Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.

Altiratinib blocks Toxoplasma gondii and Plasmodium falciparum development by selectively targeting a spliceosome kinase.

The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include Toxoplasma gondii, Plasmodium, and Cryptosporidium spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against T. gondii. We have identified TgPRP4K of T. gondii as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for TgPRP4K and for its Plasmodium falciparum counterpart, PfCLK3. Our data identified structural features critical for binding of the other PfCLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum. Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development.

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional 'single target-single drug' approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3'-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC50 and EC50 values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.

A brain cyst load-associated antigen is a Toxoplasma gondii biomarker for serodetection of persistent parasites and chronic infection.

BACKGROUND: Biomarker discovery remains a major challenge for predictive medicine, in particular, in the context of chronic diseases. This is true for the widespread protozoan Toxoplasma gondii which establishes long-lasting parasitism in metazoans, humans included. This microbe successively unfolds distinct genetic programs that direct the transition from high to low replicative potential inside host cells. As a slow-replicating cell, the T. gondii bradyzoite developmental stage persists enclosed in a cyst compartment within tissues including the nervous system, being held by a sustained immune equilibrium which accounts for the prolonged clinically silent phase of parasitism. Serological surveys indicate that nearly one third of the human population has been exposed to T. gondii and possibly host bradyzoites. Because any disruption of the immune balance drives the reverse transition from bradyzoite to fast replicating tachyzoite and uncontrolled growth of the latter, these people are at risk for life-threatening disease. While serological tests for discriminating recent from past infection are available, there is yet no immunogenic biomarker used in the serological test to allow ascertaining the presence of persistent bradyzoites. RESULTS: Capitalizing on genetically engineered parasites induced to produce mature bradyzoites in vitro, we have identified the BCLA/MAG2 protein being restricted to the bradyzoite and the cyst envelope. Using laboratory mice as relevant T. gondii host models, we demonstrated that BCLA/MAG2 drives the generation of antibodies that recognize bradyzoite and the enveloping cyst structure. We have designed an ELISA assay based on a bacterially produced BCLA recombinant polypeptide, which was validated using a large collection of sera from mice of different genetic backgrounds and infected with bcla+ or bcla-null cystogenic and non-cystogenic T. gondii strains. To refine the design of the ELISA assay, we applied high-resolution BCLA epitope mapping and identified a specific combination of peptides and accordingly set up a selective and sensitive ELISA assay which allowed the detection of anti-BCLA/MAG2 antibodies in the sera of human patients with various forms of toxoplasmosis. CONCLUSIONS: We brought proof of principle that anti-BCLA/MAG2 antibodies serve as specific and sensitive serological markers in the perspective of a combinatorial strategy for detection of persistent T. gondii parasitism.

Target Identification of an Antimalarial Oxaborole Identifies AN13762 as an Alternative Chemotype for Targeting CPSF3 in Apicomplexan Parasites.

Boron-containing compounds represent a promising class of molecules with proven efficacy against a wide range of pathogens, including apicomplexan parasites. Following lead optimization, the benzoxaborole AN13762 was identified as a preclinical candidate against the human malaria parasite, yet the molecular target remained uncertain. Here, we uncovered the parasiticidal mechanisms of AN13762, by combining forward genetics with transcriptome sequencing and computational mutation discovery and using Toxoplasma gondii as a relevant model for Apicomplexa. AN13762 was shown to target TgCPSF3, the catalytic subunit of the pre-mRNA cleavage and polyadenylation complex, as the anti-pan-apicomplexan benzoxaborole compound, AN3661. However, unique mutations within the TgCPSF3 catalytic site conferring resistance to AN13762 do not confer cross-protection against AN3661, suggesting a divergent resistance mechanism. Finally, in agreement with the high sequence conservation of CPSF3 between Toxoplasma and Cryptosporidium, AN13762 shows oral efficacy in cryptosporidiosis mouse model, a disease for which new drug development is of high priority.

Toxoplasma gondii Parasitophorous Vacuole Membrane-Associated Dense Granule Proteins Orchestrate Chronic Infection and GRA12 Underpins Resistance to Host Gamma Interferon.

Toxoplasma gondii evades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronic Toxoplasma infection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cysts in vivo without affecting the parasite growth rate or the ability to differentiate into cysts in vitro Acute virulence of the PruΔgra2, PruΔgra3, and PruΔgra4 mutants was reduced but not abolished. In contrast, the PruΔgra12 mutant was avirulent in mice and PruΔgra12 parasites failed to establish a chronic infection. High-virulence type I strain RHΔgra12 parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-γ)-activated macrophages, type I RHΔgra12 and type II PruΔgra12 parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RHΔku80 and type II PruΔku80 strains, respectively. Despite this resistance, Δgra12 PVs ultimately succumbed to IFN-γ-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-γ and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCEToxoplasma gondii cysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cysts in vivo A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-γ). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.

High-content imaging assay to evaluate Toxoplasma gondii infection and proliferation: A multiparametric assay to screen new compounds.

Toxoplasma gondii is an intracellular protozoan parasite widely distributed in animals and humans. Infection of host cells and parasite proliferation are essential steps in Toxoplasma pathology. The objective of this study was to develop and validate a novel automatic High Content Imaging (HCI) assay to study T. gondii infection and proliferation. We tested various fluorescent markers and strategies of image analysis to obtain an automated method providing results comparable to those from gold standard infection and proliferation assays. No significant difference was observed between the results obtained from the HCI assay and the standard assays (manual fluorescence microscopy and incorporation of [3H]-uracil). We developed here a robust and time-saving assay. This automated technology was then used to screen a library of compounds belonging to four classes of either natural compounds or synthetic derivatives. Inhibition of parasite proliferation and host cell toxicity were measured in the same assay and led to the identification of one hit, a thiosemicarbazone that allows important inhibition of Toxoplasma proliferation while being relatively safe for the host cells.

Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii.

Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.