Development and validation of a hedgehog heparin-binding assay for high-throughput screening
Sonic hedgehog (Shh) is a morphogenic protein with critical roles in embryogenesis and the development of some cancers. Hence, identifying inhibitors of the Shh pathway is of great therapeutic value. Heparin and HSPGs act as crucial modulators of Shh activity. To identify molecules that antagonize S...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-01-01
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| Series: | MethodsX |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215016120304271 |
| _version_ | 1818968860982771712 |
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| author | David R. Lamson Mark A. Hughes Audrey F. Adcock Ginger R. Smith Kevin P. Williams |
| author_facet | David R. Lamson Mark A. Hughes Audrey F. Adcock Ginger R. Smith Kevin P. Williams |
| author_sort | David R. Lamson |
| collection | DOAJ |
| description | Sonic hedgehog (Shh) is a morphogenic protein with critical roles in embryogenesis and the development of some cancers. Hence, identifying inhibitors of the Shh pathway is of great therapeutic value. Heparin and HSPGs act as crucial modulators of Shh activity. To identify molecules that antagonize Shh binding to heparin we have developed a solid-phase plate-based assay. The N-terminal domain of Shh (ShhN) protein is first coated in 384-well plates and the direct binding of fluorescein-labeled heparin (flu-heparin) assessed by measuring the fluorescence signal after incubation and wash steps. Binding of ShhN protein to the 384-well plates was confirmed and optimized by a standard ELISA using a monoclonal antibody recognizing folded ShhN. The assay was validated using whole plate minimum and maximum signal wells with a Z’ of 0.68–0.75 determined. Herein, we describe the development and validation of a high throughput screen to identify small molecule antagonists of Shh heparin binding.Overall, this method • Results in an optimized and validated assay for hedgehog heparin binding. • Delivers a cost effective high-throughput screen format for hedgehog heparin antagonist screening |
| first_indexed | 2024-12-20T14:11:25Z |
| format | Article |
| id | doaj.art-b7668e3874d54d878013dd3217d4b70a |
| institution | Directory Open Access Journal |
| issn | 2215-0161 |
| language | English |
| last_indexed | 2024-12-20T14:11:25Z |
| publishDate | 2021-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | MethodsX |
| spelling | doaj.art-b7668e3874d54d878013dd3217d4b70a2022-12-21T19:38:07ZengElsevierMethodsX2215-01612021-01-018101207Development and validation of a hedgehog heparin-binding assay for high-throughput screeningDavid R. Lamson0Mark A. Hughes1Audrey F. Adcock2Ginger R. Smith3Kevin P. Williams4Biomanufacturing Research Institute and Technology Enterprise, United StatesBiomanufacturing Research Institute and Technology Enterprise, United StatesBiomanufacturing Research Institute and Technology Enterprise, United StatesBiomanufacturing Research Institute and Technology Enterprise, United StatesBiomanufacturing Research Institute and Technology Enterprise, United States; Department of Pharmaceutical Sciences, North Carolina Central University, Durham, NC 27707, United States; Corresponding author at: Biomanufacturing Research Institute and Technology Enterprise, United States.Sonic hedgehog (Shh) is a morphogenic protein with critical roles in embryogenesis and the development of some cancers. Hence, identifying inhibitors of the Shh pathway is of great therapeutic value. Heparin and HSPGs act as crucial modulators of Shh activity. To identify molecules that antagonize Shh binding to heparin we have developed a solid-phase plate-based assay. The N-terminal domain of Shh (ShhN) protein is first coated in 384-well plates and the direct binding of fluorescein-labeled heparin (flu-heparin) assessed by measuring the fluorescence signal after incubation and wash steps. Binding of ShhN protein to the 384-well plates was confirmed and optimized by a standard ELISA using a monoclonal antibody recognizing folded ShhN. The assay was validated using whole plate minimum and maximum signal wells with a Z’ of 0.68–0.75 determined. Herein, we describe the development and validation of a high throughput screen to identify small molecule antagonists of Shh heparin binding.Overall, this method • Results in an optimized and validated assay for hedgehog heparin binding. • Delivers a cost effective high-throughput screen format for hedgehog heparin antagonist screeninghttp://www.sciencedirect.com/science/article/pii/S2215016120304271Hedgehog heparin HTS assay |
| spellingShingle | David R. Lamson Mark A. Hughes Audrey F. Adcock Ginger R. Smith Kevin P. Williams Development and validation of a hedgehog heparin-binding assay for high-throughput screening MethodsX Hedgehog heparin HTS assay |
| title | Development and validation of a hedgehog heparin-binding assay for high-throughput screening |
| title_full | Development and validation of a hedgehog heparin-binding assay for high-throughput screening |
| title_fullStr | Development and validation of a hedgehog heparin-binding assay for high-throughput screening |
| title_full_unstemmed | Development and validation of a hedgehog heparin-binding assay for high-throughput screening |
| title_short | Development and validation of a hedgehog heparin-binding assay for high-throughput screening |
| title_sort | development and validation of a hedgehog heparin binding assay for high throughput screening |
| topic | Hedgehog heparin HTS assay |
| url | http://www.sciencedirect.com/science/article/pii/S2215016120304271 |
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