BR102018009345A2 - METHOD OF IDENTIFICATION AND USE OF THE SERPENTINE RECEIVER PFSR25 - Google Patents

Abstract

method of identification and use of the pfsr25 serpentine receiver. The present invention relates to a method of identifying and using the pfsr25 serpentine receptor to prepare a parasiticidal medicine, for example, for the treatment of malaria. this invention proves that pfsr25 parasites are more sensitive to death induced by chloroquine and to stress induced by the removal of supplementation from the medium than wt (wild-type) cells, as well as showing greater inhibition in the formation of hemozoin after treatment with piperaquine compared to parasites wt.

Description

METHOD OF IDENTIFICATION AND USE OF THE SERPENTINE RECEIVER PFSR25 FIELD OF INVENTION

[001] The present invention falls within the field of biotechnology and describes a method of identification and use of the serpentine receptor PfSR25 to prepare a parasiticidal drug, for example, for the treatment of malaria or the treatment of veterinary diseases such as babesiosis, anaplasmosis and neosporosis, among others.

[002] Additionally, the present invention demonstrates that PfSR25 parasites are more sensitive to piperaquine-induced death and stress induced by the removal of supplementation from the medium than wt (wild) cells, as well as showing greater inhibition of hemozoin formation after piperaquine treatment compared to wt parasites.

FUNDAMENTALS OF THE INVENTION

[003] Malaria is responsible for 2 to 3 million deaths annually. The etiologic agent of the most aggressive form of the disease, Plasmodium falciparum, invades red blood cells and resides within the parasitophorous vacuole, undergoing several changes during its development.

[004] After nuclear division, merozoites (a stage of the cell cycle of P. falciparum) are generated, which, after release into the blood, invade new red blood cells.

[005] In the course of infection, P. falciparum is exposed to different microenvironments with different ionic composition. According to previous studies, the Plasmodium vacuolar membrane is known to have the ability to form a microenvironment around the parasites with a Ca2+ concentration sufficient to allow Ca2+ signaling to persist in the intracellular stage.

[006] In addition to changes in Ca2+ levels during the period of infection in mammals, Plasmodia must also deal with dramatic changes in K+ concentration, ie, from 5 mM, concentration found in the bloodstream, to 140 mM, concentration found in the cytosol of host cells.

[007] Over the past few years, evidence has been presented indicating that changes in K+ concentration across different microenvironments may regulate various parasite functions (Kumar, Garcia et al. 2007, Singh, Alam et al. 2010).

[008] For example, exposure of P. berghei parasites to high concentrations of K+ stimulates cellular invasion of hepatocytes by the rodent malaria sporozoite (Kumar, Garcia et al. 2007). In the same vein, reducing the K+ concentration from 140 mM to 5 mM K+ in P. falciparum merozoites causes an increase in the concentration of Ca2+, leading to micronema secretion and egress of merozoites from the red cell (Singh, Alam et al. 2010) .

[009] The micronema is an important parasite organelle for the invasion process and it has been suggested that the release of its content depends on the increase in the concentration of Ca2+ through the activation of phospholipase C (PLC), but the molecular mechanisms that trigger these effects K+ dependents in Plasmodia remain largely undefined.

[010] In the present invention, it is possible to demonstrate that the activation of PfSR25 (PlasmoDB number = PF3D7_0713400), a putative member of the family of G protein-coupled receptors (GPCRs), which further includes PfSR1, PfSR10, PfSR12 and PfSR25 (identified in Plasmodium), couples changes in K+ levels, such as intracellular mobilization of Ca2+ through PLC activation.

[011] This GPCR, the only one in any type of eukaryotic cell, has been shown to respond to changes in K+ concentration. Thus, PfSR25 parasites are more sensitive to chloroquine-induced death (antimalarial) and stress induced by the removal of supplementation from the medium than wt (wild) cells, as well as showing greater inhibition of hemozoin formation after treatment with piperaquine, in comparison to parasites wt.

[012] Thus, here it is proposed a method of identification and use of the serpentine receptor PfSR25 to prepare a parasiticidal drug, for example, for the treatment of malaria.

[013] SR25 is not present in mammals or humans. The primary sequence alignment of SR25 with other pathogenic organisms revealed the presence of homologous genes to SR25 in organisms such as Babesia bovis, Neospora caninum, Hammondia hammondi, Theileria annulata and Toxoplasma gondii.

[014] The profitability of livestock farming can be greatly reduced by the negative effects of pathogens that affect livestock and, in order to develop sustainable policies that reduce this impact, innovative methodologies and research are needed, such as that involving the effects of resistance to parasiticides for veterinary use.

[015] Thus, babesiosis are diseases caused by protozoa of the genus Babesia, which has 73 species that parasitize different hosts, such as cattle, goats, sheep, dogs and rodents. In Brazil and other Latin American countries, bovine babesiosis is caused by two species, Babesia bovis and B. bigemina, and anaplasmosis is caused by Anaplasma marginale. These diseases are popularly known as Bovine Parasitic Sadness (BPD).

[016] Neosporosis in cattle is a disease caused by the protozoan Neospora caninum. Serological studies from several countries have shown Neospora as the major parasite causing abortions in dairy herds. The economic importance of bovine neosporosis is mainly attributed to the costs associated with abortion, the value of fetuses, artificial insemination and reduced milk production. It is important to emphasize that there is no treatment for Neospora caninum in cattle and the diagnosis can be made by Elisa's test and also by immunofluorescence.

[017] The economic losses resulting from parasitism in cattle make it urgent to discover new targets for the development of veterinary drugs. Thus, SR25 is also a potential candidate in this search, given its fundamental role in the cycle of the parasite Plasmodium falciparum (also belonging to the phylum Apicomplexa as Babesia, Toxoplasma and Neospora).

TECHNICAL STATUS

[018] Some prior art documents investigate the role of PfSR25 in the plasmodium cell cycle through knockout parasites for this receptor and verify potential antimalarial drugs from similarities to known targets, such as calcium-dependent protein kinases.

[019] Among these documents, stand out "Screening of ligands for candidate receptors of seven transmembrane domains in the parasite Plasmodium falciparum" (Budu, 2013), "Research of PfSR25, putative serpentine receptor of Plasmodium falciparum" (Chaves, 2014) and article “In silico prediction of antimalarial drug target candidates” (Ludin et al., 2012).

[020] As previously reported, the malaria parasite Plasmodium falciparum perceives the environment in which it is found and elaborates appropriate cellular responses, which involve protein secretion, cell growth and differentiation.

[021] Factors related to the generation of second messengers and cell signaling effector proteins are described in the literature. However, the role of GPCR-like receptors responsible for the perception of extracellular stimuli in the parasite is a little explored topic.

[022] Thus, the in silico identification of receptors of seven putative transmembrane domains in the genome of P. falciparum, carried out according to the present invention, enabled the exploration of their function.

[023] The theses of Alexandre Budu and Gepoliano Chaves present studies of PfSR25 and the potential role of phosphatases / kinases as proteins modulated by the activation of PfSR25, but the study was conducted, for the most part, in mammalian cells. The exception occurs with the receptor protein expression data, which was demonstrated by Alexandre Budu, in three erythrocytic phases of P. falciparum (ring, trophozoite and schizont).

[024] The work also suggests that KCl modulates cytosolic calcium in P. falciparum and that knockout parasites for PFSR25 are unable to modulate cytosolic calcium in response to KCl. Furthermore, modulation of PfSR25 by KCl is able to activate kinases / phosphatases, leading to the activation of effector molecules. However, in the documents mentioned above, characteristics or information are not presented that allow identifying the use of the microorganism to prepare a drug to treat malaria or parasitic diseases of cattle. Furthermore, the method used in the above mentioned above differs from the present invention due to the fact that buffers with different ionic compositions were used (in particular, in the concentration of K+), thus resulting in the identification of K+ as the agonist of the PfSR25 receptor .

[025] Thus, the present invention shows that PfSR25 is responsible for the survival of the parasite P. falciparum to stress, showing potential use to prepare a drug with parasiticidal action, for example, to treat malaria, once the parasites become more sensitive to drug-induced death and stress induced by removal of supplementation from the medium compared to wild-type cells.

SUMMARY OF THE INVENTION

[026] The invention aims to propose a method of identification and use of the serpentine receptor PfSR25 to prepare a parasiticidal drug, for example, for the treatment of malaria or the treatment of veterinary diseases such as babesiosis, anaplasmosis and neosporosis, among others.

[027] It also demonstrates that the said receptor is a monovalent cation sensor capable of modulating Ca2+ signaling in parasites, thus making them more sensitive to death induced by drugs against malaria, such as chloroquine and piperaquine.

BRIEF DESCRIPTION OF THE FIGURES

[028] In order to obtain a complete and complete visualization of the object of this invention, the figures which are referred to are presented, as follows.

[029] FIG. 1 is the flowchart of the methodology applied to the identification of the PfSR25 receptor and its ligand.

[030] FIG. 2 represents the intraerythrocytic expression of PfSR25 and construction of the knockout lineage, where (A) is the expression of proteins by western blot (R = ring, T = trophozoite, S = schizont and SS = segmented schizont); (B) is IFA (incomplete Freund's adjuvant) of PfSR25 during the intraerythrocytic stages; (C) is the PfSR25 gene knockout strategy; (D) is PCR analysis of the PfSR25 deletion; (E) is southern blot analysis of PfSR25 deletion in 3D7 wt parasites and PfSR25-(clone 26) parasites and (F) is western blot analysis of PfSR25 protein from wt and clone 26 parasites (26) with antibody rabbit anti-PfSR25.

[031] FIG. 3 graphically shows how KCl and PfSR25 are involved in increasing the Ca2+ concentration in late trophozoites isolated from P. falciparum, where (A) shows the cytosolic Ca2+ level detected by flow cytometry in wild-type 3D7 parasites, (B) shows the level of cytosolic Ca2+ detected by flow cytometry in PfSR25- parasites, (C), (D), (E), (F), (G) and (H) are representative histograms of 3D7 wt parasites when transferred from high to low KCl concentration.

[032] FIG. 4 graphically represents (A) the growth curve of P. falciparum wt and PfSR25-, (B) the number of merozoites generated by P. falciparum wt or PfSR25-, (C) the changes in parasite volume (wt or PfSR25- ) after exposure to hyperosmotic buffer and (D) changes in the FSC flow cytometry parameter in P. falciparum parasites incubated in isosmotic buffer (ISO).

[033] FIG. 5 represents: (A) parasite viability, (B) PfMCA1 analysis by quantitative RT-PCR, (C) parasitemia of Wt and PfSR25- parasites when treated with SNP (D) and CQ (E).

[034] FIG. 6 depicts: (A) determination of parasitemia after reintroduction of supplementation after 0 to 6 days, (B) representative histogram of fluorescence intensity in FIG. 6A, (C) the determination of parasitemia after reintroduction of supplementation after 10 days and (D) the graph showing parasitemia during the experiment performed in FIG. 6C.

[035] FIG. 7 represents the percentage inhibition of hemozoin crystal growth, where (A) are the trophozoites of both parasites (wt and PfSR25-) compared to the control and (B) is a light microscope image of the P-infected red cell falciparum (arrow indicates hemozoin crystal).

[036] FIG. 8 graphically depicts the decrease in in vitro parasitemia of P. falciparum in the presence of anti-PfSR25 antibody.

[037] FIG. 9 graphically represents the calcium response in P. falciparum trophozoites.

DETAILED DESCRIPTION OF THE INVENTION

[038] The present invention relates to a method of identification and use of the serpentine receptor PfSR25 to prepare a parasiticidal drug, for example, for the treatment of malaria or the treatment of veterinary diseases such as babesiosis, anaplasmosis and neosporosis, among others.

[039] More specifically, said receptor (characterized by the PlasmoDB number = PF3D7_0713400), which is a putative member of the family of G protein-coupled receptors (GPCRs), is a monovalent cation sensor capable of modulating Ca2+ signaling in parasites, thus making them more sensitive to death induced by anti-malarial drugs such as chloroquine and piperaquine.

[040] Changes in KCl concentration from high (140 mM) to low (5.4 mM) trigger an increase in the concentration of Ca2+ (Cytosolic Ca2+) in isolated parasites and this increase in Ca2+ is blocked by inhibition of phospholipase C (PLC ) or by depleting the parasite's internal Ca2+ stocks.

[041] However, when the PfSR25 gene is knocked out, no effect on increasing the concentration of Ca2+ is observed in response to changing the concentration of KCl in the knocked out parasite (PfSR25-).

[042] DNA manipulations were performed following protocols described in the literature (Sambrook, Russell et al. 2006). The SR25 gene DNA fragment was amplified with specific primers by polymerase chain reaction (PCR). The amplicons obtained were purified and cloned into a vector for transfection of Plasmodium pCAM. The PfSR25/pCAM constructs were used to transfect P. falciparum 3D7 and subsequent selection of positive clones. P. falciparum 3D7 parasites transfected with the PfSr25-pCAM-BSD construct were subjected to several cycles of treatment with the drug blasticidin (2.5 µg/ml) to select the vector inserted parasites every two weeks. Blasticidin-resistant P. falciparum parasites that reappeared in the culture after several weeks were tested by PCR to assess their phenotype to see if integration occurred at the target locus.

• a) Cultura de célula, sincronização e preparação de parasitas isolados de Plasmodium falciparum;
• b) Extração e detecção da proteína por western blot;
• c) Códon-otimização do gene PfSR25 e transfecção de células HEK293;
• d) Medida dos níveis intracelulares de cálcio;
• e) Construção de P. falciparum nocaute para o gene PfSR25.

[043] The method of identification of the serpentine receiver PfSR25 was performed according to the steps and description below, being presented in a summarized form in FIG. 1:

• a) Cell culture, synchronization and preparation of isolated Plasmodium falciparum parasites;
• b) Extraction and detection of the protein by western blot;
• c) Codon-optimization of the PfSR25 gene and transfection of HEK293 cells;
• d) Measurement of intracellular calcium levels;
• e) Construction of P. falciparum knockout for the PfSR25 gene.

[044] In step (a), P. falciparum was kept in culture according to the methodology already described by Trager and Jensen (1976). The parasites were isolated by the saponin method and labeled with the calcium indicator Fluo-4AM, in order to assess the intracellular calcium dynamics (Cruz, Juliano et al. 2012). Red cells infected with P. falciparum at the late trophozoite/young schizont stage were centrifuged (2000 rpm, 5 min) and the 1 ml pellet was washed and isolated with 0.05% saponin in the respective buffer containing high (140 mM) or low (5.4 mM) K+ concentration. Then, the parasites were labeled with 10 µM Fluo4-AM for 30 min at 37°C in high K+ buffers (140 mM KCl, 5.4 mM NaCl, 0.8 MgSO4, 5.5 D-glucose, 50 MOPS, 2 mM CaCl2 pH 7.2); low K+ (5.4 mM KCl, 140 mM NaCl, 0.8 MgSO4, 5.5 D-glucose, 50 MOPS, 2 CaCl2, pH 7.2); K-gluconate (K+-140 mM gluconate, 5 mM Na+-gluconate, 0.8 MgSO4, 5.5 D-glucose, 50 MOPS, 2 mM CaCl2, pH 7.2) and Na+-gluconate (5.4 mM K+-gluconate, 140 mM Na+-gluconate, 0.8 MgSO4, 5.5 D-glucose, 50 MOPS, 2 mM CaCl2, pH 7.2, respectively. The isolated parasites were then washed three times in the respective buffer and 5x107 parasites were transferred to the flask containing high or low K+ before the experiment. For U73122 and U73343, parasites were pretreated for 1 min after labeling with Fluo4-AM. The change in Fluo4-AM fluorescence was measured for 2 minutes in each experiment by flow cytometry (FACSCalibur, Becton Dickinson, USA). Fluo4-AM was excited with a 488 nm argon laser and the fluorescence emission was collected at 520-530 nm. All data were analyzed using FlowJo software. Cytosolic calcium dynamics was also monitored in a Shimadzu spectrofluorophotometer (RF5301PC, Japan) in parasites labeled with Fluo4-AM.

[045] In step (b), the total protein extract of P. falciparum was extracted. To analyze the specific stage of PfSR25 expression, cultures were synchronized with two consecutive incubations in 5% sorbitol and proteins were collected: a) rings were collected 6 hours after synchronization, b) a mixture of trophozoites and young schizonts were collected 24 hours after synchronization, c) a mixture of mature schizonts and rings was collected 36 hours after synchronization.

[046] At each point, the isolated parasites were resuspended in lysis buffer comprising (in mM) 50 Tris-HCl, pH 8.0, 150 NaCl, 5 EDTA and 0.5% Nonidet P40 supplemented with the following mixture of protease inhibitors : 1 mM PMSF, 0.01 mM benzamidine, 10 µg ml-1 aprotinin, 10 µg ml-1 leupeptin, 10 µg ml-1 pepstatin, 10 µg ml-1 chymotastine, 0.1 mM Na3VO4, and 20 µM fluoride sodium.

[047] The particulate matter was removed by centrifugation at 14000 g for 15 minutes and the proteins were separated in 10% SDS-PAGE under reducing conditions and transferred to PVDF (polyvinylidene difluoride) membrane. The membrane was blocked for 1 hour in 5% milk / TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20) and incubated with specific antibodies at a 1:2000 dilution in 5% milk / TBS -T overnight. After extensive washes, blots were incubated with 1:10000 anti-rabbit antibody conjugated to HRP for 1 hour, washed and visualized using Plus ECL.

[048] The extracted proteins were used in the detection of PfSR25 by western blot. Anti-PfSR25 polyclonal antibodies were used in the experiment, in which the presence of a specific band for the PfSR25 receptor, of approximately 43 kDa, was observed, a size corresponding to the prediction for this protein. Furthermore, it is clear from the western blot that PfSR25 is expressed throughout the erythrocytic cycle (as seen in FIG. 2). Proteins were separated on 10% SDS-PAGE gels under reducing conditions, transferred to nitrocellulose and blocked with 5% powdered milk. Polyclonal antibody specific for PfSR25 was added to the membrane (epitope sequence GTGEVKW) at a 1:2000 dilution. After washes, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (GE Healthcare) at a dilution of 1:10000 for 1 h. Again membranes were washed and bands visualized using Plus ECL (GE Healthcare).

[049] In step (c), for heterologous expression in mammalian cells, the gene encoding the PfSR25 receptor was synthesized in a codon-optimized version. For this, the codons of the original gene (rich in adenine and thymine) were modified (optimized) for their better expression in mammalian cells. This codon-optimization was performed by the company GenScript, using a specific algorithm. However, other commercially available software can be used. The plasmids containing the codon-optimized gene were used in the transfection of HEK293 cells, followed by experiments for the analysis of calcium dynamics, 48 hours after transfection. For these experiments, the transfected HEK293 cells were transferred to a 96-well plate where they grew for 48 hours. Afterwards, the cells were labeled with Ca2+ indicator and exposed to several compounds and drugs and the Ca2+ dynamic was measured.

[050] In step (d), HEK293 cells transfected with the PfSR25 gene were subjected to high-throughput screening (high throughput screening) to identify possible PfSR25 ligands. For this, several compounds were tested, such as: adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), xanthurenic acid, melatonin, cAMP, n-acetyl cysteine, serotonin and NaCl. identify whether PfSR25 could be activated by ions. As previously described, the PfSR25 receptor was synthesized in a codon-optimized version and used in the transfection of HEK293 cells, followed by experiments for the analysis of calcium dynamics, 48 hours after transfection (Table 1).

[051] After extensive analysis, it was concluded that PfSR25 was activated by K+, and the Ca2+ response was not activated by other ions. Thus, PfSR25 became the first K+ receptor identified to date. After identification of the SR25 ligand, the Ca2+ to K+ response in P. falciparum labeled with Fluo-4AM was analyzed. When exposed to changes in the K+ level in the medium, P. falciparum responded with an intracellular increase in K+, thus indicating the presence of a receptor for this ion in the parasite.

[052] In step (e), in order to confirm the results obtained previously, a transgenic version of the P. falciparum parasite in which the pfsr25 gene was deleted was constructed. The exposure of P. falciparum SR25 knockout parasites to buffers with different K+ concentrations (140mM and 5 mM KCl) was not able to generate a Ca2+ response in the parasite, thus confirming that the PfSR25 receptor is responsive to the K+ ion ( as can be seen in Fig. 3). For this Parasites synchronized in the trophozoite stage respond to KCl in a dose-dependent manner, a response that is maintained even when the extracellular Ca2+ was chelated with 2 mM EGTA. In addition, experiments performed in the presence of 5 μM thapsigargin or 10 μM cyclopiazonic acid in order to induce the release of Ca2+ from internal parasite stores led to inhibition of the response to KCl, thus indicating that internal calcium stores present an important role as a positive regulator in the response triggered by KCl, an observation confirmed by the inhibition of PLC activity with 1 μM U73122. The addition of its inactive analogue, U73343 did not alter the response to KCl. On the other hand, KCl did not promote an increase in Ca2+ in trophozoites isolated from parasites that knocked out pfsr25, however, the addition of thapsigargin after KCl demonstrated the existence of calcium in the internal stores capable of being mobilized.

[053] Thus, when we incubated the P. falciparum PfSR25 knockout parasites and the wild parasites to the antimalarial piperaquine, we observed that the knockout parasites are more susceptible to the presence of the drug.

[054] This fact, of extreme medical importance, indicates that PfSR25 has a role in the performance of drugs against malaria and, more importantly, can be used as a target in the development of new strategies for the eradication of malaria.

[055] The tests below were performed according to protocols known from the state of the art and prove the activity of the serpentine receptor PfSR25 in the treatment of malaria or the treatment of veterinary diseases such as babesiosis, anaplasmosis and neosporosis, among others.

- PfSR25 expression during the intraerythrocytic cycle of P. falciparum

[056] By bioinformatics analysis four potential GPCRs were identified in the genome of Plasmodium falciparum and antibodies against these proteins were generated. The sequences of these receptors have the 7 transmembrane domains characteristic of these receptors that are responsible for their anchoring in the membrane. In particular, Western blot analysis with a specific anti-PfSR25 polyclonal antibody revealed the expression of an approximately 43 kDa band (consistent with the predicted molecular mass of PfSR25) in the intraerythrocytic stages of the parasite (R, T and S).

[057] Furthermore, this band was more intense in more mature parasites, that is, in the T and S stages, and very weak in the R stage (see FIG. 2A). Analysis of immunofluorescence images at different points in the cycle revealed the presence of PfSR25 in R and T stage parasites with a diffuse labeling pattern. In order to analyze the expression of PfSR25 throughout the intraerythrocytic cycle, P. falciparum parasites synchronized in different phases were isolated and total protein was collected and western blot experiments were carried out. The PfSR25 protein was detected at the height of 43kDa and its expression can be observed throughout the cycle, being more abundant in the trophozoite and schizont phases.

[058] Interestingly, a particularly intense marking has been detected in schizonts, ie, the stage prior to the parasite's exit from the red cell. Finally, PfSR25 was clearly detected in the invasive form of the parasite, ie, the merozoite. Co-localization with the surface protein of Plasmodium MSP-1 reveals the expression of SR25 on the parasite membrane (see FIG. 2B).

[059] To investigate the functional role of PfSR25 in P. falciparum, knockout parasite clones were generated and characterized (see FIGs. 2C-F). Gene disruption was verified by PCR (see FIG. 2D), Southern blot (see FIG. 2E) and protein expression by Western blot (see FIG. 2F). As expected, neither the SR25 gene nor the protein was detected in the PfSR25- samples.

[060] P. falciparum 3D7 parasites transfected with the PfSr25-pCAM-BSD construct were subjected to several cycles of treatment with the drug blasticidin (2.5 µg/ml) to select the parasites with the vector inserted every two weeks. Blasticidin-resistant P. falciparum parasites that reappeared in the culture after several weeks were tested by PCR to assess their phenotype to see if integration occurred at the target locus. First, genomic DNA from cultures of 3D7 parasites (control) and transfected culture (PfSr25) resistant to blasticidin were isolated as previously described and submitted to PCR with specific primers designed for this purpose. Then, the cultures with transfected parasites were subjected to limiting dilution in 96-well plates (0.25 to 1 parasite/well). The initial scan consisted of detecting in which wells there were parasites and, accordingly, the protocol described by Khoma et al 2007 was followed. For this purpose, the cultures present in the wells were analyzed using the LDH assay, using two solutions to detect and measure the activity of the LDH enzyme, the first solution is Malstat composed of Triton X-100, L-lactate, Tris and 3-acetylpyridine adenine dinucleotide (APAD) and the second, the NBT/PES reagent prepared with nitro-blue tetrazolium and phenazine ethosulfate. Then, 100 μl of Malstat solution and 25 μl of NBT/PES solution were added in another 96-well plate and, finally, 15 μl of the minicultures were added, starting the lactate dehydrogenase reaction. The color development of the LDH reaction on the plate was monitored colorimetrically at 620nm with a plate reader after a one hour light-free incubation. In addition to PCR and Southern blot, we also performed Western blot and immunofluorescence experiments in order to confirm the knockout for the PfSR25 protein. From FIG. 2F we can observe that these clones do not present the band corresponding to PfSR25, which can be observed in wild 3D7 parasites, thus confirming the knockout of PfSR25, similar information can be obtained when observing immunofluorescence images for these clones.

- Changes in KCl concentration and Ca2+ signaling in P. falciparum

[061] In order to discover the serpentine receptor agonist PfSR25, a high-throughput plate fluorimeter assay was performed. For this, knockout parasites of the PfSR25 receptor were constructed and calcium tests with addition of K were carried out.

[062] To functionally characterize the role of PfSR25, the response of wt and PfSR25- parasites to a number of different stimuli was compared, in particular with regard to Ca2+ signaling from parasites in the mature trophozoite/young schizont stage.

[063] The effect on cytosolic Ca2+ concentration by changing the extracellular K+ concentration was observed. For this purpose, the late trophozoite stage parasites were isolated from red blood cells, labeled with the fluorescent Ca2+ indicator Fluo4-AM and incubated in the respective buffers with high K+ / low Na+ (140 mM KCl, 5.4 mM NaCl) and low K+ / high Na+ (5.4 mM KCl, 140 mM NaCl) supplemented with 2 mM CaCl2.

[064] The change in fluorescence was observed when the parasites were transferred from a buffer with high K+ to low K+, but no change was observed in the opposite condition, ie, low K+ to high K+ in wt parasites (see FIG. 3A, Ç). The same protocol was then employed for the PfSR25- parasite, however these parasites did not respond to changes in KCl concentration when switched from a high to low K+ buffer (see FIG. 3B, G).

[065] When PfSR25- parasites were transferred from high to low K+ and treated with 5 μM thapsigargin (THG), an increase in cytosolic Ca2+ concentration was observed, indicating that the internal Ca2+ reserves are normally filled (see FIG. 3H). In this assay, red cells infected with P. falciparum at the late trophozoite/young schizont stage were centrifuged (2000 rpm, 5 min) and the 1 ml pellet was washed and isolated with 0.05% saponin in the respective high-containing buffer (140 mM ) or low (5.4 mM) concentration of K.

[066] The parasites were then transferred to the flask containing high or low K+ before the experiment. For U73122 and U73343, parasites were pretreated for 1 min after labeling with Fluo4-AM. The change in Fluo4-AM fluorescence was measured for 2 minutes in each experiment by flow cytometry.

[067] Next, in order to investigate whether the increase in cytosolic Ca2+ concentration after the change in KCl depends on the influx of Ca2+ from the extracellular medium or on the release of intracellular Ca2+ stores (or both), the same experiment shown in FIG. 3C was performed in buffer without extracellular Ca2+ (and in the presence of 2 mM EGTA).

[068] The KCl-induced increase in cytosolic Ca2+ concentration persisted in these latter conditions, clearly indicating that changes in KCl concentration in the extracellular medium mobilize Ca2+ from intracellular stores (see FIG. 3E). To confirm this conclusion, the parasites were treated with cyclopiazonic acid (CPA, 10 µM) to deplete internal Ca2+ stores and no difference was observed in high or high to low KCl concentration.

[069] Treatment with CPA was sufficient to completely prevent the KCl-induced increase in Ca2+ concentration (see FIG. 3F). In the same vein, the PLC inhibitor U73122 abolished the increase in cytosolic Ca2+ concentration caused by KCl (see FIG. 3D). On the other hand, the inactive analogue U73343 did not modify the cytosolic Ca2+ concentration response to KCl (see FIG. 3A).

[070] The increase in cytosolic Ca2+ concentration does not depend on osmotic effects or on the Cl- ion concentration, since: (i) it is not induced by the addition of an equivalent dose of Na-gluconate (see FIG. 3A); (ii) the effect was indistinguishable using KCl or K-gluconate (see FIG. 3A). The effect is specific for K+, as changes in the concentration of Ca2+ or Mg2+ had no effect on the concentration of cytosolic Ca2+ up to 5 mM.

[071] Furthermore, when increasing the concentration of KCl to 50 mM in parasites incubated in 5 mM of KCl, we still observed an increase in the concentration of cytosolic Ca2+, but, similarly, increases in the concentration of another monovalent cation, Na+ , did not result in an increase in cytosolic Ca2+ concentration (as can be seen in FIG. 9). The dose-response curve using KCl is bell-shaped as it was maximum at about 30 mM and then decreased at higher concentrations.

- PfSR25 expression and parasite volume regulation

[072] We then investigated whether PfSR25- had other functional effects on the parasites. In particular, it appears that: i) the growth rate of PfSR25- parasites in red blood cells is similar to that observed for wt parasites (see FIG. 4A); ii) the number of merozoites produced by schizont is similar between the two parasite strains (see FIG. 4B) and iii) the RBC invasion is not affected in the PfSR25- parasites. These results were based on experiments in which the wild type 3D7 wild strain was compared with the SR25 receptor knockout parasites.

[073] On the other hand, some differences were observed between the wt and PfSR25- parasites in terms of volume regulation when exposed to hyperosmotic buffer. FIG. 4C shows that a decrease in volume was clearly observed after exposure to the hyperosmotic medium in wt parasites, whereas this was largely blocked in PfSR25- parasites. To further explore this result, light scattering characteristics (FSC) were used to quantify cell volume in isosmotic buffer as illustrated in FIG. 4D. Under these conditions, significant differences were observed in direct dispersion between wt and PfSR25- parasites, the volume of mutant parasites being much larger than that of wt parasites.

- Modulation of stress-induced parasite death

[074] To determine the role of PfSR25 in resistance to toxic agents, the parasites P. falciparum, 3D7 wt and PfSR25- synchronized were treated with the NO donor, sodium nitroprusside (SNP) and the parasitemia was determined by flow cytometry .

[075] Exposure of P. falciparum to increasing doses of SNP (0 - 0.50 mM) demonstrated that PfSR25- is more sensitive to SNP toxicity than wt parasites (see FIGs. 5A and E). Giemsa stained smears confirmed the decrease in parasitemia after SNP treatment (see supplementary FIGs. 4A and B).

[076] Chloroquine has been shown to be able to stimulate NO synthesis in human monocytes as well as in endothelial cells. The parasitemia observed in PfSR25- after exposure to chloroquine for 24 hours was consistently less than that produced by wt parasites (see FIGs. 5C and E), an observation confirmed by giemsa-stained smears indicating decreased parasite viability.

[077] Reduced parasitemia may result from decreased cell proliferation and/or increased cell death. Furthermore, analysis of metacaspase levels after treating the parasites for 3 hours with 0.5 mM SNP indicated that the PfSR25- parasites showed a 72% increase in metacaspase expression compared to the wt parasites (see FIG. 5B).

[078] To demonstrate that the action of the SNP in P. falciparum was actually due to the release of NO and not the presence of other metabolic products, we observed the ability of other NO donors, at a similar concentration, to induce cell death in P. . falciparum.

[079] The effects of nitrosothiol derivative (SNAP) - NO donor - on P. falciparum wt and PfSR25- were shown in FIG. 5A. Treatment of parasites with SNAP, at micromolar concentrations, decreased parasitemia by 80% and 59%, respectively, in KO parasites compared to wt parasites, confirming the data obtained using SNP. These findings suggest that PfSR25 may influence the parasite's susceptibility to SNP toxicity (as seen in FIG. 5).

[080] Nicotinamide adenine dinucleotide phosphate (NADPH) is a critical product of the glucose-6-phosphate dehydrogenase (G6PD) pathway and is of central importance for the regulation of the antioxidant system. Many studies have shown that G6PD deficiency makes cells extremely sensitive to oxidative stress. Therefore, we were interested in studying the impact of PfSR25 on G6PD activity. G6PD activity was measured in samples of wt and PfSR25- parasites.

[081] G6PDH activity levels were measured per the G6PD Assay Kit manufacturer's instructions (ab102529, Abcam, Cambridge, MA, USA) using trophozoite stage parasites (hematocrit: 2.5%, parasitemia: 5%) The basal level of G6PD activity in PfSR25- was comparable to wt parasites, indicating that PfSR25 parasites respond under basal conditions with the same degree of NADPH production as wt parasites (see FIG. 5C).

[082] In order to better understand how PfSR25- parasites manage to survive under stressful conditions, we measured the growth of PfSR25- and wt parasites after supplementation deprivation. For these experiments, the PfSR25- and wt parasites were synchronized and maintained in medium with supplementation for 24 h and then subjected to medium without supplementation for 72 h (without changing the medium during this interval). To assess whether all remaining parasites were in fact dead, the percentages of parasites that continued to proliferate after returning to supplemented conditions were analyzed. The parasites were transferred back to the supplemented medium for another 72 h. The medium was not renewed during the 72 hour incubation. We observed that parasitemia was significantly lower in the PfSR25- parasites after being exposed to these stress conditions compared to the wt parasites (see FIGs. 6A-B and FIG. 5D).

[083] However, parasite survival after supplementation deprivation was even better when the medium was changed daily, although the observed difference in parasitemia between PfSR25- and wt was still maintained (see FIGs. 6C-D).

[084] The direct role of SR25, when treated with an antimalarial drug, would provide a better perspective, as its function is still unclear. We treated both PfSR25- and wt trophozoites (32-34 hours after infection) with piperaquine (PQ) for 2 hours and measured hemozoin formation. Interestingly, we found that the PfSR25- parasites were very susceptible to PQ treatment and, after 2 hours, the hemozoin size was inhibited by approximately 69.9 ± 2.1 % (see FIG. 7).

[085] To confirm that PfSR25 is important in the invasion of erythrocytes by the parasite, P. falciparum-infected red cells were incubated for 72 hours in medium containing different concentrations of anti-PfSR25 antibody. As can be seen, at the highest antibody concentrations, there was a decrease in parasite growth (see FIG. 8).

[086] This fact shows that PfSR25 has a fundamental role in the asexual cycle of the parasite, and can thus be a target in the development of new drugs.

[087] Thus, the use of this receptor is shown as an alternative to prepare a drug with parasiticidal activity, such as antimalarials and veterinary drugs, in addition to presenting potential application to improve the effectiveness of drugs and reduce the toxicity of treatment.

[088] The profitability of the livestock activity can be increased if the issue of the infectivity of cattle with fatal pathogens is remedied. Thus, SR25 appears as a potential candidate in this search for new pharmaceutical alternatives, given its fundamental role in the cycle of the parasite Plasmodium falciparum (which also belongs to the phylum Apicomplexa as Babesia, Toxoplasma and Neospora).

[089] Those skilled in the art will value the knowledge presented herein and may reproduce the invention in the presented modalities and in other variants, covered by the scope of the appended claims.

References

[090] Cruz, L.N., M.A. Juliano, A. Budu, L. Juliano, A.A. Holder, M.J. Blackman and C.R. Garcia (2012). "Extracellular ATP triggers proteolysis and cytosolic Ca(2)(+) rise in Plasmodium berghei and Plasmodium yoelii malaria parasites." Malar J 11: 69.

[091] Kumar, K.A., C.R. Garcia, V.R. Chandran, N. Van Rooijen, Y. Zhou, E. Winzeler and V. Nussenzweig (2007). "Exposure of Plasmodium sporozoites to the intracellular concentration of enhances infectivity and reduces cell passage activity." Mol Biochem Parasitol 156(1): 32-40.

[092] Sambrook, J., D.W. Russell and J. Sambrook (2006). The condensed protocols from Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press.

[093] Singh, S., M.M. Alam, I. Pal-Bhowmick, J.A. Brzostowski and C.E. Chitnis (2010). "Distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites." PLoS Pathhog 6(2): e1000746.

Claims (9)

Method of identification of the PfSR25 serpentine receiver, comprising the step of:

• a) Cell culture, synchronization and preparation of isolated Plasmodium falciparum parasites; characterized by the fact that it still understands the steps of:
• b) Protein extraction and detection by western blot using anti-PfSR25 polyclonal antibodies;
• c) Codon-optimization of the PfSR25 gene and transfection of HEK293 cells;
• d) Measurement of intracellular calcium levels;
• e) Construction of P. falciparum knockout for the PfSR25 gene.

Method according to claim 1, characterized in that, in step (b), for preparing the blots, the cultures are synchronized and the proteins are collected, resuspended in lysis buffer, centrifuged and incubated with specific antibodies. Method according to claim 2, characterized in that rings are collected 6 h after synchronization, trophozoites and young schizonts are collected 24 h after synchronization, and mature schizonts and rings are collected 36 h after synchronization. Method, according to any one of claims 1 and 3, characterized in that it presents a specific band of 43 kDa for the PfSR25 receptor. Method according to claim 1, characterized in that, in step (c), the experiments for the analysis of calcium dynamics are carried out 48 hours after transfection in HEK293 cells. Method according to claim 1, characterized in that, in step (d), the measurement of intracellular calcium levels is performed by high-throughput screening. Method according to claim 1, characterized in that, in step (e), the P. falciparum SR25 knockout parasites are exposed to buffers with 5 Mm and 140 Mm of KCl. Use of the serpentine receptor PfSR25, identified by the PlasmoDB number PF3D7_0713400, characterized by the fact that it is in the preparation of a drug for the treatment of malaria or the treatment of veterinary diseases. Use according to claim 7, characterized in that veterinary diseases are mainly babesiosis, anaplasmosis and neosporosis.

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