Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening
High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-neg...
| Main Authors: | , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2020
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| Online Access: | https://hdl.handle.net/1721.1/126269 |
| _version_ | 1826206626440282112 |
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| author | Seebald, Leah Madec, Amael Imperiali, Barbara |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Seebald, Leah Madec, Amael Imperiali, Barbara |
| author_sort | Seebald, Leah |
| collection | MIT |
| description | High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a KD value of 7.2 μm with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches. |
| first_indexed | 2024-09-23T13:36:03Z |
| format | Article |
| id | mit-1721.1/126269 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T13:36:03Z |
| publishDate | 2020 |
| publisher | Wiley |
| record_format | dspace |
| spelling | mit-1721.1/1262692022-09-28T14:56:51Z Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening Seebald, Leah Madec, Amael Imperiali, Barbara Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a KD value of 7.2 μm with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches. National Institutes of Health (Grants GM097241, GM131627) 2020-07-20T21:12:32Z 2020-07-20T21:12:32Z 2019-11 2020-07-16T17:56:43Z Article http://purl.org/eprint/type/JournalArticle 1439-4227 1439-7633 https://hdl.handle.net/1721.1/126269 Seebald, Leah et al. "Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening." ChemBioChem 21, 1-2 (January 2020): 108-112 © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim en http://dx.doi.org/10.1002/cbic.201900671 ChemBioChem Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Wiley Prof. Imperiali via Courtney Crummett |
| spellingShingle | Seebald, Leah Madec, Amael Imperiali, Barbara Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening |
| title | Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening |
| title_full | Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening |
| title_fullStr | Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening |
| title_full_unstemmed | Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening |
| title_short | Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening |
| title_sort | deploying fluorescent nucleoside analogues for high throughput inhibitor screening |
| url | https://hdl.handle.net/1721.1/126269 |
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