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Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery.
BACKGROUND:Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel. METHODS:Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD+/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis. RESULTS:For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline. CONCLUSIONS:The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified
Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway
In a previous study, the Schistosoma mansoni Rho1 protein was able to
complement Rho1 null mutant Saccharomyces cerevisiae cells at
restrictive temperatures and under osmotic stress (low calcium
concentration) better than the human homologue (RhoA). It is known that
under osmotic stress, the S. cerevisiae Rho1 triggers two distinct
pathways: activation of the membrane 1,3-b-glucan synthase enzymatic
complex and activation of the protein kinase C1 signal transduction
pathway, promoting the transcription of response genes. In the present
work the SmRho1 protein and its mutants smrhoE97P, smrho1L101T, and
smrhoE97P, L101T were used to try to clarify the basis for the
differential complementation of Rho1 knockout yeast strain by the human
and S. mansoni genes. Experiments of functional complementation in the
presence of caffeine and in the presence of the osmotic regulator
sorbitol were conducted. SmRho1 and its mutants showed a differential
complementation of the yeast cells in the presence of caffeine, since
smrhoE97P and smrhoE97P, L101T mutants showed a delay in the growth
when compared to the yeast complemented with the wild type SmRho1.
However, in the presence of sorbitol and caffeine the wild type SmRho1
and mutants showed a similar complementation phenotype, as they allowed
yeast growth in all caffeine concentrations tested