Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, K_i = 0.0937 ± 0.01 nM and k_inact/K_i = 8 730 000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is highly homologous. Our investigations revealed that gallinamide and its analogs potently inhibit cruzain and are exquisitely toxic toward T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC₅₀ = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for the treatment of Chagas disease.

Hexafluoropropylene oxide-dimer acid (HFPO-DA) is one of the emerging replacements for the "forever" carcinogenic and toxic long-chain PFAS. HFPO-DA is a polymerization aid used for manufacturing fluoropolymers, whose global distribution and undetermined toxic properties are a concern regarding human and ecological health. To assess embryotoxic potential, zebrafish embryos were exposed to HFPO-DA at concentrations of 0.5-20,000 mg/L at 24-, 48-, and 72-h post-fertilization (hpf). Heart rate increased significantly in embryos exposed to 2 mg/L and 10 mg/L HFPO-DA across all time points. Spinal deformities and edema phenotypes were evident among embryos exposed to 1000-16,000 mg/L HFPO-DA at 72 hpf. A median lethal concentration (LC₅₀) was derived as 7651 mg/L at 72 hpf. Shallow RNA sequencing analysis of 9465 transcripts identified 38 consistently differentially expressed genes at 0.5 mg/L, 1 mg/L, 2 mg/L, and 10 mg/L HFPO-DA exposures. Notably, seven downregulated genes were associated with visual response, and seven upregulated genes were expressed in or regulated the cardiovascular system. This study identifies biological targets and molecular pathways affected during animal development by an emerging, potentially problematic, and ubiquitous industrial chemical.

Background

Endothelial lipase (EL) regulates the metabolism of HDL cholesterol (HDL-C). However, the role of EL in regulating plasma HDL-C concentrations and EL's potential involvement in atherosclerosis in humans has not been fully investigated due to the lack of reliable assays for EL mass. We developed an ELISA system for serum EL mass.

Methods

Human recombinant EL proteins, purified from cultured media of human EL-transfected Chinese hamster ovary cells, were used as antigen and calibrator. Two specific monoclonal antibodies were generated in mice against recombinant EL protein for a sandwich ELISA. We measured EL mass in human serum using EL recombinant protein as a calibration standard.

Results

The EL antibodies did not cross-react with lipoprotein lipase and hepatic triglyceride lipase. The detection limit of the ELISA was 20 pg/mL, which is approximately 10 times lower than that of previous ELISA systems. Recovery of spiked EL in serum was 90%-105%. Assay linearity was intact with a >4-fold dilution of serum. Intra- and interassay CVs were <5%. The serum EL mass in 645 human subjects was [mean (SE)] 344.4 (7.7) pg/mL (range 55.2-1387.7 pg/mL). Interestingly, serum EL mass was increased in patients with diagnosed cardiovascular disease and inversely correlated with serum HDL-C concentrations. There was no difference in EL mass between pre- and postheparin plasma samples.

Conclusions

This ELISA should be useful for clarifying the impact of EL on HDL metabolism and EL's potential role in atherosclerosis.

A predicted rapid growth in science, technology, engineering, and math (STEM) careers demands a vast and talented workforce, but students most commonly abandon STEM majors within the first 2 years of college. Performance in introductory courses, scientific literacy, and the ability to critically reason are main predictors of retention in STEM, highlighting the importance of precollege and early college experience. The Life Science Research Immersion Program (LSRIP) is a novel science education model that focuses on the development of scientific research skills, thus preparing students for introductory college courses and beyond. To evaluate the efficacy of the LSRIP, pre- and postprogram assessments and surveys were administered to three precollege student cohorts. Scientific reasoning assessment scores improved by 4.70% in Summer 2019 (P < 0.01), 9.44% in Fall 2019 (P < 0.05), and 0.97% in Winter 2020 cohorts, with two of five questions showing statistically significant improvement. Surveyed attitudes toward science improved in 62.9% of questions across all cohorts. These results suggest that research immersion experiences are an effective educational instrument for improving and promoting scientific reasoning and attitudes among precollege students. To better prepare students for success in STEM higher education and careers, we recommend implementing LSRIPs to complement traditional precollege science curricula.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

Although toxoplasmosis is one of the most common parasitic infections worldwide, therapeutic options remain limited. Cathepsins, proteases that play key roles in the pathogenesis of toxoplasmosis and many other protozoan infections, are important potential therapeutic targets. Because both TgCPB and TgCPL play a role in T. gondii invasion, we evaluated the efficacy of the potent, irreversible vinyl sulfone inhibitor, K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl). The inhibitor's toxicity and pharmacokinetic profile have been well-studied because of its in vitro and in vivo activity against a number of parasites. We found that it inhibited both TgCPB (EC50 = 114 nM) and TgCPL (EC50 = 71 nM) in vitro. K11777 also inhibited invasion of human fibroblasts by RH tachyzoites by 71% (p = 0.003) and intracellular replication by >99% (p<0.0001). In vivo, a single dose of K11777 led to 100% survival of chicken embryos in an model of acute toxoplasmosis (p = 0.015 Cox regression analysis). Therefore, K11777 shows promise as a novel therapeutic agent in the treatment of toxoplasmosis, and may prove to be a broadly effective anti-parasitic agent.

Background

Chagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice.

Methodology/principal findings

Both acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart.

Conclusions/significance

The positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life.

Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide(1), resulting in ~70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole(2), which has adverse effects(3), and potential resistance of E. histolytica to the drug is an increasing concern(4,5). To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.