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20
Figure 1
BMAL1
CLOCK
complex
formation
PER1/2
CRY1/2
repression
activation
PER1/2
Per1/2
CRY1/2
Cry1/2
HN
CRY2
FBXL3
ubiquitination
and
degradation
CRY1
new compound
CRY2
Bmal1-dLuc U2OS
Period
1000
Period change (h)
0 µM
0.9 µM
3 µM
8 µM
1500
Per2-dLuc U2OS
600
400
200
6 Bmal1-dLuc
Per2-dLuc
0 12 24 36 48 60 72 84 96108
Relative
reporter activity
0 12 24 36 48 60 72 84 96108
1.0
0.5
0.0
Time (h)
-9 -8 -7 -6 -5
log[conc] (M)
Per2::Luc lung
0 µM
3 µM
24 µM
8 µM
DMSO
KL201
KL201
24
Conc (µM)
336
312
288
Intensity
Relative
Per2::Luc activity
Period
264
216
Time (h)
192
168
144
96
120
72
48
24
-48
-24
Period change (h)
Relative
luminescence
-9 -8 -7 -6 -5
Intensity
240
Luminescence
500
both CRY1/2
CRY1
Br
KL001
KL201
HO
1.0
0.5
0.0
24
Conc (µM)
Figure 2
1.0
0.5
-5
-7
log[conc] (M)
-6
-5
-5
0.0
1.0
1.0
0.5
0.5
0.0
Cry1
KO
-7
-6
-7
-6
-5
0.5
0.5
0.0
0.0
Cry2
KO
0.0
-5
-7
log[conc] (M)
-6
-5
KL201
0 0.3 0.9 2.7 8
24 µM
WB: anti-Flag
CRY1
WB: anti-HA
CRY2
3 CRY1
CRY2
-6
-5
log[conc] (M)
Cry2 KO
-6
Cry1 KO
-7
Cry1/Cry2
KO
Dbp
1.0
Wild type
0.0
Wild
type
Per2
1.0
Cry1/Cry2 KO
0.5
Relative mRNA levels
0.5
-5.4
Relative intensity
Relative Per2::Luc activity
1.0
-5.2
-5.6
1.0
-5.0
Cry2 KO
-6
-4.8
Cry1 KO
-7
Cry2
KO
NA
NA
-4.6
1.0
0.5
Wild Cry1/Cry2 Cry1
type
KO
KO
Wild type
1.5
CRY2
Cry1/Cry2 KO
2.5 CRY1
2.0 KL201
KL001
1.5
log[EC50] (M)
Relative half-life
Figure 3
HN
HN
R3
R1
R2
Name
R1
R2
log[EC2h] (M)
KL201
TH201
TH202
TH203
TH204
Br
Cl
OMe
Cl
-5.63
-5.26
-4.25
inactive
-4.24
Name
R3
TH205
log[EC2h] (M)
inactive
TH215
inactive
inactive
TH214
HN
TH206
-4.49
HN
HN
R6
R7
R5
Name
R4
R5
log[EC2h] (M)
Name
R6
R7
log[EC2h] (M)
TH207
TH208
TH209
TH210
Br
Cl
Cl
-4.79
-4.74
-4.27
inactive
TH211
TH212
TH213
Cl
Cl
-5.18
-4.66
inactive
size dependent
HN
non-essential
Br
essential
Figure 4
N-term
CRY1-KL201
CRY1-KL201
CRY2-KL001
CRY2-FBXL3
M424
D423
M424
D423
90°
T427
P426
M425
M425
P426
T427
W428
H355
H355
W292
F296
3.0 Å
I392
Q289
S396
W397
R358
F381
CRY1-KL201
W292
W399
F296
L400
F296
H2O
H 2O
I392
A362
A388
L385
W292
H359
H355
36
Hydrophobic Region 1
Hydrophobic Region 2
Affinity Region
W399
H2O
A388
I392
Q289
R358
W397
H355
L400
H359
A362
W397
R358
L385
32
FBXL3 si
28 Control si
24
A388
Br
L385
W399
S396
H355
NH
S396
Bmal1-dLuc
Period (h)
Hydrophobic Region 1
Hydrophobic Region 2
Affinity Region
D203
F381
CRY1-KL201
CRY2-KL001
Per2-dLuc activity
W428
C-term
-7
-6
Per2-dLuc
150
100
50
∞ -7 -6 -5
log[conc] (M)
...
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