornithine-Nifurtimox combination • Suitable i.v. ori.v.Reduced for i.m. sec-or i.m. cost andotherapy or in 35–95%with nifur- de- 35–95% depending on country, resistance • First-lineagainst treatment. T.b. somiasis. Ef- in India. stibogluconate ond-stage• First-lineduration disease. comparedtreat- combination pendingtimox. on • • Highrhodesiense resistance.in some regions offective India. in mon- Reducedment.to cost monotherapy. and with nifur- country, re- NECT Eflornithine-Nifurtimox combination • duration• Toxicity.Suitable compared for sec-timox. otherapy or in Leishmaniases • High resistance in sistance in In- to monotherapy.ond-stage disease. combination Sodium • somei.v. or regionsi.m. of In- • dia.35–95% de- Leishmaniases • Reduced cost and with nifur- stibogluconateSodium • i.v.• or i.m.dia.First-line treat-• 35–95% depending on duration compared timox. stibogluconate • First-line• Toxicity.ment. treat- pending on country, re- to monotherapy. ment.• High resistancecountry, in •resistance in In- Meglumine an- • i.v. or i.m. 35–95% de- Leishmaniases High• resistance in sistance in In- timonate some regionsFirst-linesome of regions In- treat- ofdia. In- pendingdia. on Sodium • i.v. or i.m. • 35–95% de- dia. ment.dia. country. • • First-line treat- pending on stibogluconate Toxicity.• HighToxicity. resistance in • i.v. or i.m.ment. • 35–95% de- country, re- MeglumineMeglumine an- an- • somei.v. or regionsi.m. of In- • 35–95% de- timonate • First-line• High treat- resistancepending in on sistance in In- timonate • dia.First-line treat- pending on ment. some regions ofcountry. In- dia. • High• resistanceToxicity.ment. in country. dia. some• regionsHigh of resistance In- in dia.• Toxicity. some regions of In- • Toxicity.• • Meglumine an- dia.i.v. or i.m. 35–95% de- • timonate • Toxicity.First-line treat- pending on ment. country. • High resistance in some regions of In- dia. • Toxicity.

Molecules 2021, 26, 4629 9 of 34

Suramin • i.v. • Effective • Toxicity. against hemolymphatic stage of Rho- desian trypanosomiasis.

Pentamidine • i.m. • Effective • Toxicity. against hemolymphatic stage of Gam- bian trypanosomiasis only. Melasoprol • i.v. • Effective • Suitable for sec- against menin- ond-stage disease. goencephalic • High toxicity. stage of both • High levels of forms of the treatment failure in disease. It is some regions. used only for Rhodesian trypanosomiasis. Eflornithine • i.v. • Most effective • Suitable for sec- against menin- ond-stage disease. goencephalic • High cost. stage of Gam- • Not efficacious bian trypano- against T.b. somiasis. Ef- rhodesiense. fective in mon- NECT Eflornithine-Nifurtimox combination • Suitable for sec- otherapy or in ond-stage disease. combination • Reduced cost and with nifur- duration compared timox. to monotherapy. Leishmaniases Sodium • i.v. or i.m. • 35–95% de- Molecules 2021, 26, 4629 11 of 41 stibogluconate • First-line treat- pending on ment. country, re- • High resistance in sistance in In- Table 1. Cont. some regions of In- dia. dia. Drug Structure• Toxicity. Comments [14,45] Efﬁcacy [45] Meglumine antimonateMeglumine an- • • i.v. ori.v. i.m. or i.m. • 35–95% de-• 35–95% depending on country. • timonate • First-lineFirst-line treatment. treat- pending on • High resistance in some regions of India. ment. country. • Toxicity. • High resistance in some regions of In- Molecules 2021, 26, 4629 dia. 10 of 34

• Toxicity.

Amphotericin B Amphotericin B • • i.v. i.v. • ~100% for VL • ~100% for VL in India. • • VeryVery effective effective in in regionsin India. with resistance regionsto pentavalent with re- antimonials. • sistanceHigh to toxicity. pentavalent antimonials. • High toxicity.

AmBisome® liposomial formula of amphotericin B • i.v. • ~95% in India ® • i.v. • ~95% in India and Asia, variable in AmBisome liposomial formula of amphotericin B • Well tolerated. and Asia, vari- • Well tolerated. Africa. • High cost. able in Africa. • High cost. Paromycin • i.m. • 93–95% in In- • Ototoxicity. dia, 65–85% in Africa.

Miltefosine • p.o. • 94–97% for VL • Very high efficacy in India, not for VL. efficient as sin-

• Teratogenic. gle dose in • Increasing treat- Asia/Africa, ment failures. not registered in many endemic countries. p.o.: per os, i.v.:intravenous infusion, i.m.: intramascular injection.

4. Drug Targets and Inhibitors 4.1. Kinetoplastid Proteasome Even if the identification of a common kinetoplastid drug target against T. brucei, T. cruzi, and Leishmania spp. looks like a unicorn of antiprotozoan chemotherapy, the kinetoplastid proteasome has recently become an attractive therapeutic target. Proteasomes are complex enzymes responsible for the turnover of short-lived, abnormal, or damaged proteins in eukaryotic cells. Kinetoplastid proteasomes resemble but distinguish those of other eukaryotes. Two separate studies have shown similar proteasome inhibitors with excellent potency and selectivity against kinetoplastid parasites, both in vitro and in vivo [48]. The Genomics Institute of the Novartis Research Foundation (GNF) has identified an azabenzoxazole compound series (GNF5343, 1 and the optimized GNF6702 derivative 2) as pan-kinetoplastid proteasome inhibitors [49]. Moreover, GSK and Dundee Drug Dis- covery have also identified a similar azabenzoxazole adduct (GSK3494245/DDD01305143, 3), which exhibits similar structural features to the GNF derivative [50]. Structural studies have revealed the binding site for both clusters of compounds in an open pocket between the proteasome subunits. The binding mode suggests a different orientation for the two- compound series, but this may need to be revised. Pyridine substitution improved the solubility of GNF6702 affording LXE408 adduct, which is now being tested in Phase I clinical trials [51]. These findings create the expectation that the kinetoplast proteasome might be the Achilles’ heel of all trypanosomids. The above chemical entities are listed in Table 2.

Molecules 2021, 26, 4629 10 of 34 Molecules 2021, 26, 4629 10 of 34

Amphotericin B • i.v. • ~100% for VL Amphotericin B • i.v. • ~100% for VL • Very effective in in India. • Very effective in in India. regions with re- regions with resistance to penta- sistance to pentavalent antimonials. valent antimonials. • High toxicity. Molecules 2021, 26, 4629 • High toxicity. 12 of 41

AmBisome® liposomial formula of amphotericin B • i.v. • ~95% in India AmBisome® liposomial formula of amphotericinTable B 1. Cont.• i.v. • ~95% in India • Well tolerated. and Asia, vari- • Well tolerated. and Asia, vari- Drug Structure• High Comments cost. [14,45]able in Africa. Efﬁcacy [45] • High cost. able in Africa. ParomycinParomycin • • i.m.i.m. • 93–95% in• In-93–95% in India, 65–85% in Africa. Paromycin • i.m. • 93–95% in In- • • Ototoxicity.Ototoxicity. dia, 65–85% in • Ototoxicity. dia, 65–85% in Africa. Africa.

Miltefosine • • p.o. • 94–97% for VL MiltefosineMiltefosine • p.o.p.o. • 94–97% for• VL94–97% for VL in India, not efﬁcient as • • VeryVery high high efﬁcacy efficacy for VL. in India, not • Very high efficacy in India, not single dose in Asia/Africa, not • Teratogenic.for VL. efficient as sin-registered in many endemic countries. • for VL. efficient as sin- • IncreasingTeratogenic. treatment failures. gle dose in • Teratogenic. gle dose in • Increasing treat- Asia/Africa, • Increasing treat- Asia/Africa, p.o.: per os, i.v.:intravenous infusion, i.m.: intramascularment failures. injection. not registered ment failures. not registered in many en- in many endemic coun- demic countries. tries. p.o.: per os, i.v.:intravenous infusion, i.m.: intramascular injection. p.o.: per os, i.v.:intravenous infusion, i.m.: intramascular injection. 4. Drug Targets and Inhibitors 4. Drug Targets and Inhibitors 4.1. Kinetoplastid Proteasome 4.1. Kinetoplastid Proteasome Even if the identification of a common kinetoplastid drug target against T. brucei, T. Even if the identification of a common kinetoplastid drug target against T. brucei, T. cruzi, and Leishmania spp. looks like a unicorn of antiprotozoan chemotherapy, the kinet- cruzi, and Leishmania spp. looks like a unicorn of antiprotozoan chemotherapy, the kinetoplastid proteasome has recently become an attractive therapeutic target. Proteasomes are oplastid proteasome has recently become an attractive therapeutic target. Proteasomes are complex enzymes responsible for the turnover of short-lived, abnormal, or damaged pro- complex enzymes responsible for the turnover of short-lived, abnormal, or damaged proteins in eukaryotic cells. Kinetoplastid proteasomes resemble but distinguish those of teins in eukaryotic cells. Kinetoplastid proteasomes resemble but distinguish those of other eukaryotes. Two separate studies have shown similar proteasome inhibitors with other eukaryotes. Two separate studies have shown similar proteasome inhibitors with excellent potency and selectivity against kinetoplastid parasites, both in vitro and in vivo excellent potency and selectivity against kinetoplastid parasites, both in vitro and in vivo [48]. The Genomics Institute of the Novartis Research Foundation (GNF) has identified an [48]. The Genomics Institute of the Novartis Research Foundation (GNF) has identified an azabenzoxazole compound series (GNF5343, 1 and the optimized GNF6702 derivative 2) azabenzoxazole compound series (GNF5343, 1 and the optimized GNF6702 derivative 2) as pan-kinetoplastid proteasome inhibitors [49]. Moreover, GSK and Dundee Drug Dis- as pan-kinetoplastid proteasome inhibitors [49]. Moreover, GSK and Dundee Drug Dis- covery have also identified a similar azabenzoxazole adduct (GSK3494245/DDD01305143, covery have also identified a similar azabenzoxazole adduct (GSK3494245/DDD01305143, 3), which exhibits similar structural features to the GNF derivative [50]. Structural studies 3), which exhibits similar structural features to the GNF derivative [50]. Structural studies have revealed the binding site for both clusters of compounds in an open pocket between have revealed the binding site for both clusters of compounds in an open pocket between the proteasome subunits. The binding mode suggests a different orientation for the two- the proteasome subunits. The binding mode suggests a different orientation for the two- compound series, but this may need to be revised. Pyridine substitution improved the compound series, but this may need to be revised. Pyridine substitution improved the solubility of GNF6702 affording LXE408 adduct, which is now being tested in Phase I clin- solubility of GNF6702 affording LXE408 adduct, which is now being tested in Phase I clinical trials [51]. These findings create the expectation that the kinetoplast proteasome might ical trials [51]. These findings create the expectation that the kinetoplast proteasome might be the Achilles’ heel of all trypanosomids. The above chemical entities are listed in Table be the Achilles’ heel of all trypanosomids. The above chemical entities are listed in Table 2. 2.

Molecules 2021, 26, 4629 13 of 41

4. Drug Targets and Inhibitors 4.1. Kinetoplastid Proteasome Even if the identification of a common kinetoplastid drug target against T. brucei, T. cruzi, and Leishmania spp. looks like a unicorn of antiprotozoan chemotherapy, the kinetoplastid proteasome has recently become an attractive therapeutic target. Proteasomes are complex enzymes responsible for the turnover of short-lived, abnormal, or damaged proteins in eukaryotic cells. Kinetoplastid proteasomes resemble but distinguish those of other eukaryotes. Two separate studies have shown similar proteasome inhibitors with excellent potency and selectivity against kinetoplastid parasites, both in vitro and in vivo [48]. The Genomics Institute of the Novartis Research Foundation (GNF) has identified an azabenzoxazole compound series (GNF5343, 1 and the optimized GNF6702 derivative 2) as pan-kinetoplastid proteasome inhibitors [49]. Moreover, GSK and Dundee Drug Discovery have also identified a similar azabenzoxazole adduct (GSK3494245/DDD01305143, 3), which exhibits similar structural features to the GNF derivative [50]. Structural studies have revealed the binding site for both clusters of compounds in an open pocket between the proteasome subunits. The binding mode suggests a different orientation for the two- compound series, but this may need to be revised. Pyridine substitution improved the solubility of GNF6702 affording LXE408 adduct, which is now being tested in Phase I clinical trials [51]. These findings create the expectation that the kinetoplast proteasome might Molecules 2021, 26, 4629 11 of 34 be the Achilles’ heel of all trypanosomids. The above chemical entities are listed in Table2.

Table 2. Kinetoplastid proteasome inhibitors. Molecules 2021, 26, 4629 Table 2. Kinetoplastid proteasome inhibitors. 11 of 34

Cmpd Cmpd StructureStructure Name Name Ref. Ref. Molecules 2021, 26, 4629 11 of 34 Table 2. Kinetoplastid proteasome inhibitors.

Cmpd Structure Name Ref. 1 GNF5343 [49] 1 Table 2. Kinetoplastid proteasome inhibitors.GNF5343 [49] Cmpd Structure Name Ref. 1 GNF5343 [49]

1 GNF5343 [49] 2 GNF6702 [49]

2 GNF6702 [49] 2 GNF6702 [49]

2 GNF6702 [49] GSK3494245 3 [50] GSK3494245DDD01305143 3 [50] DDD01305143

GSK3494245 3 GSK3494245 [50] 3 DDD01305143 [50] 4.2. T. brucei DDD01305143 4.2. T. brucei 4.2.1. RNA-Editing Ligase 1 (REL1) 4.2.1. RNA-Editing Ligase 1 (REL1) Novel drug approaches aim to target the trypanosome’s survival and replication in 4.2. T.Novel brucei drug approaches aim to target the trypanosome’s survival and replication in order to prevent its spread throughout the body. A key enzyme participating in trypano- 4.2.1.order RNA-Editing to prevent its Ligase spread 1 throughout(REL1) the body. A key enzyme participating in trypanosome’ssome’s survival survival in in both both insect and and bloodstream bloodstream form form is the is RNA-editingthe RNA-editing ligase ligase1 (REL1). 1 (REL1). Novel drug approaches aim to target the trypanosome’s survival and replication in 4.2. T. brucei Thus,Thus, REL1 REL1 is is considered considered as aa promising promising target target for forT. brucei T. brucei’s elimination.’s elimination. Computational Computational order to prevent its spread throughout the body. A key enzyme participating in trypano- 4.2.1. RNA-Editing Ligasestudies 1 have (REL1) been applied by Amaro et al., suggesting molecular dynamics simulations studiessome’s have survival been in appliedboth insect by and Amaro bloodstream et al., suggestingform is the RNA-editing molecular dynamicsligase 1 (REL1). simulations on the REL1, in order to take account of the protein’s flexibility in the drug discovery Novel drug approachesonThus, the REL1,REL1 aim is in considered toorder target to as take thea promising account trypanosome’s target of the for protein’s T. survivalbrucei’s flexibilityelimination. and replication in Computational the drug discovery process [52]. Docking studies, following the molecular dynamic (MD) simulations, were processstudies [52]. have Docking been applied studies, by Amaro following et al., thesuggesting molecular molecular dynamic dynamics (MD) simulationssimulations, were in order to prevent itsperformed spread to throughoutdifferent conformations the body. of the A enzyme key enzymeand revealed participating 30 compounds inas com- performedon the REL1, to different in order toconformations take account ofof thethe protein’s enzyme flexibilityand revealed in the 30 drug compounds discovery as com- trypanosome’s survivalpetent in inhibitors both insect of REL1. and The bloodstream compounds that form presented is thethe strongest RNA-editing binding affinity lig- to petentprocess inhibitors [52]. Docking of REL1. studies, The following compounds the molecular that presented dynamic the (MD) strongest simulations, binding were affinity to the protein’s active site were further evaluated through MD simulations. The final scoring ase 1 (REL1). Thus, REL1performed is considered to different conformations as a promising of the targetenzyme forandT. revealed brucei 30’s compounds elimination. as com- thepinpointed protein’s activeeight ligands site were that further were examined evaluated through through in vitro MD biologicalsimulations. assays. The In final fact, scoring Computational studiespetent have inhibitors been applied of REL1. The by Amarocompounds et that al., suggestingpresented the strongest molecular binding dynam- affinity to pinpointedtwo of the eightproposed ligands compounds that were 4, 5 indicatedexamined important through selectivity in vitro andbiological low micromolar assays. In fact, the protein’s active site were further evaluated through MD simulations. The final scoring ics simulations on thetwoactivity REL1,of the against proposed in order REL1 compounds [53]. to take account 4, 5 indicated of the important protein’s selectivity ﬂexibility and in low the micromolar drug discovery processpinpointed [52]. Dockingeight ligands studies, that were following examined through the molecular in vitro biological dynamic assays. (MD) In fact, activitytwo of againstthe proposed REL1 compounds [53]. 4, 5 indicated important selectivity and low micromolar 4.2.2. UDP-Glucose 4′-Epimerase (GalE) activity against REL1 [53]. 4.2.2. UDP-GlucoseComputer-aided 4′-Epimerase studies have (GalE) identifi ed Uridine Diphosphate Galactose 4′-Epi- 4.2.2.meraseComputer-aided UDP-Glucose inhibitors as 4 ′novel-Epimerase studies therapy have (GalE) for identifiAfri can edsleeping Uridine sickness. Diphosphate TbGalE, also Galactose known as 4′-Epi- UDP-glucose 4′-epimerase, participates in galactose metabolism of the parasite T. brucei. meraseComputer-aided inhibitors as novel studies therapy have identififor Afriedcan Uridine sleeping Diphosphate sickness. GalactoseTbGalE, also4′-Epi- known as Galactose is an essential component of surface glycoproteins that plays a key role in the merase inhibitors as novel therapy for African sleeping sickness. TbGalE, also known as UDP-glucoseevasion of the 4′ -epimerase,parasite in host participates cells. Thus, inTb galactGalE couldose metabolism also serve as ofa potentthe parasite target for T. brucei. UDP-glucose 4′-epimerase, participates in galactose metabolism of the parasite T. brucei. GalactoseHAT therapy. is an Durrant’sessential researchcomponent group of identified surface TbGalEglycoproteins inhibitors that using plays a combination a key role in the Galactose is an essential component of surface glycoproteins that plays a key role in the evasionof docking of the and parasite MD studies. in host Twenty-four cells. Thus, cluste TbrsGalE of the could protein also were serve identified as a potent and used target for evasion of the parasite in host cells. Thus, TbGalE could also serve as a potent target for as input for the docking studies. The trajectory analysis of the protein offers more chances HATHAT therapy. therapy. Durrant’s Durrant’s research research group group identified identified TbGalE TbGalE inhibitors inhibitors using ausing combination a combination for discovering effective ligands. The compounds that presented the strongest binding of ofdocking docking and and MDMD studies. Twenty-four Twenty-four cluste clusters ofrs the of proteinthe protein were identifiedwere identified and used and used affinities in the in silico calculations of UDP-glucose were further evaluated experimen- as asinput input for for the the dockingdocking studies. studies. The The trajectory trajectory analysis analysis of the of protein the protein offers moreoffers chances more chances tally. In vitro assays of the ligands indicated that the fused ring systems of the structures, forfor discovering discovering effectiveeffective ligands.ligands. The The comp compoundsounds that thatpresented presented the strongest the strongest binding binding even though they contribute to ligand binding, do not reinforce the selectivity of the com- affinitiesaffinities in in the the inin silico calculationscalculations of ofUDP-glucose UDP-glucose were were further further evaluated evaluated experimen- experimen- pounds, thus they could possibly be replaced by an equivalent group. This study suggests tally. In vitro assays of the ligands indicated that the fused ring systems of the structures, tally.taking In vitro advantage assays of ofthe the presence ligands of indicateda thiol-group that in theposition fused C266 ring of systems the protein of inthe order structures, even though they contribute to ligand binding, do not reinforce the selectivity of the com- evento amelioratethough they the contributeligands’ selectivity, to ligand because binding, this structuraldo not reinforce characteristic the selectivity (thiol-group) of theis compounds, thus they could possibly be replaced by an equivalent group. This study suggests pounds, thus they could possibly be replaced by an equivalent group. This study suggests taking advantage of the presence of a thiol-group in position C266 of the protein in order takingto ameliorate advantage the ligands’of the presence selectivity, of because a thiol-gr thisoup structural in position characteristic C266 of (thiol-group) the protein is in order to ameliorate the ligands’ selectivity, because this structural characteristic (thiol-group) is

Molecules 2021, 26, 4629 14 of 41

simulations, were performed to different conformations of the enzyme and revealed 30 compounds as competent inhibitors of REL1. The compounds that presented the strongest binding afﬁnity to the protein’s active site were further evaluated through MD simulations. The ﬁnal scoring pinpointed eight ligands that were examined through in vitro biological assays. In fact, two of the proposed compounds 4, 5 indicated important selectivity and low micromolar activity against REL1 [53].

4.2.2. UDP-Glucose 40-Epimerase (GalE) Computer-aided studies have identified Uridine Diphosphate Galactose 40-Epimerase inhibitors as novel therapy for African sleeping sickness. TbGalE, also known as UDP- glucose 40-epimerase, participates in galactose metabolism of the parasite T. brucei. Galac- tose is an essential component of surface glycoproteins that plays a key role in the evasion of the parasite in host cells. Thus, TbGalE could also serve as a potent target for HAT therapy. Durrant’s research group identified TbGalE inhibitors using a combination of docking and MD studies. Twenty-four clusters of the protein were identified and used as input for the docking studies. The trajectory analysis of the protein offers more chances for discovering effective ligands. The compounds that presented the strongest binding affinities in the in silico calculations of UDP-glucose were further evaluated experimentally. In vitro assays of the ligands indicated that the fused ring systems of the structures, even though they contribute to ligand binding, do not reinforce the selectivity of the compounds, thus they could possibly be replaced by an equivalent group. This study suggests taking advantage of the presence of a thiol-group in position C266 of the protein in order to ameliorate the ligands’ selectivity, because this structural characteristic (thiol-group) is absent in the corresponding protein of humans [54]. According to this investigation, compound 6 indicates the most favored inhibitory activity against TbGalE.

4.2.3. Pteridine Reductase-1 (PTR1) Another signiﬁcant enzyme for trypanosome’s survival that could serve as a target for the treatment of HAT is pteridine reductase-1 (PTR1) [55]. Drugs bearing aminobenzothia- zole and aminobenzimidazole scaffolds presented promising inhibitory activity against PTR1. Virtual screening of a fragment library was performed in order to discover the structural properties of potent PTR10s inhibitors. Docking studies in PTR10s crystal structure (PDB: 3BME) indicated the required ligands’ characteristics for the exertion of inhibitory activity. Computational studies were followed by complexes’ crystallization so as to discover the most accurate binding mode of the ligands inside PTR10s cavity. Furthermore, among all the compounds that were tested, compound 7 was evaluated as a selective inhibitor with low micromolar potency [56].

4.2.4. Histone Deacetylase Enzymes (HDAC) Several anticancer therapies have been investigated for their use against parasitic diseases. According to Murphy et al., CTLA-4 blockers enhance host resistance against Leishmania donovani [57], while the same effects have been observed for PDL-1 blockers by Habib et al. [58]. Histone deacetylase enzymes (HDAC) play a significant role in the modulation of parasites’ gene expression, and hence they could serve as potent targets for HAT therapy [59]. Many inhibitors of these enzymes (class I/II HDAC inhibitors and sirtuin inhibitors) have been discovered and approved for anticancer therapy. Engel et al. identified four HDAC inhibitors that presented important inhibitory activity towards trypanosomal parasites. Even if these compounds could effectively inhibit HDAC enzymes (cmpd 8), they exerted significant cytotoxicity on the cell lines. The most potent compounds for HDAC inhibition proved to be sulphonylpiperazines and in particular compound 8 (IC50 = 34 nM), which bears an heteroaryl ring attached to the piperazine scaffold. This adduct not only caused parasitic death within 4 h after treatment, but also its dosage for administration was quite low (2 µg/mL). Furthermore, hydroxamate-based HDAC inhibitors were tested for their potency to inhibit the proliferation of T.b. brucei in the Molecules 2021, 26, 4629 15 of 41

bloodstream. These are clinically approved anticancer agents that indicated moderate potency against the parasites. Unfortunately, these derivatives were further tested as parental compounds for co-administration with standard treatment (pentamidine, suramin, melarsoprol, nifurtimox), with no promising results for antiparasitic activity [60].

4.2.5. N-Myristoyltransferase (NMT) A popular enzyme that can be targeted for the treatment of HAT is N-myristoyltransferase (NMT) [61–63]. High-throughput screening was performed against NMT and generated a series of compounds that are active in a mouse model of the first stage HAT. NMT inhibitors cause the death of trypanosomes both in mice and cell lines, by binding strongly to NMT’s cavity and inhibiting the enzyme’s function. The only problem is that NMT also exists in humans, although this problem could easily be resolved since T. brucei indicated more Molecules 2021, 26, 4629 sensitivity towards the protein’s inhibition. NMT also served as13 a of target 34 for the treatment of second stage HAT. The treatment of the disease in the second stage is quite challenging, since the drug needs to penetrate the BBB without causing any toxicity to the cells [64]. drugs interact with the trypanothione4.2.6. Trypanothione system (Figure System—Trypanothione 2). Specifically, three Reductase enzymes, (TR) glutathionyl spermidine synthetase, trypanothioneThe trypanothione synthetase, system isand central trypanothione for any thiol regenerationreductase, in trypanosomatids. The absence of trypanothione metabolism from the mammalian cells makes TR a promising emerge as main targets of rational drug design [65]. Trypanothione reductase (TR) is a target for the development of novel, potent antitrypanosomal drugs. Several existing drugs flavoenzyme which is of interest interactfor its withessential the trypanothione role in controlling system (Figure the redox2). Speciﬁcally, homeostasis three enzymes, glutathionyl of T. cruzi. Its crystal structure wasspermidine solved in synthetase, complex trypanothione with NADPH synthetase, or inhibitors and trypanothione or alone reductase, emerge as [66–68]. TR is a homodimer thatmain includes targets an of rationalFAD-binding drug design domain, [65]. Trypanothione an NADPH-binding reductase (TR) is a ﬂavoenzyme domain, and an interface domain.which The is enzyme of interest catalyzes for its essential the NADPH-dependent role in controlling the redoxreduc- homeostasis of T. cruzi. Its crystal structure was solved in complex with NADPH or inhibitors or alone [66–68]. tion of dithiol trypanothione, representingTR is a homodimer high selectivity that includes [69]. an FAD-binding domain, an NADPH-binding domain, and an interface domain. The enzyme catalyzes the NADPH-dependent reduction of dithiol trypanothione, representing high selectivity [69].

Glutamate + Cysteine

γ –Glutamyl γ –Glutamyl Cysteine cysteine synthetase

Glutathione synthetase Glutathione

Glutathionyl spermidine synthetase Glutathionyl Spermidine

Trypanothione synthetase Trypanothione

(TSH2) Trypanothione reductase Trypanothione Disulfide

(TS2)

Figure 2. Trypanothione biosynthesis pathway. Figure 2. Trypanothione biosynthesis pathway.The binding mode of the inhibitors under study varies between T. cruzi and T. brucei. Docking studies revealed that the inhibitors bind to the hydrophobic “wall” of TR’s The binding mode of the inhibitorsactive site. under The study crystallization varies between of T.b. protein T. cruzi with and BTCP T. brucei (1-(1-(benzo[b]thiophen-2-. Docking studies revealed that the inhibitors bind to the hydrophobic “wall” of TR’s active site. The crystallization of T.b. protein with BTCP (1-(1-(benzo[b]thiophen-2-yl)cyclohexyl)piperidine) inhibitor in the active site led the drug design investigation, conducted by Persch et al. BTCP analogues were designed in silico and further assessed by docking studies. The binding pocket “Z-site” was described by the sidechains of Phe396′, Pro 398′, and Leu399 and the PDB code: 1BZL was used for these calculations. The analogues that presented strong binding to the enzyme’s cavity were synthesized and enzymatically evaluated. Among all the structures that were generated, the most favorable uncompetitive inhibition constant (Kui) was calculated for derivative 7 against T. brucei. According to this study, the disubstituted methyl thiazole structural part is crucial for exerting antitrypanosomal activity [70]. Additional in silico studies were performed for more BTCP analogues to TR enzyme (PDB:1BZL) by Raoul De Gasparo et al. This study revealed new structural features of TR inhibitors. The addition of one indole group at the structural skeleton indicated improved activity against the TR enzyme of T. cruzi, while the addition of both benzyl and indole groups presented the strong inhibition of TR in T. brucei [67]. Finally, another study oriented towards the drug discovery of TR inhibitors was conducted by Patterson et al. in 2009. According to this investigation, cell-based enzymatic assays were performed for a variety of BTCP analogues and the compounds that presented low micromolar activity against TR enzyme were revealed [63]. The crystal structure of trypanothione with co-crystallazed ligands is illustrated in Figure 3.

Molecules 2021, 26, 4629 16 of 41

yl)cyclohexyl)piperidine) inhibitor in the active site led the drug design investigation, conducted by Persch et al. BTCP analogues were designed in silico and further assessed by docking studies. The binding pocket “Z-site” was described by the sidechains of Phe3960, Pro 3980, and Leu399 and the PDB code: 1BZL was used for these calculations. The analogues that presented strong binding to the enzyme’s cavity were synthesized and enzymatically evaluated. Among all the structures that were generated, the most favorable uncompetitive inhibition constant (Kui) was calculated for derivative 7 against T. brucei. According to this study, the disubstituted methyl thiazole structural part is crucial for exerting antitrypanosomal activity [70]. Additional in silico studies were performed for more BTCP analogues to TR enzyme (PDB:1BZL) by Raoul De Gasparo et al. This study revealed new structural features of TR inhibitors. The addition of one indole group at the structural skeleton indicated improved activity against the TR enzyme of T. cruzi, while the addition of both benzyl and indole groups presented the strong inhibition of TR in T. brucei [67]. Finally, another study oriented towards the drug discovery of TR inhibitors was Molecules 2021, 26, 4629 14 of 34 conducted by Patterson et al. in 2009. According to this investigation, cell-based enzymatic assays were performed for a variety of BTCP analogues and the compounds that presented low micromolar activity against TR enzyme were revealed [63]. The crystal structure of trypanothioneThe abobe withnovel co-crystallazed chemical agents ligands against is T. illustrated brucei are in presented Figure3. in Table 3.

FigureFigure 3. 3. TrypanothioneTrypanothione crystal crystal structures structures with co-cryst co-crystallizedallized ligands. The residues ofof allall thethe activeac- tivesites sites are indicatedare indicated in each in each ﬁgure. figure. (a) T. (a brucei) T. bruceitrypanothione trypanothione (b) T. (b cruzi) T. cruzitrypanothione trypanothione (c) L. ( infantumc) L. infantum trypanothione. trypanothione.

TheTable abobe 3. Targets novel chemicaland related agents agents against against T.ΗΑΤ brucei. are presented in Table3. Cmpd Structure Target Ref.

4 REL1 [53]

5 REL1 [53]

6 TbGalE [54]

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Molecules 2021, 26, 4629 14 of 34 The abobe novel chemical agents against T. brucei are presented in Table 3.

The abobe novel chemical agents against T. brucei are presented in Table 3.

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The abobe novel chemical agents against T. brucei are presented in Table 3.

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Figure 3. Trypanothione crystal structures with co-crystallized ligands. The residues of all the active sites are indicated in each figure. (a) T. brucei trypanothione (b) T. cruzi trypanothione (c) L. Table 3. Targetsinfantum and trypanothione. related agents against HAT. Figure 3. Trypanothione crystal structures with co-crystallized ligands. The residues of all the active sitesTable are indicated 3. Targets in and each related figure. agents (a) T. bruceiagainst trypanothione ΗΑΤ. (b) T. cruzi trypanothione (c) L. Cmpdinfantum trypanothione. Structure Target Ref. Cmpd Structure Target Ref. Table 3. Targets and related agents against ΗΑΤ.

Cmpd Structure Target Ref. 4 Figure 3. Trypanothione crystal structures with co-crystallized ligands. The residuesREL1 of all the[53] active sites are indicated in each figure. (a) T. brucei trypanothione (b) T. cruzi trypanothione (c) L. 4 infantum trypanothione. REL1 [53]

4 Table 3. Targets and related agents against ΗΑΤ. REL1 [53]

Cmpd Structure Target Ref.

5 4 REL1REL1 [53] [53]

5 REL1 [53] 5 REL1 [53]

5 REL1 [53] 6 TbGalE [54]

6 TbGalE [54]

6 6 TbGalETbGalE [54] [54] Molecules 2021, 26, 4629 15 of 34

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7 PTR1 [56] 7 PTR1 [56] 7 PTR1 [56] 7 PTR1 [56]

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8 HDAC [60]

7 PTR1 [56] 8 HDAC [60] 8 8 HDACHDAC [60] [60 ]

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9 8 HDAC [60] NMT [64]

9 7 PTR1 [56] NMT [64]

9 9 NMT [64] NMT [64] 9 NMT [64]

10 TR [70] 8 HDAC [60] 10 TR [70] 10 TR [70]

10 TR [70] 11 9 NMT [70][64] 11 10 TR [70] [70]

4.3. T. cruzi

11 4.3.1. Ergosterol Biosynthesis Pathway [70] Sterols10 are constituents of the cellular membranes that are vital for theirTR structure[70] and 4.3. T. cruzi function. In mammalian cells, the main sterol is cholesterol, whereas in members of the Trypanosomatidae family, the main sterol is ergosterol [71]. Cholesterol and ergosterol have 4.3.1.11 Ergosterolminor Biosynthesis structural differences. Pathway An important metabolic pathway in T. cruzi is the sterol bio- [70] synthesis (SB) pathway, shown in Figure 4. The products of this pathway are ergosterol 4.3.Sterols T. cruzi are and constituents other11 24-sterols, whichof the are cellularessential for parasitememb multiplication.ranes that Therefore, are vital the[70] ergos- for their structure and function. In11 mammalianterol biosynthesis cells, pathway the is a promisingmain sterol target for isdrug cholesterol, development as the whereas survival of in members of[ 70the] 4.3.1. ErgosterolT. cruzi Biosynthesis is dependent on sterols Pathway production [72,73]. Trypanosomatidae family, the main sterol is ergosterol [71]. Cholesterol and ergosterol have Sterols are4.3. constituents T. cruzi of the cellular membranes that are vital for their structure and 4.3. minorT. cruzi structural 4.3.1.differences. Ergosterol Biosynthesis An important Pathway metabolic pathway in T. cruzi is the sterol bio- function. In mammalian cells, the main sterol is cholesterol, whereas in members of the 4.3.1.synthesis Ergosterol (SB) Biosynthesispathway,Sterols are shown constituents Pathway in of Figure the cellular 4. memb Theranes products that are vital for of their this structure pathway and are ergosterol Trypanosomatidaefunction. family, In mammalian the main cells, thesterol main sterol is ergosterol is cholesterol, whereas[71]. Cholesterolin members of the and ergosterol have and other 24-sterols,Trypanosomatidae which family,are essential the main sterol for is ergosterol parasite [71]. Cholesterol multiplication. and ergosterol Therefore,have the ergos- terolSterolsminor biosynthesis structural are constituentsminor pathway differences. structural differences.ofis athe Anpromising cellular Animportant important targmemb metabolic metabolicet for pathwayranes drug inpathway that T. development cruzi areis the insterolvital T. bio- cruzi for as theirthe is the survival structure sterol ofbio- and synthesis (SB) pathway, shown in Figure 4. The products of this pathway are ergosterol function.T. synthesiscruzi In is mammaliandependent (SB) pathway,and other on 24-sterols, cells,sterols shown whichthe production are inmain essential Figure sterol for [72,73]. parasite4. The is multiplication. cholesterol,products Therefore, of this whereas the ergos-pathway in aremembers ergosterol of the Trypanosomatidaeand other 24-sterols, family,terol biosynthesis which the mainpathway are essentialissterol a promising is fortargergosterolet parasite for drug development [71].multiplication. Cholesterol as the survival ofTherefore, and ergosterol the ergos- have T. cruzi is dependent on sterols production [72,73]. minorterol structural biosynthesis differences. pathway An is aimportant promising metabolictarget for drug pathway development in T. cruzi as the is thesurvival sterol of bio- synthesisT. cruzi (SB) is dependentpathway, onshown sterols in production Figure 4. [72,73].The products of this pathway are ergosterol

and other 24-sterols, which are essential for parasite multiplication. Therefore, the ergosterol biosynthesis pathway is a promising target for drug development as the survival of T. cruzi is dependent on sterols production [72,73].

Molecules 2021, 26, 4629 18 of 41

4.3. T. cruzi 4.3.1. Ergosterol Biosynthesis Pathway Sterols are constituents of the cellular membranes that are vital for their structure and function. In mammalian cells, the main sterol is cholesterol, whereas in members of the Trypanosomatidae family, the main sterol is ergosterol [71]. Cholesterol and ergosterol have minor structural differences. An important metabolic pathway in T. cruzi is the Molecules 2021, 26, 4629 16 of 34 sterol biosynthesis (SB) pathway, shown in Figure4. The products of this pathway are ergosterol and other 24-sterols, which are essential for parasite multiplication. Therefore, the ergosterol biosynthesis pathway is a promising target for drug development as the survival of T. cruzi is dependent on sterols production [72,73].

Acetyl-CoA

Mevalonate 3-hydroxy-3- methylglutaryl-CoA pathway HMG-CoA reductase Mevalonate

Farnesyl diphospate synthase (FPPS) Farnesyl diphosphate

Squalene synthase (SQS) Squalene

Oxidosquarene cyclase (QSC) Lanosterol Sterol biosynthesis Sterol-14-alpha dimethylate pathway (CYP51) Zymosterol

Sterol-24-c-methyltransferase (Tc24SMT) Ergosterol

Figure 4. Ergosterol biosynthesis pathway. Figure 4.Furthermore, Ergosterol biosynthesis the inhibition pathway. of enzymes of the ergosterol biosynthesis pathway plays a crucial role in antitrypanosomal drugs development. More specifically, Squalene synthase (SQS),Furthermore, Oxidosqualene the inhibition cyclase (OSC),of enzymes Sterol of 14-alpha the ergosterol demethylase biosynthesis (CYP51) pathway and Sterol plays 24-c- a crucialmethyltransferase role in antitrypanosomal (Tc24SMT) are drugs important development. targets for More novel specifically, drug development Squalene [71 syn-,74]. thase (SQS), Oxidosqualene cyclase (OSC), Sterol 14-alpha demethylase (CYP51) and SterolSterol 24-c-methyltransferase 14-α-Demethylase (CYP51) (Tc24SMT) are important targets for novel drug development [71,74].The T. cruzi sterol 14-alpha demethylase (CYP51) is a crucial enzyme for parasite survival. The enzyme is responsible for catalyzing the conversion of lanosterol to zy- Sterolmosterol. 14-α-Demethylase CYP51 has been(CYP51) found as a significant target for the development of antitrypanosomal,The T. cruzi antileishmanial, sterol 14-alpha and demethylase antifungal (CYP51) drugs. Azoles is a crucial were enzyme identified for as parasite a new potentsur- vival.class The of enzyme inhibitors. is responsible Specifically, for bifonazole catalyzing ( 12the), conversion clotrimazole of (lanosterol13), econazole to zymosterol. nitrate (14 ), CYP51miconazole has been (15 found), and tioconazoleas a significant (16) target as imidazoles for the development as well as itraconazole of antitrypanosomal, (17) and keto- antileishmanial,conazole (18) asand triazoles antifungal demonstrated drugs. Azoles high were IC50 identifiedvalues (IC as50 a= new 0.003–0.3 potentµ M)class as of CYP51 inhibitors.inhibitors Specifically, [75]. Molecular bifonazole docking (12), andclotrimazolein vitro studies (13), econazole showed that nitrate arylsubstituted (14), miconazole deriva- (15tives), and could tioconazole act as antiprotozoal (16) as imidazoles agents as in well the active as itraconazole site of CYP51. (17) Theand ligandketoconazole19 was ( found18) as totriazoles exhibit demonstrated no toxicity according high IC50 to values the study (IC50 conducted = 0.003–0.3 with μM) theas CYP51 MRC-5 inhibitors cell line and [75]. the Molecularbest selectivity docking index and againstin vitro T.studies cruzi, showedT.b. brucei that, and arylsubstitutedT.b. rhodesiense derivatives[76]. could act as antiprotozoal agents in the active site of CYP51. The ligand 19 was found to exhibit no toxicity according to the study conducted with the MRC-5 cell line and the best selectivity index against T. cruzi, T.b. brucei, and T.b. rhodesiense [76].

4.3.2. Trypanothione Reductase (TR) As it has already been described (Section 4.2.6), the TR enzyme is an attractive target for antitrypanosomal drugs. Clomipramine (20), a tricyclic antidepressant, has been shown to be active against infected mice in the chronic cardiac phase of CD, as it lessened cardiac damage [72]. Quinoxaline derivatives showed antitrypanosomal activity by inhibiting TR [8]. In silico studies showed that quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives 21, 22, and 23 exhibit high anti-T. cruzi activity. Derivative 21 and 22 presented

Molecules 2021, 26, 4629 19 of 41

4.3.2. Trypanothione Reductase (TR) As it has already been described (Section 4.2.6), the TR enzyme is an attractive target for antitrypanosomal drugs. Clomipramine (20), a tricyclic antidepressant, has been shown to be active against infected mice in the chronic cardiac phase of CD, as it lessened cardiac damage [72]. Quinoxaline derivatives showed antitrypanosomal activity by inhibiting TR [8]. In silico studies showed that quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives 21, 22, and 23 exhibit high anti-T. cruzi activity. Derivative 21 and 22 presented higher values of lysis on both strains of T. cruzi than Nfx and Bzn [73]. In silico studies of Arguelles et al. proposed quebrachamine (24), cephalotaxine (25), cryptolepine (26), (22S,25S)-tomatidine, (22R,25S)-solanidine, and (22R,25R)-solasodine as novel alkaloid scaffolds for the development of novel and potent TR inhibitors [77].

4.3.3. Enolase Enolase (2-phospho-D-glycerate hydrolase) has an essential role in glycolysis and glycogenesis. It is a metalloenzyme that catalyzes the reversible dehydration of D-2- phosphoglycerate (PGA) to phosphoenolpyruvate (PEP). It is found in a variety of or- ganisms, from bacteria to mammals, including trypanosomatids [78]. Enolase requires magnesium for both catalysis and dimer stabilization. Additionally, enolase acts on the cell surface of pathogens as a plasminogen receptor [71]. Consequently, enolase represents an attractive target for the development of drugs against T. cruzi. A combination of computational studies concluded that etidronate could be a promising drug of Chagas disease as TcENO inhibitor, since it presented high binding energy and low RMSD values [79]. Etidronate (27) is a drug currently used in the treatment of Paget’s disease, osteoporosis, and heterotropic ossiﬁcation with rare side effects [80]. In silico and in vitro studies demonstrated that butenolides could present interesting scaffolds for the synthesis of derivatives with potent activity against T. cruzi. Biological studies have showed that isolated compounds from Nectandra established antibacterial activity and parasitic infection [81]. Particularly, isolinderanolide D (28) and isolinderanolide E (29) were isolated from Nectandra oppositifolia. These two compounds were effective against the trypomastigote and amastigotes forms of T. cruzi, without any mammalian cytotoxicity [82].

4.3.4. Cruzain (Cz) Cruzain or Cruzipain (Cz) is the major papain-like cysteine protease of Trypanosoma cruzi. Cz is vital for the survival and the multiplication of this parasite [83]. The enzyme contains a catalytic domain and a C-terminal extension [84]. Vinyl sulfone (K777) 30 was the representative candidate cruzain inhibitor [85]. K777 is a peptide derivative which binds irreversibly to cruzain while its oral bioavailability was low, and its half-life was short. Additionally, pre-clinical studies using K777 identiﬁed that this drug candidate was highly hepatotoxic [86]. Yepes et al. suggested chalcones–quinoline conjugates as potential inhibitors of T. cruzi Cz. The core structure of chalcones has been used as an effective scaffold for the development of antichagastic drugs [87]. The quinoline moiety also presents biological properties and is active against T. cruzi by inhibiting cruzain. The results of this study demonstrated that the most active chalcones–quinoline conjugates displayed promising in vitro inhibition (EC50 values range between 7.0–36 µM) in comparison with Bnz (EC50: 40.3 ± 6.92 µM). The combination of computational approaches showed that compounds 31 and 32 had the optimal drug-like properties [88]. Computational studies also demonstrated that thiophen-2-iminothiazolidine derivatives could be used as cruzain inhibitors. Many thiophene compounds have been reported for their activity against T. cruzi. Thiazolidine compounds have recently become a medicinal target. The most active derivative against cruzain was thiophene–thiazolidine hybrids 33 with IC50 values of 2.4 µM[89]. Molecules 2021, 26, 4629 20 of 41

4.3.5. Ribose 5-Phosphate Isomerase Enzyme (Rpi) The ribose 5-phosphate isomerase (Rpi) enzyme plays a key role in the pentose phosphate pathway (PPP). It is responsible for the production of nucleotide precursors and NADPH. Rpi catalyzes the reversible isomerization reaction between D-ribose-5- phosphate (R5P) and D-ribulose-5-phospate (Ru5P) [90]. The inhibition of Rpi enzyme is an attractive target for drug development against T. cruzi. Molecular docking and molecular dynamic simulations revealed that two compounds, ZINC36975961 and ZINC43763931, had good performance and made interactions with key residues of the active site for the total simulation time. The compounds ZINC36975961 and ZINC43763931 are potential inhibitors of T. cruzi Rpi [80].

4.3.6. Isocitrate Dehydrogenase 2 (IDH2)

Two isocitrate dehydrogenases (IDH1 and IDH2) are displayed in T. cruzi. IDHs enzymes catalyze the oxidative decarboxylation of isocitrate, producing 2-oxoglutarate, CO2 and NADH or NADPH [91]. Sertraline (SERT) (34), a serotonin reuptake inhibitor, revealed antitrypanosomal activity against T. cruzi. Homology modeling and molecular docking studies showed that the presence of a hydrophobic pocket could provide hints for the structural optimization of sertraline in order to design novel TcIDH2 inhibitors [92].

4.3.7. Dihydrofolate Reductase–Thymidylate Synthase (DHFR-TS) Dihydrofolate reductase–thymidylate synthase (DHFR-TS) is a bifunctional enzyme that catalyzes the reduction of folate and the subsequent synthesis of thymidylate in DNA synthesis in T. cruzi [93]. According to Juárez-Saldivar et al., DHFR-TS could serve as a useful target to apply drug discovery against Chagas disease. In this study, Juárez- Saldivar’s research group has developed a strategy which combines docking studies and the statistical analysis of protein–ligand interactions so as to design compounds with strong inhibitory activity. FDA-approved drugs and food compounds were evaluated as potent DHFR inhibitors and the most prominent were evaluated in vitro. Biological assays pinpointed compound 35 as a novel therapeutic agent against Changas disease [94]. Another computational study, conducted by Schormann et al., identiﬁed the pharmacophore structure that derived from 3D QSAR studies of a ligand library to the DHFR-TS enzyme. All ligands in the library contained the 2,4-diamino-pyrimidine substructure which is characteristic for DHFR inhibitors, and they were crystallized with DHFR. This study revealed that the binding pocket of the enzyme is almost exclusively hydrophobic and the binding afﬁnity of the ligands depends on the structure of the ring fused to the 2,4- diamino-pyrimidine substructure 36 [95]. A new protocol called mt-QSAR-MLP uses QSAR for the application of multi-target drug discovery against parasitic diseases and aims to predict inhibitors that target proteins of various parasites, simultaneously. This protocol afforded derivatives, e.g., compound 37 that successfully inhibit DHFR-TS, as well as enzymes of other parasites [96].

4.3.8. Pteridine Reductase Pteridine reductase, an essential enzyme for the survival of T. cruzi, could also serve as a potent target for drug design against Chagas disease. Nine quinazoline derivatives were synthesized by Mendoza-Martínez’s research group and evaluated as potent anti- trypanosomatid agents. Docking studies have been performed to pteridine reductase, followed by in vitro biological evaluation of the compounds. Quinazoline derivatives 38 presented low micromolar activity against pteridine reductase of T. cruzi, while three of the compounds under study exhibited better results than Nfx and Bzn, which were used as reference drugs [97].

4.3.9. Farnesyl Diphosphate Synthase (FPPS) Farnesyl diphosphate synthase (FPPS) is an enzyme that catalyzes the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to farnesyl diphosphate (FPP) in Molecules 2021, 26, 4629 21 of 41

T. cruzi. Eight metal complexes were evaluated by Demoro et al. in order to evaluate their potency to serve as FPPS inhibitors. Biophosphonates, alendronate and pamidronate (pam), which complexed with the following metals: Cu, Co, Mn, and Ni, were crystallized and evaluated for their activity against FPPS of T. cruzi. These metal complexes presented strong inhibition against FPPS of T. cruzi in contrast to FPPS in mammalian cells. A representative complex 39 is illustrated in Table4[98]. Molecules 2021, 26, 4629 19 of 34 Molecules 2021, 26, 4629 19 of 34 Molecules 2021, 26, 4629 19 of 34

Molecules 2021, 26, 4629 19 of 34 Molecules 2021, 26, 4629 19 of 34 4.3.10. Sirtuins 4.3.10. Sirtuins Silent information regulator 2 (Sir2) or sirtuin inhibition could be an attractive target 4.3.10.Silent Sirtuins information regulator 2 (Sir2) or sirtuin inhibition could be an attractive target for4.3.10. drug Sirtuins development against CD. There are two sirtuins in T. cruzi: TcSir2rp1 and for drugSilent development information regulator against 2 CD.(Sir2) There or sirtuin are inhibition two sirtuins could bein anT. attractivecruzi: TcSir2rp1 target and TcSir2rp3.Silent informationTcSir2rp1 isregulator localized 2 (Sir2) in theor sirtuin cytosol inhibition and TcSir2rp3 could be an in attractive the mitochondrion. target TcSir2rp3.for drug developmentTcSir2rp1+ is against localized CD. inThere the arecytosol two sirtuinsand TcSir2rp3 in T. cruzi in: TcSir2rp1the mitochondrion. and Sirtuinsfor drug are development NAD+-dependent against CD.enzymes, There whichare two are sirtuins responsible in T. cruzi for: manyTcSir2rp1 cells and functions SirtuinsTcSir2rp3. are TcSir2rp1 NAD+-dependent is localized enzymes, in the cytosol which and are TcSir2rp3 responsible in thefor mitochondrion.many cells functions suchTcSir2rp3. as gene TcSir2rp1 silencing, is DNAlocalized damage in the repair, cytosol and and several TcSir2rp3 metabolic in the processes.mitochondrion. Sirtuins cat- such as gene silencing,+ DNA damage repair, and several metabolic processes. Sirtuins cat- suchSirtuins as gene are NADsilencing,+-dependent DNA damageenzymes, repair, which andare responsible several +metabolic for many processes. cells functions Sirtuins cat- alyzeSirtuins the aredeacetylation NAD -dependent of lysine enzymes, in the whichpresence are ofresponsible NAD+-producing for many cellsnicotinamide functions and O- alyzesuch theas gene deacetylation silencing, DNA of lysine damage in repair,the presence and several of NAD metabolic+-producing processes. nicotinamide Sirtuins cat- and O- acetyl-ADP-ribosealyzesuch theas gene deacetylation silencing, [99]. DNA ofThe lysine damage studies in repair,the of presenceBastos and several et ofal. NAD focusedmetabolic-producing on processes. derivatives nicotinamide Sirtuins of cardol,cat- and car- O- acetyl-ADP-ribosealyze the deacetylation [99]. of The lysine studies in the presenceof Bastos of et NAD al. focused+-producing on nicotinamidederivatives ofand cardol, O- car- acetyl-ADP-ribosealyze the deacetylation [99]. of The lysine studies in the presenceof Bastos of et NAD al. focused+-producing on nicotinamidederivatives ofand cardol, O- cardanol,acetyl-ADP-ribose and anacardic [99]. acid. The studiesTheir resultsof Bastos demonstrated et al. focused onthat derivatives natural compoundsof cardol, car- isolated danol,acetyl-ADP-ribose and anacardic [99]. acid. The studiesTheir resultsof Bastos demonstrated et al. focused onthat derivatives natural compoundsof cardol, car- isolated fromdanol, cashew and anacardic nut (Anacardium acid. Their occidentale,results demonstrated L. Anacardiaceae that natural) presented compounds anti- isolatedT. cruzi activ- fromdanol, cashew and anacardic nut (Anacardium acid. Their occidentale,results demonstrated L. Anacardiaceae that natural) presented compounds anti- isolatedT. cruzi activ- ityfrom by cashewinhibiting nut sirtuin(Anacardium [100]. occidentale, L. Anacardiaceae) presented anti-T. cruzi activ- ityfrom by cashewinhibiting nut sirtuin(Anacardium [100]. occidentale, L. Anacardiaceae) presented anti-T. cruzi activ- ityity by by inhibiting inhibiting sirtuinsirtuin [100]. [100]. Molecules 2021, 26, 4629 ity by inhibiting sirtuin [100]. 22 of 41 The new antichagastic agents are listed in Table 4. TheThe newnew antichagastic agents agents are are listed listed in Table in Table 4. 4. The new antichagastic agents are listed in Table 4. Table 4. Targets and related agents against CD. Table Table4. Targets 4. Targets and related and related agents agents against against CD. CD. TableTable 4. TargetsTargets and and related related agents agents against against CD. CD. Table 4. Targets and related agents against CD. Cmpd Structure Target Ref. CmpdCmpdCmpd StructureStructure Target Target Target Ref. Ref. Ref. Cmpd12Cmpd StructureStructure Sterol-14-a-dimethylase Target Target Ref. Ref.[75] 12 12 12 Sterol-14-a-dimethylaseSterol-14-a-dimethylaseSterol-14-a-dimethylase (CYP51) [75] [75] [75] 12 12 Sterol-14-a-dimethylaseSterol-14-a-dimethylase [75] [75] (CYP51)(CYP51) (CYP51)(CYP51)

13 Sterol-14-a-dimethylase [75] 13 13 13 Sterol-14-a-dimethylaseSterol-14-a-dimethylaseSterol-14-a-dimethylase (CYP51) [75] [75] [75] 13 Sterol-14-a-dimethylase(CYP51) [75] (CYP51)(CYP51) (CYP51)

14 Sterol-14-a-dimethylase [75] 14 Sterol-14-a-dimethylase [75] 14 14 Sterol-14-a-dimethylaseSterol-14-a-dimethylase(CYP51) (CYP51) [75] [75] 14 Sterol-14-a-dimethylase(CYP51) [75] 14 Sterol-14-a-dimethylase(CYP51) [75] (CYP51)

15 Sterol-14-a-dimethylase [75] 15 Sterol-14-a-dimethylase [75] (CYP51) (CYP51) 15 15 Sterol-14-a-dimethylaseSterol-14-a-dimethylase (CYP51) [75] [75] 15 Sterol-14-a-dimethylase(CYP51) [75] (CYP51)

16 Sterol-14-a-dimethylase [75] 16 Sterol-14-a-dimethylase [75] (CYP51) (CYP51) 16 Sterol-14-a-dimethylase [75] 16 16 Sterol-14-a-dimethylaseSterol-14-a-dimethylase (CYP51) [75] [75] 16 Sterol-14-a-dimethylase(CYP51) [75] (CYP51)

17 Sterol-14-a-dimethylase [75] 17 Sterol-14-a-dimethylase [75] (CYP51) (CYP51)

17 Sterol-14-a-dimethylase [75] 17 Sterol-14-a-dimethylase(CYP51) [75] (CYP51)

Molecules 2021, 26, 4629 19 of 34

4.3.10. Sirtuins Silent information regulator 2 (Sir2) or sirtuin inhibition could be an attractive target for drug development against CD. There are two sirtuins in T. cruzi: TcSir2rp1 and TcSir2rp3. TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. Sirtuins are NAD+-dependent enzymes, which are responsible for many cells functions such as gene silencing, DNA damage repair, and several metabolic processes. Sirtuins catalyze the deacetylation of lysine in the presence of NAD+-producing nicotinamide and O- acetyl-ADP-ribose [99]. The studies of Bastos et al. focused on derivatives of cardol, cardanol, and anacardic acid. Their results demonstrated that natural compounds isolated from cashew nut (Anacardium occidentale, L. Anacardiaceae) presented anti-T. cruzi activity by inhibiting sirtuin [100].

The new antichagastic agents are listed in Table 4.

Table 4. Targets and related agents against CD.

Cmpd Structure Target Ref. 12 Sterol-14-a-dimethylase [75] (CYP51)

13 Sterol-14-a-dimethylase [75] (CYP51)

14 Sterol-14-a-dimethylase [75] (CYP51)

15 Sterol-14-a-dimethylase [75] Molecules 2021, 26, 4629 (CYP51) 23 of 41

16 Sterol-14-a-dimethylase [75] (CYP51) Table 4. Cont.

Cmpd Structure Target Ref.

17 Sterol-14-a-dimethylase [75] 17Molecules 2021, 26, 4629 Sterol-14-a-dimethylase (CYP51)20 of 34 [75] Molecules 2021, 26, 4629 (CYP51) 20 of 34

Molecules 2021, 26, 4629 20 of 34

18 Sterol-14-a-dimethylase [75] 18 18 Sterol-14-a-dimethylaseSterol-14-a-dimethylase(CYP51) (CYP51) [75] [75] (CYP51) (CYP51)

19 Sterol-14-a-dimethylase [76] 19 Sterol-14-a-dimethylase (CYP51) [76] 19 (CYP51)Sterol-14-a-dimethylase [76] (CYP51)

20 Trypanothione Enzyme (TR) [72]

20 20 TrypanothioneTrypanothione Enzyme (TR)Enzyme (TR) [72] [72]

21 Trypanothione Enzyme (TR) [73]

21 Trypanothione Enzyme (TR) [73] 22 Trypanothione Enzyme (TR) [73]

23 Trypanothione Enzyme (TR) [73] 22 Trypanothione Enzyme (TR) [73]

24 Trypanothione Enzyme (TR) [77]

23 Trypanothione Enzyme (TR) [73]

25 Trypanothione Enzyme (TR) [77]

24 Trypanothione Enzyme (TR) [77] 24 Trypanothione Enzyme (TR) [77]

25 Trypanothione Enzyme (TR) [77]

Molecules 2021, 26, 4629 20 of 34

18 Sterol-14-a-dimethylase [75] (CYP51)

Molecules 2021,, 2626,, 46294629 20 of 34 Molecules 2021, 26, 4629 20 of 34 Molecules 2021, 26, 4629 20 of 34

18 Sterol-14-a-dimethylase [75] 1918 Sterol-14-a-dimethylase [76][75] 18 Sterol-14-a-dimethylase(CYP51) [75] (CYP51) (CYP51)

19 Sterol-14-a-dimethylase [76] 19 Sterol-14-a-dimethylase [76] 19 Sterol-14-a-dimethylase(CYP51) [76] (CYP51) (CYP51) 20 Trypanothione Enzyme (TR) [72]

Molecules 2021, 26, 4629 24 of 41 2120 Trypanothione Enzyme (TR) [73][72] 20 Trypanothione Enzyme (TR) [72] 20 Trypanothione Enzyme (TR) [72]

Table 4. Cont.

Cmpd Structure Target Ref.

21 22 TrypanothioneTrypanothione Enzyme (TR)Enzyme (TR) [73] [73] 21 Trypanothione Enzyme (TR) [73] 21 Trypanothione Enzyme (TR) [73] 21 Trypanothione Enzyme (TR) [73]

22 23 Trypanothione Enzyme (TR) [73] 22 TrypanothioneTrypanothione Enzyme (TR)Enzyme (TR) [73] [73] 22 Trypanothione Enzyme (TR) [73] 22 Trypanothione Enzyme (TR) [73]

24 Trypanothione Enzyme (TR) [77] 23 23 TrypanothioneTrypanothione Enzyme (TR)Enzyme (TR) [73] [73] 23 Trypanothione Enzyme (TR) [73] 23 Trypanothione Enzyme (TR) [73]

25 Trypanothione Enzyme (TR) [77]

24 Trypanothione Enzyme (TR) [77] 24 24 TrypanothioneTrypanothione Enzyme (TR)Enzyme (TR) [77] [77] 24 Trypanothione Enzyme (TR) [77]

25 Trypanothione Enzyme (TR) [77] 25 Trypanothione Enzyme (TR) [77] 25 25 TrypanothioneTrypanothione Enzyme (TR)Enzyme (TR) [77] [77]

Molecules 2021, 26, 4629 25 of 41

Molecules 2021, 26, 4629 21 of 34 Molecules Molecules 2021, 26 2021, 4629, 26 , 4629 Table 4. Cont. 21 of 3421 of 34 Molecules 2021, 26, 4629 21 of 34 Molecules 2021, 26, 4629 21 of 34 Molecules 2021, 26, 4629 21 of 34 Cmpd Structure Target Ref.

26 26 TrypanothioneTrypanothione Enzyme (TR)Enzyme (TR) [77] [77] 26 Trypanothione Enzyme (TR) [77] 26 Trypanothione Enzyme (TR) [77] 26 Trypanothione Enzyme (TR) [77]

27 27 EnolaseEnolase [80] [80] 27 Enolase [80] 27 Enolase [80] 27 Enolase [80]

28 Enolase [82] 28 28 EnolaseEnolase [82] [82] 28 28 EnolaseEnolase [82] [82] 28 Enolase [82] 28 Enolase [82]

29 Enolase [82] 29 29 EnolaseEnolase [82] [82] 29 Enolase [82] 29 Enolase [82]

30 Cruzain (Cz) [86] 30 30 CruzainCruzain (Cz) (Cz) [86] [86] 30 30 Cruzain (Cz) [86] 30 CruzainCruzain (Cz) (Cz) [86] [86] 30 Cruzain (Cz) [86]

31 Cruzain (Cz) [88] 31 Cruzain (Cz) [88] 31 Cruzain (Cz) [88] 31 31 Cruzain (Cz)Cruzain (Cz) [88] [88]

32 Cruzain (Cz) [88] 32 Cruzain (Cz) [88] 32 Cruzain (Cz) [88] 32 Cruzain (Cz) [88]

33 Cruzain (Cz) [89] 33 Cruzain (Cz) [89] 33 Cruzain (Cz) [89] 33 Cruzain (Cz) [89]

34 IsocitrateIsocitrate dehydrogenasedehydrogenase 22 [92][92] 34 Isocitrate dehydrogenase 2 [92] 34 Isocitrate(TClDH2)(TClDH2) dehydrogenase 2 [92] 34 Isocitrate(TClDH2) dehydrogenase 2 [92] (TClDH2) (TClDH2)

Molecules 2021, 26, 4629 21 of 34 Molecules 2021, 26, 4629 21 of 34 Molecules 2021, 26, 4629 21 of 34

26 Trypanothione Enzyme (TR) [77] 26 Trypanothione Enzyme (TR) [77]

27 Enolase [80] 27 Enolase [80]

28 Enolase [82] 28 Enolase [82]

29 Enolase [82] 29 Enolase [82]

30 Cruzain (Cz) [86] 30 Cruzain (Cz) [86]

Molecules 2021, 26, 4629 26 of 41 31 Cruzain (Cz) [88] 31 Cruzain (Cz) [88]

Table 4. Cont.

Cmpd Structure Target Ref.

32 32 Cruzain (Cz)Cruzain (Cz) [88] [88] 32 Cruzain (Cz) [88]

33 33 Cruzain (Cz)Cruzain (Cz) [89] [89] 33 Cruzain (Cz) [89]

34 Isocitrate dehydrogenase 2 [92] 34 34 IsocitrateIsocitrate dehydrogenase dehydrogenase 2 2 [92] [92] (TClDH2) (TClDH2) (TClDH2)

Molecules 2021, 26, 4629 22 of 34

35 35 Dihydrofolate Reductase-thy- [92] midylate synthase (DHFR-TS)

Dihydrofolate Reductase-thymidylate [92] synthase (DHFR-TS)

36 Dihydrofolate Reductase-thy- [95] midylate synthase (DHFR-TS)

37 Dihydrofolate Reductase-thy- [96] midylate synthase (DHFR-TS)

38 Pteridine reductase [97]

39 [Co(Pam)2(H2O)2] Farnesyl diphosphate synthase [98]

4.4. Leishmania spp 4.4.1. Purine Salvage Pathway Leishmania parasites do not present the essential enzymes for the de novo biosynthesis of purines and they exhibit retrieving mechanisms to obtain the purines from the mammalian host cells [34]. The purine salvage systems of the parasites consist of unique transporters that might be drug targets, even if it is not easy to be selectively inhibited. Phos- phoribosyltransferases (PRTs) play a crucial role in the purine salvage pathway [44] and more than one PRT must be targeted to successfully inhibit the biosynthesis of purines, because of the existence of alternative pathways for the retrieval of purines. LdNT1/2 (Leishmania dovani Nucleotide Tranpsporters 1/2) are also druggable targets, since they transfer nucleoside analogs that inhibit cell growth. Immucillins 40, 41 are nucleoside analogs that inhibit the nucleoside hydrolase (NH36), which belong to purine salvage enzymes [101]. Nucleoside diphosphate kinases (NDKs) are significant in the purine salvage pathway. A pyrrole–indolinone adduct SU11652 42 binds to NDKs of Leishmania spp. and generates a new series of NDK inhibitors with antileishmanial activity [102]. In contrast to purines, pyrimidines are synthesized de novo or through the pyrimidine salvage pathway. Consequently, 5-fluorouracil (43) and cytarabine (44) pyrimidine analogs inhibit the corresponding enzymes of the folate pathway and exhibit antiproliferative activity against promastigotes and intracellular amastigotes of Leishmania parasites [34,103].

MoleculesMolecules 2021 2021, ,26 26, ,4629 4629 2222 of of 34 34

3535 DihydrofolateDihydrofolate Reductase-thy- Reductase-thy- [92][92] midylate synthase (DHFR-TS) Molecules 2021, 26, 4629 midylate syntha22se of 34(DHFR-TS)

35 Dihydrofolate Reductase-thy- [92] midylate synthase (DHFR-TS) Molecules 2021, 26, 4629 27 of 41

Table 4. Cont.

36 Dihydrofolate Reductase-thy- [95] Cmpd36 StructureDihydrofolate Target Reductase-thy- [95] Ref. midylate synthase (DHFR-TS) 36 36 Dihydrofolatemidylate Reductase-thy- syntha[95]se (DHFR-TS) midylate synthase (DHFR-TS)

Dihydrofolate Reductase-thymidylate [95] synthase (DHFR-TS)

37 Dihydrofolate Reductase-thy- [96] 37 3737 Dihydrofolatemidylate synthaDihydrofolateDihydrofolatese Reductase-thymidylate (DHFR-TS) Reductase-thy- Reductase-thy- [96][96] [96] synthase (DHFR-TS) midylatemidylate syntha synthasese (DHFR-TS) (DHFR-TS)

38 Pteridine reductase [97]

38 3838 PteridinePteridinePteridine reductase reductase reductase [97] [97] [97] 39 [Co(Pam)2(H2O)2] Farnesyl diphosphate synthase [98]

4.4. Leishmania spp 4.4.1. Purine Salvage Pathway Leishmania parasites do not present the essential enzymes for the de novo biosynthe- 39 [Co(Pam)2(H2O)2] Farnesyl diphosphate synthase [98] 39 sis[Co(Pam) of purines and2(H they2O) exhibit2] retrieving mechanisms to obtainFarnesyl the purines diphosphate from the mam- synthase [98] 39 [Co(Pam)2(H2O)2] Farnesyl diphosphate synthase [98] malian host cells [34]. The purine salvage systems of the parasites consist of unique trans- 4.4.4.4.porters Leishmania Leishmania that might spp spp be drug targets, even if it is not easy to be selectively inhibited. Phos- phoribosyltransferases (PRTs) play a crucial role in the purine salvage pathway [44] and 4.4.1.4.4.1.more Purine Purine than one Salvage Salvage PRT must Pathway Pathway be targeted to successfully inhibit the biosynthesis of purines, becauseLeishmania of the existence parasites of alternative do not presentpathways thefor theessential retrieval enzymesof purines. LdNT1/2for the de novo biosynthe- (LeishmaniaLeishmania dovani parasites Nucleotide do Tranpsporters not present 1/2) the are essentialalso druggable enzymes targets, sincefor the they de novo biosynthe- sissis of transferof purines purines nucleoside and and theyanalogs they exhibit exhibit that inhibit retrieving retrieving cell growth. me me Immucillinschanismschanisms 40 to, to41 obtain obtainare nucleoside the the purines purines an- from from the the mam- mam- malianmalianalogs host thathost inhibit cells cells the[34]. [34]. nucleoside The The purine purine hydrolase salvage salvage (NH36), syst syst whichemsems belong of of the the to purineparasites parasites salvage consist consist en- of of unique unique trans- trans- porterszymes that[101]. might be drug targets, even if it is not easy to be selectively inhibited. Phos- portersNucleoside that might diphosphate be drug kinases targets, (NDKs) even are ifsignificant it is not in easythe purine to be salvage selectively path- inhibited. Phos- phoribosyltransferasesphoribosyltransferasesway. A pyrrole–indolinone (PRTs) adduct(PRTs) SU11652 play play a 42a crucial crucialbinds to NDKsrole role in ofin Leishmaniathe the purine purine spp. salvage andsalvage gen- pathway pathway [44] [44] and and moremoreerates than than a new one one series PRT PRT of mustNDK must inhibitors be be targeted targeted with an to tileishmanialto succe successfullyssfully activity inhibit inhibit [102]. In the thecontrast bios bios toynthesisynthesis of of purines, purines, becausepurines, ofpyrimidines the existence are synt hesizedof alternative de novo or paththroughways the pyrimidinefor the retrievalsalvage path- of purines. LdNT1/2 becauseway. Consequently, of the existence 5-fluorouracil of alternative (43) and cytarabine path (ways44) pyrimidine for the anal retrievalogs inhibit theof purines. LdNT1/2 (Leishmania(Leishmaniacorresponding dovanidovani enzymes NucleotideNucleotide of the folate pathway TranpsportersTranpsporters and exhibit 1/2)antiproliferative1/2) areare alsoalso activity druggabledruggable against targets,targets, sincesince theythey transfertransferpromastigotes nucleoside nucleoside and intracellular analogs analogs amastigotesthat that inhibit inhibit of Leishmania cell cell growth. growth. parasites Immucillins Immucillins [34,103]. 40 40, ,41 41 are are nucleoside nucleoside an- an- alogsalogs that that inhibit inhibit the the nucleoside nucleoside hydrolase hydrolase (NH36), (NH36), which which belong belong to to purine purine salvage salvage en- en- zymeszymes [101]. [101]. NucleosideNucleoside diphosphate diphosphate kinases kinases (NDKs) (NDKs) are are significant significant in in the the purine purine salvage salvage path- path- way.way. A A pyrrole–indolinone pyrrole–indolinone adduct adduct SU11652 SU11652 42 42 binds binds to to NDKs NDKs of of Leishmania Leishmania spp. spp. and and gen- gen- erateserates aa newnew series series ofof NDK NDK inhibitorsinhibitors with with anantileishmanialtileishmanial activityactivity [102].[102]. InIn contrastcontrast toto purines,purines, pyrimidinespyrimidines areare syntsynthesizedhesized dede novonovo oror throughthrough thethe pyrimidinepyrimidine salvagesalvage path-pathway.way. Consequently, Consequently, 5-fluorouracil 5-fluorouracil ( 43(43) )and and cytarabine cytarabine ( 44(44) )pyrimidine pyrimidine anal analogsogs inhibit inhibit the the correspondingcorresponding enzymes enzymes of of the the folate folate pathway pathway an andd exhibit exhibit antiproliferative antiproliferative activity activity against against promastigotespromastigotes and and intracellular intracellular amastigotes amastigotes of of Leishmania Leishmania parasites parasites [34,103]. [34,103].

Molecules 2021, 26, 4629 28 of 41

4.3.10. Sirtuins Silent information regulator 2 (Sir2) or sirtuin inhibition could be an attractive target for drug development against CD. There are two sirtuins in T. cruzi: TcSir2rp1 and TcSir2rp3. TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. Sirtuins are NAD+- dependent enzymes, which are responsible for many cells functions such as gene silencing, DNA damage repair, and several metabolic processes. Sirtuins catalyze the deacetylation of lysine in the presence of NAD+-producing nicotinamide and O-acetyl-ADP-ribose [99]. The studies of Bastos et al. focused on derivatives of cardol, cardanol, and anacardic acid. Their results demonstrated that natural compounds isolated from cashew nut (Anacardium occidentale, L. Anacardiaceae) presented anti-T. cruzi activity by inhibiting sirtuin [100]. The new antichagastic agents are listed in Table4.

4.4. Leishmania spp 4.4.1. Purine Salvage Pathway Leishmania parasites do not present the essential enzymes for the de novo biosynthesis of purines and they exhibit retrieving mechanisms to obtain the purines from the mammalian host cells [34]. The purine salvage systems of the parasites consist of unique transporters that might be drug targets, even if it is not easy to be selectively inhibited. Phosphoribosyltransferases (PRTs) play a crucial role in the purine salvage pathway [44] and more than one PRT must be targeted to successfully inhibit the biosynthesis of purines, because of the existence of alternative pathways for the retrieval of purines. LdNT1/2 (Leishmania dovani Nucleotide Tranpsporters 1/2) are also druggable targets, since they transfer nucleoside analogs that inhibit cell growth. Immucillins 40, 41 are nucleoside analogs that inhibit the nucleoside hydrolase (NH36), which belong to purine salvage enzymes [101]. Nucleoside diphosphate kinases (NDKs) are signiﬁcant in the purine salvage pathway. A pyrrole–indolinone adduct SU11652 42 binds to NDKs of Leishmania spp. and generates a new series of NDK inhibitors with antileishmanial activity [102]. In contrast to purines, pyrimidines are synthesized de novo or through the pyrimidine salvage pathway. Consequently, 5-ﬂuorouracil (43) and cytarabine (44) pyrimidine analogs inhibit the corresponding enzymes of the folate pathway and exhibit antiproliferative activity against promastigotes and intracellular amastigotes of Leishmania parasites [34,103].

4.4.2. Mitochondrial Electron Chain and Cytochromes The electron transport chain is crucial for the growth of the Leishmania parasites into the host cells. Blocking the electron transport chain reduces pathogen survival because of the ROS that produce toxic byproducts into eucaryote cells [104]. The enzyme that catalyzes the electron transfer from NADH to ubiquinone is known as type 2 NADH dehydrogenase and is a promising molecular target. A 6-methoxyquinalidine derivative 45 was proven as a potent inhibitor for L. infantum NADH dehydrogenase (LiNDH2), exhibiting antileishmanial activity in a nanomolar range [105]. Oleanolic acid (46) also acts on the electron chain as it interacts with CYP51 and inhibits promastigote and amastigote forms of L. infantum. Consequently, triterpenoids, such as oleanolic acid, are plausible antileishmanial agents [106].

4.4.3. Polyamine Biosynthesis Pathway Polyamines such as putrescine, spermidine and spermine are vital for the differentiation of Leishmania parasites and cell survival. There are several enzymes that are involved in the biosynthesis of polyamines and might be considered as molecular targets [44]. Arginase converts L-arginine to L-ornithine and urea and is one of the most important enzymes of the polyamine biosynthesis pathway. A shortage of arginase in the extracellular environment of Leishmania spp. provokes apoptosis. The benzimidazole analog 47 binds into the catalytic pocket of arginase exhibiting inhibitory activity and becomes a promising lead compound in the antileishmanial drug design [104]. The polyamine spermidine, along with two glutathione molecules, composes trypanothione, which is the only compound of trypanosomes that does not exist in mammalian cells. Molecules 2021, 26, 4629 29 of 41

Therefore, enzymes that are involved in the synthesis and metabolism of trypanothione should be targeted by selective antileishmanial drugs. Trypanothione reductase (TR) is considered as one of the most important targets of the trypanothione pathway, and many studies reveal several compounds 48, 49, 50 that act as inhibitors [34,44,104]. The crystallographic structure of trypanothione reductase led to the identification of several classes of inhibitors that bind on specific sites of the enzyme [107]. Acridine derivative 51 binds to the active cavity (mepacrine binding site/MBS) of TR and acts as inhibitor. 3,4-Dihydroqyinazoline analogs of 52 also inhibit TR, binding to MBS. Derivatives of 1-(1-(benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) are also promising competitive inhibitors of TbTR, due to their drug-like properties, such as low molecular weight and blood–brain barrier crossing. Several structural modifications of the BTCP scaffold led to indole–thiazole derivative 53, which targets a hydrophobic sub-pocket near the catalytic cysteines in the TR active site [107]. Metalloid-based drugs, such as pentavalent antimonials, are currently used to treat leishmaniasis, despite having severe side effects and resistance phenomena. It is known that these drugs, at least in part, act on TR by binding catalytic cysteines. Antimonial derivatives of 54 exhibited antileishmanial activity against amastigotes, effecting the binding pocket of trypanothione reductase. Besides antimony, silver and gold were proven to bind TR in a similar way but even more efficiently. Auranofin (55), a gold-containing antirheumatic tested on leishmanial TR and parasites, was found to be 10-fold more potent than Sb on TR. Apart from the Au complex, the thiosugar moiety of auranofin binds the inner part of the TR catalytic site, resulting to a double inhibition mechanism [107,108].

4.4.4. Folate Metabolism Trypanosome parasites are folate and pterin auxotrophs, as they rely completely on pteridine salvage from their hosts. The key enzyme of folate metabolism is dihydrofolate reductase (DHFR), a target for the antifolates. Antifolates are exploited to treat several diseases such as malaria, bacterial infections, and various cancer types. An upcoming problem for the use of antifolates as trypanocides is parasite resistance to these agents. Pteridine reductase 1 (PTR1) is unique in these parasites and provides a bypass for DHFR inhibition. Since trypanosomatids are able to overcome DHFR inhibition by overexpressing the pteridine reductase 1, the inhibition of both PR1 and DHFR might provide a new therapeutical target [104,109]. Quinolone derivatives of 56 bind on the active site of PTR1, acting as inhibitors. Chromenone derivatives of 57 form a new class of PTR1 inhibitors, forming various interactions with amino acid residues of the active site. Phenyl-linked oxadiazole–phenylhydrazone hybrids of 8 are also potent PTR1 inhibitors, acting against the promastigote form of Leishmania spp. A series of analogs of compounds 59, bearing a triazole ring fused with pyrazine moiety, showed signiﬁcant antileishmanial activity by inhibiting PTR1 [110–113].

4.4.5. Protein Kinases Protein kinases are a promising target for antileishmanial drugs as they play an important role in the cell cycle of Leishmania spp. Cyclic-dependent kinases (CDKs) are an evolving drug target and CRK3 is the functional homologue of CDK1 in Leishmania. CRK3 is active in both amastigote and promastigote stages of Leishmania parasites and participates in all cell division processes. Two chemical classes, 2,6,9-trisubstituted purines of 60 and indirubines of 61, have been identified as efficient inhibitors of CRK3 [44,114]. Mitogen-activated kinases (MAPKs) have been characterized and studied widely in L. Mexicana. They have an important role in the intracellular survival and infectivity of the parasite. The L. mexicana MAPK3 (LmxMPK3) was found to be involved in the regulation of flagellar length in promastigotes and can be a possible drug target. Flavonoids, such as genistein (62) and chrysin (63), proved to be MPK3 inhibitors and represent a new series of phytochemicals against leishmaniasis. Genistein also has antioxidant properties and eliminates DNA damage caused by classic antileishmanials, such as meglumine antimonate [115,116]. The novel antileishmanial agents are summarized in Table5. Molecules 2021, 26, 4629 24 of 34 Molecules 2021, 26, 4629 24 of 34

inhibition. Since trypanosomatids are able to overcome DHFR inhibition by overexpress- inhibition. Since trypanosomatids are able to overcome DHFR inhibition by overexpressing the pteridine reductase 1, the inhibition of both PR1 and DHFR might provide a new ing the pteridine reductase 1, the inhibition of both PR1 and DHFR might provide a new therapeutical target [104,109]. Quinolone derivatives of 56 bind on the active site of PTR1, therapeutical target [104,109]. Quinolone derivatives of 56 bind on the active site of PTR1, acting as inhibitors. Chromenone derivatives of 57 form a new class of PTR1 inhibitors, acting as inhibitors. Chromenone derivatives of 57 form a new class of PTR1 inhibitors, forming various interactions with amino acid residues of the active site. Phenyl-linked forming various interactions with amino acid residues of the active site. Phenyl-linked oxadiazole–phenylhydrazone hybrids of 8 are also potent PTR1 inhibitors, acting against oxadiazole–phenylhydrazone hybrids of 8 are also potent PTR1 inhibitors, acting against the promastigote form of Leishmania spp. A series of analogs of compounds 59, bearing a the promastigote form of Leishmania spp. A series of analogs of compounds 59, bearing a triazole ring fused with pyrazine moiety, showed significant antileishmanial activity by triazole ring fused with pyrazine moiety, showed significant antileishmanial activity by inhibiting PTR1 [110–113]. inhibiting PTR1 [110–113]. 4.4.5. Protein Kinases 4.4.5. Protein Kinases Protein kinases are a promising target for antileishmanial drugs as they play an im- Protein kinases are a promising target for antileishmanial drugs as they play an important role in the cell cycle of Leishmania spp. Cyclic-dependent kinases (CDKs) are an portant role in the cell cycle of Leishmania spp. Cyclic-dependent kinases (CDKs) are an evolving drug target and CRK3 is the functional homologue of CDK1 in Leishmania. CRK3 evolving drug target and CRK3 is the functional homologue of CDK1 in Leishmania. CRK3 is active in both amastigote and promastigote stages of Leishmania parasites and partici- is active in both amastigote and promastigote stages of Leishmania parasites and participates in all cell division processes. Two chemical classes, 2,6,9-trisubstituted purines of 60 pates in all cell division processes. Two chemical classes, 2,6,9-trisubstituted purines of 60 and indirubines of 61, have been identified as efficient inhibitors of CRK3 [44,114]. Mito- and indirubines of 61, have been identified as efficient inhibitors of CRK3 [44,114]. Mito- gen-activated kinases (MAPKs) have been characterized and studied widely in L. Mexi- gen-activated kinases (MAPKs) have been characterized and studied widely in L. Mexi- cana. They have an important role in the intracellular survival and infectivity of the para- cana. They have an important role in the intracellular survival and infectivity of the parasite. The L. mexicana MAPK3 (LmxMPK3) was found to be involved in the regulation of site. The L. mexicana MAPK3 (LmxMPK3) was found to be involved in the regulation of flagellar length in promastigotes and can be a possible drug target. Flavonoids, such as flagellar length in promastigotes and can be a possible drug target. Flavonoids, such as genistein (62) and chrysin (63), proved to be MPK3 inhibitors and represent a new series genistein (62) and chrysin (63), proved to be MPK3 inhibitors and represent a new series of phytochemicals against leishmaniasis. Genistein also has antioxidant properties and of phytochemicals against leishmaniasis. Genistein also has antioxidant properties and eliminates DNA damage caused by classic antileishmanials, such as meglumine antimo- eliminates DNA damage caused by classic antileishmanials, such as meglumine antimo- Molecules 2021, 26, 4629 nate [115,116]. 30 of 41 nate [115,116]. The novel antileishmanial agents are summarized in Table 5. The novel antileishmanial agents are summarized in Table 5. Table 5. Targets and related agents against Leishmaniases. Table 5. Targets and related agents against Leishmaniases. Table 5. Targets and related agents against Leishmaniases. CmpdCmpd StructureStructure Target Target Ref. Ref. Cmpd Structure Target Ref. 40 40 NucleosideNucleoside hydrolase hydrolase NH36 NH36 [101] [101] 40 Nucleoside hydrolase NH36 [101]

41 Nucleoside hydrolase NH36 [101] 41 41 NucleosideNucleoside hydrolase hydrolase NH36 NH36 [101] [101]

Molecules 2021, 26, 4629 25 of 34 Molecules 2021, 26, 4629 25 of 34

42 42 Nucleoside diphosphate kinase NDK [102] 42 NucleosideNucleoside diphosphate diphosphate kinase kinase NDK NDK [102] [102]

43 Nucleoside transporter 1 NT1 [34] 43 43 NucleosideNucleoside transporter transporter 1 NT1 1 NT1 [34] [34]

44 Nucleoside transporter 1 NT1 [34] 44 Nucleoside transporter 1 NT1 [34]

45 NADH dehydrogenase 2 [105] 45 NADH dehydrogenase 2 [105]

46 CYP51 [106] 46 CYP51 [106]

47 L-arginase [104] 47 L-arginase [104]

48 Trypanothione reductase TR1 [117] 48 Trypanothione reductase TR1 [117]

49 Trypanothione reductase TR1 [118] 49 Trypanothione reductase TR1 [118]

Molecules 2021, 26, 4629 25 of 34 Molecules 2021, 26, 4629 25 of 34 Molecules 2021, 26, 4629 25 of 34 Molecules 2021, 26, 4629 25 of 34 Molecules 2021, 26, 4629 25 of 34

42 Nucleoside diphosphate kinase NDK [102] 42 Nucleoside diphosphate kinase NDK [102] 42 Nucleoside diphosphate kinase NDK [102] 42 Nucleoside diphosphate kinase NDK [102] 42 Nucleoside diphosphate kinase NDK [102] Molecules 2021, 26, 4629 31 of 41

43 Nucleoside transporter 1 NT1 [34] 43 Table 5. Cont.Nucleoside transporter 1 NT1 [34] 43 Nucleoside transporter 1 NT1 [34] 43 Nucleoside transporter 1 NT1 [34] Cmpd43 StructureNucleoside transporter Target 1 NT1 [34] Ref.

44 44 NucleosideNucleoside transporter transporter 1 NT1 1 NT1 [34] [34] 44 Nucleoside transporter 1 NT1 [34] 44 Nucleoside transporter 1 NT1 [34] 44 Nucleoside transporter 1 NT1 [34] 44 Nucleoside transporter 1 NT1 [34]

45 NADH dehydrogenase 2 [105] 45 45 NADHNADH dehydrogenase dehydrogenase 2 2 [105] [105] 45 NADH dehydrogenase 2 [105] 45 NADH dehydrogenase 2 [105] 45 NADH dehydrogenase 2 [105]

46 CYP51 [106] 46 CYP51 [106] 46 46 CYP51CYP51 [106] [106] 46 CYP51 [106] 46 CYP51 [106]

47 L-arginase [104] 47 L-arginase [104] 47 L-arginase [104] 47 L-arginase [104] 47 47 L-arginaseL-arginase [104] [104]

48 Trypanothione reductase TR1 [117] 48 Trypanothione reductase TR1 [117] 48 Trypanothione reductase TR1 [117] 48 Trypanothione reductase TR1 [117] 48 48 TrypanothioneTrypanothione reductase reductase TR1 TR1 [117] [117]

49 Trypanothione reductase TR1 [118] 49 Trypanothione reductase TR1 [118] 49 Trypanothione reductase TR1 [118] 49 Trypanothione reductase TR1 [118] 49 Trypanothione reductase TR1 [118]

Molecules 2021, 26, 4629 25 of 34

42 Nucleoside diphosphate kinase NDK [102]

43 Nucleoside transporter 1 NT1 [34]

44 Nucleoside transporter 1 NT1 [34]

45 NADH dehydrogenase 2 [105]

46 CYP51 [106]

47 L-arginase [104]

48 Trypanothione reductase TR1 [117] Molecules 2021, 26, 4629 32 of 41

Table 5. Cont.

Cmpd Structure Target Ref.

49 49 TrypanothioneTrypanothione reductase reductase TR1 TR1 [118] [118]

Molecules 2021, 26, 4629 26 of 34 Molecules 2021, 26, 4629 26 of 34 Molecules 2021, 26, 4629 26 of 34 Molecules 2021, 26, 4629 26 of 34

50 50 TrypanothioneTrypanothione reductase reductase TR1 TR1 [119] [119] 50 Trypanothione reductase TR1 [119] 50 Trypanothione reductase TR1 [119] 50 Trypanothione reductase TR1 [119]

51 Trypanothione reductase TR1 [107] 51 51 TrypanothioneTrypanothione reductase reductase TR1 TR1 [107] [107] 51 Trypanothione reductase TR1 [107] 51 Trypanothione reductase TR1 [107]

52 Trypanothione reductase TR1 [107] 52 Trypanothione reductase TR1 [107] 52 52 TrypanothioneTrypanothione reductase reductase TR1 TR1 [107] [107] 52 Trypanothione reductase TR1 [107]

53 Trypanothione reductase TR1 [107] 53 Trypanothione reductase TR1 [107] 53 Trypanothione reductase TR1 [107] 53 53 TrypanothioneTrypanothione reductase reductase TR1 TR1 [107] [107]

54 Trypanothione reductase TR1 [108] 54 Trypanothione reductase TR1 [108] 5554 Trypanothione reductase TR1 [107][108] 5554 Trypanothione reductase TR1 [107][108] 55 Trypanothione reductase TR1 [107] 55 Trypanothione reductase TR1 [107]

56 Pteridine reductase PTR1 [110] 56 Pteridine reductase PTR1 [110] 56 Pteridine reductase PTR1 [110] 56 Pteridine reductase PTR1 [110]

57 Pteridine reductase PTR1 [112] 57 Pteridine reductase PTR1 [112] 57 Pteridine reductase PTR1 [112] 57 Pteridine reductase PTR1 [112]

58 Pteridine reductase PTR1 [111] 58 Pteridine reductase PTR1 [111] 58 Pteridine reductase PTR1 [111] 58 Pteridine reductase PTR1 [111]

Molecules 2021, 26, 4629 26 of 34 Molecules 2021, 26, 4629 26 of 34 Molecules 2021, 26, 4629 26 of 34 Molecules 2021, 26, 4629 26 of 34 Molecules 2021, 26, 4629 26 of 34

50 Trypanothione reductase TR1 [119] 50 Trypanothione reductase TR1 [119] 50 Trypanothione reductase TR1 [119] 50 Trypanothione reductase TR1 [119] 50 Trypanothione reductase TR1 [119]

51 Trypanothione reductase TR1 [107] 51 Trypanothione reductase TR1 [107] 51 Trypanothione reductase TR1 [107] 51 Trypanothione reductase TR1 [107] 51 Trypanothione reductase TR1 [107]

52 Trypanothione reductase TR1 [107] 52 Trypanothione reductase TR1 [107] 52 Trypanothione reductase TR1 [107] 52 Trypanothione reductase TR1 [107] 52 Trypanothione reductase TR1 [107]

Molecules 2021, 26, 4629 33 of 41

53 Trypanothione reductase TR1 [107] 53 Trypanothione reductase TR1 [107] 53 Trypanothione reductase TR1 [107] 53 Trypanothione reductase TR1 [107] 53 Trypanothione reductase TR1 [107] Table 5. Cont.

Cmpd Structure Target Ref.

54 54 TrypanothioneTrypanothione reductase reductase TR1 TR1 [108] [108] 54 Trypanothione reductase TR1 [108] 5554 TrypanothioneTrypanothione reductase reductase TR1 TR1 [107] [108] 55 5554 TrypanothioneTrypanothione reductase reductase TR1 TR1 [107][108] [107] 5554 TrypanothioneTrypanothione reductase reductase TR1 TR1 [107] [108] 55 Trypanothione reductase TR1 [107] 55 Trypanothione reductase TR1 [107]

56 Pteridine reductase PTR1 [110] 56 Pteridine reductase PTR1 [110] 56 56 PteridinePteridine reductase reductase PTR1 PTR1 [110] [110] 56 Pteridine reductase PTR1 [110] 56 Pteridine reductase PTR1 [110]

57 Pteridine reductase PTR1 [112] 57 Pteridine reductase PTR1 [112] 57 Pteridine reductase PTR1 [112] 57 57 PteridinePteridine reductase reductase PTR1 PTR1 [112] [112] 57 Pteridine reductase PTR1 [112]

58 Pteridine reductase PTR1 [111] 58 Pteridine reductase PTR1 [111] 58 Pteridine reductase PTR1 [111] 58 Pteridine reductase PTR1 [111] 58 58 PteridinePteridine reductase reductase PTR1 PTR1 [111] [111]

Molecules 2021, 26, 4629 27 of 34

59 59 PteridinePteridine reductase reductase PTR1 PTR1 [113] [113]

60 CRK3 [114]

61 CRK3 [114]

62 MAPK3 [115]

63 MAPK3 [115]

4.5. Targeting Death Mechanisms of Kinetoplastid Protozoans Regulated cell death (RCD) in kinenoplastids is a precise biochemical and molecular processes involved with regulatory steps which are still unknown. This limitation points out the controversy of using the term RCD and therefore makes the term “apoptosis-like” more appropriate. The “autophagic cell death” is also employed when the degradation of damaged structures, macromolecules or organelles is carried out through autophagy [120]. The molecular machinery of RCD comprises various subroutine forms (apoptosis, autophagy, necroptosis, etc.), which might uncover new therapeutic targets [121]. Various RCD types are impaired in the parasite metabolic pathways during differentiation processes of their life cycle. For instance, autophagy has been reported during L. Mexicana differentiation of metacyclic promastigotes into amastigotes [122]. The identification of parasite-specific regulators of RCD represent an attractive target for rational drug design for these protozoan infections [123].

4.6. Future Perspective of Drug Discovery Over the last decade, there are many examples of applications of machine learning (ML) and artificial intelligence (AI) regarding NTDs [124]. As the estimated cost of developing new drugs increases and problems such as significant toxicity, low efficacy, and emerging resistance of the current regimen against NTDs remain unresolved, ML and AI are emerging as a new approach to overcome these challenges [125].

Molecules 2021, 26, 4629 27 of 34 Molecules 2021, 26, 4629 Molecules 2021, 26, 4629 27 of 34 34 of 41 Molecules 2021, 26, 4629 27 of 34 Molecules 2021, 26, 4629 27 of 34

59 Pteridine reductase PTR1 [113] 59 Pteridine reductase PTR1 [113] 59 Pteridine reductase PTR1 [113] 59 Table 5. Cont. Pteridine reductase PTR1 [113] Cmpd Structure Target Ref.

60 60 CRK3CRK3 [114] [114] 60 CRK3 [114] 60 CRK3 [114] 60 CRK3 [114]

61 CRK3 [114] 61 61 CRK3CRK3 [114] [114] 61 CRK3 [114] 61 CRK3 [114]

62 MAPK3 [115] 62 MAPK3 [115] 62 62 MAPK3MAPK3 [115] [115] 62 MAPK3 [115]

63 MAPK3 [115] 63 MAPK3 [115] 63 MAPK3 [115] 63 63 MAPK3MAPK3 [115] [115]

4.5. Targeting Death Mechanisms of Kinetoplastid Protozoans 4.5. Targeting Death Mechanisms of Kinetoplastid Protozoans 4.5. TargetingRegulated Death cell deathMechanisms (RCD) of in Kinetoplastid kinenoplastids Protozoans is a precise biochemical and molecular 4.5. TargetingRegulated Death cell deathMechanisms (RCD) of in Kinetoplastid kinenoplastids Protozoans is a precise biochemical and molecular processesRegulated involved cell deathwith regulatory (RCD) in kinenoplastids steps which are is still a precise unknown. biochemical This limitation and molecular points processesRegulated involved cell deathwith regulatory (RCD) in kinenoplastids steps which are is stilla precise unknown. biochemical This limitation and molecular points outprocesses the controversy involved withof using regulatory the term steps RCD whic and htherefore are still unknown.makes the termThis limitation“apoptosis-like” points processesout the controversy involved withof using regulatory the term steps RCD whic andh therefore are still unknown.makes the Thisterm limitation“apoptosis-like” points moreout the appropriate. controversy The of using“autophagic the term cell RCD death” and istherefore also employed makes the when term the “apoptosis-like” degradation of outmore the appropriate. controversy The of using “autophagic the term cell RCD death” and thereforeis also employed makes the when term the “apoptosis-like” degradation of damagedmore appropriate. structures, The macromolecules “autophagic cell or death” organelles is also isemployed carried whenout through the degradation autophagy of moredamaged appropriate. structures, The macromolecules “autophagic cell ordeath” organelles is also employedis carried whenout through the degradation autophagy of [120].damaged The structures,molecular machinerymacromolecules of RCD or comp organellesrises various is carried subroutine out through forms (apoptosis,autophagy damaged[120]. The structures,molecular machinerymacromolecules of RCD or comp organellesrises various is carried subroutine out through forms (apoptosis,autophagy autophagy,[120]. The molecular necroptosis, machinery etc.), which of RCD might comp uncoverrises newvarious therapeutic subroutine targets forms [121]. (apoptosis, Various [120].autophagy, The molecular necroptosis, machinery etc.), which of RCD might comp uncoverrises newvarious therapeutic subroutine targets forms [121]. (apoptosis, Various RCDautophagy, types arenecroptosis, impaired etc.), in the which parasite might meta uncoverbolic newpathways therapeutic during ta differentiationrgets [121]. Various pro- autophagy,RCD types necroptosis,are impaired etc.), in the which parasite might meta uncoverbolic newpathways therapeutic during ta differentiationrgets [121]. Various pro- cessesRCD types of their are life impaired cycle. Forin the instance, parasite auto metaphagybolic has pathways been reported during duringdifferentiation L. Mexicana pro- RCDcesses types of their are lifeimpaired cycle. Forin the instance, parasite auto metaphagybolic has pathways been reported during duringdifferentiation L. Mexicana pro- differentiationcesses of their lifeof metacyclic cycle. For instance,promastigotes autophagy into amastigotes has been reported [122]. The during identification L. Mexicana of cessesdifferentiation of their lifeof metacycliccycle. For instance,promastigotes autophagy into amastigotes has been reported [122]. The during identification L. Mexicana of parasite-specificdifferentiation of regulators metacyclic of promastigotes RCD represent in anto attractiveamastigotes target [122]. for Therational identification drug design of differentiationparasite-specific of regulators metacyclic of promastigotes RCD represent in anto attractiveamastigotes target [122]. for Therational identification drug design of forparasite-specific these protozoan regulators infections of RCD[123]. represent an attractive target for rational drug design parasite-specificfor these protozoan regulators infections of RCD[123]. represent an attractive target for rational drug design for these protozoan infections [123]. for these protozoan infections [123]. 4.6. Future Perspective of Drug Discovery 4.6. Future Perspective of Drug Discovery 4.6. FutureOver thePerspective last decade, of Drug there Discovery are many examples of applications of machine learning 4.6. FutureOver thePerspective last decade, of Drug there Discovery are many examples of applications of machine learning (ML)Over and artificialthe last decade,intelligence there (AI) are regardin many examplg NTDses [124]. of applications As the estimated of machine cost oflearning devel- (ML)Over and artificialthe last decade,intelligence there (AI) are regardin many examplg NTDses [124].of applications As the estimated of machine cost oflearning developing(ML) and new artificial drugs increasesintelligence and (AI) problems regardin suchg NTDs as significant [124]. As the toxicity, estimated low costefficacy, of devel- and (ML)oping and new artificial drugs increasesintelligence and (AI) problems regardin suchg NTDs as significant [124]. As the toxicity, estimated low costefficacy, of devel- and emergingoping new resistance drugs increases of the current and problems regimen suchagainst as NTDssignificant remain toxicity, unresolved, low efficacy, ML and and AI opingemerging new resistance drugs increases of the current and problems regimen suchagainst as NTDssignificant remain toxicity, unresolved, low efficacy, ML and and AI areemerging emerging resistance as a new of approachthe current to regimen overcome against these NTDschallenges remain [125]. unresolved, ML and AI emergingare emerging resistance as a new of approachthe current to regimen overcome against these NTDschallenges remain [125]. unresolved, ML and AI are emerging as a new approach to overcome these challenges [125]. are emerging as a new approach to overcome these challenges [125].

Molecules 2021, 26, 4629 35 of 41

4.5. Targeting Death Mechanisms of Kinetoplastid Protozoans Regulated cell death (RCD) in kinenoplastids is a precise biochemical and molecular processes involved with regulatory steps which are still unknown. This limitation points out the controversy of using the term RCD and therefore makes the term “apoptosis-like” more appropriate. The “autophagic cell death” is also employed when the degradation of damaged structures, macromolecules or organelles is carried out through autophagy [120]. The molecular machinery of RCD comprises various subroutine forms (apoptosis, autophagy, necroptosis, etc.), which might uncover new therapeutic targets [121]. Various RCD types are impaired in the parasite metabolic pathways during differentiation processes of their life cycle. For instance, autophagy has been reported during L. Mexicana differentiation of metacyclic promastigotes into amastigotes [122]. The identiﬁcation of parasite-speciﬁc regulators of RCD represent an attractive target for rational drug design for these protozoan infections [123].

4.6. Future Perspective of Drug Discovery Over the last decade, there are many examples of applications of machine learning (ML) and artificial intelligence (AI) regarding NTDs [124]. As the estimated cost of developing new drugs increases and problems such as significant toxicity, low efficacy, and emerging resistance of the current regimen against NTDs remain unresolved, ML and AI are emerging as a new approach to overcome these challenges [125]. ML and AI have improved significantly in recent years due to rapid growth in computer-processing power, the development of advanced algorithms and the tendency to be implemented in R&D. ML is the study of computer algorithms which is automatically improved through the experience and use of scientific data, and AI is the ability of a digital computer to perform tasks with intellectual processes that characterize humans such as logic, learning, meaning discovery, and experience acquisition [126]. Chemical and biological information has become more and more available in public databases, offering new strategies in biomolecule structure prediction, mechanism of action elucidation, inverse drug design interpretation, organ or issue targeting of compounds, derivative properties, and multiobjective optimization. ML algorithms and AI can be used in a variety of applications of drug research and development and improve the current process, which is described by the failure of 9 out of 10 candidate therapies between Phase I clinical trials and regulatory approval [127]. There are plenty examples of AI/ML applications against kinetoplastid protozoan infections. ML chemoinformatics utilize high-throughput screens of the Broad Institute against T. cruzi and highlight active in vivo molecules [125]. Public databases such as TriTrypDB, TDR Targets, the Crystal Structures Database, and the T. cruzi RNA Sequence Database (in progress) provide a wealth of information that can be analyzed and validated by AI/ML methods. These approaches screen millions of compounds to predict those that bind and potentially inhibit protein function of T. cruzi and further present proper PK/PD properties to enter clinical trials. DNDi and other PDPs are funding these activities to procced on the scale required for drug discovery. DNDi is currently collaborating with Atomwise Inc., to advance drug discovery applying AI [128]. The new approaches overcome toxicity and resistance, the major drawback of the current regimen against the NTDs, focusing on specific proteins of the parasites. For instance, Jamal et al. analyzed a dataset of 292,470 available compounds of PubChem as pyruvate kinase inhibitors, using four classifier algorithms: Naïve Bayes (NB), Random Forest (RF), J48 and Sequential Minimization Optimization (SMO). The correlation of the aforementioned method scores with the docking method results predicted more possible active derivatives from a very large dataset and accelerated the discovery of new chemical entities against NTDs [129]. ML techniques have been used in combating drug resistance which has emerged in NTD pathologies. It is known that protein transporters (P-gp, ABC transporter, etc.) play a critical role in the decreased uptake or increased efflux of the drug from the parasites and generate related drug resistance [130]. Computer approaches Molecules 2021, 26, 4629 36 of 41

identified parasite-specific motifs of the responsible protein transporters in Leishmania major and facilitated the allosteric modulation of miltefosine transporter and subsequently reduced the parasite resistance [131]. The progress of data science, ML and AI, and their applications in biosciences is a near future perspective. ML and AI are still in ongoing progress and their limitations concerning the data validation might influence the quality of their models to generate novel chemical space and effective drug design. This danger of irrelevant endpoints and targets will be optimized in due course by the increasing use and the development of successful achievements. Challenges and limitations should be wisely carried out and draw a path for improvement in the field of drug design and chemotherapy of NTDs, without biased and distorted results.

5. Conclusions WHO has prioritized the intervention against the NTDs as a cornerstone of the vision of universal health coverage. While the course of the current COVID-19 pandemic remains uncertain, many programs and activities against the NTDs have been disrupted or delayed. This hindrance will impact on the gradual rise of these diseases towards the pre-intervention level. Remedial action should prompt an opportunity of innovation as a catch-up policy [132]. The application of scientiﬁc innovation offers new approaches against NTDs. The drug design and development against kinetoplastid protozoan infections should be accelerated over the next decade. The unmet need for new effective and safe drugs should lead the change for the treatment for trypanosomid diseases. The lack of well- validated trypanosomatid molecular targets is a drawback that should be overcome. The challenge that remains for drug discovery is the development of completely new classes of therapeutic agents with reduced host toxicity and improved administration regimens. The countdown to 2030 has begun, according to the General Director of WHO.

Funding: This research received no external funding. Acknowledgments: The authors would like to thank the graphic designer, Mirto Altani, for generat- ing the Graphical Abstract and Figure1. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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