High-Throughput Screening to Identify Inhibitors of Plasmodium falciparum Importin α.

The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in Plasmodium species and Toxoplasma gondii threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from P. falciparum and T. gondii have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit P. falciparum IMPα binding to a P. falciparum NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to P. falciparum IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of P. falciparum IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both P. falciparum and T. gondii. The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.

Haematological response in experimental human Plasmodium falciparum and Plasmodium vivax malaria.

BACKGROUND: Malaria-associated anaemia, arising from symptomatic, asymptomatic and submicroscopic infections, is a significant cause of morbidity worldwide. Induced blood stage malaria volunteer infection studies (IBSM-VIS) provide a unique opportunity to evaluate the haematological response to early Plasmodium falciparum and Plasmodium vivax infection. METHODS: This study was an analysis of the haemoglobin, red cell counts, and parasitaemia data from 315 participants enrolled in IBSM-VIS between 2012 and 2019, including 269 participants inoculated with the 3D7 strain of P. falciparum (Pf3D7), 15 with an artemisinin-resistant P. falciparum strain (PfK13) and 46 with P. vivax. Factors associated with the fractional fall in haemoglobin (Hb-FF) were evaluated, and the malaria-attributable erythrocyte loss after accounting for phlebotomy-related losses was estimated. The relative contribution of parasitized erythrocytes to the malaria-attributable erythrocyte loss was also estimated. RESULTS: The median peak parasitaemia prior to treatment was 10,277 parasites/ml (IQR 3566-27,815), 71,427 parasites/ml [IQR 33,236-180,213], and 34,840 parasites/ml (IQR 13,302-77,064) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. The median Hb-FF was 10.3% (IQR 7.8-13.3), 14.8% (IQR 11.8-15.9) and 11.7% (IQR 8.9-14.5) in those inoculated with Pf3D7, PfK13 and P. vivax, respectively, with the haemoglobin nadir occurring a median 12 (IQR 5-21), 15 (IQR 7-22), and 8 (IQR 7-15) days following inoculation. In participants inoculated with P. falciparum, recrudescence was associated with a greater Hb-FF, while in those with P. vivax, the Hb-FF was associated with a higher pre-treatment parasitaemia and later day of anti-malarial treatment. After accounting for phlebotomy-related blood losses, the estimated Hb-FF was 4.1% (IQR 3.1-5.3), 7.2% (IQR 5.8-7.8), and 4.9% (IQR 3.7-6.1) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. Parasitized erythrocytes were estimated to account for 0.015% (IQR 0.006-0.06), 0.128% (IQR 0.068-0.616) and 0.022% (IQR 0.008-0.082) of the malaria-attributable erythrocyte loss in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. CONCLUSION: Early experimental P. falciparum and P. vivax infection resulted in a small but significant fall in haemoglobin despite parasitaemia only just at the level of microscopic detection. Loss of parasitized erythrocytes accounted for < 0.2% of the total malaria-attributable haemoglobin loss.

Synthesis, Molecular Docking and Antiplasmodial Activities of New Tetrahydro-ß-Carbolines.

Malaria is still one of the most dangerous infectious diseases and the emergence of drug resistant parasites only worsens the situation. A series of new tetrahydro-ß-carbolines were designed, synthesized by the Pictet-Spengler reaction, and characterized. Further, the compounds were screened for their in vitro antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Moreover, molecular modeling studies were performed to assess the potential action of the designed molecules and toxicity assays were conducted on the human microvascular endothelial (HMEC-1) cell line and human red blood cells. Our studies identified N-(3,3-dimethylbutyl)-1-octyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b] indole-3-carboxamide (7) (a mixture of diastereomers) as the most promising compound endowed with the highest antiplasmodial activity, highest selectivity, and lack of cytotoxicity. In silico simulations carried out for (1S,3R)-7 provided useful insights into its possible interactions with enzymes essential for parasite metabolism. Further studies are underway to develop the optimal nanosized lipid-based delivery system for this compound and to determine its precise mechanism of action.

An Open-Source Toolkit To Expand Bioinformatics Training in Infectious Diseases.

As access to high-throughput sequencing technology has increased, the bottleneck in biomedical research has shifted from data generation to data analysis. Here, we describe a modular and extensible framework for didactic instruction in bioinformatics using publicly available RNA sequencing data sets from infectious disease studies, with a focus on host-parasite interactions. We highlight lessons learned from adapting this course for virtual learners during the coronavirus disease 2019 (COVID-19) pandemic.

Protease Inhibition-An Established Strategy to Combat Infectious Diseases.

Therapeutic agents with novel mechanisms of action are urgently needed to counter the emergence of drug-resistant infections. Several decades of research into proteases of disease agents have revealed enzymes well suited for target-based drug development. Among them are the three recently validated proteolytic targets: proteasomes of the malarial parasite Plasmodium falciparum, aspartyl proteases of P. falciparum (plasmepsins) and the Sars-CoV-2 viral proteases. Despite some unfulfilled expectations over previous decades, the three reviewed targets clearly demonstrate that selective protease inhibitors provide effective therapeutic solutions for the two most impacting infectious diseases nowadays-malaria and COVID-19.

Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria.

OBJECTIVE: Nigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients collected in August 2014, aged 1-61 years old from South-west Nigeria. RESULTS: Two Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the PATPase6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes although this could not be validated with independent isolation or additional methods. Our findings call for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for Covid-19.

Antiplasmodial Activity of p-Substituted Benzyl Thiazinoquinone Derivatives and Their Potential against Parasitic Infections.

Malaria is a life-threatening disease and, what is more, the resistance to available antimalarial drugs is a recurring problem. The resistance of Plasmodium falciparum malaria parasites to previous generations of medicines has undermined malaria control efforts and reversed gains in child survival. This paper describes a continuation of our ongoing efforts to investigate the effects against Plasmodium falciparum strains and human microvascular endothelial cells (HMEC-1) of a series of methoxy p-benzyl-substituted thiazinoquinones designed starting from a pointed antimalarial lead candidate. The data obtained from the newly tested compounds expanded the structure-activity relationships (SARs) of the thiazinoquinone scaffold, indicating that antiplasmodial activity is not affected by the inductive effect but rather by the resonance effect of the introduced group at the para position of the benzyl substituent. Indeed, the current survey was based on the evaluation of antiparasitic usefulness as well as the selectivity on mammalian cells of the tested p-benzyl-substituted thiazinoquinones, upgrading the knowledge about the active thiazinoquinone scaffold.