WO2024126363A1 - Image-based high-content screening methods for identifying compounds targeting apicomplexan parasites - Google Patents

Abstract

Apicomplexa phylum includes numerous obligate intracellular protozoan parasites that are life threatening for humans and animals. Apicomplexan parasites have a highly polarized morphology, with their apical end containing specific secretory organelles named rhoptries and micronemes, which depend on the unique receptor and transporter sortilin TgSORT for their biogenesis. In the present study, the inventors took advantage of the subcellular polarity of the parasite to engineer a clonal transgenic Toxoplasma line that expresses simultaneously the green fluorescent protein TgSORT-GFP in the post-Golgi-endosome-like compartment and the red fluorescent protein rhoptry ROP1-mCherry near the apical end. The inventors utilized this dually fluorescent transgenic T. gondii to develop a miniaturized image-based phenotype assay coupled to an automated image analysis. By applying this methodology to 1,120 compounds, they identified 12 that are capable of disrupting the T. gondii morphology and inhibiting intracellular replication. The findings highlight the advantage of comparative and targeted phenotypic analysis involving two related parasite species as a means of identifying molecules with a conserved mode of action.

Description

IMAGE-BASED HIGH-CONTENT SCREENING METHODS FOR IDENTIFYING

COMPOUNDS TARGETING APICOMPLEXAN PARASITES

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular parasitology.

BACKGROUND OF THE INVENTION:

The phylum Apicomplexa includes about 5,000 intracellular protozoan parasites that infect humans and animals, such as Plasmodium falciparum (the causative agent of human malaria), Toxoplasma gondii (an important opportunistic pathogen associated with AIDS and congenital birth defects. This phylum also contains harmful infections in animals, Eimeria (a causative agent of deadly coccidiosis in poultry) and Cryptosporidium (an opportunistic intestinal pathogen causing severe diarrhea). Malaria is an ancient vector-borne infectious disease primarily occurring in developing countries that is responsible for about 600,000 deaths per year, most of which occur in children (WHO, 2021). On the other hand, toxoplasmosis is less deadly, but in utero exposure can lead to devastating outcomes, including congenital malformations such as blindness, lack of normal development of intellectual capacities and hydrocephaly in newborns. In addition, T. gondii is recognized as the advent of AIDS and is a highly opportunistic pathogen (Montoya and Liesenfeld, 2004; Halonen and Weiss, 2013; Milne et al., 2020).

Toxoplasmosis is presently treated with a combination of drugs, such as pyrimethamine and sulfadiazine, and the affected individuals are typically prescribed folinic acid to prevent pyrimethamine from suppressing bone marrow formation (Hill and Dubey, 2002). As these drugs have severe side effects, such as neutropenia, leucopenia, severe reduction in platelet count, thrombocytopenia, and hypersensitivity (Porter and Sande, 1992; Rajapakse et al., 2013), clindamycin may be used as an alternative. However, if patients are unable to tolerate either sulfonamides or clindamycin, atovaquone can be prescribed (Dunay et al., 2018), whereas spiramycin is recommended for pregnant women due to its bioavailability in the placenta. In seropositive AIDS patients, trimethoprim in combination with sulfamethoxazole has been shown to prevent cerebral toxoplasmosis. Nonetheless, most of these drugs are poorly tolerated and their long-term use results in cytotoxicity, or fails to deliver expected outcomes. Likewise, malaria is currently treated via an artemisinin-based combination therapy, which is increasingly ineffective as a result of the growing resistance of the malaria parasite P. falciparum to the available drugs (Balikagala et al., 2021).

Apicomplexa are complex single-celled eukaryotes containing classical nucleus, mitochondrion, endoplasmic reticulum and Golgi, as well as specific secretory organelles — rhoptries and micronemes — that are located at the apical end of the parasites, whose contents are required for host cell attachment, invasion and virulence. Extant research indicates that many rhoptry proteins are kinases or pseudokinases capable of defining virulence factors that can be secreted in different compartments of the infected host cells, including the nucleus. For example, ROP16 can phosphorylate STAT3/6 and thus control the transcription level of numerous genes involved in the host’s immune response (Saeij et al., 2006; Taylor et al., 2006; Saeij et al., 2007; Yamamoto et al., 2009; Ong et al., 2010; Butcher et al., 2011). In contrast, the microneme MIC proteins are defined as attachment factors required by the parasite to recognize the receptors on the host cell surface during entry (Huynh et al., 2003; Huynh and Carruthers, 2006). In addition, ROP and MIC proteins can act synergistically to form the moving junction necessary for the host cell invasion (Alexander et al., 2005; Lamarque et al., 2011). Available evidence further indicates that apicomplexan parasites, including T. gondii, are 10 pm long eukaryotic organisms with a highly polarized secretory pathway. Several authors have also noted that proteins and probably lipids destined for the apical secretory organelles (i.e., rhoptries and micronemes) are synthesized in the ER and move to the Golgi apparatus before reaching the post-Golgi and endosomal-like compartment (Pelletier et al., 2002; He, 2007; Tomavo et al., 2013). ROP and MIC proteins are also synthesized as proproteins that are cleaved at the N-terminus during maturation, leading to properly folded and functionally active proteins (Bradley and Boothroyd, 2001; Harper et al., 2006; Brydges et al., 2008). In our earlier work, we demonstrated that the transport and maturation of ROP and MIC proteins required the presence of an essential sortilin homologue, which we denoted as T. gondii sortilin-like receptor (TgSORT) (Sloves et al., 2012). In addition, we showed that the rhoptry and microneme biogenesis depends on TgSORT. These findings were subsequently confirmed by Sangare et al. (2016) who reported that the luminal domain of the receptor binds rhoptry and microneme proteins, while the cytosolic tail recruits partners to enable anterograde and retrograde receptor transport. This evidence indicates that TgSORT is a key receptor for the biogenesis of secretory organelles in T gondii as well as P. falciparum, and its homologue PfSortilin in malaria was later found to control transport of proteins to form rhoptries (Hallee et al., 2018a, 2018b). Because the homologues of TgSortilin are present in all apicomplexan parasites whose genome has been sequenced (VEuPathDB), we hypothesize that this receptor is a key factor that allows apicomplexan parasites to build the complex apical structure composed of a functional conoid containing rhoptries and micronemes. We further posit that protein trafficking is a critical parameter in parasite multiplication and dissemination. Therefore, identifying molecules that target this process could enrich the therapeutic arsenal.

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to image-based high-content screening methods for identifying compounds targeting apicomplexan parasites.

DETAILED DESCRIPTION OF THE INVENTION:

Apicomplexa phylum includes numerous obligate intracellular protozoan parasites that are life threatening for humans and animals. In this context, Plasmodium falciparum and Toxoplasma gondii are of particular interest, as they are responsible for malaria and toxoplasmosis, respectively, for which efficient vaccines are presently lacking and therapies need to be improved. Apicomplexan parasites have a highly polarized morphology, with their apical end containing specific secretory organelles named rhoptries and micronemes, which depend on the unique receptor and transporter sortilin TgSORT for their biogenesis. In the present study, the inventors took advantage of the subcellular polarity of the parasite to engineer a clonal transgenic Toxoplasma line that expresses simultaneously the green fluorescent protein TgSORT-GFP in the post-Golgi-endosome-like compartment and the red fluorescent protein rhoptry ROPl-mCherry near the apical end. The inventors utilized this dually fluorescent transgenic T. gondii to develop a miniaturized image-based phenotype assay coupled to an automated image analysis. By applying this methodology to 1,120 compounds, they identified 12 that are capable of disrupting the T. gondii morphology and inhibiting intracellular replication. Analysis of the selected compounds confirmed that all 12 are kinase inhibitors and intramembrane pumps, with some exhibiting potent activity against Plasmodium falciparum. The findings highlight the advantage of comparative and targeted phenotypic analysis involving two related parasite species as a means of identifying molecules with a conserved mode of action.

Definitions: As used herein, the term “apicomplexan parasite” has its general meaning in the art and represents a large phylum of parasitic alveolates. Most of apicomplexan parasite possess a unique form of organelle that comprises a type of non-photosynthetic plastid called an apicoplast, and an apical complex structure. The organelle is an adaptation that the apicomplexan applies in penetration of a host cell. The Apicomplexa are unicellular and sporeforming. All species are obligate endoparasites of animals. Many of the apicomplexan parasites are important pathogens of human and domestic animals. The Apicomplexa are a diverse group that includes organisms such as the coccidia, gregarines, piroplasms, haemogregarines, and plasmodia. Examples of apicomplexan parasites include Toxoplasma gondii and Plasmodium falciparum.

As used herein, the term “transgenic apicomplexan parasite” refers to an apicomplexan parasite in which one or more cells are altered by or receive a recombinant polynucleotide.

As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.

As used herein, the term “sortilin-like receptor polypeptide” or “SORTER polypeptide” has its general meaning in the art and refers to a type I transmembrane cargo transporter located in the post-Golgi and endosome related compartments of apicomplexan parasites. SORTER polypeptides are essential to the biogenesis of secretory organelles and in turn to motility, invasion and egress of the parasites (Sloves, Pierre-Julien, et al. "Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection." Cell host & microbe 11.5 (2012): 515-527f Genomic sequences for SORTER polypeptides are well known in the art and are typically accessible from the VEuPathDB data base (https://veupathdb.org/veupathdb/app/). An exemplary amino acid sequence for the Toxoplasma gondii SORTER polypeptide is shown as SEQ ID NO: 1.

SEQ ID NO : 1 Protein Sequence of Toxoplasma gondii Sortilin ( TgSORT ) 1033 aa

MYTSRTPHGGLRRWPLSRSSVSAVASRVLSRSVAFHRRLASFLLSRKKTPLCALSSRPSGLQAS SGVQT VTMAHSFARGRPPAAGRTLAVFLAFLAVLSPSLLPEFFSEDAQKRESSVLVA AASYANKKVSVSEVSF DSHIEDIQWCGTDHRTILLKTRRGRLYRSQDGGKSWTEITDLLKSSEAATGTVAVDSI IVSPVDKRWL IVGSKRNHFI SEDSAATFRRLKYKNTIHNFHFHPTRPKYAILSTWTDACYSGSGTASRAQSQQDCNHQL FYTRDLGRSFKLVADYWQFSWGDKKLGNTDHI FFTQHRGRSGDQPRYGGWSKNVDLMYTPDFGATITR LVYRGNKFLLSNGYFFVAKVKDAAKQTVSLLVSTDGGKSFQMAKLPVEIEERSYTVLDTSEDAIMLHVN HGHDNKGDTGNVYI SDAKGVRYSLSLPNNIRTSTGECEFDKVLSLEGVYLANFKDSVDSSASVDGGQQG DLEKLEEEIEEEAEGVQVDLEKKHKSVATRSRQEEVIRTVI SFDKGGVWSYLKAPKVDSRGQKI DCPPD RCWLHLNGITRFSDFAPFYSVENAVGI IMGTGNVGSYLRPEKDEANTYLSRDGGVSWIEAHKGAFIYEM GDHGGLLVMADDTKKTNQWFSWNEGQSWYDFELGAAPLFVDNIVIEPNASSVEFLLYGKREQDSAGVL FHLDFNALNQQQCKGIWAADSVSSDYETWSPSDGRAGGERCILGKHITYTRRKQTSECFNGRDFDRPKV SKVCPCTMEDYECEFGFTRAIGSTQCVATDAAAAAAATATGLAQFADESDAAAAAACTSSSFFYTSAYR KVPGDVCEGGWMPEKVAVPCPAHSPVSRGGKTVLLLLLFIVWMWINYLAKTGRLKKFFRNAGFDSFA NVSYGLVGASAGGPGGWLDQEAGESRRGEELGERSKYEPELGFIEAEQDENEEDAPTLMNYGNAAGGQR

TSGMSSRSPKPTEDFELDDSRPLFPSHVSSRETQGSSGLHPRSTSTENEPI PRLAPPRFDEDNVELL

As used herein, the term “rhoptry polypeptide” or “ROP polypeptide” has its general meaning in the art and refers to polypeptides contained in the rhoptry that is a specialized secretory organelle of apicomplexan parasites. In particular, rhoptries are club-shaped organelles connected by thin necks to the extreme apical pole of the parasite. These organelles, like micronemes, are characteristic of the motile stages of Apicomplexa protozoans. They can vary in number and shape and contain numerous enzymes that are released during the penetration process. The rhoptry proteins are important in the interaction between the host and the parasite, including the formation of the parasitophorous vacuole. Extant research indicates that many rhoptry proteins are kinases or pseudokinases capable of defining virulence factors that can be secreted in different compartments of the infected host cells, including the nucleus. An exemplary amino acid sequence for the Toxoplasma gondii ROP1 polypeptide is shown as SEQ ID NO:2

SEQ ID NO : 2 Protein Sequence of Toxoplasma Rhoptry protein 1 ( ROP1 ) 456 aa MACRQLLCSVQNLLFFFLRDIYCTDFDTMEQRLPI ILLVLSVFFSSTPSAALSSHNGVPAYPSYAQVSL SSNGEPRHRGIRGSFLMSVKPHANADDFASDDNYEPLPSFVEAPVRGPDQVPARGEAALVTEETPAQQP AVALGSAEGEGTSTTESASENSEDDDTFHDALQELPEDGLEVRPPNAQELPPPNVQELPPPNVQELPPP TEQELPPPTEQELPPPTEQELPPPTEQELPPSTEQELPPPVGEGQRLQVPGEHGPQGPPYDDQQLLLEP TEEQQEGPQEPLPPPPPPTRGEQPEGQQPQGPVRQNFFRRALGAARSRFGGARRHVSGVFRRVRGGLNR IVGGVRSGFRRAREGWGGVRRLTSGASLGLRRVGEGLRRSFYRVRGAVSSGRRRAADGASNVRERFVA AGGRVRDAFGAGLTRLRRRGRTNGEEGRPLLGEGREQDDGSQ

As used herein, the term “identity” refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8: 155- 165, 1992; and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994). Altschul et al., Nat. Genet., 6: 119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266: 131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.

As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one). As used herein, the term “fusion protein” refers to a single polypeptide chain having at least two polypeptide domains that are not normally present in a single, natural polypeptide. Thus, naturally occurring proteins are not “fusion proteins”, as used herein.

As used herein, the term “linker” refers to a sequence of at least one amino acid that links the polypeptide of the invention to the expression tag. Such a linker may be useful to prevent steric hindrances. Typically a linker comprises 2, 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; or 20 amino acids.

As used herein, the term “labelling polypeptide” refers to a polypeptide that is detectable with or without instrumentation, for example, by visual inspection, spectroscopy, or a photochemical, biochemical, immunochemical or chemical reaction. Exemplary labeling polypeptide (non-limiting) include fluorescent polypeptide, enzymes (such as those commonly used in an ELISA), and binding polypeptides. For example, a labeling polypeptide can generate a measurable signal such as fluorescent light in a sample.

As used herein, the term "subject" or "subject in need thereof", is intended for a human or non-human mammal. Typically the patient is affected or likely to be infected by an apicomplexan parasite.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

Transgenic apicomplexan parasites:

The first object of the present invention relates to a transgenic apicomplexan parasite that expresses simultaneously i) a first fusion protein wherein a sortilin-like receptor (SORTER) polypeptide is fused to a first labelling polypeptide (i.e. the “SORTER fusion protein”) and ii) a second fusion protein wherein a rhoptry polypeptide is fused to a second labelling polypeptide (i.e. the “rhoptry fusion protein”).

In some embodiments, the apicomplexan parasite is a Toxoplasma gondii strain as described in the EXAMPLE.

In some embodiments, the SORTLR polypeptide comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

In some embodiments, the rhoptry polypeptide comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:2.

Typically, the labelling polypeptide is a fluorescent polypeptide. For instance, fluorescent or otherwise detectable proteins that can be used to practice the invention include Blue/UV Proteins, Cyan Proteins, Green proteins, Yellow Proteins, Orange Proteins and Red proteins. In some embodiments, the fluorescent polypeptide is a green fluorescent protein (GFP), an mCherry protein, a td Tomato protein, an E2 Crimson protein, a Cerulean protein or an mBanana protein, or any equivalent fluorescent or otherwise detectable protein. For example, the green fluorescent protein (GFP) used to practice this invention comprises 238 amino acid residues, at 26.9 kDa, that exhibits a bright green fluorescence when exposed to light in the blue to ultraviolet range. Equivalent green fluorescent proteins can have a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm.; an emission peak can be at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. For example, in alternative embodiments, a GFP from the jellyfish Aequorea victoria or a GFP from the sea pansy Renilla reniformis is used having a single major excitation peak at 498 nm. Equivalent fluorescent proteins that can be used to practice the invention include any red fluorescent protein, e.g., as derived from Discosoma sp. In some embodiments, another red fluorescent protein mCherry protein is used: it is a monomeric fluorescent construct with peak absorption/emission at 587 nm and 610 nm, respectively. It is resistant to photobleaching and is stable. It matures quickly, with a to 0.5 of 15 minutes, allowing it to be visualized soon after translation. In some embodiments, a tdTomato used to practice this invention is an exceptionally bright red fluorescent protein. tdTomato's emission wavelength of 581 nm and brightness make it ideal for live animal imaging studies. The tdTomato fluorescent protein is equally photostable to mCherry. In some embodiments, E2-Crimson used to practice this invention is a bright far-red fluorescent protein initially designed for in vivo applications involving sensitive cells such as primary cells and stem cells. E2-Crimson was derived from DsRed-Express2, and retains its rapid maturation (half time of 26 minutes at 37° C ), high photostability, high solubility, and low cytotoxicity (Takara Holdings Inc., Kyoto, Japan).

In some embodiments, the SORTER polypeptide is fused to GFP polypeptide.

In some embodiments, the Rhoptry polypeptide is fused to a mCherry polypeptide.

In some embodiments, the SORTER polypeptide and the rhoptry polypeptide are fused directly to their corresponding labelling polypeptide or via a linker. The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678. Other examples are poly-alanine linker sequences such as Ala-Ala-Ala. Further preferred examples of linker sequences are Gly/Ser linkers of different length. The transgenic apicomplexan parasite of the present invention is generated according to any well-known method, such as described in the EXAMPLE. For instance, tachyzoites of the selected strain are transfected with an amount of vectors (e.g. plasmids) that incorporate the polynucleotides that encodes for the fusion proteins. After selection, stable parasites are cloned and positive clones are screened by indirect immunofluorescence assay (IF A).

Methods of screening:

The transgenic apicomplexan parasites of the present invention are particularly suitable for preparing assays suitable for screening a plurality of test substances for their ability to kill the parasite. Selected test substances may be then used as anti-parasite compounds that could be used for the treatment of diseases mediated by said parasite (e.g. malaria).

According to the present invention, in normal conditions, the SORTER fusion protein is expressed in the post-Golgi-endosome-like compartment and the rhoptry fusion protein is expressed near the apical end. The mislocalisation of the rhoptry fusion protein indicates that the test substance is capable of compromising parasite’s polarity, morphology and/or intracellular replication of the parasite.

Thus a further object of the present invention relates to a method for screening a plurality of test substances for their ability to kill an apicomplexan parasite comprising the steps of i) contacting the plurality of the test substances with a population of cells infected by the transgenic apicomplexan parasite, ii) detecting the subcellular localisation of the fusion proteins and iii) positively selecting the test substances capable of disrupting the distinct subcellular localization of the rhoptry fusion protein.

In some embodiments, the method of the present invention implements an automated image analysis that comprises the steps of a) subjecting the infected cells to imaging to collect a plurality of images of the individual infected cells present and b) applying an algorithm configured to detect fluorescence emitted by the fusion proteins from said plurality of images, c) detecting the subcellular localisation of the fusion proteins and d) positively selecting test substances that induces mislocalisation of the rhoptry fusion protein. In particular, the algorithm implements one or more photometric parameters that are suitable for detecting the subcellular localisation of the fusion proteins. Typically, the photometric parameters can include fluorescence mean intensity and fluorescence median intensity. Therefore the algorithm involves processing the image data collected to determine if any of the imaged cells in the population exhibit one or more parameters associated with a mislocalisation of the rhoptry fusion protein. Since parasites are gathered in vacuoles, the vacuole population can be split into positive and negative groups. Thus in some embodiments, the algorithm is suitable to detect the vacuoles and classify them as “positive” or “negative”. For instance, “positive” vacuoles that are considered to express the expected mislocalisation phenotype can thus be selected using a multiparameter threshold, including the ratio of Rhoptry fusion protein (e.g. ROPl-mCherry) intensity between the center and the border of the vacuole and the mean fluorescent signal (e.g. mCherry) intensity in the vacuole. In some embodiments, to be classified as “positive”, vacuoles have to fulfill the following two criteria: (1) the ratio of intensity between the center and the border of the vacuole must exceed 1.4, and (2) the mean intensity in the vacuole must be below 500. The percentage of “positive” vacuoles is thus used as a parameter that reflects the vacuole disruption phenotype. Then, it is possible to compare the percentage of “positive” vacuoles with a predetermined reference value. For instance, said predetermined reference value can be the percentage of “positive” vacuoles obtained in the absence of a test substance (i.e. in presence of the vehicle) (i.e. “negative control”). Alternatively, the predetermined reference value can be the percentage of “positive” vacuoles that are obtained with a reference test substance, is known to induce mislocalisation of the rhoptry fusion protein (i.e. “positive control”). Then any test substances that lead to a percentage of “positive” vacuoles that is higher than the predetermined value can thus be positively selected. It should however be understood that any image analysis methods or software packages can be implemented to apply the concepts disclosed herein, and the preferred image analysis software package that is disclosed in the EXAMPLE is intended to be exemplary, rather than limiting of the concepts disclosed herein.

The test substance of the invention may be selected from a library of substances previously synthesised, or a library of substances for which the structure is determined in a database, or from a library of substances that have been synthesised de novo. The test substance may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) organic or chemical substances. In some embodiments, the screening method of the invention further comprises the step consisting in determining whether the selected substance is capable of compromising parasite’s polarity, morphology and/or intracellular replication of the parasite. In some embodiments, the screening method of the present invention further comprises a step that consists in positively selecting the test substance capable of inhibiting the replication of said parasite in said host cell. In some embodiments, the method comprises the steps consisting of i) infecting said host cell with the parasite of interest and ii) culturing said infected cell in presence of the test substance, iii) comparing the replicating capacity of the parasite with the replication capacity determined in the absence of the test substance and iv) positively selecting the test substance that provides a decrease in the replication capacity of the parasite.

Methods of treatment:

A further object of the present invention relates to a method for treating a disease caused by an apicomplexan parasite in a subject in need thereof comprising administering to the patient a therapeutically effective amount of at least one compound of Table 1 and/or Figure 8.

In some embodiments, the subject can be human or any other animal (e.g., birds and mammals) susceptible to infection by apicomplexan parasites (e.g. domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.). Typically said subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult.

In some embodiments, the subject is infected by Toxoplasma gondii.

In some embodiments, the subject is infected by a. Plasmodium parasite selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium simiovale, Plasmodium brazilianum, Plasmodium schwetzi and Plasmodium simium, and more preferably from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovate, Plasmodium knowlesi and Plasmodium malariae, and more preferably from the group consisting of Plasmodium falciparum and Plasmodium vivax. In some embodiments, said parasite is Plasmodium falciparum, in particular the Palo Alto I strain of Plasmodium falciparum.

As used herein, the term "therapeutically effective amount" of the drug of the present invention is meant a sufficient amount of the compound to block the transmission of the Apicomplexan parasite at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated, the potential positive impact of treatment for the local or general human community, and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically, the drug of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Galenic adaptations may be done for specific delivery in the small intestine or colon. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol ; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising the compound of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compound of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifusoluble agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. The compound of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered. In addition to compound formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations ; time release capsules ; and any other form currently used.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Generation of transgenic T. gondii expressing TgSORT-GFP/ROPl-mCherry. Panel A corresponds to the vector that allows the knock-in of ROPl-mCherry in the parasites. One clone was selected and the red color indicates the R0P1 signal located at the apical end of four intracellular dividing tachyzoites (Panel B). Thus, the vector expressing the TgSORT-GFP was transfected (Panel C) and one positive clone (green signal), which replicates into eight intracellular tachyzoites (Panel D) for the expression of this receptor in the post-Golgi and endosome-like compartment (ELC) is shown. Bar scale = 3 pm.

Figure 2. Assay workflow showing all steps of the library screening protocol, namely cell culture, parasite infection, sample processing and labeling, and image acquisition followed by automated image analysis. MOI: multiplicity of infection.

Figure 3. Typical images and image analysis workflow (described in detail in the Columbus analysis script): (1) Raw image acquisition from the reader; (2) Segmentation on the blue channel to detect the HFF nuclei (stained with DAPI reagent), followed by false detection removal based on the intensity/morphology properties in order to retain true nuclei only; (3) HFF cytosol delimitation around nuclei using predefined algorithm based on the use of the DAPI channel, which also stains cytosol (producing a signal of lower intensity); 4) Parasite detection within the cytosol region allowing the DAPI-labeled plots to be segmented into parasite nuclei; (5) Vacuole determination by the total proximal parasites nuclei area (as parasites grow in the vacuole the nucleus area can be mathematically expanded around them); (6) mCherry intensity, enabling determination of disrupted vacuoles. In order for a vacuole to be considered as positive, the mean of the mCherry fluorescence intensity should not be too high and the ratio of mCherry intensity between the vacuole center and the periphery must exceed 1.4.

Figure 4. Pre-screen optimization using pyrimethamine, the well-known anti-Toxoplasma drug as control. (A) Checker-board layout; (B) Determination of the parasite number per HFF cells for DMSO and Pyr at 10-pM concentration. VAL1 and VAL2 correspond to the two analyzed plates.

Figure 5. Plate heat map for each of the three screening plates (P37, P43 and P49) for each well, where each square corresponds to a well. DMSO was dispensed in the 1^st eight rows of the 1^st column and the last eight rows of the 21^st column, while the two compounds — SB 239063 (Cpd2) and SB 208 (Cpd3) — were placed in the last eight rows of the 1^st and the first eight rows of the 21^st column, respectively. For each well, the Z-score determined on the percentage of vacuoles displaying disruption is given, with Z-score > 3 indicating a compound impacting vacuole disruption.

Figure 6. Data dot plot for each of the three screening plates — P37 (circles), P43 (diamonds) and P49 (triangles). The percentage of positive vacuoles is shown before normalization on the Y-axis. Red color corresponds to the negative control (DMSO), and blue and pink denote the positive controls — SB 239063 (Cpd2) and SB 208 (Cpd3) — in each plate, while yellow color is used for the tested compounds and black for non-infected wells.

Figure 7. Screening hits. Five hits were obtained using our automatic image-based screening protocol and these fluorescence images (featuring necrotized intracellular parasites) illustrate the disruption of classical apical location of ROP1 signal compared to the negative DMSO control that shows intracellular dividing tachyzoites with normal morphology.

Figure 8. Chemical structures of all 12 hits with their corresponding names in the Tocris library, along with the nomenclature used in the present study (Cpd) and with the number corresponding to the order of identification.

Figure 9. Dose-dependent inhibitory activities on the 12 compounds using P-galactosidase assays. Panel A shows the assays performed on the first six compounds (including Cpd5, which probably represents a false positive). Panel B illustrates the six remaining compounds. These two panels correspond to single-dose assays and all p values are below 0.0001. Panel C pertains to a mix of three compounds (p value = 0.0024), while Panel D shows a dose-dependent inhibition of plaque formation by T. gondii after nine days of drug exposure. As two independent experiments yielded identical results, one is illustrated here.

Figure 10. Dose-dependent inhibitory effects of the studied drugs on P. falciparum growth in red blood cells (RBCs). Panel A, Panel B and Panel C respectively show ring stage, trophozoite and schizonte counts after drug treatments based on flow cytometry, with chloroquine as positive control (p < 0.0001). Panel D shows a blood smear used to visualize ring stages of the DMSO control versus selected drugs in all used concentrations.

EXAMPLE:

Material & Methods

Parasite strains and reagents

The following T. gondii strains were used in this work: RH strain, RHAKU80, and RH containing Lac Z (clone 2F1) expressing P-galactosidase, which were kindly provided by Prof. Vem Carruthers (University of Michigan, USA) and P. falciparum 3D7 strain procured from the Institut de Recherche et de Developpement (IRD-Benin). The following reagents were also used: DAPI, (D9542, Sigma-Aldrich), crystal violet (C0775, Sigma Aldrich), Kit RAL 555 (RAL Diagnostics, Labelians, Belgium), 5 ’-fluorodeoxyuridine (5’FUDR, Merck), pyrimethamine (Merck), mycophenolic acid (MPA, Calbiochem), Xanthine (XAN, Merck), chlorophenol red-P-D-galactopyranoside (CPRG, Merck), Hoechst- Thiazole Orange (Sigma Aldrich) and Tocriscreen Total library (Cat. No. 2884, Tocris Biotechne).

Intracellular growth of T. gondii

The tachyzoites of T. gondii strains used in the present study were routinely cultured on a human fibroblast foreskin (HFF) monolayer in Dulbecco’s Modified Eagles Medium (DMEM, PAN Biotech, Dutscher, France) supplemented with heat-inactivated 10% fetal bovine serum (PAN Biotech, Dutscher, France), 2 mM of glutamine and 50 pg/mL of penicillin/ streptomycin (PAN Biotech, Dutscher, France). After three hours, the infected cells were washed with culture medium and the intracellular parasites were allowed to grow at 37°C for 2-3 days to ensure complete lysis. Freshly lysed tachyzoites were purified using 0.33 pm filter (Millipore) to remove cell debris before counting, and the obtained parasites were used for transfection or for drug assays.

Plasmid constructs and generation of transgenic T. gondii

Transgenic ROPl-mCherry parasites were obtained via a knock-in strategy using a 2283 bp DNA fragment as well as KI-R0P1 forward primer (TAC TTC CAA TCC AAT TTA ATG CTG GGC TCG CAC CAA TAG CAC) and KI-R0P1 reverse primer (TCC TCC ACT TCC AAT TTT AGC TTG CGA TCC ATC ATC CTG CTC). This DNA fragment was cloned into the pLIC-mCherry-HXGPRT (hypoxanthine-xanthine-guanine phosphoribosyltransferase selectable marker) plasmid (Huynh and Carruthers, 2009), and was linearized using BstBI restriction enzyme. Tachyzoites (5* 10⁶ parasites) of the RHAKu80 strain were transfected with 25 pg of linearized plasmids. After two selections with 25 pg/mL MPA and 50 pg/mL XAN, stable parasites were cloned and positive clones were screened by indirect immunofluorescence assay (IF A), whereby three individual clones were selected for further confocal imaging. One of the positive clones was then knocked out for the uracil phosphoribosyl transferase (UPRT) gene using a second plasmid containing TgSORT-GFP as previously described Sloves et al. (2015), whereby the plasmid containing the HA tag was replaced by GFP. All aforementioned plasmids were checked for accuracy by full DNA sequencing before use. Transgenic tachyzoites (5xl0⁶) expressing rhoptry ROPl-mCherry were transfected with 25 pg of linearized promSORT-TgSORT-GFP plasmid and were subsequently transferred to the monolayer HFF for 4h, and were washed with culture medium. This was followed by a 24-hour growth, after which 5 pM of 5’FUDR was added. After three days, selection was repeated twice before cloning the emerging resistant parasites. After screening by IF A, one positive clone was selected for drug screening using the Tocris library as described below.

Toxoplasma strains and cell infection for the miniaturized high-throughput assay

T. gondii RH-AKu80 expressing ROPl-mCherry/TgSORT-GFP (clone D6) tachyzoites were maintained by growth on HFF cell monolayers as previously described, whereby HFF cells at 12 sub-cultured dilution cycles kept in liquid nitrogen prior to their use, and were cultivated in complete DMEM medium after thawing in a 75-cm² flask. Three days later, the cells were trypsinized, resuspended to complete the DMEM medium, and transferred to a 175-cm² flask until confluency had been attained. Prior to infection, the culture medium was removed and HFF cells were washed twice with sterile phosphate buffered saline (PBS) after which 3 mL of trypsin-EDTA was added and the sample was incubated for 4 minutes at 37 °C. After verifying cell detachment by microscope, 25 mL of growth medium was added and HFF cells were then counted using a TALI Cytometer (Invitrogen) and were infected at MOI (multiplicity of infection) 2.5 with freshly lysed sourced from a 25-cm² flask containing infected HFF cells and filtered through 3 pM membrane (polycarbonate, Whatman). After parasite counting using Malassez hematocytometer, the infected cells were grown for 4 hours at 37°C, and the HFF cell monolayer was washed with PBS, trypsinized, and resuspended with complete culture medium before counting. The contents were diluted to obtain a final concentration of 3.25* 10⁵ cells per mL and these infected cells were immediately used for drug library screening.

Compound screening and image acquisition

Tested compounds were obtained from the Tocriscreen Total library (Cat. No. 2884, Tocris), which contains 1,120 biologically active compounds solubilized in pure DMSO at 10 mM. Compound solutions were stored in specific plates to allow Acoustic Droplet Emission transfers, which were performed using an Echo550® (Labcyte), allowing us to dispense 40 nL of each compound to reach a 10 pM concentration in the 40 pL volume used for the test, with DMSO serving as negative control (1% final concentration in the assay). Initially, assays were performed using pyrimethamine (Pyr) as a positive control (at 0.6 pM and 10 pM concentrations), as this drug is known to efficiently and rapidly kill T. gondii at very low concentrations. After the initial identification, SB 203580 (Cpdl) and SB 208 (Cpd3) were used as positive controls. For compound screening, 40 pL (corresponding to 1.3* 10⁵ cells) of infected-cell suspension was dispensed in each well of 384-well plates containing compounds, which were prepared and kept for a few minutes at room temperature before use. These plates were incubated at 37°C in 5% CO2 and 95% humidity atmosphere for 24 hours before further processing.

Sample fixation and staining

After 24-hour of incubation, infected cells were subjected to drug effects, and the cell culture media and drugs were removed, after which 20 pL of PBS was added to each well using a washer-dispenser device (E1406® from Biotek), whereby 20 pL of 10% formalin solution was placed on top of the PBS. Plates were kept at room temperature for 20 minutes and were washed once with PBS before staining with DAPI prepared at a 2 pg/mL concentration in PBS containing 0.1% of Triton X-100, 100 mM of glycine and 5% fetal bovine serum (FBS). After incubating at room temperature for 20 minutes, plates were washed with 50 pL of PBS, and 40 pL of PBS containing 1% FBS was added to each well, after which the plate was sealed and stored at 4°C until required for image acquisition, performed using an InCell Analyzer 6000® (GE Healthcare). For this purpose, six fields per well were acquired from the Al well and were placed in the P24 well in a horizontal serpentine mode of acquisition with the 60* objective and the following exposure parameters:

Laser wavelength (in nm) Emission Filter (in nm) Exposition time (in ms)

405 455 200

488 525 500

561 605 200

Images from all three channels (hereafter denoted as blue, green and red, respectively) were captured in confocal mode, using the closed aperture option in order to reduce background noise.

Image analysis

Images captured by the InCell Analyzer 6000® were analyzed using Columbus software. Both cell- and parasite-based analyses were performed, and were supplemented by vacuole structure and organization assessments in certain cases. First, a local maxima detection algorithm was applied to the DAPI channel to detect the nuclei, which were then thresholded by size, roundness and DAPI mean intensity to avoid any false detection (as parasite vacuoles could be misidentified as nuclei). Next, cytosols were detected based on the DAPI background signal in this region, after which a spot detection on the DAPI channel was processed to detect parasites in the entire cell layer. Based on this parameter, parasite count per field or per HFF cell monolayer was obtained. In parallel, as parasites were gathered in vacuoles, the vacuole population was split into positive and negative groups. Given that positive vacuoles were considered to express the expected mislocalisation phenotype, they were selected using a multiparameter threshold, including the ratio of ROPl-mCherry intensity between the center and the border of the vacuole and the mean mCherry intensity in the vacuole. To be labelled as positive, vacuoles had to fulfill the following two criteria: (1) the ratio of intensity between the center and the border of the vacuole must exceed 1.4, and (2) the mean intensity in the vacuole must be below 500. The percentage of positive vacuoles was thus used as a parameter that reflects the vacuole disruption phenotype. The data were normalized using Z-score.

Indirect immunofluorescence using confocal microscopy Intracellular parasites were fixed with 4% paraformaldehyde prepared in PBS for 30 minutes at room temperature, and were washed three times with PBS before staining with DAPI. Samples were observed with a Zeiss confocal microscope and the obtained images were processed using Image J software. When antibodies were utilized, the samples were permeabilized with 0.2% Triton X-100 prepared in PBS with 100 mM of glycine to block free aldehyde groups, incubations were done for 30 min at room temperature all along the experiment. After blocking with 10% of FBS in PBS containing 0.1 % Triton X-100, monoclonal or polyclonal antibodies were added at 1 :500 dilutions and incubated at 37°C for 30 min. After three washes, Alexa 488 nm or 560 nm secondary antibodies were added to the same buffer with DAPI and blue Evans, and after incubation at 37°C for 30 min, and three washes with PBS containing 0.1% of Triton X-100, the coverslips were mounted with Mowiol and dried at 37°C before confocal microscopy observations.

P-galactosidase assay

Purified tachyzoites (2/ I 0⁵ parasites) of the wild type T. gondii RH (clone 2F1) strain were used to infect confluent HFF cell monolayer in 24 cm²-well plates for 4 hours and, after washing once with culture medium, the infected cells were incubated with compounds at different concentrations for 48 hours. After recovery by scraping, materials were centrifuged at 5,000 rpm for 10 minutes at 4 °C. The pellets were washed once with PBS and lysed for 60 minutes at 50°C in 150 pL of a buffer containing of 1% Triton X-100, 5 mM DTT, 1 mM MgSCh and 100 mM HEPES as described previously (Seeber and Boothroyd, 1996; McFadden et al., 1997). After centrifugation at 10,000 rpm for 5 minutes at 4°C, 50 pL of the supernatant was diluted to 50% with lysis buffer. After adding 100 pL of 2 mM chlorophenol red-P-D- galactopyranoside (CPRG), the resulting mixtures were incubated at 30°C overnight and the optical density was measured at k = 570 nm.

Plaque formation

Plaque assays were performed using 24-well plates containing at least 5 day-confluent HFF cells infected with 10³ parasites per well in media, with DMSO as negative control, or in DMSO combined with drugs at 25 pM or 50 pM concentration. Nine days post-infection, the controls and infected HFF cells were stained with crystal violet, as previously described (Sloves et al., 2012; Sangare et al., 2016). Two independent experiments were performed with identical results. In vitro growth of P. falciparum

P. falciparum 3D7 strain was used at the Institut de Recherche et de Developpement (IRD- Benin) to obtain parasite cultures from group 0+ red blood cells (RBCs) of healthy adult volunteers. RBCs were washed twice in PBS IX and then in complete malaria culture medium (CMCM) composed of RPMI 1640 (PAN Biotech, Dutscher, France), 0.8% Albumax, 25 mM HEPES, 0.4 mM hypoxanthine, 0.05 mg/mL gentamicin and 2 mM L-glutamine before storage at 4°C. RBCs infected with P. falciparum 3D7 were grown at 5% hematocrit at 37°C in an atmosphere comprising 5% CO2, 1% O2 and 94% N2. The culture medium was renewed daily and samples were checked for evidence of parasitemia. Prior to drug testing, the infected RBCs were synchronized with 5% sorbitol for 15 minutes at room temperature, and a second synchronization was performed after 48 hours to eliminate persistent mature forms. Compounds were then tested in 96-well flat-bottom plates at a hematocrit of 5% and an initial ring parasitemia of 1% in 250 pL of CMCM. The following concentrations were tested: 5 pM, 25 pM, 50 pM, 100 pM and 150 pM drugs solubilized by DMSO and prepared in CMCM. The experiments were done in quadruplicate and DMSO was used as the negative control. Chloroquine, a well-known anti-plasmodial drug used at 0.005-150 pM served as the positive control. Plates were incubated for 48 hours at 37°C under the same conditions. Thin smears were created by spreading 6 pL of parasitized RBCs (pRBCs) on a glass slide, which was fixed and stained using the RAL 555 kit before using the AxioCam MRc (color) CCD Rev3 camera of ZEISS microscope at X63 to observe and photograph the RBC smears and process the data using ImageJ software.

Flow cytometry analysis

Both untreated and drug-treated infected RBCs (2.5 pL) were collected from each well and were incubated at 37°C for 60 minutes in a Hoechst 33258-Thiazole Orange mixture at 0.001 mg/mL and 0.005 mg/mL final concentrations, respectively (Grimberg et al., 2008). Samples were analyzed using BD FACS Canto flow cytometer, whereby FlowJO was used for data analysis and statistical analyses were conducted using GraphPad Prism.

Statistical analyses

Statistical analyses involved two-way ANOVA followed by Tukey’s multiple comparisons test, which were performed using GraphPad Prism version 8.3.0 (GraphPad Software, San Diego, California USA), with p < 0.05 indicating statistical significance. The results are reported as mean of parasite growth percentage ± SD as a function of the logarithm of inhibitor concentration.

Results

Generation of transgenic T. gondii TgSORT-GFP/ROPl-mCherry

With the aim of screening small molecules on a large scale, we created a transgenic T. gondii strain that simultaneously expresses ROPl-mCherry (red fluorescent signal) and TgSORT-GFP (green fluorescent signal) as schematically depicted in Figure 1A and 1C. For this purpose, we chromosomally appended an encoded mCherry to the rhoptry protein 1 (R0P1) using the plasmid mCherry -LIC-HXGPRT (as explained in the Materials and Methods section and shown in Figure 1A). This knock-in strategy was adopted, as it ensures steady-state levels of epitopetagged protein expression via homologous promoters. As indicated in Figure IB, it resulted in a clonal T. gondii strain expressing R0P1 -mCherry at the apical end of intracellular dividing tachyzoites. Next, we transfected a second vector that expresses TgSORT-GFP, thereby disrupting the uracil phosphoribosyl transferase (UPRT) gene by double homologous recombination and conferring the resistance to 5’FUDR (Figure 1C). The UPRT gene was chosen because it is non-essential and its knockout has no phenotypic consequences for the parasite, allowing it to salvage uracil from the infected host cells (Fox and Bzik, 2002).

As shown in Figure ID, we also selected a positive clone for TgSORT-GFP (green signal) in the background of the transgenic T. gondii already expressing ROP1 -mCherry (red signal). These intracellular dividing transgenic tachyzoites of T. gondii simultaneously exhibited TgSORT-GFP in the post-Golgi/endosomal-like compartment and ROPl-mCherry at the apical end of the parasites.

Image-based miniaturized assay

For the assay, HFF cells were first infected with a pre-culture of T. gondii expressing both TgSORT-GFP and ROPl-mCherry, as shown in Figure 2 depicting assay workflow. The infected cells were subsequently harvested onto a compound containing 384-well plates and were incubated for 24 hours, after which the samples were fixed and stained with nuclear label - DAPI. Next, confocal images were acquired using an automated microscope and well-based analysis was performed using Columbus software (Figure 3). To establish the assay, the number of HFF cells, the multiplicity of infection (MOI), and the DMSO impact have to be determined to ensure a homogenous T. gondii-m cXe HFF layer in each well at read-out. We have chosen pyrimethamine (Pyr) for this purpose, as this well-known drug is frequently used in toxoplasmosis treatment (Hill and Dubey, 2002; Halonen and Weiss, 2013; Dunay et al., 2018) and is an effective parasite growth inhibitor in assays. A dose response of Pyr was determined and the number of parasites per field was quantified. For DMSO, 18 parasites were noted on average per field, declining to 5 in the presence of 0.6 pM Pyr. Thus, an ECso of 0.6 pM could be inferred (Figure 4A), concurring with the previously reported values (Touquet et al., 2018).

Small molecule screen for identifying known compounds that inhibit T. gondii intracellular growth

As a part of the present study, a pre-screen optimization was also performed by filling two 384- well plates with two Pyr doses (0.6 pM and 10 pM) in a checkerboard style (Figure 4A). For the DMSO negative control, 20 parasites were noted on average per HFF cell, declining to about 2 in cells containing 10 pM Pyr (Figure 4B). Next, the first 384-well plate from the Tocris library was analyzed under the conditions described above. For this purpose, we generated a data processing script utilizing the signal emitted by the ROPl-mCherry and correlating it with the mislocalisation of the rhoptry signal. We first tested 300 compounds of Tocris library and the initial results pertaining to the wells corresponding to a compound yielding positive results were visually validated to concur with the findings related to the rhoptry marker mislocalisation. For the DMSO negative control, the rhoptry fluorescence signal was in the apical form of very distinct spots, while it was largely diffuse for the SB 239063 (Cpd2) and SB 208 (Cpd3) compounds, which correlated with a disruption phenotype. Cpd2 and Cpd3 were identified as p38-MAP kinase and TGF-P type I receptor inhibitors, which were reported to reduce T. gondii growth by several authors (Wei et al., 2002; Wiley et al., 2010), indicating that our screen is robust. Thus, Cpd2 and Cpd3 were used as positive controls for the automatic screening of the 900 remaining small molecules distributed in three additional 384-well plates, as shown in Figure 5. For the image analysis of these plates, the percentage of positive vacuoles that correlate with the rhoptry disruption phenotype was chosen as the key parameter, and the corresponding data plot is shown in Figure 6. These automatic image-based screenings identified nine further compounds that exhibited the expected phenotypic disruption fluorescence, as demonstrated in Figure 7. Unlike the negative DMSO control showing viable well-shaped intracellular tachyzoites with distinct green and red signal, these drug-treated wells contained necrotized intracellular parasites. When our visual and automatic screening methods were combined, 12 such compounds were identified, and their chemical structures are shown in Figure 8, revealing inhibitors of one CB2 receptor, two ATP-competitive TGF-PRI, one Cav3.x pump and one estrogen receptor. The remaining seven compounds are kinase inhibitors, five of which inhibit p38-MAP kinase, and two inhibit ERK and Src family kinases, respectively (Table 1).

Dose-dependent inhibitory effects of the small molecules on T gondii growth

The inhibitory activity of the compounds identified through image-based screening was validated by another enzymatic method and the findings revealed a highly significant difference (p < 0.0001) in parasite growth depending on the inhibitor presence and concentration used, reflecting an important effect of inhibitors on the intracellular replication of T. gondii using P- galactosidase assays (Figure 9A and 9B). As compound 5 failed to produce inhibitory effect (Figure 9A), it was excluded from the validation process. When the positive compounds were placed in groups of three, their joint IC50 decreased from 15-60 pM to 1.5-2.5 pM, and the concentrations were below the IC50 of each individual compound (Figure 9C). Nonetheless, the difference observed between the results obtained by applying inhibitor mixes to T. gondii and the untreated parasites was significant (p = 0.0024), indicating beneficial synergistic effect. Moreover, when infected confluent HFF cells were subjected to drug inhibition for nine days to allow plaque formation defines by several rounds of invasion, egress and reinvasion, parasite growth was completely abolished for all six compounds tested , i.e., Cpd2, Cpd3, Cpd4, Cpd7, CpdlO and Cpdl l (Figure 9D). Even at high concentrations (25 and 50 pM), the confluent monolayer of HFF cells remained intact, as demonstrated by homogenous crystal violet staining at these doses. These results confirm that these drugs are not detrimental to human fibroblasts under the experimental conditions employed in the present study.

Effect of the identified compounds on P. falciparum

To test the inhibitory effects of eight compounds identified during our image-based screen on P. falciparum growth, we used infected red blood cells (RBCs) synchronized twice with sorbitol and subjected them to flow cytometry analysis. The findings indicated that four compounds (Cpd3, Cpd4, CpdlO and Cpdl 1) were very active against P falciparum and the infected RBC cultures were cleared of ring stages in a manner similar to the conventional anti-plasmodia drug chloroquine (Figure 10A). Moreover, increasing inhibitor concentrations resulted in a significant (p < 0.0001) decrease in P. falciparum growth in RBCs. When trophozoite and schizonte forms of P falciparum were counted by flow cytometry (Figure 10B and 10C), identical inhibitory effect of these four compounds was affirmed. The remaining four compounds inhibited P falciparum growth, but their activity was below that noted for Cpd3, Cpd4, CpdlO and Cpdl l (Figures 10A-10C). In all cases, inhibitory activity and absence of ring stage in the infected RBCs were confirmed by direct observation of stained red blood smears, as shown in Figure 10D. Only the negative DMSO controls exhibited numerous ring stages, confirming that these compounds are capable of directly targeting parasites, since P. falciparum was grown in non-nucleated red blood cells characterized by reduced metabolism and lack of intracellular organelles.

Discussion:

T. gondii and P. falciparum share evolutionary history, as both contain orthologous proteins often associated with similar cellular processes and likely exhibit similar sensitivity to inhibitors that affect their unique life stage events. Guided by these findings, we developed a powerful and efficient image-based screening protocol involving transgenic T. gondii simultaneously expressing TgSORT-GFP (green signal) and ROPl-mCherry (red signal) at two distinct subcellular locations, which can be applied to the extant library of 1,120 compounds. Our aim was to identify drugs capable of disrupting this distinct subcellular localization of the green and red signals, thereby compromising parasite’s polarity, morphology and intracellular replication. For this purpose, we employed experimental conditions that would allow us to identify compounds that not only inhibit but also disrupt parasite growth and its classical apical rhoptry signal. Our findings revealed that, in addition to the DAPI signal enabling detection of both HFF and parasite nuclei, the red signal was sufficient to identify the disruption phenotype during the screening process. This protocol was thus adopted in automated image analysis, which resulted in the identification of seven kinase inhibitors that mostly inhibited p38-MAP kinase, and two of which — Cpd2 (SB 203580) and Cpd3 (SB 208) — were previously reported to reduce T. gondii growth (Wei et al., 2002; Wiley et al., 2010). It should be emphasized that the compound library used in our analyses was used by Ditmar et al. (2016) to assess inhibition of T. gondii growth based on P-galactosidase assay and these authors identified 94 compounds and a significant number of small molecules known to impact dopamine or estrogen signaling. One of the compounds identified in our study (Cpdl2, ZK 164015) is an estrogen receptor antagonist. It is also worth noting that Touquet et al. (2018) developed an automated imagebased strategy for screening a library of compounds belonging to four classes of either natural compounds or synthetic derivatives. Next, we established the kinetics of T. gondii death for the identified 12 active compounds using enzymatic assay based on the P-galactosidase activity. We further confirmed that some of these compounds are also capable of inhibiting or eliminating P falciparum grown in red blood cells. Interestingly, four of these compounds (potent ATP-competitive TGF-PRI inhibitor, CB2 receptor inverse agonist, p38-MAPK and Src family kinase inhibitor) were at micromolar potency, as established using flow cytometry method. These observations suggest that the effects of these compounds may be specific to intracellular parasites, which develop in nucleated mammalian cells. It is interesting to note that our screen revealed several kinase inhibitors, which is relevant given that both P. falciparum and T. gondii contain a large number of proteins that are phosphorylated through action of several kinases, including tyrosine kinases (Treeck et al., 2011). The secreted proteins included an expanded, lineage-specific family of protein kinases termed rhoptry kinases (ROPKs), several of which have been shown to be key virulence factors. For example, ROP16 targets the nucleus and phosphorylates STAT3/6, and regulates the immune responses (Saeij et al., 2006; Taylor et al., 2006; Saeij et al., 2007; Yamamoto et al., 2009; Ong et al., 2010; Butcher et al., 2011). Thus, it is likely that the kinase inhibitors identified during our screen may have some of these parasite kinases as targets. Consequently, the identified compounds may be useful for clinical trials, as repurposing drugs with known safety profiles presents attractive and significant advantages in terms of drug development, including safety, toxicity testing and clinical evaluation. Although p38-MAP kinase inhibitors appear to be toxic, as indicated by adverse effects during previous clinical trials (Patnaik et al., 2016), other drugs (unrelated to kinase inhibitors) identified as a part of the present study can be considered. Based on the obtained findings, we postulate that a combination of drugs acting via multiple effector mechanisms might be superior to a single drug or a group of drugs with the same mechanism of action, because developing parasite resistance against multiple effector mechanisms simultaneously would be extremely hard to achieve. Further work on target identification and mechanism analysis is thus required to facilitate the development of anti-parasitic compounds with cross-species efficacy. Establishing the linkages between unique chemical scaffolds and their resultant cellular phenotypes on two evolutionarily related yet distinct parasites will provide an avenue for conducting detailed mechanistic studies with the organism of choice. In particular, biochemical and/or genetic approaches should be employed to identify the drug targets for further design of new parasite-specific inhibitors. Table 1. Chemical names and known functions of compounds identified.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS: A transgenic apicomplexan parasite that expresses simultaneously i) a first fusion protein wherein a sortilin-like receptor (SORTLR) polypeptide is fused to a first labelling polypeptide (i.e. the “SORTLR fusion protein”) and ii) a second fusion protein wherein a rhoptry polypeptide is fused to a second labelling polypeptide (i.e. the “rhoptry fusion protein”). The transgenic apicomplexan parasite of claim 1 that is a Toxoplasma gondii strain. The transgenic apicomplexan parasite according to claim 1 or 2 wherein the SORTLR polypeptide comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1. The transgenic apicomplexan parasite according to claim 1 or 2 wherein the rhoptry polypeptide comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:2. The method according to any one of claims 1 to 4 wherein the SORTLR polypeptide is fused to a GFP polypeptide. The method according to any one of claims 1 to 4 wherein the Rhoptry polypeptide is fused to a mCherry polypeptide. A method for screening a plurality of test substances for their ability to kill an apicomplexan parasite comprising the steps of i) contacting the plurality of the test substances with a population of cells infected by the transgenic apicomplexan parasite according to any one of claims 1 to 6, ii) detecting the subcellular localisation of the fusion proteins and iii) positively selecting the test substances capable of disrupting the distinct subcellular localization of the rhoptry fusion protein. The method of claim 7 that implements an automated image analysis that comprises the steps of a) subjecting the infected cells to imaging to collect a plurality of images of the individual infected cells present and b) applying an algorithm configured to detect fluorescence emitted by the fusion proteins from said plurality of images, c) detecting the subcellular localisation of the fusion proteins and d) positively selecting test substances that induces mislocalisation of the rhoptry fusion protein. A method for treating a disease caused by an apicomplexan parasite in a subject in need thereof comprising administering to the patient a therapeutically effective amount of at least one compound of Table 1 and/or Figure 8.