A Screening Pipeline for Antiparasitic Agents Targeting Cryptosporidium Inosine Monophosphate Dehydrogenase

Persistent diarrhea is a leading cause of illness and death among impoverished children, and a growing share of this disease burden can be attributed to the parasite Cryptosporidium. There are no vaccines to prevent Cryptosporidium infection, and the treatment options are limited and unreliable. Critically, no effective treatment exists for children or adults suffering from AIDS. Cryptosporidium presents many technical obstacles for drug discovery; perhaps the most important roadblock is the difficulty of monitoring drug action. Here we have developed a set of methods to accelerate the drug discovery process for cryptosporidiosis. We exploit the opportunities for experimental manipulation in the related parasite Toxoplasma to genetically engineer a Cryptosporidium model. This new model parasite mirrors the metabolism of Cryptosporidium for a particularly promising drug target that supplies the building blocks for DNA and RNA. Drug effectiveness can be assayed through simple fluorescence measurements for many candidates. Using this assay as an initial filter, and adapting other assays to a high throughput format, we identify several novel chemical compounds that exhibit markedly improved anti-cryptosporidial activity and excellent selectivity

The Cryptosporidium parvum Kinome

<p>Abstract</p> <p>Background</p> <p>Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the <it>Cryptosporidium parvum </it>kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.</p> <p>Results</p> <p>The <it>C</it>. <it>parvum </it>kinome comprises over 70 members, some of which may be promising drug targets. These <it>C. parvum </it>protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of <it>Cryptosporidium spp</it>. Comparison of specific kinases with their <it>Plasmodium falciparum </it>and <it>Toxoplasma gondii </it>orthologues revealed some distinct characteristics within the <it>C. parvum </it>kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening <it>Cp</it>CDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of <it>Cp</it>CDPK1. In addition, structural analysis of <it>Cp</it>CDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.</p> <p>Conclusions</p> <p>Identification and comparison of the <it>C. parvum </it>protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.</p

Net primary productivity estimates and environmental variables in the Arctic Ocean: An assessment of coupled physical-biogeochemical models

The relative skill of 21 regional and global biogeochemical models was assessed in terms of how well the models reproduced observed net primary productivity (NPP) and environmental variables such as nitrate concentration (NO3), mixed layer depth (MLD), euphotic layer depth (Zeu), and sea ice concentration, by comparing results against a newly updated, quality-controlled in situ NPP database for the Arctic Ocean (1959-2011). The models broadly captured the spatial features of integrated NPP (iNPP) on a pan-Arctic scale. Most models underestimated iNPP by varying degrees in spite of overestimating surface NO3, MLD, and Zeu throughout the regions. Among the models, iNPP exhibited little difference over sea ice condition (ice-free vs. ice-influenced) and bottom depth (shelf vs. deep ocean). The models performed relatively well for the most recent decade and towards the end of Arctic summer. In the Barents and Greenland Seas, regional model skill of surface NO3 was best associated with how well MLD was reproduced. . Regionally, iNPP was relatively well simulated in the Beaufort Sea and the central Arctic Basin, where in situ NPP is low and nutrients are mostly depleted. Models performed less well at simulating iNPP in the Greenland and Chukchi Seas, despite the higher model skill in MLD and sea ice concentration, respectively. iNPP model skill was constrained by different factors in different Arctic Ocean regions. Our study suggests that better parameterization of biological and ecological microbial rates (phytoplankton growth and zooplankton grazing) are needed for improved Arctic Ocean biogeochemical modeling

First mesocosm experiments to study the impacts of ocean acidification on plankton communities in the NW Mediterranean Sea (MedSeA project)

There is a growing international interest in studying the effects of ocean acidification on plankton communities that play a major role in the global carbon cycle and in the consumption of atmospheric CO2 via the so-called biological pump. Recently, several mesocosm experiments reported on the effect of ocean acidification on marine plankton communities, although the majority were performed in eutro- phic conditions or following nutrient addition. The objective of the present study was to perform two mesocosm experiments in the oligo- to meso-trophic Northwestern Mediterranean Sea during two seasons with contrasting environmental conditions: in summer 2012 in the Bay of Calvi (Corsica, France) and in winter 2013 in the Bay of Villefranche (France). This paper describes the objectives of these ex- periments, the study sites, the experimental set-up and the environmental and experimental conditions during the two experiments. The 20-day experiment in the Bay of Calvi was undoubtedly representative of summer conditions in the Northwestern Mediterranean Sea with low nutrient and chlorophyll a concentrations, warm waters and high surface solar irradiance. In contrast, the winter experiment, which was reduced to 12 days because of bad weather conditions, failed to reproduce the mesotrophic con- ditions typical of the wintertime in this area. Indeed, a rapid increase in phytoplankton biomass during the acidification phase led to a strong decrease in nitrate concentrations and an unrealistic N and P co- limitation at this period of the year. An overview of the 11 other papers related to this study and pub- lished in this special issue is provided.MEDiterranean Sea Acidification in a changing climat