Development of a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) for high-throughput screening of KRASG12C inhibitors
KRAS, the most frequently mutated oncogene in human cancers, was considered “undruggable” until the identification of small molecules that bind irreversibly to the mutant reactive cysteine at residue 12. Despite the encouraging anticancer activity of KRASG12C inhibitors in clinical trials, identific...
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| Format: | Article |
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Elsevier
2022-03-01
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| Series: | SLAS Discovery |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2472555222000053 |
| _version_ | 1811304775104331776 |
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| author | Shuang Liu Junfen Shi Hongjuan Li Jijun Li Yan Zhu Binghui Li Yinghui Sun |
| author_facet | Shuang Liu Junfen Shi Hongjuan Li Jijun Li Yan Zhu Binghui Li Yinghui Sun |
| author_sort | Shuang Liu |
| collection | DOAJ |
| description | KRAS, the most frequently mutated oncogene in human cancers, was considered “undruggable” until the identification of small molecules that bind irreversibly to the mutant reactive cysteine at residue 12. Despite the encouraging anticancer activity of KRASG12C inhibitors in clinical trials, identification of more potent drugs is expected to achieve the maximal clinical benefit, which is hindered by the low sensitivity or throughput of current biochemical approaches. To overcome these limitations, a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) based on the competitive interaction of biotin-labeled probe and the test compound with KRASG12C was developed. Compared with reported assays, less protein was used in BA-ELISA, which significantly improves the resolution of inhibitor potency, thus contributing to the identification of highly potent inhibitors. Furthermore, BA-ELISA can also be expanded to determine the cellular potency of the inhibitors using KRASG12C mutant living cells. Using three previously disclosed compounds, ARS-1620, AMG 510, and MRTX849, we demonstrated that BA-ELISA is a highly sensitive, specific, and robust method for high-throughput screening of KRASG12C inhibitors. |
| first_indexed | 2024-04-13T08:13:31Z |
| format | Article |
| id | doaj.art-55f6d9221cc340e28adac8eace267cf5 |
| institution | Directory Open Access Journal |
| issn | 2472-5552 |
| language | English |
| last_indexed | 2024-04-13T08:13:31Z |
| publishDate | 2022-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | SLAS Discovery |
| spelling | doaj.art-55f6d9221cc340e28adac8eace267cf52022-12-22T02:54:53ZengElsevierSLAS Discovery2472-55522022-03-01272107113Development of a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) for high-throughput screening of KRASG12C inhibitorsShuang Liu0Junfen Shi1Hongjuan Li2Jijun Li3Yan Zhu4Binghui Li5Yinghui Sun6Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing, China; Discovery Biology Department, Shouyao Holdings (Beijing) Co., Ltd., Beijing, ChinaDiscovery Biology Department, Shouyao Holdings (Beijing) Co., Ltd., Beijing, ChinaDiscovery Biology Department, Shouyao Holdings (Beijing) Co., Ltd., Beijing, ChinaMedicinal Chemistry Department, Shouyao Holdings (Beijing) Co., Ltd., Beijing, ChinaMedicinal Chemistry Department, Shouyao Holdings (Beijing) Co., Ltd., Beijing, ChinaDepartment of Biochemistry and Molecular Biology, Capital Medical University, Beijing, China; Corresponding author at Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, China.Discovery Biology Department, Shouyao Holdings (Beijing) Co., Ltd., Beijing, China; Corresponding author at. Discovery Biology Department, Shouyao Holdings (Beijing) Co., Ltd., Beijing 100195, China.KRAS, the most frequently mutated oncogene in human cancers, was considered “undruggable” until the identification of small molecules that bind irreversibly to the mutant reactive cysteine at residue 12. Despite the encouraging anticancer activity of KRASG12C inhibitors in clinical trials, identification of more potent drugs is expected to achieve the maximal clinical benefit, which is hindered by the low sensitivity or throughput of current biochemical approaches. To overcome these limitations, a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) based on the competitive interaction of biotin-labeled probe and the test compound with KRASG12C was developed. Compared with reported assays, less protein was used in BA-ELISA, which significantly improves the resolution of inhibitor potency, thus contributing to the identification of highly potent inhibitors. Furthermore, BA-ELISA can also be expanded to determine the cellular potency of the inhibitors using KRASG12C mutant living cells. Using three previously disclosed compounds, ARS-1620, AMG 510, and MRTX849, we demonstrated that BA-ELISA is a highly sensitive, specific, and robust method for high-throughput screening of KRASG12C inhibitors.http://www.sciencedirect.com/science/article/pii/S2472555222000053BA-ELISAKRASG12C inhibitorsHigh-throughput screeningCancer drug discovery |
| spellingShingle | Shuang Liu Junfen Shi Hongjuan Li Jijun Li Yan Zhu Binghui Li Yinghui Sun Development of a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) for high-throughput screening of KRASG12C inhibitors SLAS Discovery BA-ELISA KRASG12C inhibitors High-throughput screening Cancer drug discovery |
| title | Development of a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) for high-throughput screening of KRASG12C inhibitors |
| title_full | Development of a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) for high-throughput screening of KRASG12C inhibitors |
| title_fullStr | Development of a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) for high-throughput screening of KRASG12C inhibitors |
| title_full_unstemmed | Development of a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) for high-throughput screening of KRASG12C inhibitors |
| title_short | Development of a biotin–streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) for high-throughput screening of KRASG12C inhibitors |
| title_sort | development of a biotin streptavidin enhanced enzyme linked immunosorbent assay ba elisa for high throughput screening of krasg12c inhibitors |
| topic | BA-ELISA KRASG12C inhibitors High-throughput screening Cancer drug discovery |
| url | http://www.sciencedirect.com/science/article/pii/S2472555222000053 |
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