Assay Report. Histone Deacetylase (HDAC) Inhibitor Assays Enzymatic Study of Compounds from Client

Transcription

1 Assay Report Histone Deacetylase (HDAC) Inhibitor Assays Enzymatic Study of Compounds from Client Page 1 of 27 Client_HDAC _Year Month Day 1

2 Client_HDAC_Year Month Year HDAC Inhibitor Assays Study Sponsor: Client Attention: Address: Study Director: Testing Facility: Henry Zhu, Ph.D. BPS Bioscience Inc. 42 Cornerstone Court West, Ste. B USA Study Period: Report Version: 1 Report Date: Month Day, Year Page 2 of 27 Client_HDAC _Year Month Day 2

3 Study Director Scientist Date Henry Zhu, Ph.D. President Date Page 3 of 27 Client_HDAC _Year Month Day 3

4 CONTENTS HISTONE DEACETYLASE (HDAC) INHIBITOR ASSAYS... 1 ENZYMATIC STUDY OF COMPOUNDS FROM CLIENT... 1 HDAC INHIBITOR ASSAYS... 2 STUDY DIRECTOR... 3 PURPOSE OF THE STUDY MATERIALS AND METHODS MATERIALS COMPOUNDS EXPERIMENTAL CONDITIONS Enzymes and Substrates Assay Conditions Data Analysis ASSAY RESULTS SUMMARY OF THE INHIBITORY EFFECTS OF THE COMPOUNDS ON HDAC AND SIRTUIN ACTIVITIES RESULTS OF THE EFFECTS OF THE COMPOUNDS ON INDIVIDUAL HDAC ACTIVITY HDAC SAHA HDAC SAHA HDAC3/NCOR SAHA HDAC TSA HDAC TSA HDAC SAHA HDAC TSA HDAC TSA HDAC TSA HDAC SAHA HDAC TSA SIRT Nicotinamide SIRT Nicotinamide SIRT Nicotinamide Page 4 of 27 Client_HDAC _Year Month Day 4

5 SIRT Nicotinamide QUALITY ASSURANCE STATEMENT Page 5 of 27 Client_HDAC _Year Month Day 5

6 Purpose of the Study The purpose of the study is to determine the effects of compounds from Client on the activities of recombinant HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC, HDAC11, Sirtuin1, Sirtuin2, Sirtuin3, and Sirtuin5 using an in vitro enzymatic assay. Page 6 of 27 Client_HDAC _Year Month Day 6

7 2. Materials and Methods 2.1 Materials SAHA is purchased from Cayman Chemicals (Ann Arbor, MI, Catalog Number ). Nicotinamide is purchased from Sigma-Aldrich (St. Louis, MO, Catalog number 723). TSA is purchased from Selleck (Houston, TX, Catalog number S45). HDAC Assay Buffer (BPS catalog number 031) HDAC Assay Developer (BPS catalog number 0) SIRT Assay Developer (BPS catalog number 089) HDAC Substrate 1 (BPS catalog number 032) HDAC Substrate 3 (BPS catalog number 037) HDAC Class 2a Substrate 1 (BPS catalog number 0) Sirtuin 5 Substrate (BPS catalog number 126) 2.2 Compounds The test compounds are supplied by AMSBIO, LLC. Compound I.D. Compound Supplied Stock Concentration Dissolving Solvent Test Range (μm) Intermediate Dilution SAHA * Solid mm DMSO TSA * Solid mm DMSO Nicotinamide * Solid 0mM H 2 O * Reference Compound % DMSO in HDAC Assay Buffer % DMSO in HDAC Assay Buffer % DMSO in HDAC Assay Buffer Page 7 of 27 Client_HDAC _Year Month Day 7

8 2.3 Experimental Conditions Enzymes and Substrates Assay Catalog # Enzyme Lot # Enzyme Used (ng) / Substrate Reaction HDAC B 7.2 µm HDAC Substrate 3 HDAC G 7.5 µm HDAC Substrate 3 HDAC3/NCOR µm HDAC Substrate 3 HDAC µM HDAC Substrate Class 2a HDAC µM HDAC Substrate Class 2a HDAC A µm HDAC Substrate 3 HDAC µM HDAC Substrate Class 2a HDAC µM HDAC Substrate Class 2a HDAC µM HDAC Substrate Class 2a HDAC B 0 µm HDAC Substrate 3 HDAC µM HDAC Substrate Class 2a SIRT µm HDAC Substrate 1 SIRT B 00 µm HDAC Substrate 1 SIRT B µm HDAC Substrate 1 SIRT µm SIRT5 Substrate Page 8 of 27 Client_HDAC _Year Month Day 8

9 2.3.2 Assay Conditions All of the compounds are dissolved in DMSO. The serial dilution of the compounds was first performed in 0% DMSO with the highest concentration at 1mM. Each intermediate compound dilution (in 0% DMSO) will then get directly diluted x fold into assay buffer for an intermediate dilution of % DMSO in HDAC assay buffer and 5µl of the dilution was added to a µl reaction so that the final concentration of DMSO is 1% in all of reactions. The enzymatic reactions for the HDAC enzymes were conducted in duplicate at 37ºC for minutes in a µl mixture containing HDAC assay buffer, 5µg BSA, an HDAC substrate (see 2.3.1), a HDAC enzyme (see 2.3.1) and a test compound (see 2.2). The enzymatic reactions for the SIRT enzymes were conducted in duplicate at 37ºC for minutes in a µl mixture containing HDAC assay buffer, 5µg BSA, 0.5mM NAD + an HDAC substrate (see 2.3.1), a SIRT enzyme (see 2.3.1) and a test compound (see 2.2). After enzymatic reactions, μl of 2 x HDAC Developer was added to each well for the HDAC enzymes and the plate was incubated at room temperature for an additional 15 minutes. After enzymatic reactions, μl of 2 x SIRT Developer was added to each well for the SIRT enzymes and the plate was incubated at room temperature for an additional 15 minutes. Fluorescence intensity was measured at an excitation of 3 nm and an emission of 4 nm using a Biotek Synergy microplate reader. Page 9 of 27 Client_HDAC_Year Month Day 9

10 2.3.3 Data Analysis HDAC activity assays were performed in duplicates at each concentration. The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (F t ) in each data set was defined as 0% activity. In the absence of HDAC, the fluorescent intensity (F 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 = (F-F b )/(F t - F b ), where F= the fluorescent intensity in the presence of the compound. 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+ ((LogEC-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 IC value was determined by the concentration causing a half-maximal percent activity. Page of 27 Client_HDAC_Year Month Day

11 3. Assay Results 3.1. Summary of the Inhibitory Effects of the Compounds on HDAC and Sirtuin Activities The IC of the compound against HDACSs and SIRTs are summarized on Table 3.1. If the IC is higher than XμM, the percentage inhibition of the compound at XμM is calculated. Table 3.1 Inhibitory Effects of the Compound on HDAC and SIRT Activities (IC ) Enzymes HDAC HDAC HDAC3/NCOR IC (μm) or Percentage Inhibition SAHA TSA Nicotinamide HDAC4 3.5 HDAC5 1.8 HDAC HDAC7 1.4 HDAC HDAC9 4.4 HDAC HDAC SIRT1 177 SIRT2 41 SIRT3 21 SIRT5 12 Page 11 of 27 Client_HDAC_Year Month Day 11

12 3.2. Results of the Effects of the Compounds on Individual HDAC Activity HDAC SAHA Table Data for the Effect of SAHA on HDAC1 Activity HDAC1 Activity SAHA No Compound Background HDAC1 Activity Substrate Conc.= µm Substrate 3 (037) SAHA, µm IC = 0.031µM Page 12 of 27 Client_HDAC_Year Month Day 12

13 HDAC SAHA Table Data for the Effect of SAHA on HDAC2 Activity HDAC2 Activity SAHA No Compound Background HDAC2 Activity Substrate Conc.= µm Substrate 3 (037) SAHA, µm IC = 0.067µM Page 13 of 27 Client_HDAC_Year Month Day 13

14 HDAC3/NCOR SAHA Table Data for the Effect of SAHA on HDAC3/NCOR2 Activity HDAC3/NCOR2 Activity SAHA No Compound Background HDAC3/NCOR2 Activity Substrate Conc.= µm Substrate 3 (037) SAHA, µm IC = 0.028µM Page 14 of 27 Client_HDAC_Year Month Day 14

15 HDAC TSA Table Data for the Effect of TSA on HDAC4 Activity HDAC4 Activity TSA No Compound Background HDAC4 Activity Substrate Conc.=2 µm Class 2a Substrate 1 (0) 1 0 IC = 3.5µM TSA, µm Page 15 of 27 Client_HDAC_Year Month Day 15

16 HDAC TSA Table Data for the Effect of TSA on HDAC5 Activity HDAC5 Activity TSA No Compound Background HDAC5 Activity Substrate Conc.=2 µm Class 2a Substrate 1 (0) 1 0 IC = 1.8µM TSA, µm Page 16 of 27 Client_HDAC_Year Month Day 16

17 HDAC SAHA Table Data for the Effect of SAHA on HDAC6 Activity HDAC6 Activity SAHA No Compound Background HDAC6 Activity Substrate Conc.= µm Substrate 3 (037) SAHA, µm IC = 0.011µM Page 17 of 27 Client_HDAC_Year Month Day 17

18 HDAC TSA Table Data for the Effect of TSA on HDAC7 Activity HDAC7 Activity TSA No Compound Background HDAC7 Activity Substrate Conc.=2 µm Class 2a Substrate 1 (0) 1 0 IC = 1.4µM TSA, µm Page 18 of 27 Client_HDAC_Year Month Day 18

19 HDAC TSA Table Data for the Effect of TSA on HDAC8 Activity HDAC8 Activity TSA No Compound Background HDAC8 Activity Substrate Conc.=2 µm Class 2a Substrate 1 (0) 1 0 IC = 0.63µM TSA, µm Page 19 of 27 Client_HDAC_Year Month Day 19

20 HDAC TSA Table Data for the Effect of TSA on HDAC9 Activity HDAC9 Activity TSA No Compound Background HDAC9 Activity Substrate Conc.=2 µm Class 2a Substrate 1 (0) 1 0 IC = 4.4µM TSA, µm Page of 27 Client_HDAC_Year Month Day

21 3.2.. HDAC SAHA Table Data for the Effect of SAHA on HDAC Activity HDAC Activity SAHA No Compound Background HDAC Activity Substrate Conc.= µm Substrate 3 (037) SAHA, µm IC = 0.082µM Page 21 of 27 Client_HDAC_Year Month Day 21

22 HDAC TSA Table Data for the Effect of TSA on HDAC11 Activity HDAC11 Activity TSA No Compound Background HDAC11 Activity Substrate Conc.=2 µm Class 2a Substrate 1 (0) 1 0 IC = 12.1µM TSA, µm Page 22 of 27 Client_HDAC_Year Month Day 22

23 SIRT Nicotinamide Table Data for the Effect of Nicotinamide on SIRT1 Activity SIRT1 Activity Nicotinamide No Compound Background SIRT1 Activity Substrate Conc.=µM Substrate 1 (032) Nicotinamide, µm IC = 177µM Page 23 of 27 Client_HDAC_Year Month Day 23

24 SIRT Nicotinamide Table Data for the Effect of Nicotinamide on SIRT2 Activity SIRT2 Activity Nicotinamide No Compound Background SIRT2 Activity Substrate Conc.=µM Substrate 1 (032) Nicotinamide, µm IC = 41µM Page 24 of 27 Client_HDAC_Year Month Day 24

25 SIRT Nicotinamide Table Data for the Effect of Nicotinamide on SIRT3 Activity SIRT3 Activity Nicotinamide No Compound Background SIRT3 Activity Substrate Conc.=µM Substrate 1 (032) Nicotinamide, µm IC = 21µM Page 25 of 27 Client_HDAC_Year Month Day 25

26 SIRT Nicotinamide Table Data for the Effect of Nicotinamide on SIRT5 Activity SIRT5 Activity Nicotinamide No Compound Background SIRT5 Activity Substrate Conc.=µM Substrate 5 (126) Nicotinamide, µm IC = 12µM Page 26 of 27 Client_HDAC_Year Month Day 26

27 4. 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 27 of 27 Client_HDAC_Year Month Day 27