The search for new antimalarial drugs with unexplored mechanisms of action is currently one of the main objectives to combat the resistance already in the clinic. New drugs should target specific mechanisms that once initiated lead inevitably to the parasite's death and clearance and cause minimal toxicity to the host. One such new mode of action recently characterized is to target the parasite's calcium dynamics. Disruption of the calcium homeostasis is associated with compromised digestive vacuole membrane integrity and release of its contents, leading to programmed cell death-like features characterized by loss of mitochondrial membrane potential and DNA degradation. Intriguingly, chloroquine (CQ)-treated parasites were previously reported to exhibit such cellular features. Using a high-throughput phenotypic screen, we identified 158 physiological disruptors (hits) of parasite calcium distribution from a small subset of approximately 3000 compounds selected from the GSK TCAMS (Tres Cantos Anti-Malarial Set) compound library. These compounds were then extensively profiled for biological activity against various CQ- and artemisinin-resistant Plasmodium falciparum strains and stages. The hits were also examined for cytotoxicity, speed of antimalarial activity, and their possible inhibitory effects on heme crystallization. Overall, we identified three compounds, TCMDC-136230, -125431, and -125457, which were potent in inducing calcium redistribution but minimally inhibited heme crystallization. Molecular superimposition of the molecules by computational methods identified a common pharmacophore, with the best fit assigned to TCMDC-125457. There were low cytotoxicity or CQ cross-resistance issues for these three compounds. IC50 values of these three compounds were in the low micromolar range. In addition, TCMDC-125457 demonstrated high efficacy when pulsed in a single-dose combination with artesunate against tightly synchronized artemisinin-resistant ring-stage parasites. These results should add new drug options to the current armament of antimalarial drugs as well as provide promising starting points for development of drugs with non-classical modes of action.
Background: Trypanosoma brucei is a blood-borne, protozoan parasite that causes African sleeping sickness in humans and nagana in animals. The current chemotherapy relies on only a handful of drugs that display undesirable toxicity, poor efficacy and drug-resistance. In this study, we explored the use of lysosomotropic drugs to induce bloodstream form T. brucei cell death via lysosome destabilization. Methods: We measured drug concentrations that inhibit cell proliferation by 50% (IC50) for several compounds, chosen based on their lysosomotropic effects previously reported in Plasmodium falciparum. The lysosomal effects and cell death induced by L-leucyl-L-leucyl methyl ester (LeuLeu-OMe) were further analyzed by flow cytometry and immunofluorescence analyses of different lysosomal markers. The effect of autophagy in LeuLeu-OMe-induced lysosome destabilization and cytotoxicity was also investigated in control and autophagy-deficient cells. Results: LeuLeu-OMe was selected for detailed analyses due to its strong inhibitory profile against T. brucei with minimal toxicity to human cell lines in vitro. Time-dependent immunofluorescence studies confirmed an effect of LeuLeu-OMe on the lysosome. LeuLeu-OMe-induced cytotoxicity was also found to be dependent on the acidic pH of the lysosome. Although an increase in autophagosomes was observed upon LeuLeu-OMe treatment, autophagy was not required for the cell death induced by LeuLeu-OMe. Necrosis appeared to be the main cause of cell death upon LeuLeu-OMe treatment. Conclusions: LeuLeu-OMe is a lysosomotropic agent capable of destabilizing lysosomes and causing necrotic cell death in bloodstream form of T. brucei.
Cell salvage is integral in perioperative blood management, reducing complications associated with perioperative anemia and allogenic blood transfusions. Complications during cell salvage such as air and fat embolism, dilutional coagulopathy, and hemolysis are uncommon. We report a rare case of hemolysis during intraoperative cell salvage arising from the use of look-alike, sound-alike (LASA) wash solutions, where collected blood was washed with hypotonic sterile water instead of isotonic 0.9% saline solution. Hemolysis was detected and confirmed prior to initiating autotransfusion hence avoiding adverse patient outcomes. Subsequent serum potassium levels in our patient remained normal and our postoperative recovery was uneventful. Root cause analysis found that 0.9% saline solution and sterile water had similar appearance and packaging, and were stored in adjacent unlabeled bins in the central storage area. Cell salvage guidelines at our institution were otherwise consistent with international best practice guidelines and postoperative inspection did not detect faults in our cell salvage device. Our experience demonstrates that dark and translucent blood collected during cell salvage should raise suspicion for hemolysis which can be confirmed by assessing collected blood potassium levels. Next, LASA drug errors might occur during cell salvage and institutions should adopt systemic measures to minimize such occurrences which can lead to significant patient morbidity and mortality.
The biopharmaceutical industry relies on selecting high-performing cell lines to meet quality and manufacturability criteria. However, this process is time- and labor-intensive. To address this, label-free multimodal multiphoton microscopy techniques were employed to characterize biopharmaceutical cell lines in early passages. Using a machine learning-assisted single-cell analysis pipeline, over 95% accuracy for monoclonal cell line classification was achieved in all passages. Additionally, Open Set Recognition allowed the differentiation of desired cell lines in polyclonal pools. The study offers a promising solution to expedite the cell line selection process, reducing time and resources while ensuring the identification of high-performance biopharmaceutical cell lines.
Abstract The large intestine harbors microorganisms playing unique roles in host physiology. The beneficial or detrimental outcome of host‐microbiome coexistence depends largely on the balance between regulators and responder intestinal CD4 + T cells. We found that ulcerative colitis‐like changes in the large intestine after infection with the protist Blastocystis ST7 in a mouse model are associated with reduction of anti‐inflammatory Treg cells and simultaneous expansion of pro‐inflammatory Th17 responders. These alterations in CD4 + T cells depended on the tryptophan metabolite indole‐3‐acetaldehyde (I3AA) produced by this single‐cell eukaryote. I3AA reduced the Treg subset in vivo and iTreg development in vitro by modifying their sensing of TGFβ, concomitantly affecting recognition of self‐flora antigens by conventional CD4 + T cells. Parasite‐derived I3AA also induces over‐exuberant TCR signaling, manifested by increased CD69 expression and downregulation of co‐inhibitor PD‐1. We have thus identified a new mechanism dictating CD4 + fate decisions. The findings thus shine a new light on the ability of the protist microbiome and tryptophan metabolites, derived from them or other sources, to modulate the adaptive immune compartment, particularly in the context of gut inflammatory disorders.
Abstract The total syntheses of antimalarial agent Falcitidin (I) as well as related N‐acyl analogues (II) are outlined and the efficiency of the latter compounds against chloroquine‐sensitive Plasmodium falciparum 3D7 is tested.
An alternative antimalarial pathway of an ‘outdated’ drug, chloroquine (CQ), may facilitate its return to the shrinking list of effective antimalarials. Conventionally, CQ is believed to interfere with hemozoin formation at nanomolar concentrations, but resistant parasites are able to efflux this drug from the digestive vacuole (DV). However, we show that the DV membrane of both resistant and sensitive laboratory and field parasites is compromised after exposure to micromolar concentrations of CQ, leading to an extrusion of DV proteases. Furthermore, only a short period of exposure is required to compromise the viability of late-stage parasites. To study the feasibility of this strategy, mice malaria models were used to demonstrate that high doses of CQ also triggered DV permeabilization in vivo and reduced reinvasion efficiency. We suggest that a time-release oral formulation of CQ may sustain elevated blood CQ levels sufficiently to clear even CQ-resistant parasites.
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