Polycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is... Show morePolycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is not well tolerated by all patients and there remains a strong need for alternative treatments. The signalling rewiring in PKD that drives cyst formation is highly complex and not fully understood. As a consequence, the effects of drugs are sometimes difficult to predict. We previously established a high-throughput microscopy phenotypic screening method for quantitative assessment of renal cyst growth. Here, we applied this 3D cyst growth phenotypic assay and screened 2320 small drug-like molecules, including approved drugs. We identified 81 active molecules that inhibit cyst growth. Multi-parametric phenotypic profiling of the effects on 3D cultured cysts discriminated molecules that showed preferred pharmacological effects above genuine toxicological properties. Celastrol, a triterpenoid from Tripterygium wilfordii, was identified as a potent inhibitor of cyst growth in vitro. In an in vivo iKspCre-Pkd1(lox,lox) mouse model for PKD, celastrol inhibited the growth of renal cysts and maintained kidney function. Show less
Polycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is... Show morePolycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is not well tolerated by all patients and there remains a strong need for alternative treatments. The signaling rewiring in PKD that drives cyst formation is highly complex and not fully understood. As a consequence, the effects of drugs are sometimes difficult to predict. We previously established a high throughput microscopy phenotypic screening method for quantitative assessment of renal cyst growth. Here, we applied this 3D cyst growth phenotypic assay and screened 2320 small drug-like molecules, including approved drugs. We identified 81 active molecules that inhibit cyst growth. Multi-parametric phenotypic profiling of the effects on 3D cultured cysts discriminated molecules that showed preferred pharmacological effects above genuine toxicological properties. Celastrol, a triterpenoid from Tripterygium Wilfordii, was identified as a potent inhibitor of cyst growth in vitro. In an in vivo iKspCre-Pkd1lox,lox mouse model for PKD, celastrol inhibited the growth of renal cysts and maintained kidney function. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes,... Show more3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting. Show less