In vitro screening model for compound interactions with human and dairy animal BCRP orthologs.

Orthologs of breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) efflux transmembrane transporter, are present in several species. The list of compounds known to interact with BCRP is growing, and many questions remain concerning species-specific variations in substrate specificity and affinity and the potency of inhibitors. As the most abundant efflux transporter known to be present in the blood-milk barrier, BCRP can increase the elimination of certain xenobiotics to milk, posing a risk for suckling offspring and dairy product consumers. Here we developed a model that can be employed to investigate species-specific differences between BCRP substrates and inhibitors. Membrane vesicles were isolated from transiently transduced human embryonic kidney (HEK) 293 cells, overexpressing BCRP, with human, bovine, caprine, and ovine cDNA sequences. To confirm BCRP transport activity in the transduced cells, D-luciferin efflux was measured and to confirm transport activity in the membrane vesicles, [3H] estrone-3-sulfate ([3H]E1S) influx was measured. We also determined the Michaelis-Menten constant (Km) and Vmax of [3H]E1S for each species. We have developed an in vitro transport model to study differences in compound interactions with BCRP orthologs from milk-producing animal species and humans. BCRP transport activity was demonstrated in the species-specific transduced cells by a reduced accumulation of D-luciferin compared with the control cells, indicating BCRP-mediated efflux of D-luciferin. Functionality of the membrane vesicle model was demonstrated by confirming ATP-dependent transport and by quantifying the kinetic parameters, Km and Vmax for the model substrate [3H]E1S. The values were not significantly different between species for the model substrates tested. This model can be insightful for appropriate inter-species extrapolations and risk assessments of xenobiotics in lactating woman and dairy animals.

Pyruvate dehydrogenase is a potential mitochondrial off-target for gentamicin based on in silico predictions and in vitro inhibition studies.

During the drug development process, organ toxicity leads to an estimated failure of one-third of novel chemical entities. Drug-induced toxicity is increasingly associated with mitochondrial dysfunction, but identifying the underlying molecular mechanisms remains a challenge. Computational modeling techniques have proven to be a good tool in searching for drug off-targets. Here, we aimed to identify mitochondrial off-targets of the nephrotoxic drugs tenofovir and gentamicin using different in silico approaches (KRIPO, ProBis and PDID). Dihydroorotate dehydrogenase (DHODH) and pyruvate dehydrogenase (PDH) were predicted as potential novel off-target sites for tenofovir and gentamicin, respectively. The predicted targets were evaluated in vitro, using (colorimetric) enzymatic activity measurements. Tenofovir did not inhibit DHODH activity, while gentamicin potently reduced PDH activity. In conclusion, the use of in silico methods appeared a valuable approach in predicting PDH as a mitochondrial off-target of gentamicin. Further research is required to investigate the contribution of PDH inhibition to overall renal toxicity of gentamicin.