Biochemistry and Chemical Biology

Identification of Suitable Target/E3 Ligase Pairs for PROTAC Development using a Rapamycin-induced Proximity Assay (RiPA)

Bikash Adhikari
Katharina Schneider
Mathias Diebold
Christoph Sotriffer
Elmar Wolf author has email address

Institute of Biochemistry, University of Kiel, Kiel, Germany
Institut für Pharmazie und Lebensmittelchemie, University of Würzburg, Würzburg, Germany

https://doi.org/10.7554/eLife.98450.1

Open access
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Figures and data

Rapamycin-induced proximity assay (RiPA) induces quantifiable degradation of target proteins
(A) Schematic illustration of scenarios where PROTAC could not induce degradation of a target protein.
(B) Scheme of target protein (I) and E3-ligase or control (II) constructs used in RiPA. The linker indicated is 2xGSSG in all constructs unless stated otherwise.
(C) Schematic describing the RiPA experimental protocol.
(D) Immunoblot of WDR5 and VHL. HEK293 cells were co-transfected with WDR5-Luc-FKBP12 and VHL-FRB or FRB in the indicated ratio and treated with 100 nM rapamycin or vehicle for 6 hr after ∼24 hr of expression. Vinculin was used as a loading control (as in all other immunoblotting experiments).
(E) WDR5 levels based on luciferase measurements. HEK293 cells were co-transfected with WDR5-Luc-FKBP12 or Luc-WDR5-FKBP12 and VHL-FRB or FRB constructs in the indicated ratio, expressed for ∼24 hr, and treated with rapamycin overnight. Bars represent mean ± s.d. of n=3 replicates.
(F) Immunoblot of WDR5 and VHL. HEK293 cells were co-transfected with a combination of WDR5-Luc-FKBP12 or Luc-WDR5-FKBP12 and VHL-FRB or FRB-VHL or FRB in the ratio of 1:10, expressed for ∼24 hr and treated with rapamycin overnight.

RiPA correctly predicts suitability of E3 ligases for WDR5 PROTACs
(A) Immunoblot of WDR5, VHL, and CRBN. HEK293 cells were transfected with WDR5-Luc-FKBP12 and VHL-FRB or CRBN-FRB or FRB in a 1:10 ratio, expressed for 24 hr, and treated with 10 nM rapamycin for 6 hr.
(B) WDR5 levels based on luciferase measurement. Luminescence was measured in HEK293 cells as described in (A) after 10 nM rapamycin treatment at specified time points. Data represent mean ± s.d. of n=3 replicates.
(C) Immunoblot of AURKA, VHL and CRBN. HEK293 cells were transfected with AURKA-Luc-FKBP12 and VHL-FRB or CRBN-FRB or FRB in a 1:10 ratio and treated with 10 nM rapamycin for 6 hr.
(D) AURKA levels based on luciferase measurement. Luminescence was measured in HEK293 cells as described in (C) after 10 nM rapamycin treatment at indicated time points. Data represent mean ± s.d. of n=3 replicates.
(E) Structure of FKBP12 and luciferase. Molecular surface representation of FKBP12 (top) and luciferase (bottom) showing lysine residues on their surface. The lysine residues are labelled and two sides for each protein are shown.
(F) WDR5 levels based on luciferase measurement. HEK293 cells were co-transfected with either WDR5-Luc-FKBP12 (WT) or WDR5-Luc-FKBP12 construct where all lysine residues on Luc and FKBP12 were mutated to arginine (K-less) and FRB, expressed for ∼24 hr and luminescence measured. Bars represent mean ± s.d. of n=3 replicates.
(G) Immunoblot of WDR5, VHL, and CRBN. HEK293 cells expressing WDR5-Luc-FKBP12 (K-less) and VHL-FRB or CRBN-FRB or FRB were treated with rapamycin as in (A).
(H) WDR5 levels based on luciferase measurement. Luminescence was measured in HEK293 cells as described in (G) after 10 nM rapamycin treatment at specified time points. Data represent mean ± s.d. of n=3 replicates.

RiPA can identify degradative E3 ligases not previously used for PROTACs
(A) Immunoblot of WDR5. HEK293 cells were transfected with WDR5-Luc-FKBP12 (K-less) and FBXL12-FRB or FRB in a ratio of 1:10 and treated with 10 nM rapamycin for 8 hr.
(B) WDR5 levels based on luciferase measurement. Luminescence of WDR5-Luc-FKBP12 (K-less) in the same cells as in (A). Bars represent mean ± s.d. of n=3 replicates.
(C) WDR5 levels based on luciferase measurement. HEK293 cells were transfected with WDR5-Luc-FKBP12 (K-less) and FBXL12-FRB or VHL-FRB or FRB in a ratio of 1:1000 and treated with 10 nM rapamycin for the indicated time point. Data represent mean ± s.d. of n=3 replicates.
(D) Model of Luc-WDR5-FKBP12 constructs. Structure of Luc-WDR5-FKBP12 with indicated linkers between WDR5 and FKBP12 bound to rapamycin and FRB. The linker between Luc-WDR5 is always 2xGSSG.
(E) Immunoblot of WDR5. HEK293 cells were transfected with Luc-WDR5-FKBP12 containing indicated linker length and FBXL12-FRB or FRB in the ratio of 1:100, expressed for ∼24 hr, and treated with 10 nM rapamycin for 6 hr.
(F) WDR5 levels based on luciferase measurement. Luminescence of Luc-WDR5-FKBP12 constructs as in cells from (E) and treated with 10 nM rapamycin for 8 hr. Bars represent mean ± s.d. of n=2 replicates.
(G) WDR5 levels based on kinetic luciferase measurement. HEK293 cells were transfected with WDR5-Luc-FKBP12 (K-less) and FBXL12-FRB or FRB in a ratio of 1:100, expressed for ∼24 hr, treated with 10 nM rapamycin, and luminescence measured for 19 hr. Data represent mean ± s.d. of n=3 replicates.

Identification of degradation-inducing E3 ligases by designing a universal substrate
(A) Model of lysine-rich luciferase. Structure of mutated luciferase with 5 additional (Luc^V1) and 12 additional (Luc^V2) lysines as compared to 7 lysine residues of wild-type luciferase. The lysine residues from WT (black), Luc^V1 (red), and Luc ^V2 (orange; red as in V1) are labeled.
(B) Scheme of wild-type luciferase (WT) or lysine-rich luciferase (V1, V2, K3, K6, and K12) containing constructs.
(C) Luciferase measurement. HEK293 cells were co-transfected with Luc-FKBP12 constructs as shown in (B) and FRB, expressed for ∼24 hr, and luminescence was compared. Bars represent mean ± s.d. of n=2 replicates.
(D) Luciferase measurement. HEK293 cells were transfected with the indicated versions of Luc-FKBP12 and FBXL12-FRB or FRB in a ratio of 1:100, expressed for ∼24 hr, and treated with 10 nM rapamycin for 8 hr. Bars represent mean ± s.d. of n=2 replicates.
(E) Kinetic luminescence measurement. HEK293 cells expressing constructs as described in (D) were treated with 10 nM rapamycin or vehicle and luminescence was monitored for 22 hr. Bars represent mean ± s.d. of n=2 replicates.

Rapamycin-induced proximity assay (RiPA) induces quantifiable degradation of target proteins
(A) Schematic illustration of rapamycin-induced dimerization of FRB and FKBP12 and structure of rapamycin.
(B) Structure of WDR5-Luc-FKBP12 and Luc-WDR5-FKBP12 fusion proteins. A flexible linker, 2xGSSG is present between each component.

RiPA can identify degradative E3 ligases not previously used for PROTACs
(A) WDR5 levels based on luciferase measurement. HEK293 cells were transfected with Luc-WDR5-FKBP12 or WDR5-Luc-FKBP12 and FBXL12-FRB or FRB in a ratio of 1:10, expressed for ∼24 hr, and treated with 10 nM rapamycin for 9 hr. Bars represent mean ± s.d. from n=3 replicates.

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