Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput.

The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.Bill & Melinda Gates Foundation FAPES

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation - a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.publishersversionpublishe

Relationship between Genetic Variability and Land Use and Land Cover in Populations of Campomanesia adamantium (Myrtaceae)

Campomanesia adamantium is an endemic plant of Cerrado biome that has potential for cultivation because its fruits have culinary and medicinal uses. However, genetic diversity studies using molecular markers with Cerrado species are scarce, and the inadequate extractive exploitation of fruits and the expansion of agricultural frontiers may also affect genetic variability. Therefore, studies in this field are of interest as they can provide sources for conservation and breeding programs. In this context, we investigated the genetic diversity of native populations of C. adamantium from different sites and the relationship between genetic variability and the land use and land cover of each site. A total of 207 plants were sampled in seven sites and characterized with seven polymorphic microsatellite markers. The use and coverage of land were mapped based on aerial images, and the land was classified into different categories. The genetic diversity was high in all populations, with low levels of differentiation due to allele sharing, mainly in Mato Grosso do Sul and Paraguay populations. The geographically closest populations were more genetically similar. The use and coverage of land indicated that intense agriculture promotes a significant decrease in genetic variability