Merits of Diazirine Photo-Immobilization for Target Profiling of Natural Products and Cofactors

Abstract

Finding the targets of natural products is of key importance in both chemical biology and drug discovery, and deconvolution of cofactor interactomes contributes to the functional annotation of the proteome. Identifying the proteins that underlie natural compound activity in phenotypic screens helps to validate the respective targets and, potentially, expand the druggable proteome. Here, we present a generally applicable protocol for the photoactivated immobilization of unmodified and microgram quantities of natural products on diazirine-decorated beads and their use for systematic affinity-based proteome profiling. We show that among 31 molecules of very diverse reported activity and biosynthetic origin, 25 could indeed be immobilized. Dose-response competition binding experiments using lysates of human or bacterial cells followed by quantitative mass spectrometry recapitulated targets of 9 molecules with <100 μM affinity. Among them, immobilization of coenzyme A produced a tool to interrogate proteins containing a HotDog domain. Surprisingly, immobilization of the cofactor flavin adenine dinucleotide (FAD) led to the identification of nanomolar interactions with dozens of RNA-binding proteins.

Figure 1

Workflow for target deconvolution using photo-immobilized affinity matrices. (a) Photo-crosslinking of natural products on diazirine-loaded beads through unselective carbene insertion. (b) Dose-dependent competition pulldown experiment of unmodified natural products and the corresponding affinity matrix coupled to quantitative mass spectrometry for protein identification and determination of apparent interaction constants (K_D^app); EC₅₀: effective concentration of natural product necessary to reduce protein binding to beads by 50%. Figure created with Biorender.com.

Figure 2

Optimization of photo-crosslinking conditions using tacrolimus as a model system. (a) Tacrolimus apparent loading on beads (blue circles) as a function of irradiation time or compound:linker ratio. 2 molar excess of tacrolimus was added to 2 μmol mL^–1 LC-SDA-loaded beads for irradiation time tests; for cpd:linker optimization, a respective mol amount of tacrolimus was titrated to 2 μmol mL^–1 LC-SDA-loaded beads. Tacrolimus final loading on beads was determined from LC–MS coupling controls (see Materials and Methods in the Supporting Information). (b) Relative amount of FKBP12 bound to beads (expressed as the fraction of FKBP12 vs total protein intensity-based absolute quantification, rIBAQ, %) as a function of linker density in the presence of DMSO (gray), in competition with 10 μM free tacrolimus (pink) and bound to beads that do not display immobilized tacrolimus (green, blocked beads). The blue circles indicate the density of tacrolimus on beads. (c) Structures of the diazirine linkers evaluated for photo-crosslinking of tacrolimus. (d) Relative amount of FKBP12 vs total protein bound to beads for the different linkers and the mixture of all linkers. (e) Residual binding of FKBP12 to beads as a function of increasing doses of free tacrolimus used as a competitor. Curve fitting was achieved using a four-parameter log-logistic regression model; the fit error is estimated with the error bars.

Figure 3

Comparison of target proteins identified by selective secondary amine vs photo-immobilization of staurosporine or Kinobeads. (a) Chemical structure of staurosporine with the secondary amine used for immobilization highlighted in blue (selective immobilization). The orange circle depicts that promiscuous UV-induced immobilization may occur anywhere in the compound. (b) Heatmap depicting apparent binding constants of protein targets obtained by photo-crosslinked or selectively immobilized staurosporine. (c) Apparent binding constants of all staurosporine kinase targets obtained by Kinobeads (KBs) profiling. The black dots indicate the targets that were only identified in Kinobeads assay, selectively immobilized staurosporine and Kinobeads (NHS, blue dots), UV-immobilized staurosporine and Kinobeads (orange), or by all three approaches (KBs, UV, and NHS, green dots).

Figure 4

Broad assessment of photo-immobilization for target deconvolution of natural molecules. (a) Immobilization efficiency upon UV irradiation for 31 natural compounds. (b) Number of proteins bound by each affinity matrix. (c) Dose–response curves for cyclosporine A binders PPIA, PPIF, and PPIB. (d) Same as panel c but for rifamycin B. (e) Same as panel c but for geldanamycin.

Figure 5

Binders of the protein cofactors CoA and acetyl-CoA. (a) Heatmap of apparent interaction constants of protein binders of CoA and acetyl-CoA. (b) Residual binding of NAA25 to CoA and acetyl-CoA beads in response to increasing concentrations of free CoA and acetyl-CoA, respectively. (c) Intensity distribution of all proteins bound to CoA or acetyl-CoA beads (dotted lines). The solid lines mark the position of NAA25 in these distributions. (d) Same as panel b but for NAA10. (e) Same as panel c but for NAA10.

Figure 6

FAD interactors. (a) Affinity ranking of all identified direct or indirect FAD interactors in a full dose competition pulldown assay. (b) Dose–response curves for identified FAD binders UBAP2L, OTUD4, and CNOT4. CNOT1 was bound by the matrix but not competed up to 100 μM. (c) Same as panel b but for MED15, TCF3, and GATA1.