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
Dauwerse, J.G.; Belzen, M. van; Haeringen, A. van; Santen, G. van; Lans, C. van de; Rahikkala, E.; ... ; Peters, D. 2016
We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional... Show moreWe identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both alleles of POLR1C in three individuals with TCS. These findings identify two additional genes involved in TCS, confirm the genetic heterogeneity of TCS and support the hypothesis that TCS is a ribosomopathy. Show less
Dauwerse, J.G.; Dixon, J.; Seland, S.; Ruivenkamp, C.A.L.; Haeringen, A. van; Hoefsloot, L.H.; ... ; Wieczorek, D. 2011
Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy in adults that is foremost characterized by progressive wasting of muscles in the upper body. FSHD is associated... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy in adults that is foremost characterized by progressive wasting of muscles in the upper body. FSHD is associated with contraction of D4Z4 macrosatellite repeats on chromosome 4q35, but this contraction is pathogenic only in certain "permissive" chromosomal backgrounds. Here, we show that FSHD patients carry specific single-nucleotide polymorphisms in the chromosomal region distal to the last D4Z4 repeat. This FSHD-predisposing configuration creates a canonical polyadenylation signal for transcripts derived from DUX4, a double homeobox gene of unknown function that straddles the last repeat unit and the adjacent sequence. Transfection studies revealed that DUX4 transcripts are efficiently polyadenylated and are more stable when expressed from permissive chromosomes. These findings suggest that FSHD arises through a toxic gain of function attributable to the stabilized distal DUX4 transcript. Show less
Lemmers, R.J.L.F.; Vliet, P.J. van der; Klooster, R.; Sacconi, S.; Camano, P.; Dauwerse, J.G.; ... ; Maarel, S.M. van der 2010
Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy in adults that is foremost characterized by progressive wasting of muscles in the upper body. FSHD is associated... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy in adults that is foremost characterized by progressive wasting of muscles in the upper body. FSHD is associated with contraction of D4Z4 macrosatellite repeats on chromosome 4q35, but this contraction is pathogenic only in certain "permissive" chromosomal backgrounds. Here, we show that FSHD patients carry specific single-nucleotide polymorphisms in the chromosomal region distal to the last D4Z4 repeat. This FSHD-predisposing configuration creates a canonical polyadenylation signal for transcripts derived from DUX4, a double homeobox gene of unknown function that straddles the last repeat unit and the adjacent sequence. Transfection studies revealed that DUX4 transcripts are efficiently polyadenylated and are more stable when expressed from permissive chromosomes. These findings suggest that FSHD arises through a toxic gain of function attributable to the stabilized distal DUX4 transcript. Show less