ChemInform Abstract: Fragment Screening by Native State Mass Spectrometry

ChemInform ◽  
2014 ◽  
Vol 45 (7) ◽  
pp. no-no
Author(s):  
Sally-Ann Poulsen
Keyword(s):  
Mass Spectrometry ◽  
Native State ◽  
Fragment Screening

scholarly journals Fragment Screening by Native State Mass Spectrometry

2013 ◽  
Vol 66 (12) ◽  
pp. 1495 ◽  
Author(s):  
Sally-Ann Poulsen
Keyword(s):  
Mass Spectrometry ◽  
Screening Method ◽  
Native State ◽  
Fragment Screening ◽  
Protein Ligand Interactions ◽  
Adoption And Implementation ◽  
High Throughput Method ◽  
Ligand Interactions ◽  
Complementary Fragment ◽  
Fragment Based Drug Discovery

Native state mass spectrometry (MS) has been recognised as a rapid, sensitive, and high throughput method to directly investigate protein-ligand interactions for some time, however there are few examples reporting this approach as a screening method to identify relevant protein–fragment interactions in fragment-based drug discovery (FBDD). In this paper an overview of native state MS will be presented, highlighting the attractive properties of this method within the context of fragment screening applications. A summary of published examples using MS for fragment screening will be described and reflection on the outlook for the future adoption and implementation of native state MS as a complementary fragment screening method will be presented.

Covalent-Fragment Screening of Brd4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

2019 ◽  
Author(s):  
Michael Olp ◽  
Daniel Sprague ◽  
Stefan Kathman ◽  
Ziyang Xu ◽  
Alexandar Statsyuk ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Site ◽  
Binding Sites ◽  
Computational Prediction ◽  
Chemical Probes ◽  
Computational Docking ◽  
Fragment Screening ◽  
Covalent Inhibitors ◽  
Bet Protein ◽  
Proof Of Principle

<p>Brd4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly-conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to non-homologous cysteine residues within the <i>C</i>-terminal Brd4 bromodomain (Brd4-BD2), we performed a mid-throughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify Brd4. Subsequent mass spectrometry, NMR and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to Brd4 among all human bromodomains. This site is orthogonal to the Brd4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays. Finally, we tethered covalent fragments to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace Brd4 from chromatin.</p>

scholarly journals Automated in-line gel filtration for native state mass spectrometry

2008 ◽  
Vol 19 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Greg M. Waitt ◽  
Robert Xu ◽  
G. Bruce Wisely ◽  
Jon D. Williams
Keyword(s):  
Mass Spectrometry ◽  
Gel Filtration ◽  
Native State

scholarly journals Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alice Douangamath ◽  
Daren Fearon ◽  
Paul Gehrtz ◽  
Tobias Krojer ◽  
Petra Lukacik ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Design ◽  
Active Site ◽  
Large Scale ◽  
Structure Based Drug Design ◽  
Dimer Interface ◽  
X Ray ◽  
Fragment Screening ◽  
Viral Proteases ◽  
Main Protease

Abstract COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

Covalent-Fragment Screening of Brd4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

2019 ◽  
Author(s):  
Michael Olp ◽  
Daniel Sprague ◽  
Stefan Kathman ◽  
Ziyang Xu ◽  
Alexandar Statsyuk ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Site ◽  
Binding Sites ◽  
Computational Prediction ◽  
Chemical Probes ◽  
Computational Docking ◽  
Fragment Screening ◽  
Covalent Inhibitors ◽  
Bet Protein ◽  
Proof Of Principle

<p>Brd4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly-conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to non-homologous cysteine residues within the <i>C</i>-terminal Brd4 bromodomain (Brd4-BD2), we performed a mid-throughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify Brd4. Subsequent mass spectrometry, NMR and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to Brd4 among all human bromodomains. This site is orthogonal to the Brd4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays. Finally, we tethered covalent fragments to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace Brd4 from chromatin.</p>

scholarly journals Pulse labeling reveals the tail end of protein folding by proteome profiling

2021 ◽  
Author(s):  
Mang Zhu ◽  
Erich R. Kuechler ◽  
Nikolay Stoynov ◽  
Joerg Gsponer ◽  
Thibault Mayor
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Heat Stress ◽  
Protein Complexes ◽  
Protein Sequences ◽  
Protein Homeostasis ◽  
Native State ◽  
Binding Motifs ◽  
Specific Subset ◽  
Β Sheet

SummaryAccurate and efficient folding of nascent protein sequences into their native state requires support from the protein homeostasis network. Herein we probed which newly translated proteins are less thermostable to infer which polypeptides require more time to fold within the proteome. Specifically, we determined which of these proteins were more susceptible to misfolding and aggregation under heat stress using pulse SILAC coupled mass spectrometry. These proteins are abundant, short, and highly structured. Notably these proteins display a tendency to form β-sheet structures, a configuration which typically requires more time for folding, and were enriched for Hsp70/Ssb and TRiC/CCT binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides were also more often components of stable protein complexes in comparison to other proteins. All evidence combined suggests that a specific subset of newly translated proteins requires more time following synthesis to reach a thermostable native state in the cell.

Application of Native-State Electrospray Mass Spectrometry to Identify Zinc-Binding Sites on Engineered Hemoglobin

2000 ◽  
Vol 284 (2) ◽  
pp. 247-255 ◽  
Author(s):  
Julie Lippincott ◽  
Timothy J. Fattor ◽  
Douglas D. Lemon ◽  
Izydor Apostol
Keyword(s):  
Mass Spectrometry ◽  
Binding Sites ◽  
Electrospray Mass Spectrometry ◽  
Zinc Binding ◽  
Native State

A Review on Computational Approaches for Analyzing HydrogenDeuterium (H/D) Exchange of Proteins

2020 ◽  
Vol 27 ◽  
Author(s):  
Thirunavukkarasu Sivaraman
Keyword(s):  
Mass Spectrometry ◽  
Residue Level ◽  
Level Fluctuation ◽  
Native State ◽  
Huge Amount ◽  
Exchange Method ◽  
Computational Tools ◽  
Computational Approaches ◽  
Hydrogen Deuterium ◽  
Exchange Data

: Native state Hydrogen-Deuterium (H/D) exchange method has been used to study the structures and the unfolding pathways for quite a number of proteins. The H/D exchange method is generally monitored using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) techniques. NMR-assisted H/D exchange methods primarily monitor the residue level fluctuation of proteins, whereas MS-assisted H/D exchange methods analyze multifold ensemble conformations of proteins. In this connection, quite a large number of computational tools and algorithms have been developed for processing and analyzing huge amount of the H/D exchange data generated from these techniques. In this review, most of the freely available computational tools associated with the H/D exchange of proteins have been comprehensively reviewed and scopes to improve/develop novel computational approaches for analyzing the H/D exchange data of proteins have also been brought into fore.

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