Supplementary data

Stereospecific PARP trapping by BMN 673 and comparison with olaparib and

rucaparib

Junko Murai, Shar-yin N. Huang, Amèlie Renaud, Yiping Zhang, Jiuping Ji, Shunichi Takeda, Joel Morris, Beverly Teicher, James H. Doroshow, and Yves Pommier

Supplementary figure 1. Stereospecific targeting of PARP by BMN 673. (A) Chemical structures of BMN 673 (left) and its inactive enantiomer, LT674 (right). The structural difference is outlined in red. (B) Differential PARP inhibition by BMN 673 and LT673 in DT40 cells. Total PAR levels were examined by Western blotting. Cells were treated for 30 min as indicated. Cell lysate from PARP1-/- DT40 cells was used as a negative control. (C) Differential PARP inhibition by BMN 673 and LT673 measured by ELISA. PAR levels without drug were set as 100%.  

O NHN

NHF

N

NNCH3

FBMN  673 LT674

Drug  concentration  (µM)

%  PAR

0.001 0.01 0.1 10

20

40

60

80

100

BMN  673

A

B C

D

O NHN

NHF

N

NNCH3

F

PARP1-­/-­ Wild  type

BMN  673 LT674(µM)

anti-­PAR

250148

986450

36

16

(kDa)0.001 0.01 0.1 0.001 0.01 0.1W

ild  type

LT674

   Supplementary figure 2. Lack of effect of PARP inhibition on ATP concentration. ATP concentration measured by the ATPlite® assay was determined in wild type DT40 cells treated with the indicated concentrations of BMN 673 for 30 min; conditions that are equivalent to Figure 1B. The ATP concentration was not influenced by catalytic inhibition of PARP. The ATP concentration of untreated cells was set as 100%. Error bars represent standard deviation (SD) (n = 3).  

ATP  activity  (%)

0 1 10 100

1000

10000

0

50

100

150

BMN  673  (nM)

Supplementary  figure  3

A

ATP

conc

entra

tion

(%)

                           

     

                      Supplementary figure 3. Flow cytometry analyses after treatment with different PARP inhibitors. DT40 (A) or DU145 (B) cells were treated with the indicated concentration of each PARP inhibitor. The cells were analyzed at different time points as indicated. 0.5 µM BMN 673 induced deleterious alternation of cell cycle in both cells and majority of cells were dead by 72 hours, while the same concentration of olaparib or rucaparib induced minimum effect.

A

B

Supplementary  figure  2

24H 48H 72HNo  drug

24H 48H 72HNo  drug

BMN  6730.5  µM

BMN  6730.5  µM

Olaparib0.5  µM

Olaparib0.5  µM

Rucaparib0.5  µM

Rucaparib0.5  µM

DT40  wile  type

DU145

Supplementary figure 4. Stereospecific poisoning of PARP by BMN 673 Viability curves of wild type, PARP1-/-, and BRCA2tr/- cells treated with the indicated concentrations of BMN 673 (left) and LT674 (right). Untreated cells were set as 100%. Data are mean ± SD (n ≥ 3). The bottom panel tabulates the IC90 of both enantiomers in wild type and BRCA2tr/-.

1

10

100

IC90  (µM)  

 

 BMN  673  LT674  

BMN  673  (µM)0.001 0.01 0.1 10

Wild  type BRCA2tr/-­

0.0040.42

LT674  (µM)

Wild  type

PARP1-­/-­

BRCA2tr/-­

Wild  typePARP1-­/-­

BRCA2tr/-­

NA 0.6

1

10

100

0.001 0.01 0.1 10%

Via

bilit

y

% V

iabi

lity

Drug