Chemoproteomic-enabled phenotypic screening

Evaluation of fully-functionalized diazirine tags for chemical proteomic applications

“…The ability of photoaffinity probes to irreversibly modify the target proteins enables the possibility to identify the binding site of small molecules, a significant advantage over label-free target identification techniques. 11 Depending on the goals of a chemical proteomics experiment, the suitability of the probe for identifying sites of labeling may be a major factor in the choice of photoaffinity tag. Using the benzhydrylazetidine-based probe series as a test, we found probes LD–F and BD–F to be the most effective, allowing us to identify modified peptides with a high degree of confidence for 11.6% and 11.3% of enriched proteins, respectively.…”

“…Along these lines, we failed to enrich many well-established targets of staurosporine, for example PRKCH, 38 with any of the tested probes, presumably due to unfavorable orientations of the diazirine which may occlude crosslinking or reduced affinities towards other kinase targets. In this regard, effects of photoaffinity tag conjugation can frequently perturb biological activities of bioactive compounds; 11 while conjugation of staurosporine to photoaffinity tags generally increased IC 50 values by two orders of magnitude, the Ar tag resulted in almost complete loss of kinase inhibition and so the biological relevance of any proteins enriched by this probe would be questionable. Together, these observations suggest that proteomic profiles of diazirine-containing probes are not only affected by differences in diazirine chemical reactivity but are also substantially influenced by tag structure.…”

“…Diazirines are widely utilized photoreactive groups and are routinely employed in FF tags, likely due to their relatively small size, and therefore minimized proteomic interactions and physicochemical perturbations, as well as their irreversible and efficient photoactivation. 6,7 Diazirine-based FF tags have been used to deconvolute the targets of drugs, 8–10 phenotypic screening hits, 11–14 as well as metabolites, 15,16 and have also been employed for fragment-based ligand discovery in cells. 17,18 Despite their utility, attachment of such tags can have consequential effects on the molecular interactions of the parent molecule, potentially convoluting proteomic analysis and even altering biological activity.…”

Compound interaction screen on a photoactivatable cellulose membrane (CISCM) identifies drug targets

“…A major challenge in such forward chemical genetic approaches is the chemoproteomics-based deconvolution and characterization of protein target and mode of action of the identified small molecule. [1][2][3] Classical affinity-based target identification (ID) involves one distinct derivatization of the compound by one linker trajectory. This requires extensive efforts by structure activity relationship (SAR) studies to obtain suitable small molecular probes for affinity-based pulldown assays --a tedious and time consuming-process which may even unintentionally exclude additional target proteins.…”

“…To confirm the efficient functionalization of the cellulose we followed each of the functionalization steps shown in Scheme 1a (bottom) using X-ray photoelectron spectroscopy (XPS, Figure 2a) and attenuated total reflection Fourier-transformed infrared (ATR-FTIR) spectroscopy (Figure 2b, Supporting Figure 1). Comparison of high resolution C 1s spectra of unmodified (1) versus oxidized cellulose (2) shows the appearance of a peak of weak intensity at 289.2 eV binding energy, which corresponds to the newly formed carboxylic group in 2. The formation of a carboxylic group could also be observed by an ATR-FTIR signal for carboxyl vibrations at 1599 cm -1 for the oxidized cellulose (2).…”