Structure and antiparasitic activity relationship of alkylphosphocholine analogues against Leishmania donovani

Miltefosine (Milt) is the only oral treatment for visceral leishmaniasis (VL) but its use is associated with adverse effects e.g. teratogenicity, vomiting, diarrhoea. Understanding how its chemical structure induces cytotoxicity, whilst not compromising its anti-parasitic efficacy, could identify more effective compounds. Therefore we systemically modified the compound’s head, tail and linker tested the in vitro activity of three alkylphosphocholines (APC) series against Leishmania donovani strains with different sensitivities to antimony. The analogue, APC12, with an alkyl carbon chain of 12 atoms, was also tested for anti-leishmanial in vivo activity in a murine VL model. All APCs produced had anti-leishmanial activity in the micromolar range (IC50 and IC90, 0.46 µM - >82.21 µM and 4.14 µM - 739.89 µM; 0.01 - >8.02 µM and 0.09 µM - 72.18 µM respectively against promastigotes and intracellular amastigotes). The analogue, APC12 was the most active, was 4-10 fold more effective than the parent Milt molecule (APC16), irrespective of the strain’s sensitivity to antimony. Intravenous administration of 40 mg/kg APC12 to L. donovani infected BALB/c mice reduced liver and spleen parasite burdens by 60 ± 11% and 60 ± 19% respectively while oral administration reduced parasite load in the bone marrow by 54 ± 34%. These studies confirm that it is possible to alter the Milt structure and produce more active anti-leishmanial compounds

Distinct roles in autophagy and importance in infectivity of the two ATG4 cysteine peptidases of leishmania major

Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite

The Public Repository of Xenografts enables discovery and randomized phase II-like trials in mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease

Structure and antiparasitic activity relationship of alkylphosphocholine analogues against Leishmania donovani

Miltefosine (Milt) is the only oral treatment for visceral leishmaniasis (VL) but its use is associated with adverse effects e.g. teratogenicity, vomiting, diarrhoea. Understanding how its chemical structure induces cytotoxicity, whilst not compromising its anti-parasitic efficacy, could identify more effective compounds. Therefore, we systemically modified the compound’s head, tail and linker tested the in vitro activity of three alkylphosphocholines (APC) series against Leishmania donovani strains with different sensitivities to antimony. The analogue, APC12, with an alkyl carbon chain of 12 atoms, was also tested for anti-leishmanial in vivo activity in a murine VL model. All APCs produced had anti-leishmanial activity in the micromolar range (IC50 and IC90, 0.46 µM - >82.21 µM and 4.14 µM - 739.89 µM; 0.01 - >8.02 µM and 0.09 µM - 72.18 µM respectively against promastigotes and intracellular amastigotes). The analogue, APC12 was the most active, was 4–10 fold more effective than the parent Milt molecule (APC16), irrespective of the strain’s sensitivity to antimony. Intravenous administration of 40 mg/kg APC12 to L. donovani infected BALB/c mice reduced liver and spleen parasite burdens by 60 ± 11% and 60 ± 19% respectively while oral administration reduced parasite load in the bone marrow by 54 ± 34%. These studies confirm that it is possible to alter the Milt structure and produce more active anti-leishmanial compounds

Protein turnover and differentiation in Leishmania

Leishmania occurs in several developmental forms and thus undergoes complex cell differentiation events during its life-cycle. Those are required to allow the parasite to adapt to the different environmental conditions. The sequencing of the genome of L. major has facilitated the identification of the parasite’s vast arsenal of proteolytic enzymes, a few of which have already been carefully studied and found to be important for the development and virulence of the parasite. This review focuses on these peptidases and their role in the cellular differentiation of Leishmania through their key involvement in a variety of degradative pathways in the lysosomal and autophagy networks

Estimating soil health in urban allotments: Integrated two-way soil quality index and free-living amoebae in nitrogen recycling

Nitrogen deficiency and contamination by potentially toxic elements (PTEs) adversely impact soil health and ecosystem services. Existing tools for assessing contaminated soils, necessary for sustainable management, remain limited. In this study, we introduce an integrated approach using geochemical constituents and biological factors to construct a comprehensive index to evaluate contaminant impacts on soil health. We collected samples in triplicate from six plots within an urban allotment with a history of PTE contamination. Selected biological and chemical characteristics of the samples were quantified to derive impact scores, with a single numerical index representing overall soil quality for each plot. Multivariate, T-statistics and Pearson Correlation analysis were used to identify relationships and differences between selected soil parameters between plots. The role of the free-living amoeba Acanthamoeba in nitrogen recycling was assessed with feeding experiments, enzymatic assays and bioinformatics analysis. The plot with the highest index value, indicative of good health, exhibited higher pH, significantly high microbial load, and a high nitrate to ammonium (NO3−:NH4+) ratio of 5.3. This turnover was associated with Acanthamoeba uptake of exogenous nitrates and secretion of ammonium, through the assimilatory/dissimilatory nitrate reduction pathway. In contrast, the lower index plot with the low nitrogen turnover of 0.69, showed elevated aluminium, low pH activity and a significantly reduced microbial load, dominated by aluminium resistant microorganisms. Our findings highlight the importance of a comprehensive soil quality index by integrating multiple characteristics to assess soil health and contamination. The approach addresses the need for improved tools to identify the direct impact of contaminants on soil biological activity, supporting more sustainable land management

The crystal structure of Leishmania major 3-mercaptopyruvate sulfurtransferase : A three-domain architecture with a serine protease-like triad at the active site

Leishmania major 3-mercaptopyruvate sulfurtransferase is a crescent-shaped molecule comprising three domains. The N-terminal and central domains are similar to the thiosulfate sulfurtransferase rhodanese and create the active site containing a persulfurated catalytic cysteine (Cys-253) and an inhibitory sulfite coordinated by Arg-74 and Arg-185. A serine protease-like triad, comprising Asp-61, His-75, and Ser-255, is near Cys-253 and represents a conserved feature that distinguishes 3-mercaptopyruvate sulfurtransferases from thiosulfate sulfurtransferases. During catalysis, Ser-255 may polarize the carbonyl group of 3-mercaptopyruvate to assist thiophilic attack, whereas Arg-74 and Arg-185 bind the carboxylate group. The enzyme hydrolyzes benzoyl-Arg-p-nitroanilide, an activity that is sensitive to the presence of the serine protease inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone, which also lowers 3-mercaptopyruvate sulfurtransferase activity, presumably by interference with the contribution of Ser-255. The L. major 3-mercaptopyruvate sulfurtransferase is unusual with an 80-amino acid C-terminal domain, bearing remarkable structural similarity to the FK506-binding protein class of peptidylprolyl cis/trans-isomerase. This domain may be involved in mediating protein folding and sulfurtransferase-protein interactions