Biology subjects

Riscoe, M. K.

Publications and source records attributed to Riscoe, M. K..

7 recordsLinked to original sources

bioRxiv2021

Babesia duncani as a model organism to study the development, virulence and drug susceptibility of intraerythrocytic parasites in vitro and in vivo

Hematozoa are a subclass of protozoan parasites that invade and develop within vertebrate red blood cells to cause the pathological symptoms associated with diseases of both medical and veterinary importance such as malaria and babesiosis. A major limitation in the study of the most prominent hematozoa, Plasmodium spp, the causative agents of malaria, is the lack of a broadly accessible mouse model to evaluate parasite infection in vivo as is the case for P. falciparum or altogether the lack of an in vitro culture and mouse models as is the case for P. vivax, P. malariae and P. ovale. Similarly, no in vitro culture system exists for Babesia microti, the predominant agent of human babesiosis. In this study, we show that human red blood cells infected with the human pathogen Babesia duncani continuously propagated in culture, as well as merozoites purified from parasite cultures, can cause lethal infection in immunocompetent C3H/HeJ mice. Furthermore, highly reproducible parasitemia and survival outcomes were established using specific parasite loads and different mouse genetic backgrounds. Using the combined in culture-in mouse (ICIM) model of B. duncani infection, we demonstrate that current recommended combination therapies for the treatment of human babesiosis, while synergistic in cell culture, have weak potency in vitro and failed to clear infection or prevent death in mice. Interestingly, using the ICIM model, we identified two new endochin-like quinolone prodrugs, ELQ-331 and ELQ-468, that alone or in combination with atovaquone are highly efficacious against B. duncani and B. microti. The novelty, ease of use and scalability of the B. duncani ICIM dual model make it an ideal system to study intraerythrocytic parasitism by protozoa, unravel the molecular mechanisms underlying parasite virulence and pathogenesis, and accelerate the development of innovative therapeutic strategies that could be translated to unculturable parasites and important pathogens for which an animal model is lacking. Author SummaryUse of model organisms is vital to the understanding of virulence and pathogenesis of a large number of human and animal pathogens. In case of hematozoan parasites that invade and develop within vertebrate erythrocytes, the studies are challenging because of the dearth of small animal model systems and the lack of continuous parasite growth in in vitro culture conditions. Here, we report a small animal model of lethal infection of Babesia duncani, one of the causative agents of human babesiosis. We show that in vitro cultured parasites and as well as parasites propagated in vivo can establish highly reproducible parasitemia which is dependent on the parasite load and also defined by different mouse genetic backgrounds. We further use this combined in culture-in mouse (ICIM) model of B. duncani infection to demonstrate the anti-babesial efficacy of two novel endochin like quinoline compounds. We propose that this ICIM dual model of B. duncani is an ideal system to get insights into protozoan intraerythrocytic parasitism, virulence, pathogenesis, and therapeutics and will open the vista to other important pathogens which are unculturable or lack an animal model.

microbiology↗

bioRxiv2021

Effective Therapy Targeting Cytochrome bc1 Prevents Babesia Erythrocytic Development and Protects from Lethal Infection

Targeting conserved metabolic processes that are essential for viability of pathogens, such as Plasmodium and Babesia that cause blood-borne diseases, is an effective strategy to eliminate malaria and babesiosis infections with no recrudescence. One interesting target is the mitochondrial cytochrome bc1 complex, which could be inhibited by drugs such as endochin-like quinolones (ELQ) and atovaquone. We used the tick-transmitted and culturable blood-borne pathogen Babesia duncani to evaluate the structure-activity relationship, safety, efficacy and mode of action of ELQs. We identified a potent and highly selective ELQ prodrug (ELQ-502), which alone or in combination with atovaquone eliminates B. microti and B. duncani infections in vitro and in mouse models of parasitemia and lethal infection. The strong efficacy at low dose, excellent safety, bioavailability and long half-life of this experimental therapy makes it an ideal clinical candidate for the treatment of human infections caused by Babesia and its closely related apicomplexan parasites.

microbiology↗

bioRxiv2020

Atypical molecular basis for drug resistance to mitochondrial function inhibitors in Plasmodium falciparum

The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc1 complex, which is an essential component of the mitochondrial electron transport chain (mtETC) necessary for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis. Despite the essentiality of this complex, resistance to a novel acridone class of compounds targeting cyt bc1 was readily attained, resulting in a parasite strain (SB1-A6) that was pan-resistant to both mtETC and DHODH inhibitors. Here we describe the molecular mechanism behind the resistance of the SB1-A6 parasite line which lacks the common cyt bc1 point mutations characteristic of resistance to mtETC inhibitors. Using Illumina whole-genome sequencing, we have identified both a copy number variation ([~]2x) and a single-nucleotide polymorphism (C276F) associated with pfdhodh in SB1-A6. We have characterized the role of both genetic lesions by mimicking the copy number variation via episomal expression of pfdhodh and introducing the identified SNP using CRISPR/Cas9 and assessed their contributions to drug resistance. Although both of these genetic polymorphisms have been previously identified as contributing to both DSM-1 (1) and atovaquone resistance (2, 3), SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the pan-resistant phenotype. This novel mechanism of resistance to mtETC inhibition has critical implications for the development of future drugs targeting the bc1 complex or de novo pyrimidine synthesis that could help guide future anti-malarial combination therapies and reduce the rapid development of drug resistance in the field.

microbiology↗

bioRxiv2020

Orally Bioavailable Endochin-like Quinolone Carbonate Ester Prodrug Reduces Toxoplasma gondii Brain Cysts

Toxoplasmosis is a potentially fatal infection for immunocompromised people and the developing fetus. Current medicines for toxoplasmosis have high rates of adverse effects that interfere with therapeutic and prophylactic regimens. Endochin-like quinolones (ELQs) are potent inhibitors of Toxoplasma gondii proliferation in vitro and in animal models of acute and latent infection. ELQ-316, in particular, was found to be effective orally against acute toxoplasmosis in mice and highly selective for the T. gondii cytochrome b over the human cytochrome b. Despite oral efficacy, the high crystallinity of ELQ-316 limits oral absorption, plasma concentrations and therapeutic potential. A carbonate ester prodrug of ELQ-316, ELQ-334, was created to decrease crystallinity and increase oral bioavailability, which resulted in a six-fold increase in both Cmax (maximum plasma concentration) and AUC (area under the curve) of ELQ-316. The increased bioavailability of ELQ-316, when administered as ELQ-334, resulted in greater efficacy than the equivalent dose of ELQ-316 against acute toxoplasmosis and had similar efficacy against latent toxoplasmosis compared to intraperitoneal administration of ELQ-316. Carbonate ester prodrugs are a successful strategy to overcome the limited oral bioavailability of ELQs for the treatment of toxoplasmosis.

pharmacology and toxicology↗

bioRxiv2020

Genetic ablation of the mitochondrial ribosome in Plasmodium falciparum sensitizes the human malaria parasite to antimalarial drugs targeting mitochondrial functions

The mitochondrion of malaria parasites contains clinically validated drug targets. Within Plasmodium spp., the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and [~] 27 small, fragmented rRNA genes in length of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum has been shown to be essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of mitoribosomes in sustaining the metabolic status of the parasite mitochondrion remains unknown. Here, among the 14 annotated mitoribosomal proteins of the small subunit of P. falciparum, we verified the localization and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, PfmtRPS18), employing a CRISPR/Cas9 mediated conditional knockdown tool. Using immuno-electron microscopy, we provided evidence that the mitoribosome is closely associated with the mitochondrial inner membrane in the parasite. Upon knockdown of the mitoribosome, the parasites became hypersensitive to inhibitors targeting the bc1 complex, dihydroorotate dehydrogenase and F1Fo ATP synthase complex. Furthermore, knockdown of the mitoribosome blocked the pyrimidine biosynthesis pathway and reduced the pool of pyrimidine nucleotides. Together, our data suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capability of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors targeting mitochondrial functions.

biochemistry↗

bioRxiv2019

ELQ-331 as a prototype for extremely durable chemoprotection against malaria

BackgroundThe potential benefits of long-acting injectable chemoprotection (LAI-C) against malaria have been recently recognized, prompting a call for suitable candidate drugs to help meet this need. On the basis of its known pharmacodynamic and pharmacokinetic profiles after oral dosing, ELQ-331, a prodrug of the parasite mitochondrial electron transport inhibitor ELQ-300, was selected for study of pharmacokinetics and efficacy as LAI-C in mice.\n\nMethodsFour trials were conducted in which mice were injected with a single intramuscular dose of ELQ-331 or other ELQ-300 prodrugs in sesame oil with 1.2% benzyl alcohol; the ELQ-300 content of the doses ranged from 2.5 to 30 mg/kg. Initial blood stage challenges with Plasmodium yoelii were used to establish the model, but the definitive study measure of efficacy was outcome after sporozoite challenge with a luciferase-expressing P.yoelii, assessed by whole-body live animal imaging. Snapshot determinations of plasma ELQ-300 concentration ([ELQ-300]) were made after all prodrug injections; after the highest dose of ELQ-331 (equivalent to 30 mg/kg ELQ-300), both [ELQ-331] and [ELQ-300] were measured at a series of timepoints from 6 hours to 5 [1/2] months after injection.\n\nResultsA single intramuscular injection of ELQ-331 outperformed four other ELQ-300 prodrugs and, at a dose equivalent to 30 mg/kg ELQ-300, protected mice against challenge with P. yoelii sporozoites for at least 4 [1/2] months. Pharmacokinetic evaluation revealed rapid and essentially complete conversion of ELQ-331 to ELQ-300, a rapidly achieved (< 6 hours) and sustained (4-5 months) effective plasma ELQ-300 concentration, maximum ELQ-300 concentrations far below the estimated threshold for toxicity, and a distinctive ELQ-300 concentration vs. time profile. Pharmacokinetic modeling indicates a high-capacity, slow-exchange tissue compartment which serves to accumulate and then slowly redistribute ELQ-300 into blood, and this property facilitates an extremely long period during which ELQ-300 concentration is sustained above a minimum fully-protective threshold (60-80 nM).\n\nConclusionsExtrapolation of these results to humans clearly predicts that ELQ-331 should be capable of meeting and far-exceeding currently published duration-of-effect goals for antimalarial LAI-C. Allometric scaling from mice to humans would predict a several-fold enhancement in the relationship between duration-of-effect and dose, and available drug engineering and formulation technologies would be expected to offer significant improvement over the simple powder in sesame oil used here. Furthermore, the distinctive pharmacokinetic profile of ELQ-300 after treatment with ELQ-331 may facilitate durable protection using a variety of delivery and formulation options, and may enable protection for far longer than 3 months. Particularly in light of the favorable pharmacodynamic profile of ELQ-300, ELQ-331 warrants consideration as a leading prototype for LAI-C.

pharmacology and toxicology↗

bioRxiv2018

Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum is dispensable and not the functional target of putative NDH2 quinolone inhibitors

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasites susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results call into question the validity of PfNDH2 as an antimalarial drug target.\n\nImportanceFor a long time, PfNDH2 has been considered an attractive antimalarial drug target. However, the conclusion that PfNDH2 is essential was based on preliminary and incomplete data. Here we generate a PfNDH2 KO (knockout) parasite in the blood stages of Plasmodium falciparum, showing that the gene is not essential. We also show that previously reported PfNDH2-specific inhibitors kill the parasites primarily via targeting the cytochrome bc1 complex, not PfNDH2. Overall, we provide genetic and biochemical data that help to resolve a long-debated issue in the field regarding the potential of PfNDH2 as an antimalarial drug target.

microbiology↗