Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes

Yogo Sakakibara; Christine S. Chow

doi:10.1039/c7ob02147j

Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes

Organic & Biomolecular Chemistry ◽

10.1039/c7ob02147j ◽

2017 ◽

Vol 15 (40) ◽

pp. 8535-8543 ◽

Cited By ~ 2

Author(s):

Yogo Sakakibara ◽

Christine S. Chow

Keyword(s):

Chemical Probes ◽

Conformational States ◽

Helix 69

Pseudouridine modifications in helix 69 of bacterial ribosomes impact aminoglycoside interactions by altering the RNA conformational states and accessibility to chemical probes.

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Probing Conformational States of Modified Helix 69 in 50S Ribosomes

Journal of the American Chemical Society ◽

10.1021/ja2005658 ◽

2011 ◽

Vol 133 (22) ◽

pp. 8396-8399 ◽

Cited By ~ 20

Author(s):

Yogo Sakakibara ◽

Christine S. Chow

Keyword(s):

Conformational States ◽

Helix 69

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The Discovery and Utility of Chemical Probes in Target Discovery

10.1039/9781839160745 ◽

2020 ◽

Keyword(s):

Chemical Probes ◽

Target Discovery

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Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra Terminal Domain

10.26434/chemrxiv.12026952 ◽

2020 ◽

Author(s):

Luke Adams ◽

Lorna E. Wilkinson-White ◽

Menachem J. Gunzburg ◽

Stephen J. Headey ◽

Martin J. Scanlon ◽

...

Keyword(s):

Binding Site ◽

Systematic Approach ◽

Structural Information ◽

Peptide Binding ◽

Chemical Diversity ◽

Chemical Probes ◽

Protein Targets ◽

Structure Activity ◽

Peptide Binding Site ◽

Selection Of

The development of low-affinity fragment hits into higher affinity leads is a major hurdle in fragment-based drug design. Here we demonstrate an approach for the Rapid Elaboration of Fragments into Leads (REFiL) applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. Upon completion of a fragment screen against Bromodomain-3 extra terminal (BRD3-ET) domain we applied the REFiL workflow, which allowed us to develop a series of tetrahydrocarbazole ligands that bind to the peptide binding site of BRD3-ET. With REFiL we were able to rapidly improve binding affinity >30-fold. The REFiL workflow can be applied readily to a broad range of protein targets without the need of a structure, allowing the efficient evolution of low-affinity fragments into higher affinity leads and chemical probes.

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

10.26434/chemrxiv.8859098 ◽

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

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 C-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.

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