Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins

IF 3.8 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY ACS Bio & Med Chem Au Pub Date : 2023-11-20 DOI:10.1021/acsbiomedchemau.3c00067
Photis Rotsides, Paula J. Lee, Nakoa Webber, Kimberly C. Grasty, Joris Beld and Patrick J. Loll*, 
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Abstract

Vancomycin’s interactions with cellular targets drive its antimicrobial activity and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycin’s interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications as compared to previous photoprobes. Using proteins fused to vancomycin’s main cell-wall target, d-alanyl-d-alanine, we used mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying vancomycin-binding proteins.

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万古霉素结合蛋白的重氮嗪光探针鉴定
万古霉素与细胞靶标的相互作用驱动其抗菌活性,也引发对抗生素的耐药性表达。万古霉素的相互作用伙伴先前已使用光亲和探针确定,这已被证明是探索万古霉素相互作用组的有用工具。这项工作旨在开发基于重氮嘧啶的万古霉素光探针,与以前的光探针相比,它具有增强的特异性和更少的化学修饰。利用蛋白质与万古霉素的主要细胞壁靶标d-丙氨酰-d-丙氨酸融合,我们使用质谱分析表明,这些光探针可以在几分钟内特异性地标记已知的万古霉素结合伙伴。作为补充,我们开发了一种针对光探针的万古霉素加合物的Western-blot策略,消除了对亲和力标签的需要,简化了光标记反应的分析。总之,这些探针和鉴定策略为鉴定万古霉素结合蛋白提供了一种新颖的流线型管道。
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来源期刊
ACS Bio & Med Chem Au
ACS Bio & Med Chem Au 药物、生物、化学-
CiteScore
4.10
自引率
0.00%
发文量
0
期刊介绍: ACS Bio & Med Chem Au is a broad scope open access journal which publishes short letters comprehensive articles reviews and perspectives in all aspects of biological and medicinal chemistry. Studies providing fundamental insights or describing novel syntheses as well as clinical or other applications-based work are welcomed.This broad scope includes experimental and theoretical studies on the chemical physical mechanistic and/or structural basis of biological or cell function in all domains of life. It encompasses the fields of chemical biology synthetic biology disease biology cell biology agriculture and food natural products research nucleic acid biology neuroscience structural biology and biophysics.The journal publishes studies that pertain to a broad range of medicinal chemistry including compound design and optimization biological evaluation molecular mechanistic understanding of drug delivery and drug delivery systems imaging agents and pharmacology and translational science of both small and large bioactive molecules. Novel computational cheminformatics and structural studies for the identification (or structure-activity relationship analysis) of bioactive molecules ligands and their targets are also welcome. The journal will consider computational studies applying established computational methods but only in combination with novel and original experimental data (e.g. in cases where new compounds have been designed and tested).Also included in the scope of the journal are articles relating to infectious diseases research on pathogens host-pathogen interactions therapeutics diagnostics vaccines drug-delivery systems and other biomedical technology development pertaining to infectious diseases.
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