Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary...

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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

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Transcript of Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary...

Page 1: Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary Figure S1.

Compound 5

Structure

hLDHA IC50

0.032 ± 0.016 µM

hLDHBIC50

2.512 ± 0.07 µM

N

HN

H2N

O

O

N

NOH3C

OCH3

HO O

GSK2619729ALDHA pIC50 = 7.6 (25 nM)LDHB pIC50 = 5.6 (2,512 nM)

Supplementary Figure S1

Page 2: Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM 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

A

conc

entr

ation

in c

ell, m

M

Cmpd 1 Cmpd 2 Cmpd 3

0

20

40

60

80

NADH Cmpd IC50

67 nM

43 nM

22 nM

12 nM

Page 3: Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary Figure S1.

13C 15N- Arg/Lys

HEK293

SILAC

Mix 1:1 Ratio

SNU398

12C 14N- Arg/Lys

Protein ConcentrationPrepare lysates

triplicate

Protein Concentrationrecomb LDHA

Spike

13C 15N- Arg/Lys

HEK293

SILAC

Supplementary Figure S3

Trypsin digest

LC-MS/MS

Trypsin digest

LC-MS/MS

B

A

Snu398

HepG2

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]

972 973 974 975 976 977 978

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

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

Rela

tive

Abun

danc

e

Rela

tive

Inte

nsity

%

0

100

973 975 977HEK293Snu398

LLIVSNPVDILTYVAWK

Page 4: Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary Figure S1.

A2780

MDA-MB-453

Snu423

NCI-H1395

CAL-27

MiaPaC

a-2

HepG2786-O

Snu398

0

1

2

3

4

LDHA

LDHB

A

C D

Snu398

MDA-MB-453

MiaPaCa-2

NCI-H1395

Snu423

U251A2780

Cal-27

HepG2

786-O

LDH

A, m

M

b-actin

Supplementary Figure S4

7=0 42

LDHA

2 3 4

trxn 2, daystrxn 1, days

WB

+ + + + + + + LDHAsi

7

E

HepG2

Kidney

Melanoma

Liver

ColonPancre

as

Breast

LDHA

b-actin

LDHB

LDH

A, m

M

B

HepG2

Kidney

Melanoma

Liver

Colon

Pancre

as

Breast

0

1

2

3

4

Page 5: Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary Figure S1.

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 g/mLoligomycin

MAS Composition:70 mM sucrose220 mM mannitol10 mM potasium phosphate5 mM magnesium chloride2 mM HEPES1 mM EGTApH 7.2

Inject 4 Mantimycin

A B DC

OCROLG = (B) - (C) OCRLeak = (C) - (D)

DM

SO M

2.5

M5

M10

M20

M40

0

20

40

60

80

**

**

*

Compound 1

OC

RO

LG

,%

of

init

ial

DM

SO M

2.5

M5

M10

M20

M40

0

20

40

60** **

Compound 1

OC

RL

ea

k,

% o

f in

itia

l

Page 6: Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary Figure S1.

0

1

2

3FB

P, n

mol

/mln

cel

ls

DMSO Cmpd 1 Cmpd 2

Supplementary Figure S6

0

1

2

3

4

5

FBP,

nm

ol/m

ln c

ells

< 0.3

DMSO Cmpd 1 Cmpd 2

A

B

Page 7: Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary Figure S1.

-9 -8 -7 -6 -5 -40

50

100

Compound 1, logM

cell

#,

% D

MS

O

-9 -8 -7 -6 -5 -40

50

100

Compound 1, logM

cell

#,

% D

MS

O

-9 -8 -7 -6 -5 -40

50

100

150

Compound 1, logM

cell

#,

% D

MS

O

-9 -8 -7 -6 -5 -40

50

100

Compound 1, logM

cell

#,

% D

MS

O

Snu398 HepG2A

+0.5nM NAD+ synthesis inhibitor +0.5nM NAD+ synthesis inhibitor

B

EC50= 3.6 1.4 M

EC50= 0.9 0.2 M

Supplementary Figure S7

Page 8: Compound 5 Structure hLDHA IC 50 0.032 ± 0.016 µM hLDHB IC 50 2.512 ± 0.07 µM Supplementary Figure S1.

-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

CA

R R

atio


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