Inhibitor mediated protein degradation

Abstract

The discovery of drugs that cause the degradation of their target proteins has been largely serendipitous. Here we report that the tert-butyl carbamate-protected arginine (Boc(3)Arg) moiety provides a general strategy for the design of degradation-inducing inhibitors. The covalent inactivators ethacrynic acid and thiobenzofurazan cause the specific degradation of glutathione-S-transferase when linked to Boc(3)Arg. Similarly, the degradation of dihydrofolate reductase is induced when cells are treated with the noncovalent inhibitor trimethoprim linked to Boc(3)Arg. Degradation is rapid and robust, with 30%-80% of these abundant target proteins consumed within 1.3-5 hr. The proteasome is required for Boc(3)Arg-mediated degradation, but ATP is not necessary and the ubiquitin pathways do not appear to be involved. These results suggest that the Boc(3)Arg moiety may provide a general strategy to construct inhibitors that induce targeted protein degradation.

Figure 1. Degradation of HA-tagged GST-α1

(A) Structures of EA and EA-Boc₃Arg and the reaction of GST with EA derivatives. (B) EA and EA-Boc₃Arg inactivate GST-α1 with similar potency. Purified recombinant GST-α1 (200 nM) was incubated with EA or EA-Boc₃Arg and assayed for activity. (C and D) The degradation of EA-Boc₃Arg-modified GST-α1. Purified recombinant C-terminally HA-tagged GST-α1 (0.2 mg/ml) was inactivated by EA or EA-Boc₃Arg (80 μM) and diluted 50-fold into HeLa cell lysates supplemented with an ATP regenerating system. Samples were analyzed by immunoblotting for the HA tag. Inosine monophosphate dehydrogenase (IMPDH) was used as a loading control. (C) A representative immunoblot. (D) Average of four experiments. Error bars show standard errors. (E) Structure of Fur-Boc₃Arg. (F) Degradation of GST-α1 by Fur-Boc₃Arg. Purified C-terminally HA-tagged GST-α1 was modified with Fur-Boc₃Arg (40 μM) and added to NIH 3T3 cell lysates supplemented with an ATP regenerating system.

Figure 2. TMP-Boc₃Arg induced degradation of eDHFR fusion proteins in cell lysates

(A) Structures of TMP and derivatives. (B and C) Purified recombinant eDHFR-HA (0.2 mg/ml) was incubated with TMP derivatives (80 μM) and diluted 50-fold into Cos-1 cell lysates supplemented with an ATP regenerating system. (B) Representative immunoblot showing degradation of eDHFR-HA in the presence of TMP and TMP-Boc₃Arg. (C) Combined data from 2 experiments after 4 h incubation.

Figure 3. Degradation of eDHFR fusion proteins in whole cells

(A and B) Global protein stability assay. HeLa cells co-express RFP and eDHFR-HA-GFP from a bicistronic construct. Red and green fluorescence was measured by flow cytometry. (A) TMP (80 μM). (B) TMP-Boc₃Arg (80 μM). (C) Quantitation of four independent GPS experiments. (D) Degradation of eDHFR-GFP was confirmed by anti-HA and anti-GFP immunoblotting. Supported by Figure S1.

Figure 4. Degradation of eDHFR fusion proteins in cycloheximide treated cells

(A) Experiment as in Figure 3, but HeLa cells were incubated with 0.2 mg/mL cycloheximide for 20 minutes prior to TMP-Boc₃Arg treatment. The rightmost panel is an independent blot. (B) Quantitation of three independent experiments.

Figure 5. EA-Boc₃Arg-induced degradation of endogenous and ectopic GST in whole cells

(A) Degradation of endogenous GST-π control when Cos-1 cells are treated with EA and EA-Boc₃Arg for 3 hrs (N = 4). (B) Degradation of eDHFR-HA-GST-α1 in HeLa cells. Cells expressing eDHFR-HA-GST-α1 were treated with EA-Boc₃Arg (80 μM) over 1.8 hours. Protein was measured by both anti-GST and anti-HA immunoblotting (N≥3). (C) HeLa cells expressing eDHFR-HA-GST-α1 were treated with: triangles EA (80 μM); squares EA-Boc₃Arg (8 μM); diamonds EA-Boc₃Arg (80 μM). At the indicated time points, the eDHFR-HA-GST-α1 was quantitated and normalized to GAPDH (D) HeLa cells expressing eDHFR-HA-GST-α1 were treated with a range of concentrations of EA-Boc₃Arg or just DMSO. After 1.2 hours, the amount of eDHFR-HA-GST-α1 was quantitated. Supported by Figure S2.

Figure 6. Mechanism of Boc₃Arg-induced degradation

(A) Effect of lactacystin (50 μM) on the TMP-Boc₃Arg-induced degradation of eDHFR in Cos-1 cell lysates supplemented with an ATP regeneration system. (B) Degradation of GST-α1 (0.2 mg/ml) was modified by EA-Fur-Boc₃Arg (40 μM) then diluted 50-fold into NIH 3T3 cell lysates. closed diamonds, standard assay buffer containing ATP regenerating system; closed squares, ATP regenerating system omitted. (C) Effect of lactacystin (50 μM) and ubiquitin aldehyde (20 μM) on the degradation of eDHFR•TMP-Boc₃Arg in Cos-1 lysates. (D) Effect of MG132 (100 μM) on the EA-Boc₃Arg-induced degradation of GST-α1 in Cos-1 cell lysates supplemented with an ATP regeneration system.