2H-Azirine-Based Reagents for Chemoselective Bioconjugation at Carboxyl Residues Inside Live Cells

Ma, Nan; Hu, Jun; Zhang, Zhimin; Liu, Wenyan; Huang, Ming; Fan, Youlong; Yin, Xinhua; Wang, Jigang; Ding, Ke; Ye, Wen‐Cai; Li, Zhengqiu

doi:10.1021/jacs.9b12116

Cited by 127 publications

(111 citation statements)

References 32 publications

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“…In recent years the scope, scale, and speed with which chemically reactive amino acids can be profiled has accelerated dramatically; chemoselective probes are now available for cysteine 4 , serine 39 , lysine 5 , methionine 6 , tyrosine 7 , aspartate/glutamate 9,40 , tryptophan 41 , and histidine 42 . Supporting this, advances in mass-spectrometry have significantly expanded the number and rate at which individual reactive sites can be profiled 43,44 , and subsequently exploited by electrophilic drug hunters.…”

Section: Discussionmentioning

confidence: 99%

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Benns

Storch

Falco

et al. 2021

Preprint

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Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.

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Section: Discussionmentioning

confidence: 99%

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Benns

Storch

Falco

et al. 2021

Preprint

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“…2H-Azirines have been described as constitutively active electrophiles to target aspartates and glutamates by initial nucleophilic attack at the azirine eventually leading to an acylated -aminoketone (Az-alkyne, 20 Fig. 4h, Supplementary Fig.…”

Section: Monitoring Aspartates and Glutamates In The Proteomementioning

confidence: 99%

Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

Zanon¹,

Yu²,

Zhang³

et al. 2021

Preprint

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<a>Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome‑wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome‑wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the N‑terminus proteome‑wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.</a>

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“…Cellular imaging experiment was conduct with uorescence microscopy as described previously to verify the utility of cel-p for imaging of potential cellular targets [23]. To track the cellular distribution of cel-p, living primary neurons were incubated with 0.8 μM cel-p for 0-6 h. The cells were xed with 4% paraformaldehyde solution for 10 min and 0.2% Triton-X 100 permeated 15 min.…”

Section: Cellular Imaging Of Cel-pmentioning

confidence: 99%

Celastrol Exerts Neuroprotective Effect via Directly Binding to Hmgb1 Protein in Cerebral Ischemic Reperfusion

Liu

Luo

et al. 2021

Preprint

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Background: Celastrol is one of the early isolated and identified chemical constituents from Tripterygium wilfordii Hook.f.. Based upon the celastrol probe that maintained the bioactivity of the parent compound, the targets of celastrol in cerebral ischemic reperfusion (I/R) were comprehensively analyzed by quantitative chemical proteomics method.Methods: We constructed primary cortical neurons model of oxygen-glucose deprivation (OGD) and rat model of middle cerebral artery occlusion (MCAO) to detect the targets of celastrol in cerebral I/R. Combining with various experimental methods such as tandem mass tags (TMT) labeling, mass spectrometry and cellular thermal shift assay (CETSA), we revealed the directly binding cellular targets of celastrol.Results: We uncovered that celastrol inhibited pro-inflammatory activity of high mobility group protein 1 (HMGB1) by directly binding to it and then blocking the binding of HMGB1 to its inflammatory receptors in the microenvironment of ischemic and hypoxia. In addition, celastrol rescued neurons from OGD injury in vitro and decreased cerebral infarction in vivo by targeting HSP70 and NF-κB.Conclusion: Celastrol exhibited neuroprotective and anti-inflammatory effects via targeting HSP70 and NF-κB and directly binding to HMGB1 in cerebral ischemic reperfusion injury.

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2H-Azirine-Based Reagents for Chemoselective Bioconjugation at Carboxyl Residues Inside Live Cells

Cited by 127 publications

References 32 publications

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

Celastrol Exerts Neuroprotective Effect via Directly Binding to Hmgb1 Protein in Cerebral Ischemic Reperfusion

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