Table 1.

Summary of in vitro JNK1 PhALC (with 10 mM GSH), cell-based JNK1 NanoBRET target engagement (HEK293) and p-c-Jun EC₅₀ measurements (SH-SY5Y MKK7 ACT cells)

	R1	PhAlc IC50 /(nM)	NanoBRET EC50/(nM)	HTRF p-c-Jun(S63) EC50/(nM)	Western blot p-c-Jun (S73) EC50/(nM)
IN-8		3805 ± 402	417 ± 38	2127 ± 135	8975 ± 2680
IN-8a		5333 ± 232	x	6792 ± 256	n.a.
JNK-IN-8		44 ± 6	10.5 ± 0.5	88 ± 8	760 ± 120
CA-IN-8		318 ± 45	166 ± 35	4970 ± 620	8624 ± 1973
1aR-IN-8		22 ± 6	11.5 ± 0.7	135 ± 15	660 ± 100
1aS-IN-8		30 ± 5	3.3 ± 1.7	65 ± 7	320 ± 120a
1a’R-IN-8		225 ± 17	1060 ± 150	747 ± 247	4320 ± 1380
1a”-IN-8		349 ± 24	650 ± 180	644 ± 110	7330 ± 3600a
2-IN-8		780 ± 159	76 ± 30	616 ± 169	3350 ± 370a
3-IN-8		254 ± 55	240 ± 70	378 ± 108	6710 ± 980a
4-IN-8		37 ± 4	9.4 ± 0.4	220 ± 25	n.a.
5S-IN-8		641 ± 126	n.a.	n.a.	n.a.
5R-IN-8		537 ± 131	n.a.	n.a.	n.a.
6S,S-IN-8		59 ± 4	n.a.	n.a.	n.a.
6 R,R-IN-8		128 ± 3	n.a.	n.a.	n.a.

The p-c-Jun EC50 values were determined with two different methods, by an HTRF based assay with a p-c-Jun(Ser63) antibody or by the classical western blot based method with a p-c-Jun(Ser73) antibody; and the Pearson coefficient (0.68) calculated with the nine data points indicated a strong positive correlation between these two methods as expected. JNK activation was induced by doxycycline treatment in both cases and inhibitors were co-administered with the inducer and the respective p-c-Jun signal was measured after 6 h.

(Data show the mean ± parameter error estimate from weighted least squares method; for NanoBRET n = 2; for other measurements n = 3, independent experiments, except for ^a where n = 1; n.a.: not available, not measured; x denotes that target engagement with NanoLuc-JNK1 could not be quantitatively measured due to lack of change in the BRET signal; Supplementary Fig. 1, 6, 7 and 8).