Synthesis and discovery of the first potent proteolysis targeting chimaera (PROTAC) degrader of AIMP2-DX2 as a lung cancer drug

Figures & data

Figure 1. PROTAC composition and mechanism of PROTAC.

Figure 2. Chemical Structures of BC-DXI-495 (1) and putative positions to link the E3 ligase ligand.

Figure 3. Representative E3 ligase ligands.

Figure 4. In vitro pull-down assay representing final selection of E3 ligase ligand.

Scheme 1. Synthesis of AIMP2-DX2 PROTACs 13–16, 18–23. Reagents and conditions: (i) 4-cyanobenzenesulfonyl chloride, TEA, THF:H₂O, 0–25 °C, overnight; (ii) 4-morphlinoaniline, HATU, DIPEA, DMF, 25 °C, overnight; (iii) LiAlH₄, THF, 0–25 °C, overnight; (iv) ADMP, DCM, DMAP, 25 °C overnight; (v) (+)-Sodium L-ascorbate, copper(II) sulphate pentahydrate, DMSO, 25 °C, overnight.

Scheme 2. Synthesis of AIMP2-DX2 PROTACs 25, 26, 28–31, and 33–34. Reagents and conditions: (i) KOH, 2-propanol, reflux, 18 h; (ii) HATU, DIPEA, DMF, 25 °C, overnight; (iii) HATU, DIPEA, DMF, 25 °C, overnight; (iv) HATU, DIPEA, DMF, 25 °C, overnight.

Scheme 3. Synthesis of AIMP2-DX2 PROTACs 36, 37, and 39–41. Reagents and conditions: (i), (ii) HATU, DIPEA, DMF, 25 °C, overnight.

Scheme 4. Synthesis of AIMP2-DX2 PROTACs 45–49. Reagents and conditions: (i) p-Toluenesulfonyl chloride, NEt₃, THF:H₂O (10:1 v/v mixture) 0–25 °C, overnight; (ii) HATU, DIPEA, DCM, 25 °C, 1 h; (iii) 4-(piperazin-1-yl) aniline, HATU, DIPEA, DMF, 60 °C, 4 h.

Scheme 5. Synthesis of AIMP2-DX2 PROTACs 51–54. Reagents and conditions: (i) 4-(piperazin-1-yl) aniline, K₂CO₃, MECN, reflux, overnight; (ii) compound 42, HATU, DIPEA, DCM, 25 °C, 1 h, overnight.

Table 1. Structures and the % inhibition AIMP2-DX2 values for PROTACs.

Compound	E3 ligand	Number of non-H linker atoms	Linker	% Inhibition of AIMP2-DX2 Luciferase^a,b
1				37.67 ± 15.68
13	A	10		36.63 ± 15.82
14	A	13		47.09 ± 6.11
15	A	16		41.48 ± 1.88
16	A	18		32.97 ± 5.23
18	B	8		34.11 ± 5.44
19	B	11		38.13 ± 1.33
20	B	14		33.79 ± 3.20
21	B	17		24.03 ± 1.17
22	B	20		29.10 ± 8.91
23	B	23		23.61 ± 18.96
25	A	8		46.16 ± 6.60
26	A	12		46.39 ± 5.37
28	B	6		35.88 ± 14.48
29	B	9		24.65 ± 5.14
30	B	12		24.37 ± 16.10
31	B	18		14.84 ± 14.91
33	B	9		32.40 ± 11.15
34	B	12		34.01 ± 5.36
36	B	4		37.50 ± 5.70
37	B	10		31.73 ± 5.42
39	B	6		40.03 ± 4.42
40	B	9		44.61 ± 4.77
41	B	12		37.80 ± 5.19

^aThe change of the luciferase-tagged AIMP-DX2 level upon treating chemicals was determined by detecting the luminescence signal. ^b4 µM, 24 h.

Table 2. Structures and the % inhibition AIMP2-DX2 values for PROTACs with different linkers.

Compound	E3 ligand	Number of non-H linker atoms	Linker	% Inhibition of AIMP2-DX2 Luciferase^a,b
44	B	4		51.10 ± 3.69
45	B	6		51.99 ± 1.89
46	B	9		45.79 ± 6.04
47	B	12		39.74 ± 3.28
48	B	15		26.11 ± 4.25
51	B	3		25.50 ± 9.90
52	B	4		35.33 ± 4.64
53	B	5		44.44 ± 8.81
54	B	6		42.53 ± 5.94

^aThe change of the luciferase-tagged AIMP-DX2 level upon treating chemicals was determined by detecting the luminescence signal. ^b4 µM, 24 h.

Table 3. IC₅₀ value of compounds against AIMP2-DX2.

Compound number	IC50 (µM)
1	2.65 ± 2.74
14	3.58 ± 0.40
15	3.31 ± 0.21
19	N.A.^a
25	3.74 ± 0.11
26	3.53 ± 2.68
36	10.4 ± 2.42
39	N.A.^a
40	N.A.^a
44	2.75 ± 1.74
45	2.37 ± 0.09
46	3.96 ± 0.77
47	N.A.^a
53	4.80 ± 1.89
54	4.41 ± 0.93

^aN.A.: not active

Figure 5. Compound 45 mediated induction of interaction between Siah1 and AIMP2-DX2. (A) Immunoprecipitation assay showing compound 45-dependent increased binding of two proteins. IP and WCL mean immunoprecipitation and whole cell lysates, respectively. Actin was used as a loading control. (B) In vitro pull-down assay confirming compound 45-mediated direct effect on binding of two proteins. GST proteins were detected by Coomassie staining. EV means empty vector.

Figure 6. Ubiquitination-mediated degradation of AIMP2-DX2 via compound 45. (A) Compound 45-mediated alteration of AIMP2-DX2 protein and mRNA level. Cells treated with compound 45 were subjected to western blotting (WB) and RT-PCR (RT). (B) Determination of compound 45-dependent ubiquitination of AIMP2-DX2. The cells expressing strep-AIMP2-DX2 were treated with compound 45 and MG132 and subjected to ubiquitination assay. Ub: ubiquitin.

Figure 7. Dependency of compound 45-mediated anti-proliferative efficacy on level of both AIMP2-DX2 and Siah1. (A) Siah1-dependency of the suppressed cell viability via compound 45. Siah1-knockdowned cells via its specific si-RNA were treated with compound and subjected to MTT assay. The results were shown as a bar graph (left) and the level of proteins were checked by immunoblotting (right). (B) Compound 45-mediated cell viability in lung cancer cell lines with the different level of AIMP2-DX2 and Siah1. Cell viability was determined as above. N.D.: not determined. (A and B) The experiments were independently repeated three times.

Figure 8. Binding modes of pomalidomide (green atom-coloured stick) and thalidomide (yellow atom-coloured stick) in Pockets 1 and 3 of the human Siah1 protein (blue ribbon). The electron density surface is shown for Pocket 3.