Figures and data
Schematic illustration of the POST-IT system for target identification. Upon the introduction of a drug-specific HaloTag ligand derivative, POST-IT tags target proteins with sPup, a tagging process known as pupylation, in live cells or organisms. Subsequently, these proteins are enriched and identified via mass spectrometry (MS) analysis.
Optimized Halo-PafA and Pup substrates efficiently promote pupylation in vitro in a proximity-dependent manner. (A) The chemical structure of DH1, an HTL derivative of dasatinib. (B) Comparison of proximity-tagging by different Halo-PafA derivatives on in vitro pupylation. (C) Comparison of different Pup substrates on in vitro pupylation with 500 nM DH1. (D) Pupylation levels by DH1 decrease with an increasing amount of dasatinib as a competitor, ranging from 0.2 μM to 10 μM. (B-D) All reactions were conducted with 1 μM of a Halo-PafA derivative, 10 μM of one of the different Pup substrates, and 0.5 μM of a purified short SRC with a 2×V5 tag, SRC(247-536)-2×V5, at 37 °C for 30 min. Pupylation levels of SRC were assessed by immunoblot (IB) using an anti-V5 antibody.
POST-IT can label target proteins in live cells.
(A) Effect of linker length between Halo8KR and PafA on target-labeling. HEK293T cells were co-transfected with HA-Halo8KR-PafA containing the specified length of linker, SBPK4R- sPupK61R, and SRC(247-536)-2×V5. (B) Introducing mutations S126A and K172R into PafA significantly enhances its proximity-tagging efficiency on target. (C-E) Comparison of labeling activity of Halo-PafA and Halo8KR-PafAS126A,K172R, a form used for POST-IT. (F, G) POST- ITDH1 mediates proximity-tagging in a DH1-dose dependent manner (F), an effect completely inhibited by competitive dasatinib, ranging from 0.025 to 25.6 μM (G). Pupylation levels of exogenously expressed short SRC (C, F, G), endogenous SRC (D), or pull-downed endogenous SRC (E) were assessed by immunoblot (IB) analysis using antibodies against V5-tag or SRC for exogenous or endogenous SRC, respectively. In all experiments, SBPK4R-sPupK61R was used for co-transfection, and cells were treated with 500 nM DH1, except in (F, G).
Identification of lysine residues for pupylation in PafA by mass spectrometry.
Dasatinib-HTL (DH) derivatives with modified and longer linkers enhances POST-IT performance.
(A) In vitro binding assay for DH derivatives via fluorescence polarization (FP) assay. Purified Myc-Halo-PafA (15 nM) and 1 nM Halo-AF488 were incubated with various DH derivatives in a serial dilution at 37 °C for 30 min prior to FP measurement. Data are shown as mean ± s.d. (B) Cellular binding assay for DH derivatives by FP measurement. HEK293T cells transfected with Myc-Halo-PafA were later incubated with 1 μM of each DH derivative or DMSO for 3 h, followed by cell lysate incubation with 2 nM of Halo-AF488 at 37 °C for 30 min. n = 4. Data are shown as mean ± s.e.m. P values were calculated by an unpaired two-sided t-test. Immunoblot presents a representative image of the input levels for each condition, demonstrating that treatment with DH derivatives did not alter the expression levels of Myc-Halo-PafA. (C) In vitro labeling activity comparison among DH derivatives. Purified recombinant Halo8KR-PafA (0.5 μM), SBPK4R-sPupK61R (10 μM), and SRC(247-536)-2×V5 (0.5 μM) were incubated with 0.5 μM of each DH derivative at 37 °C for 30 min. (D, E) In cellular labeling activity comparison among DH derivatives. HA-Halo8KR-PafA and SBPK4R-sPupK61R were used for co-transfection, and cells were treated with 250 nM of various DH derivatives. Pupylation levels of purified short SRC (C), endogenous SRC (D), or pull-downed endogenous SRC (E) were assessed by immunoblot (IB) using anti-V5 or anti-SRC antibodies for exogenous or endogenous SRC, respectively.
Target-ID by POST-ITDH5.
(A) Volcano plot displaying the relative fold change (FC) of DH5-binding proteins compared to those with dasatinib competition. Known target proteins for dasatinib were highlighted in red, and a newly identified target protein, SEPHS2, was marked in blue. (B, C) MST analysis demonstrates a direct interaction between DH5 and Cy5-labeled SEPHS2 (B) or between dasatinib and Cy5-labeled SEPHS2 (C), yielding Kd values of 11.6 ± 5.7 µM and 9.1 ± 0.8 µM, respectively. Data are shown as mean ± s.d. (D) Molecular docking binding pose between dasatinib and SEPHS2. The yellow box indicates the binding pocket, expanded for a closer view. The binding pocket of dasatinib precisely fits into the active sites of SEPHS2. (E) Diagram of 2D molecular docking interaction between SEPHS2 and dasatinib. Red boxes highlight the active sites, and blue boxes indicate residues among HUB nodes.
Target-ID for HCQ by POST-ITDC661-H1.
(A) Rank plot analysis of SILAC ratio values of proteins in three biological replicates. Autophage-related proteins ranked highly are marked in red. Heavy-labeled cells were treated with 200 nM DC661-H1, whereas light-labeled cells underwent incubation with both DC661-H1 (200 nM) and competitive DC660 (2 µM). (B, C) MST analyses demonstrate direct interactions of DC660 (B) and HCQ (C) with VPS37C, yielding Kd values of 21.5 ± 9.8 nM and 16.9 ± 8.7 µM, respectively. Data are shown as mean ± s.d. (D, E) Immunoblot results indicate that the VPS37C-V5 protein from cells treated with DC661-H1 was significantly enriched after streptavidin pulldown. The competition between DC661-H1 and either DC660 (D) or HCQ (E) nearly completely abolished VPS37C binding. HEK293T cells were co-transfected with HA- Halo8KR-PafAS126A,K172R, SBPK4R-sPupK61R, and VPS37C-V5. After 24 h, cells were incubated with 200 nM of DC661-H1, with or without 2 µM of DC660 (D) or HCQ (E). Twenty-four hours later, cells were collected for further analysis. (F, G) CETSA results demonstrate that VPS37C becomes thermostable when exposed to DC660 (F) or HCQ (G). Selective stabilization of VPS37C is evident at temperatures of 53 °C or higher, a phenomenon not observed in β-actin.
POST-IT enables in vivo target-ID.
(A) Schematic of experimental design for applying POST-IT in zebrafish. A plasmid construct containing the POST-IT system was injected into embryos at the one-cell stage. The injected embryos were dechorionated 24 h later, and then treated with DH5 or DMSO for an additional 24 h. Embryos lysates were collected and analyzed by immunoblot. (B) Representative images of injected embryos. Bright field (BF) images reveal no overt toxicity from POST-IT expression. EGFP images display robust expression of POST-IT. Scale bar, 500 µm. (C) Immunoblot analysis demonstrates that POST-IT exhibits significant enrichment of SRC in the presence of DH5 but not DMSO.
