Pantothenate and CoA biosynthesis in Apicomplexa and their promise as antiparasitic drug targets

The Apicomplexa phylum comprises thousands of distinct intracellular parasite species, including coccidians, haemosporidians, piroplasms, and cryptosporidia. These parasites are characterized by complex and divergent life cycles occupying a variety of host niches. Consequently, they exhibit distinct adaptations to the differences in nutritional availabilities, either relying on biosynthetic pathways or by salvaging metabolites from their host. Pantothenate (Pan, vitamin B5) is the precursor for the synthesis of an essential cofactor, coenzyme A (CoA), but among the apicomplexans, only the coccidian subgroup has the ability to synthesize Pan. While the pathway to synthesize CoA from Pan is largely conserved across all branches of life, there are differences in the redundancy of enzymes and possible alternative pathways to generate CoA from Pan. Impeding the scavenge of Pan and synthesis of Pan and CoA have been long recognized as potential targets for antimicrobial drug development, but in order to fully exploit these critical pathways, it is important to understand such differences. Recently, a potent class of pantothenamides (PanAms), Pan analogs, which target CoA-utilizing enzymes, has entered antimalarial preclinical development. The potential of PanAms to target multiple downstream pathways make them a promising compound class as broad antiparasitic drugs against other apicomplexans. In this review, we summarize the recent advances in understanding the Pan and CoA biosynthesis pathways, and the suitability of these pathways as drug targets in Apicomplexa, with a particular focus on the cyst-forming coccidian, Toxoplasma gondii, and the haemosporidian, Plasmodium falciparum.

Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

The Neglected Angio-Neurotrophic Parasite Gurltia Paralysans (Nematoda: Angiostrongylidae): Northernmost South American Distribution, Current Knowledge, and Future Perspectives

Gurltia paralysans is a rare metastrongyloid nematode in South America that has begun to gain relevance in feline internal medicine as a differential diagnosis of progressive degenerative myelopathy disorders. The parasite life cycle has not been fully elucidated but probably involves invertebrate gastropod fauna as obligate intermediate hosts; thus, G. paralysans remaining an extremely neglected parasitosis. Feline gurltiosis intra vitam diagnosis is highly challenging due to lack of evidence in the excretion of G. paralysans eggs and larvae, neither in feces nor in other body secretions because environmental stages and the transmission route of the parasite remain unknown. Unfortunately, no experimental trials for the treatment of feline gurltiosis have been conducted to date. However, there are some reports of the successfully antiparasitic drugs used with different effectiveness and clinical improvement results in diagnosed cats. Further studies are needed to evaluate the parasite occurrence among domestic cats and the neotropical wild felid species distributed within Colombia in addition to the gastropod fauna that may harbor the developing larvae (L1–L3) stages of this underestimated parasite.

Small molecule modulation of the Drosophila Slo channel elucidated by cryo-EM

Slowpoke (Slo) potassium channels display extraordinarily high conductance, are synergistically activated by a positive transmembrane potential and high intracellular Ca2+ concentrations and are important targets for insecticides and antiparasitic drugs. However, it is unknown how these compounds modulate ion translocation and whether there are insect-specific binding pockets. Here, we report structures of Drosophila Slo in the Ca2+-bound and Ca2+-free form and in complex with the fungal neurotoxin verruculogen and the anthelmintic drug emodepside. Whereas the architecture and gating mechanism of Slo channels are conserved, potential insect-specific binding pockets exist. Verruculogen inhibits K+ transport by blocking the Ca2+-induced activation signal and precludes K+ from entering the selectivity filter. Emodepside decreases the conductance by suboptimal K+ coordination and uncouples ion gating from Ca2+ and voltage sensing. Our results expand the mechanistic understanding of Slo regulation and lay the foundation for the rational design of regulators of Slo and other voltage-gated ion channels.

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90–100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.

Overcoming the Prokaryote/Eukaryote Barrier in Tuberculosis Treatment: A Prospect for the Repurposing and Use of Antiparasitic Drugs

Antimicrobial resistance, the so-called silent pandemic, is pushing industry and academia to find novel antimicrobial agents with new mechanisms of action in order to be active against susceptible and drug-resistant microorganisms. In the case of tuberculosis, the need of novel anti-tuberculosis drugs is specially challenging because of the intricate biology of its causative agent, Mycobacterium tuberculosis. The repurposing of medicines has arisen in recent years as a fast, low-cost, and efficient strategy to identify novel biomedical applications for already approved drugs. This review is focused on anti-parasitic drugs that have additionally demonstrated certain levels of anti-tuberculosis activity; along with this, natural products with a dual activity against parasites and against M. tuberculosis are discussed. A few clinical trials have tested antiparasitic drugs in tuberculosis patients, and have revealed effective dose and toxicity issues, which is consistent with the natural differences between tuberculosis and parasitic infections. However, through medicinal chemistry approaches, derivatives of drugs with anti-parasitic activity have become successful drugs for use in tuberculosis therapy. In summary, even when the repurposing of anti-parasitic drugs for tuberculosis treatment does not seem to be an easy job, it deserves attention as a potential contributor to fuel the anti-tuberculosis drug pipeline.

E-NTPDases: Possible Roles on Host-Parasite Interactions and Therapeutic Opportunities

Belonging to the GDA1/CD39 protein superfamily, nucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP) and inorganic phosphate (Pi). Several NTPDase isoforms have been described in different cells, from pathogenic organisms to animals and plants. Biochemical characterization of nucleotidases/NTPDases has revealed the existence of isoforms with different specificities regarding divalent cations (such as calcium and magnesium) and substrates. In mammals, NTPDases have been implicated in the regulation of thrombosis and inflammation. In parasites, such as Trichomonas vaginalis, Trypanosoma spp., Leishmania spp., Schistosoma spp. and Toxoplasma gondii, NTPDases were found on the surface of the cell, and important processes like growth, infectivity, and virulence seem to depend on their activity. For instance, experimental evidence has indicated that parasite NTPDases can regulate the levels of ATP and Adenosine (Ado) of the host cell, leading to the modulation of the host immune response. In this work, we provide a comprehensive review showing the involvement of the nucleotidases/NTPDases in parasites infectivity and virulence, and how inhibition of NTPDases contributes to parasite clearance and the development of new antiparasitic drugs.

Lipid and fatty acid metabolism in trypanosomatids

Trypanosomiases and leishmaniases are neglected tropical diseases that have been spreading to previously non-affected areas in recent years. Identification of new chemotherapeutics is needed as there are no vaccines and the currently available treatment options are highly toxic and often ineffective. The causative agents for these diseases are the protozoan parasites of the Trypanosomatidae family, and they alternate between invertebrate and vertebrate hosts during their life cycles. Hence, these parasites must be able to adapt to different environments and compete with their hosts for several essential compounds, such as amino acids, vitamins, ions, carbohydrates, and lipids. Among these nutrients, lipids and fatty acids (FAs) are essential for parasite survival. Trypanosomatids require massive amounts of FAs, and they can either synthesize FAs de novo or scavenge them from the host. Moreover, FAs are the major energy source during specific life cycle stages of T. brucei, T. cruzi, and Leishmania. Therefore, considering the distinctive features of FAs metabolism in trypanosomatids, these pathways could be exploited for the development of novel antiparasitic drugs. In this review, we highlight specific aspects of lipid and FA metabolism in the protozoan parasites T. brucei, T. cruzi, and Leishmania spp., as well as the pathways that have been explored for the development of new chemotherapies.

152. Use of Antimicrobials among Suspected COVID-19 Patients at Selected 12 Hospitals in Bangladesh: Findings from the First Wave of COVID-19 Pandemic

Background Antimicrobials are empirically used in COVID-19 patients resulting in inappropriate stewardship and increased antimicrobial resistance. Our objective was to assess antimicrobial use among suspected COVID-19 in-patients while waiting for the COVID-19 test report. Methods From March to August 2020, we collected data from in-patients of 12 tertiary-level hospitals across Bangladesh. We identified suspected COVID-19 patients; collected information on antimicrobial received within 24 h before and on hospitalization; and tested nasopharyngeal swab for SARS-CoV-2 using rRT-PCR. We used descriptive statistics and a regression model for data analysis. Results Among 1188 suspected COVID-19 patients, the median age was 34 years (IQR:2–56), 69% were male, 40% had comorbidities, 53% required oxygen, and 1% required ICU or ventilation support after admission. Antibiotics were used in 92% of patients, 47% within 24 h before, and 89% on admission. Patients also received antiviral, mostly favipiravir (1%) and antiparasitic drugs particularly ivermectin (3%). Third-generation cephalosporin use was the highest (708;60%), followed by macrolide (481;40%), and the majority (853;78%) who took antibiotics were SARS-CoV-2 negative. On admission, 77% mild and 94% moderately ill patients received antibiotics. Before admission, 3% patients had two antibiotics, and on admission, 27% received two to four classes of antibiotics at the same time. According to WHO AWaRe classification, the Watch group antibiotics were mostly used before (43%) as well as on admission (80%). Reserve group antibiotic particularly linezolid was used in 1% patients includes mild cases on admission. Antibiotic use on admission was higher among severely ill patients (AOR = 11.7;95%CI:4.5–30.1) and those who received antibiotics within 24 h before hospital admission (AOR = 1.6;95%CI:1.0–2.5). Antimicrobials used among suspected COVID-19 patients and SARS-CoV-2 positive and negative patients 24 h before and on hospital admission at 12 selected hospitals in Bangladesh, March–August 2020 Antimicrobials used on admission among suspected COVID-19 patients according to disease severity at 12 selected hospitals in Bangladesh, March–August 2020 Conclusion Antimicrobial use was highly prevalent among suspected COVID-19 in-patients in Bangladesh. Initiating treatment with Watch group antibiotics like third-generation cephalosporin and azithromycin among mild to moderately ill patients were common. Promoting antimicrobial stewardship with monitoring is essential to prevent blanket antibiotic use, thereby mitigating antimicrobial resistance. Disclosures All Authors: No reported disclosures