Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors
Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of...
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Elsevier
2024-12-01
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| Series: | SLAS Discovery |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2472555224000601 |
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| author | Scott B. Ficarro Zachary H. Marto Nicholas M. Girardi Dingyu Deng Isabella Jaen Maisonet Guillaume Adelmant Laura E. Fleming Mona Sharafi Isidoro Tavares Andrew Zhao HyoJeon Kim Hyuk-Soo Seo Sirano Dhe-Paganon Sara J. Buhrlage Jarrod A. Marto |
| author_facet | Scott B. Ficarro Zachary H. Marto Nicholas M. Girardi Dingyu Deng Isabella Jaen Maisonet Guillaume Adelmant Laura E. Fleming Mona Sharafi Isidoro Tavares Andrew Zhao HyoJeon Kim Hyuk-Soo Seo Sirano Dhe-Paganon Sara J. Buhrlage Jarrod A. Marto |
| author_sort | Scott B. Ficarro |
| collection | DOAJ |
| description | Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple Python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC50 < 5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling. |
| format | Article |
| id | doaj-art-4332138746db43fda20fad84946e548d |
| institution | OA Journals |
| issn | 2472-5552 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | SLAS Discovery |
| spelling | doaj-art-4332138746db43fda20fad84946e548d2025-08-20T01:58:27ZengElsevierSLAS Discovery2472-55522024-12-0129810019810.1016/j.slasd.2024.100198Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitorsScott B. Ficarro0Zachary H. Marto1Nicholas M. Girardi2Dingyu Deng3Isabella Jaen Maisonet4Guillaume Adelmant5Laura E. Fleming6Mona Sharafi7Isidoro Tavares8Andrew Zhao9HyoJeon Kim10Hyuk-Soo Seo11Sirano Dhe-Paganon12Sara J. Buhrlage13Jarrod A. Marto14Department of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Emergent Drug Targets, Dana-Farber Cancer Institute, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Emergent Drug Targets, Dana-Farber Cancer Institute, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USADepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USADepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USADepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA; Center for Emergent Drug Targets, Dana-Farber Cancer Institute, Boston, MA, USADepartment of Cancer Biology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Emergent Drug Targets, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA; Corresponding author at: Department of Cancer Biology, Dana-Farber Cancer Institute, 360 Longwood Avenue, LC 2205, Boston, MA 02215-5450, USA.Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple Python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC50 < 5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling.http://www.sciencedirect.com/science/article/pii/S2472555224000601Deubiquitinating enzymesUCHL1Open-source hardware and softwareCovalent fragment screeningElectrophilic fragmentsIntact protein LC–MS screening |
| spellingShingle | Scott B. Ficarro Zachary H. Marto Nicholas M. Girardi Dingyu Deng Isabella Jaen Maisonet Guillaume Adelmant Laura E. Fleming Mona Sharafi Isidoro Tavares Andrew Zhao HyoJeon Kim Hyuk-Soo Seo Sirano Dhe-Paganon Sara J. Buhrlage Jarrod A. Marto Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors SLAS Discovery Deubiquitinating enzymes UCHL1 Open-source hardware and software Covalent fragment screening Electrophilic fragments Intact protein LC–MS screening |
| title | Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors |
| title_full | Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors |
| title_fullStr | Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors |
| title_full_unstemmed | Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors |
| title_short | Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors |
| title_sort | open source electrophilic fragment screening platform to identify chemical starting points for uchl1 covalent inhibitors |
| topic | Deubiquitinating enzymes UCHL1 Open-source hardware and software Covalent fragment screening Electrophilic fragments Intact protein LC–MS screening |
| url | http://www.sciencedirect.com/science/article/pii/S2472555224000601 |
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