Kinase off-target screening results.
CM11 was screened at 1 μM concentration against a panel of 50 kinases (Dundee MRC-PPU Express Screen, http://www.kinase-screen.mrc.ac.uk/services/express- screen). The remaining kinase activity was recorded at the end of the assay. The data is reported as average % activity remaining of assay duplicates for each kinase tested (± standard deviation in brackets), ranked from highest to lowest.
Plate Barcode CM11
D6769PM 1uM
% activity (s.d.)
RIPK2 130 (37)
VEG-FR 126 (17)
MKK1 126 (2)
IGF-1R 118 (14)
HER4 115 (11)
CHK2 114 (3)
MARK3 113 (3)
SYK 113 (28)
MLK3 112 (2)
TAK1 112 (2)
TBK1 112 (8)
BTK 112 (5)
MST2 111 (3)
Lck 111 (2)
HIPK2 111 (9)
EF2K 110 (9)
PDK1 109 (8)
SmMLCK 109 (3)
JAK3 108 (2)
JNK1 108 (1)
ROCK 2 108 (8)
IRAK4 107 (5)
CK1δ 107 (4)
PIM1 107 (1)
MSK1 106 (8)
NEK6 106 (4)
EPH-A2 105 (11)
CAMK1 105 (5)
PRK2 104 (7)
LKB1 104 (5)
PKD1 103 (10)
p38a MAPK 102 (1)
GSK3b 101 (2)
DYRK1A 100 (4)
AMPK (hum) 100 (11)
CK2 100 (3)
CAMKKb 99 (3)
PKBa 97 (8)
SRPK1 97 (12)
PLK1 97 (4)
S6K1 97 (7)
RSK1 96 (6)
Aurora B 96 (8)
Src 95 (10)
PKCa 95 (1)
SGK1 95 (5)
PKA 90 (5)
PAK4 90 (10)
TTK 89 (2)
TrkA 89 (4)
In cell validation
Potency in cells:
DC50
10-100 nM
Potency assay (cells):
Western blot
Target engagement assay (cells):
Western Blot
Potency assay, off target (cells):
To evaluate the specificity of homo-PROTAC-induced degradation, and identify potential off-targets, we performed isobaric tagging mass spectrometry proteomics to quantify degradation at the proteome level in an unbiased fashion. Amongst the 6,450 detected proteins that passed filtering criteria no proteins other than Cul2 was substantially depleted
by homo-PROTACs CM09, CM10 or
6
compared to DMSO or VHL inhibitor treatment. Crucially, no effect on protein levels of other Cullins (Cul1, Cul3, Cul4A, Cul4B, Cul5 and Cul7) or CRL-associated subunits was observed. Together, the data demonstrate that CM11 mainly induce depletion of pVHL30 and Cul2 but not other proteins. The proteomics data further evidenced no increase in protein levels of HIF-1α with homo-PROTACs (but a small decrease with CM09) relative to DMSO (Supplementary Fig. 6). In contrast, treatment with 150 μM VH032 led to increased HIF-1α levels, as expected.