A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation

Chen, Yuanyuan; Tang, Hong; Seibel, William; Papoian, Ruben; Li, Xiaoyü; Lambert, Nevin A.; Palczewski, Krzysztof

doi:10.1167/iovs.14-16298

Cited by 17 publications

(51 citation statements)

References 90 publications

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“…NIH3T3 (WTopsin/GFP), NIH3T3 (P23Hopsin/GFP) and NIH3T3 (GFP) stable cell lines were generated by viral infection with pMiLRO, pMiLRO23 and pMXs-IG constructs, as previously published [19]. Mouse opsin or P23H opsin was co-expressed with GFP.…”

Section: Methodsmentioning

confidence: 99%

“…Immunostaining and fluorescence imaging followed published procedures [19]. Briefly, cells were seeded in a 384-well plate at 5,000 cells/well on day 1.…”

Section: Methodsmentioning

confidence: 99%

“…Compound 1 (an isoquinoline-2(3 H )-hexanamide) and compound 2 (4-(5-chlorothiophen-2-yl)furan-2(5H)-one) were selected by a cell-based HTS of small molecules from the University of Cincinnati 2.5 K Diversity Set of Small-Molecules Library [19]. To identify small molecular compounds which rescue the P23H opsin mutant from ER retention, we performed a HTS with a β-galactosidase fragment complementation assay as described in reference [19].…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively, increasing the degradation of mutant opsin and leaving the WT rhodopsin allele to preserve visual function could be an equally viable therapeutic strategy for adRP. Thus, we developed and performed two sets of cell-based, small-molecule high-throughput screens (HTSs) to identify compounds that either improve the stability of the P23H opsin mutant or enhance its degradation [19]. Even though a valid photoreceptor cell line is unavailable, mammalian cells have been commonly used to study the biosynthesis of rhodopsin and to screen for drug candidates because the pre-ciliary biosynthesis of rhodopsin is generally shared by mammalian cells and rod photoreceptor cells [14, 20, 21].…”

Section: Introductionmentioning

confidence: 99%

“…Even though a valid photoreceptor cell line is unavailable, mammalian cells have been commonly used to study the biosynthesis of rhodopsin and to screen for drug candidates because the pre-ciliary biosynthesis of rhodopsin is generally shared by mammalian cells and rod photoreceptor cells [14, 20, 21]. In mammalian cell cultures, heterologously expressed WT opsin is located on the plasma membrane whereas P23H opsin accumulates in the endoplasmic reticulum (ER) due to its structural instability [14-16, 19-21]. …”

Section: Introductionmentioning

confidence: 99%

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Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa

Chen

Brooks

Gieser

et al. 2017

Pharmacological Research

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Mammalian cells are commonly employed in screening assays to identify active compounds that could potentially affect the progression of different human diseases including retinitis pigmentosa (RP), a class of inherited diseases causing retinal degeneration with compromised vision. Using transcriptome analysis, we compared NIH3T3 cells expressing wildtype (WT) rod opsin with a retinal disease-causing single P23H mutation. Surprisingly, heterologous expression of WT opsin in NIH3T3 cells caused more than a 2-fold change in 783 out of 16,888 protein coding transcripts. The perturbed genes encoded extracellular matrix proteins, growth factors, cytoskeleton proteins, glycoproteins and metalloproteases involved in cell adhesion, morphology and migration. A different set of 347 transcripts was either up- or down-regulated when the P23H mutant opsin was expressed suggesting an altered molecular perturbation compared to WT opsin. Transcriptome perturbations elicited by drug candidates aimed at mitigating the effects of the mutant protein revealed that different drugs targeted distinct molecular pathways that resulted in a similar phenotype selected by a cell-based high-throughput screen. Thus, transcriptome profiling can provide essential information about the therapeutic potential of a candidate drug to restore normal gene expression in pathological conditions.

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Section: Methodsmentioning

confidence: 99%

“…Immunostaining and fluorescence imaging followed published procedures [19]. Briefly, cells were seeded in a 384-well plate at 5,000 cells/well on day 1.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa

Chen

Brooks

Gieser

et al. 2017

Pharmacological Research

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Rhodopsin Oligomerization and Aggregation

Park

2019

J Membrane Biol

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Misfolded rhodopsin mutants display variable aggregation properties

Gragg

Park

2018

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental.

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A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation

Cited by 17 publications

References 90 publications

Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa

Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa

Rhodopsin Oligomerization and Aggregation

Misfolded rhodopsin mutants display variable aggregation properties

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