Small molecule target identification using photo-affinity chromatography

Abstract

Identification of the protein targets of bioactive small molecules is a routine challenge in chemical biology and phenotype-based drug discovery. Recent years have seen an explosion of approaches to meeting this challenge, but the traditional method of affinity pulldowns remains a practical choice in many contexts. This technique can be used as long as an affinity probe can be synthesized, usually with a crosslinking moiety to enable photo-affinity pulldowns. It can be applied to varied tissue types and can be performed with minimal specialized equipment. Here, we provide our protocol for photo-affinity pulldown experiments, with notes on making this method generally applicable to varied target identification challenges.

Comparative structure activity and target exploration of 1,2-diphenylethynes in Haemonchus contortus and Caenorhabditis elegans

“…Possibly, orthogonal approaches, aimed at validating the protein targets identified here, could illuminate the genuine mode(s) of action of UMW-9729 in a nematode model. Complementary protein-focussed investigations, such as isothermal dose-response fingerprinting ( Jafari et al, 2014 ) or affinity-based assays ( Him et al, 2009 ; Seo and Corson, 2019 ), or genomics-directed studies, such as RNA interference ( Blanchard et al, 2018 ; Hou et al, 2023 ), CRISPR/Cas9 genome editing (cf. Waaijers et al, 2013 ; Quinzo et al, 2022 ) or resistance-based studies utilising either H. contortus (see Kaminsky et al, 2008 ) or C. elegans (see Burns et al, 2006 ), could be employed to identify and/or validate drug-protein interactions.…”

Investigating Antiprotozoal Chemotherapies with Novel Proteomic Tools – Chances and Limitations. A Critical Review

“…The cell lysates are then separated via SDS-PAGE. The fluorescent bands are isolated and the proteins identified by LC-MS/MS [103]. Bio-orthogonal studies using photoactive derivatives have identified binding proteins of various antimalarials, such as albitiazolium [104], artemisinin [105], diaminoquinazoline [106], or, more recently, probes for tagging plasmepsins [107].…”

Chemo‐proteomics in antimalarial target identification and engagement

“…A major disadvantage of traditional AfBPPs is that their effectiveness is dependent on the activity or affinity of the probe as well as the abundance of the protein target 100 . UV‐mediated covalent photo‐crosslinking or photo‐affinity labeling (PAL) has been developed as a method to circumvent this problem 119 . PAL involves adding a photo‐reactive tag to the probe structure and upon UV irradiation, a reactive radical is generated that allows covalent linkage to proteins in close proximity to the chemical probe—ideally a protein for which the probe has the highest affinity.…”

“… 100 UV‐mediated covalent photo‐crosslinking or photo‐affinity labeling (PAL) has been developed as a method to circumvent this problem. 119 PAL involves adding a photo‐reactive tag to the probe structure and upon UV irradiation, a reactive radical is generated that allows covalent linkage to proteins in close proximity to the chemical probe—ideally a protein for which the probe has the highest affinity. The photo‐reactive tag consists of groups that can generate reactive diradicals, carbenes, or nitrenes yielded from benzophenones, diazirine, and aryl azide, respectively (Figure 3 ).…”