Protozoa are a wide group of unicellular organisms that can be found in multiple ecosystems and are often non-pathogenic for humans. However, a small subset of protozoa has evolved as lethal intracellular parasites posing serious global health challenges. Among the most deadly intracellular protozoan parasites are those of the genus Plasmodium, Leishmania and Trypanosoma, which are collectively responsible for millions of death every year. Due to the emergence and spread of drug resistance (Dondorp et al. 2009; Tun et al. 2015), treatment options are often limited with reduced efficacy (Wongsrichanalai and Meshnick 2008). Drug R&D have been largely inexistent for these three parasites until early 2000’ when initiatives from leading pharmaceutical industries, largely supported by philanthropic and public nonprofit organizations emerged as a new business model to develop drugs for neglected diseases. These product development partnerships (PDP) have brought promising candidates for malaria in a record time.

Plasmodium

There are four species of Plasmodium that cause malaria in human. Collectively, they are responsible for 584,000 deaths and more than 200 million cases every year. The most common species are Plasmodium falciparum and Plasmodium vivax. The discovery and development of KAE609; an investigational drug to treat malaria is the most notable example of an antimalarial drug resulting from the effort of a PDP. The spiroindolone KAE609 was discovered in a phenotypic screen designed to identify small-molecules that rapidly clear intracellular P. falciparum from human red blood cells (Plouffe et al. 2008). Owing to its high potency in the cellular assay, low toxic liability and an early proof of efficacy in infected animals (Rottmann et al. 2010; Yeung et al. 2010), the spiroindolone series was optimized to improve overall properties. A reverse genetic approach was employed to identify the Na(+) efflux ATPase PfATP4 as the molecular target of the spiroindolone series (Rottmann et al. 2010; Spillman et al. 2013). Having demonstrated a remarkable efficacy in a phase 2a study at low dose, the optimized clinical candidate KAE609 has the potential to revolutionize malaria treatment (White et al. 2014; Held et al. 2015). The road is still uncertain before the introduction of KAE609 to clinical practice, but the program is a paradigm of how a PDP together with international collaborations can revolutionize the drug discovery process. Besides the discovery of KAE609, the same phenotypic screen also screen deliver KAE609, but also the novel class of imidazolopiperazines (IP) (Meister et al. 2011; Derbyshire et al. 2012), which is under clinical development (Leong et al. 2014).

A similar forward chemical genetics approach was conducted by two independent teams to identify hundreds of novel chemotypes active against drug resistant P. falciparum (Gamo et al. 2010; Guiguemde et al. 2010). The release of the chemical structures in the public domain is yet another example on how open innovation can accelerate drug discovery for neglected diseases. In an elegant reverse chemical genetics approach targeting 61 proteins and enzymes of P. falciparum, Crowther et al. identified the putative target of several promising hits using a thermal shift assay (Crowther et al. 2009). Even though validation experiments are yet to be performed to ascertain target engagement, their preliminary results set a solid foundation in spearheading translational research. Since the release of the chemical structures, several lead series have been evaluated for further evaluation (Sanz et al. 2011; Jimenez-Diaz et al. 2014; Vaidya et al. 2014). Interestingly, the promising dihydroisoquinolines compound (+)-SJ733 compound act through PfATP4 to mediate host clearance of the parasite (Jimenez-Diaz et al. 2014), which is also the target of mechanism shared with KAE609 (Spillman et al. 2013).

A most challenging subpopulation of Plasmodium to eradicate is the liver form of the disease. Plasmodium infect hepatocytes as sporozoites that first undergo a phase of maturation before emerging in the blood to cause disease manifestations. Targeting the early exoerythrocytic form of the parasite could be prophylactic since it would eradicate Plasmodium before the onset of the disease. In a technical tour de force, Meister et al. developed a high-content assay to quantify replication of P. yeollii inside human hepatocytes (Meister et al. 2011). An image-based approach is particularly well adapted for screening since only 1 % of the hepatocytes are infected by the parasites. By screening a collection of more than 4000 commercially available compounds that have activity against blood stage P. falciparum, the authors identified several chemicals that kill exoerythrocytic parasites. The most advanced drug candidate GNF179 provided protection against a challenge with P. berghei sporozoite, demonstrating in vivo activity against the early-liver stage of the disease. The putative target for GNF179 is pfcar1, an uncharacterized protein that is postulated to be involved in protein folding that is assumed to be essential for the biology of both the liver and blood stages of Plasmodium infection (Jonikas et al. 2009). More recently, a team at AstraZeneca utilized another high-throughput imaging assay to identify 2 more novel classes of fast-acting antiplasmodial agents; the N-aryl-2-aminobenzimidazoles, which targets the asexual blood stages of P. falciparum (Ramachandran et al. 2014), and the triaminopyrimidines (TAPs), which may potentially be used for single-dose treatment of malaria when used in multidrug combination therapy (Hameed et al. 2015). Exhibiting potent and wide-spectrum antimalarial activity against multiple life-cycle stages of P. falciparum, DDD107498 is another optimized lead derived from a 2, 6-disubstituted quinolone-4-carboxamide scaffold previously identified from a previous phenotypic screen (Baragana et al. 2015).

A key challenge in quest to eradicate malaria is the lack of potent drugs active against exoerythrocytic Plasmodium vivax. P. vivax is the most common form of malaria in South-east Asia. Although less deadly than P. falciparum, this species can survive in a dormant form as hypnozoites for extended period of time, leading to recurrent relapse (Wells et al. 2010). The identification of drug candidates active against hypnozoites is challenging due to the lack of predictive ex vivo models amenable to large-scale phenotypic screens. (Wells et al. 2010). Recent technical developments bring about cautious but yet resurgent optimism for the eventual discovery for an inherent cure for vivax malaria, through the complete eradication of all forms of Malaria parasites from the body. Using a low-throughput assay designed to quantify the number of hypnozoites and schizont forms of Plasmodium cynomolgi (a model for P. vivax) inside rhesus hepatocytes, the drug candidate KAI407 was identified for activity in reducing the formation of hypnozoites (Zou et al. 2014). In vivo prophylactic efficacy suggests that KAI407 may indeed represent a radical cure for vivax malaria. In prospect, the recent development of transgenic fluorescent P. cynomolgi (Voorberg-van der Wel et al. 2013), and of platforms that recapitulate the hepatic stage of P. vivax (Chattopadhyay et al. 2010; March et al. 2013) will probably be instrumental in the coming year to the identification and development of drugs targeting P. vivax hypnozoites.

Other kinetoplastids

Parasites of the genus Trypanosoma and Leishmania are kinetoplastid protozoan parasites that cause trypanosomiasis and leishmaniasis, respectively. These diseases, prevalent in tropical and subtropical countries, cause significant morbidity and mortality. No vaccines are available; and the current chemotherapies available for these neglected tropical diseases (NTDs) are limited with multiple shortcomings including potentially severe side effects, lengthy drug regimens and variable efficacy.

Chagas disease, caused by Trypanosoma cruzi, is the major cause of heart failure in Latin America. The only 2 available chemotherapies are benznidazole and nifurtimox, which have been shown to be largely ineffective and toxic, commonly causing drug resistance (Filardi and Brener 1987; Viotti et al. 2006). Drug R&D for Chagas disease is challenging due to the complex infection cycle of T. cruzi that can persist for many years in infected patients as trypomastigotes and amastigotes. The availability of engineered reporter gene expressing-parasites (Bettiol et al. 2009; Canavaci et al. 2010) have triggered the development of phenotypic assays suitable for HTS, as well as the establishment of new in vivo protocols (Canavaci et al. 2010; Rodriguez and Tarleton 2012) that allow faster evaluation of experimental therapeutic options.

Automated high content microscopy approaches (Engel et al. 2010; Moon et al. 2014) have been used to identify new parasitic inhibitors. To mimic the intracellular life cycle of T. cruzi for drug screening, Engel et al. developed and validated a flexible cell-based, high-throughput 96-well plate assay that could be used with a variety of untransfected T. cruzi isolates and host cells (Engel et al. 2010). This allowed the simultaneous measurement of both efficacies against the intracellular amastigote stage and host cell toxicity. Validation of the HTS assay enabled the identification of 55 hits upon screening a library of 909 bioactive compounds. Further drug testing narrowed the list down to 17 compounds that showed at least 5-fold selectivity between the inhibition of T. cruzi and host cell toxicity (Engel et al. 2010). Since these confirmed hits were selected from a library of clinical drugs, they could potentially be repurposed for T. cruzi treatment and used for mode of action and target identification studies. In a similar vein, a team from the Institut Pasteur Korea customized a high content image-based and HTS algorithm for the quantification of infection ratio and intracellular T. cruzi amastigote in human cell line in response to drug treatment (Moon et al. 2014). Based solely on DNA staining and single-channel images, the algorithm precisely segments and identifies the nuclei and cytoplasm of mammalian host cells as well as the intracellular parasites infecting the cells to produce various statistical parameters that can be used to assess both drug responses and compound cytotoxicity (Moon et al. 2014).

The Genomics Institute of the Novartis Foundation (GNF) has also initiated a drug discovery program for Chagas disease using phenotypic screens that measure the inhibition of proliferation of various kinetoplastid parasites to identify hits among low molecular mass compounds (Bustamante et al. 2011). To date, GNF has screened around 700,000 small molecules against the bloodstream form of T. brucei and the Leishmania donovani axenic amastigotes, which yielded around 2,000 confirmed hits in total. 44 % of these hits were also found to be active against intracellular T. cruzi (IC50 < 4 µM). Several of these T. cruzi-active compounds that possess a favorable profile have been selected for medicinal chemistry exploration with the goal of identifying lead scaffolds that could be further optimized. These early efforts have resulted in the discovery of analogs with sub-nanomolar potency against T. cruzi (Bustamante et al. 2011). These screens have also been recently extended to a larger 1.8 million compound library to identify T. cruzi-active compounds (Bustamante et al. 2011). It is expected that these screening efforts by GNF will eventually yield a single pool of anti-T. cruzi compounds that will be further validated and chemically optimized to yield preclinical candidates that could address this crucial gap in drug discovery for Chagas disease.

Leishmaniasis has been ranked among the most neglected of tropical diseases. As a group of diseases caused by trypanosomatids from the genus, cutaneous and mucosal leishmaniases are the predominant pathologies produced by Leishmania infection. Several drugs are currently available to treat cutaneous leishmaniasis, but each has limitations (Cruz et al. 2009). While target-based screening approaches for anti-leishmanial drug discovery has yielded little progress for a variety of reasons (Freitas-Junior et al. 2012), the development and implementation of HTS phenotypic assays by individual academic groups, consortia and public–private partnerships have generated several potential starting points for drug development. There is a general consensus for an urgent need for more reliable phenotypic in vitro screening that would mimic as closely as possible the definitive host environment. Therefore, genetically modified parasites expressing easily detectable reporters represent promising tools for phenotypic screening (Reguera et al. 2014).

Yet another evident advancement in this field is the development and validation of a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages without the need for a reporter gene (Siqueira-Neto et al. 2012). The in vitro infection protocol was adapted to a 384-well-plate format, thus enabling acquisition of a large amount of readouts by automated confocal microscopy. This assay has enabled the screening of up to 300,000 compounds to obtain 350 hits (Siqueira-Neto et al. 2012; Reguera et al. 2014). Since the same study also established that only 4 % of the hits identified by using Leishmania promastigotes display efficacy against intracellular amastigote forms (Siqueira-Neto et al. 2012), intracellular amastigote-based phenotypic screening is reinforced as the most suitable approach to be developed.

An exciting approach that is midway between in vitro cell infections and experimental infections in mice consists of the use of ex vivo explants of Leishmania-infected organs. Target-infected organs are harvested from fluorescent or bioluminescent Leishmania-infected rodents for development of ex vivo explant culture. These splenic or lymph node ex vivo infected explants are advantageous over in vitro systems by including the whole cellular population involved in the host-parasite interaction: macrophages, CD3 + and CD4 + T cells, B lymphocytes and granulocytes; which could affect the therapeutic effect of the tested compound. Since a single infected spleen can yield up to four 96-well plates, the use of these ex vivo explants will also drastically reduce the number of animals used for screening, whilst still enabling medium-throughput screening capabilities. Using this approach, 4,035 compounds were screened at a single-dose concentration against luciferase-transfected L. donovani-infected ex vivo hamster spleens, revealing more than 200 active hits (Osorio et al. 2011). More recently, the same group has also validated a lymph node ex vivo explant model using the same bioluminescent reporter in a L. major strain (Peniche et al. 2014).

Furthermore, lead compounds can be scaled up to in vivo preclinical trials using rodent models of infection monitoring parasite loads by means of advanced bioimaging devices. The use of quick and reproducible fluorescent and bioluminescent readouts would greatly reduce the number of animals used for these trials and allow for an earlier stage detection of the infective process as compared with classical methods (Reguera et al. 2014). A total of near half million compounds have been screened for visceral leishmaniasis treatment through a series of cutting-edge technologies combined with optimized assays (Freitas-Junior et al. 2012). However, since more systemic approaches to develop new chemical entities for leishmaniasis have started only recently, the late discovery process is still in its infancy.

Human African trypanosomiasis (HAT), also known as sleeping sickness, is yet another example of a vector-transmitted disease caused by 2 T. brucei subspecies namely, T. b. gambiense and T. b. rhodesiense. Untreated HAT leads to a fatal outcome, causing significant morbidity and mortality. With no vaccine available, current chemotherapy against HAT relies on four drugs that possess several limitations and occasional severe side-effects. Whole-cell assays in HTS format for T. brucei are relatively new, with the development of luciferase and resazurin-based cell viability assays a 384-well format (Mackey et al. 2006; Sykes and Avery 2009a, b). HTS resazurin-based assays have been recently used to screen 87,296 compounds, resulting in 6 hits from 5 new chemical classes with activity confirmed against the causative species of HAT (Sykes et al. 2012). Being intensively used in antimalarial drug discovery as well (Smilkstein et al. 2004; Johnson et al. 2007; Izumiyama et al. 2009; Vossen et al. 2010), SYBR Green whole-cell assays, which are an indirect assessment of cell number based on quantitative detection of nuclei acids, have also been found to be applicable to T. brucei. To aid in drug discovery efforts for HAT, both assays were semi-automated to screen a library of 4,000 putative kinase inhibitors (Faria et al. 2015). The compounds with the most potent anti-trypanosomal activity could be grouped into 13 structural clusters. Several of the identified compounds had IC 50 <1 μM coupled with high selectivity toward the parasite, thus providing promising starting points for lead optimization.

The limitation for the development of novel lead candidates for kinetoplastid parasites remains in target identification. Early identification of the candidate target and demonstration of target engagement would indeed accelerate drug development by using enzymatic assays or biophysical methods to rationally optimize drug candidates. While efforts can be further expanded for drug discovery for these NTDs, it is anticipated that novel drug candidates are on the horizon for the treatment of trypanosomiasis and leishmaniasis. On the model of P. falciparum, selection of escape mutants followed by whole-genome sequencing is probably the most straightforward approach to identify the target, or at least mechanisms of resistance.