Supplementary Figure S1
8
Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM N HN H 2 N O O N N O H 3 C O CH 3 HO O G SK2619729A Supplementary Figure S1
description
Supplementary Figure S1. A. concentration in cell, m M. Cmpd 1. Cmpd 2. Cmpd 3. B. NADH. Cmpd IC50. 120 m M. 67 nM. 100. 60 m M. 43 nM. 30 m M. 22 nM. 75. 5 m M. 12 nM. % inhibition. 50. 25. -9. -8. -7. -6. -5. Compound 2. Supplementary Figure S2. A. 13 C. - PowerPoint PPT Presentation
Transcript of Supplementary Figure S1
Compound 5
Structure
hLDHA IC50
0.032 ± 0.016 µM
hLDHBIC50
2.512 ± 0.07 µM
N
HN
H2N
OO
N
NOH3C
OCH3
HO O
GSK2619729ALDHA pIC50 = 7.6 (25 nM)LDHB pIC50 = 5.6 (2,512 nM)
Supplementary Figure S1
Supplementary Figure S2
Compound 2-9 -8 -7 -6 -5
% in
hibi
tion
25
50
75
100
5 mM
30 mM
60 mM
120 mM
B
Aco
ncen
trati
on in
cell,
mM
Cmpd 1 Cmpd 2 Cmpd 3
0
20
40
60
80
NADH Cmpd IC50
67 nM
43 nM
22 nM
12 nM
13C 15N- Arg/LysHEK293
SILAC
Mix 1:1 Ratio
SNU398
12C 14N- Arg/Lys
Protein ConcentrationPrepare lysates
triplicate
Protein Concentrationrecomb LDHA
Spike
13C 15N- Arg/LysHEK293
SILAC
Supplementary Figure S3
Trypsin digestLC-MS/MS
Trypsin digestLC-MS/MS
B
A
Snu398HepG2
LDHA copies/cell
LDHAcell concentration
4.7 x 106
2.9 x 106
2.6
1.5
C
Hek293 3.8 x 106 5.4
mM
973 975 977
Rela
tive
Inte
nsity
%
0
100LDH-A_Titrate_5uL_load1of25_102011 #14003-14210 RT: 97.57-99.24 AV: 74 NL: 2.33E6F: FTMS + p NSI Full ms [400.00-2000.00]
972 973 974 975 976 977 978m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
rLDHA HEK293
LDH-A_HEK_Snu_mix_replicate1_load_2uL_of_24_100611 #13605-13634 RT: 100.32-100.45 AV: 4 NL: 2.46E6F: FTMS + p NSI Full ms [400.00-2000.00]
972 973 974 975 976 977 978 979 980m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ative
Abu
ndan
ce
Rela
tive
Inte
nsity
%
0
100
973 975 977HEK293Snu398
LLIVSNPVDILTYVAWK
A2780
MDA-MB-453
Snu423
NCI-H1395
CAL-27
MiaPaC
a-2HepG2
786-O
Snu398
0
1
2
3
4
LDHA
LDHB
A
C D
Snu398
MDA-MB-453
MiaPaCa-2
NCI-H1395
Snu423
U251A2780
Cal-27
HepG2786-O
LDHA
, mM
b-actin
Supplementary Figure S4
7=0 42
LDHA
2 3 4
trxn 2, daystrxn 1, days
WB
+ + + + + + + LDHAsi
7
E
HepG2Kidney
Melanoma
Liver
ColonPancre
as
Breast
LDHA
b-actin
LDHB
LDHA
, mM
B
HepG2Kidney
Melanoma
Liver
Colon
Pancre
asBreast
0
1
2
3
4
Supplementary Figure S5
Exchange DMEM medium tomitochondrial assay medium (MAS)10 mM pyruvate/10 mM malate/4 mM ADPwith 2 nM PMP (permeabilizing agent)
Start Seahorse runt = 0
Inject Compound 1or DMSO
Inject 2 mg/mLoligomycin
MAS Composition:70 mM sucrose220 mM mannitol10 mM potasium phosphate5 mM magnesium chloride2 mM HEPES1 mM EGTApH 7.2
Inject 4 mMantimycin
A B DC
OCROLG = (B) - (C) OCRLeak = (C) - (D)
DM
SO Mm2.
5
Mm5
Mm10
Mm20
Mm40
0
20
40
60
80
**
**
*
Compound 1
OC
RO
LG,
% o
f ini
tial
DM
SO Mm2.
5
Mm5
Mm10
Mm20
Mm40
0
20
40
60** **
Compound 1
OC
RLe
ak,
% o
f ini
tial
0
1
2
3FB
P, n
mol
/mln
cells
DMSO Cmpd 1 Cmpd 2
Supplementary Figure S6
0
1
2
3
4
5
FBP,
nm
ol/m
ln ce
lls
< 0.3
DMSO Cmpd 1 Cmpd 2
A
B
-9 -8 -7 -6 -5 -40
50
100
Compound 1, logM
cell
#, %
DM
SO
-9 -8 -7 -6 -5 -40
50
100
Compound 1, logM
cell
#, %
DM
SO
-9 -8 -7 -6 -5 -40
50
100
150
Compound 1, logM
cell
#, %
DM
SO
-9 -8 -7 -6 -5 -40
50
100
Compound 1, logM
cell
#, %
DM
SO
Snu398 HepG2A
+0.5nM NAD+ synthesis inhibitor +0.5nM NAD+ synthesis inhibitor
B
EC50= 3.6 1.4 mM
EC50= 0.9 0.2 mM
Supplementary Figure S7
-15
-10
-5
0
5
10
15re
lativ
e pa
thw
ay a
ctivi
tyHepG2Snu398
pentose phosphateglycolysisfatty acid synthesisglutaminolysis
Supplementary Figure S8
A
B
Snu398 HepG20
2
4
6***
OC
RA
TP/E
CAR
Rat
io
