Assay Report. Poly (ADP-ribose) Polymerase (PARP) Inhibitor Assays Enzymatic Study of the compounds from Client

Transcription

1 Assay Report Poly (ADP-ribose) Polymerase (PARP) Inhibitor Assays Enzymatic Study of the compounds from Client Page 1 of 24 Client_PARP _mmddyyyy 1

2 Client_PARP_mmddyyyy PARP Inhibitor Assays Study Sponsor: Client Attention: Address: Study Director: Testing Facility: Henry Zhu, Ph.D. BPS Bioscience Inc Cornerstone Court West, Ste. B USA Study Period: Report Version: 1 Report Date: mm/dd/yyyy Page 2 of 24 Client_PARP _mmddyyyy 2

3 Study Director Principal Scientist Date Henry Zhu, Ph.D. President Date Page 3 of 24 Client_PARP _mmddyyyy 3

4 CONTENTS 1. PURPOSE OF THE STUDY MATERIALS AND METHODS MATERIALS COMPOUNDS EXPERIMENTAL CONDITIONS Enzymes and Substrates Assay Conditions Data Analysis ASSAY RESULTS SUMMARY OF THE EFFECTS OF THE COMPOUNDS ON PARP ACTIVITIES RESULTS OF EFFECTS OF THE COMPOUNDS ON PARPS PARP PARP PARP TNKS TNKS PARP PARP PARP PARP PARP PARP PARP PARP STORAGE AND RETENTION OF RECORDS QUALITY ASSURANCE STATEMENT Page 4 of 24 Client_PARP _mmddyyyy 4

5 1. Purpose of the Study The purpose of the study is to determine the effects of the compounds from Company on the enzymatic activities of recombinant human PARP enzymes by using an in vitro enzymatic assay. Page 5 of 24 Client_PARP _mmddyyyy 5

6 2. Materials and Methods 2.1 Materials BPS PARP assay kits are used in the assay 2.2 Compounds The test compound is supplied by Client Compound I.D. Compound Supplied Stock Concentration Dissolving Solvent Test Range Intermediate Dilution Solid 40 mm DMSO 0.03 nμ 50 µm 10 % DMSO (aq) Page 6 of 24 Client_PARP _mmddyyyy 6

7 2.3 Experimental Conditions Enzymes and Substrates Assay PARP1 PARP2 PARP3 TNKS1 TNKS2 PARP6 Catalog # (Lot #) ( ) ( P4) (130111G) ( ) (130314G2) (120614) Enzyme Used/ Reaction 10 ng 25 ng 80 ng 30 ng 10 ng 500 ng PARP7 (160302) 100 ng PARP8 (130301) 300 ng PARP10 PARP11 PARP12 PARP14 PARP (120208) (150820B) (151102A) (141121) (130103) 50 ng 800 ng 400 ng 150 ng 70 ng Substrate Activated DNA 0.xx mg/ml 0.xx mg/ml 0.xx mg/ml N/A N/A 0.xx mg/ml N/A N/A N/A N/A N/A N/A N/A Page 7 of 24 Client_PARP _mmddyyyy 7

8 2.3.2 Assay Conditions In general, all assays were done by following the BPS PARP or TNKS assay kit protocols. The enzymatic reactions were conducted in duplicate at room temperature for 1 hour in a 96 well plate coated with histone substrate. 50 µl of reaction buffer contains NAD +, biotinylated NAD + (see 2.3.1), activated DNA (see 2.3.1), a PARP enzyme (see 2.3.1) and the test compound (see 2.2). After enzymatic reactions, 50 μl of Streptavidin-horseradish peroxidase was added to each well and the plate was incubated at room temperature for an additional 30 min. 100 μl of developer reagents were added to wells and luminescence was measured using a BioTek Synergy TM 2 microplate reader Data Analysis PARP or TNKS activity assays were performed in duplicates. The luminescence data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the luminescence (L t ) in each data set was defined as 100% activity. In the absence of the PARP or TNKS, the luminescence (L b ) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity = [(L- L b )/(L t - L b )] 100, where L= the luminescence in the presence of the compound, L b = the luminescence in the absence of the PARP or TNKS, and L t = the luminescence in the absence of the compound. The percent inhibition was calculated according to the following equation: % inhibition = % activity. The percentage inhibition of the compounds is shown in a bar graph. The values of % activity versus a series of compound concentrations were then plotted using non-linear regression analysis of Sigmoidal dose-response curve generated with the equation Y=B+(T-B)/1+10 ((LogEC50-X) Hill Slope), where Y=percent activity, B=minimum percent activity, T=maximum percent activity, X= logarithm of compound and Hill Slope=slope factor or Hill coefficient. The IC50 value was determined by the concentration causing a half-maximal percent activity. Page 8 of 24 Client_PARP_mmddyyyy 8

9 3. Assay Results 3.1. Summary of the effects of the compounds on PARP Activities The % inhibition of the compounds against PARP are summarized in Table 3.1 Table 3.1 Inhibitory Effects of the Compounds on PARP Activities PARPs PARP1 PARP2 PARP3 IC nm 0.2 nm 41.0 nm TNKS1 1.8 µm TNKS2 1.2 µm PARP6 3.7 µm PARP7 750 nm PARP8 4.9 µm PARP µm PARP11 >% 50 µm PARP µm PARP14 >50 µm PARP µm Page 9 of 24 Client_PARP_mmddyyyy 9

10 3.2. Results of effects of test compounds on PARPs PARP1 Table Data for the Effect of on No Compound Background 7 7 PARP IC50=0.9 nm [Log(nM)] Page 10 of 24 Client_PARP_mmddyyyy 10

11 3.2.2 PARP2 Table Data for the Effect of on No Compound Background PARP IC50=0.2 nm [Log(nM)] Page 11 of 24 Client_PARP_mmddyyyy 11

12 3.2.3 PARP3 Table Data for the Effect of on No Compound Background PARP IC50=41.0 nm [Log(nM)] Page 12 of 24 Client_PARP_mmddyyyy 12

13 3.2.4 TNKS1 Table Data for the Effect of on PARP Activit No Compound Background TNKS IC50=1.8 µm [Log(nM)] Page 13 of 24 Client_PARP_mmddyyyy 13

14 3.2.5 TNKS2 Table Data for the Effect of on No Compound Background TNKS IC50=1.2 µm [Log(nM)] Page 14 of 24 Client_PARP_mmddyyyy 14

15 3.2.6 PARP6 Table Data for the Effect of on No Compound Background PARP IC50=3.7 µm [Log(nM)] Page 15 of 24 Client_PARP_mmddyyyy 15

16 3.2.7 PARP7 Table Data for the Effect of on No Compound Background PARP IC50=750 nm [Log(nM)] Page 16 of 24 Client_PARP_mmddyyyy 16

17 3.2.8 PARP8 Table Data for the Effect of on No Compound Background PARP IC50=4.9 µm [Log(nM)] Page 17 of 24 Client_PARP_mmddyyyy 17

18 3.2.9 PARP10 Table Data for the Effect of Olcaparib on No Compound Background PARP IC50=1.5 µm [Log(nM)] Page 18 of 24 Client_PARP_mmddyyyy 18

19 PARP11 Table Data for the Effect of on No Compound Background PARP11 1 >% inhibition at 50 µm [Log(nM)] Page 19 of 24 Client_PARP_mmddyyyy 19

20 PARP12 Table Data for the Effect of on No Compound Background PARP IC50=6.6 µm [Log(nM)] Page 20 of 24 Client_PARP_mmddyyyy 20

21 PARP14 Table Data for the Effect of on No Compound Background PARP IC50>50 µm [Log(nM)] Page 21 of 24 Client_PARP_mmddyyyy 21

22 PARP15 Table Data for the Effect of on No Compound Background PARP IC50=12.2 µm [Log(nM)] Page 22 of 24 Client_PARP_mmddyyyy 22

23 4. Storage and Retention of Records The original final report provided to the sponsor will be kept by the sponsor under its sole responsibility. Page 23 of 24 Client_PARP_mmddyyyy 23

24 5. Quality Assurance Statement I certify that the results presented in this report were generated using the materials and methods mentioned and that these results reflect the Raw Data. Henry Zhu, Ph.D. President Date Page 24 of 24 Client_PARP_mmddyyyy 24