RapidFire Selected Therapeutic Areas and Screening Techniques

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1 RapidFire Selected Therapeutic Areas and Screening Techniques RapidFire: SPE/MS/MS Life Sciences Group William A. LaMarr, Ph.D. Senior R&D Manager, RapidFire

2 RapidFire Mass Spectrometry Ultra-fast autosampler & online SPE system Replaces LC in LC/MS Reusable SPE cartridge Integrates with standard ESI MS instruments Cycle time = 7-13 s/sample Compatible with various matrices Sub-cellular fractions Cell culture supernatants Cell / tissue extracts Biological fluids

3 Advantages of mass spectrometry True label-free detection Direct, quantitative measurements P 4 Native reaction substrates & products (no radioactivity, no surrogate analytes, no indirect or secondary components) Functional biochemical assays instead of : P 4 (rather than target binding assays)

4 Limitations of MS Molecules must be charged Desalting step required Sample purification is Serial slow Instrumentation is expensive, not easily scalable to meet demand

5 Applications of the RapidFire Platform 1) Native Analyte Detection - surrogate substrates can introduce confounding factors, effect enzyme kinetics, and produce data artifacts 2) Replace Intractable Assays - assays may present challenges in workflow, may be resource intensive, may be cost prohibitive, may present regulatory issues (radioactivity) 3) Enable Target Classes - multiple modification events on the same substrate are impossible to track by many common optical and radioactive methodlogies

6 ADS Customer Base Biotech Large Pharma Multiple Target Customers ne Target Customers Biopharma High Profile Targets Unique Targets Primary Secondary Screening Screening (27x) Primary Screening Secondary Screening (6x) & Support

7 Targeted Therapeutic Areas Metabolic Disorder Anti-Infectives ncology ther Inflammation Epigenetics Inflammation Cardiovascular Disease Metabolic Disorder

8 Metabolic Disorder

9 Metabolic Disorders Class of genetic disease that encompasses varied conditions Inborn errors of metabolism Congenital metabolic disease Most are due to a single enzyme mutation effecting conversion of substrate to product and often result in: accumulation of toxic substances or reduced ability to synthesize essential compounds Because of the diverse nature of the diseases in the group, accurate numbers for incidence are difficult to determine. ne Canadian study* found that approximately 15% of single gene disorders in the population are considered metabolic disorders. * Applegarth DA, Toone JR, Lowry RB (January 2000). "Incidence of inborn errors of metabolism in British Columbia, ". Pediatrics 105 (1): e10

10 Enzymes and associated disorder(s) An example of enzymes discussed in today s presentation: Enzyme Role Associated Metabolic Disorder(s) Serine palmitoyltransferase Involved in sphingolipid biosynthesis Hereditary sensory neuropathy type 1 Acetyl CoA Carboxylase Involved in fatty acid synthesis Type 2 Diabetes ATP citrate lyase GM3 Synthase Stearoyl CoA Desaturase Diacylglycerol Acyltransferase Crucial in many biosynthetic pathways including lipogenesis and cholesterolgenesis Involved in the biosynthesis of complex gangliosides Important for the desaturation of fatty acids Catalyzes the synthesis of triglycerides from digylcerides Fatty liver disease, type 2 diabetes Infantile seizure/epilepsy disorder besity, liver disease obesity

11 Acetyl-Coenzyme A Carboxylase + KH 14 C 3 AT P Acetyl Coenzyme A 2 14 C H Malonyl Coenzyme A

12 Acetyl-Coenzyme A AssayConditions 50 mm HEPES, ph mm MgCl 2 2 mm tripotassium citrate 2 mm DTT 0.75 mg/ml BSA 4 mm ATP 12.5 mm KHC 3 20 mm Acetyl-Coenzyme A *ACC assay conditions based on previously published 14C-incorporation assay protocol: Harwood, H.J., Jr. et al. J. Biol. Chem. (2003), 278 (39):

13 Acetyl-Coenzyme A (m.w. 809) Malonyl-Coenzyme A (m.w. 853)

14 * Sumper, M. Eur. J. Biochem. (1974), 49 (2): * Harwood, H.J., Jr. et al. Curr. pin. Investig. Drugs (2004), 5 (3):

15 Stearoyl-Coenzyme A Desaturase NH 2 3 H 3 H S H N H N H H P - P - NH 4 + NH 4 + N N N N H P H - NH 4 + NH 2 S + H N H N H H N N N N P P - NH NH 4 H P H - - NH H 3 H

16 Stearoyl-Coenzyme A leoyl-coenzyme A * Soulard, P., et al. Analytica Chimica Acta (2008), 627 (1):

17 Z' Score > 200,000 wells screened Plate Number

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19 Diacylglycerol Acyltransferase Diolein 14 C 14 C 14 C H N NH 2 N H N N P H P H P H H N H H N S leoyl-coenzyme A H Triolein 14 C 14 C 14 C

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21 IC 50 = 49.1 nm Z Score = 0.77

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23 Epigenetics

24 Protein Methyltransferase Assay Strategies ThioGlo Assay SAH detection AlphaScreen Masoud Vedadi Pan- methylation or selective antibodies are needed for multiple states Amy Quinn F. Liu et.al., Journal of Medicinal Chemistry. 2009:

25 Protein Methyltransferase Assay Strategies Fluorescence Polarization Transcreener Epigen methyltransferase assay principle. Klink T A et al. J Biomol Screen 2011;17:59-70

26 Methylation-Sensitive Proteolysis Assay on EZ Reader II Wigle et al, Chemistry & Biology,

27 Co-Product Measurements -Histone Methyltransferases (HMTs) S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH) -Histone Acetyltransferases (HATs) Acetyl-coenzyme A (ACoA) to Coenzyme A (CoA) -Sirtuin Deacetylases (SIRTs) Nicotinamide adenine dinucleotide (NAD + ) to nicotinamide acetyl-adp-ribose (AADPr)

28 Direct measurement of the AADPr product of sirtuin reactions SIRT1, SIRT2, SIRT3 & SIRT5 & SIRT6? Acetylated Protein De-Acetylated Protein NH 2 N N NH 2 N N P H P - N + NH 2 N N N N P H P - H + N NH 2 H H H H H H H NAD acetyl-adp-ribose Nicotinamide

29 SIRT1 - Enzyme Titration Timecourse Reaction Conditions: 50 mm Tris ph mm NaCl 2.7 mm KCl 1 mm MgCl % BSA 5 mm DTT 100 mm NAD + 10 mm p53 peptide (Anaspec cat # 62121) 2 --acetyl-adp-ribose Internal Standard ~12.5 minute analysis time

30 Initial Velocity (V 0 ) SIRT1 - Enzymatic Parameters (Linearity, K m & IC 50 ) 2 --acetyl-adp-ribose Based Analysis 1.2E E E E E E E+00 R² = Enzyme Dilution K m = 28 ± 5 mm K m = 54 ± 1 mm IC 50 = 77 ± 1 mm

31 Comparison of Peptide Based and 2 --acetyl- ADP-ribose Based Assay Parameters Peptide Read K m of NAD + K m of peptide IC 50 of nicotinamide 2AADPr Read Peptide Read 2AADPr Read Peptide Read 2AADPr Read SIRT1 38 ± 4 mm 54 ± 1 mm 25 ± 6 mm 28 ± 5 mm 62 ± 1 mm 77 ± 1 mm SIRT2 46 ± 11 mm 50 ± 8 mm 8 ± 3 mm 12 ± 1 mm 10 ± 1 mm 11 ± 1 mm SIRT3 118 ± 44 mm 144 ± 21 mm 4 ± 1 mm 6 ± 1 mm 31 ± 1 mm 39 ± 1 mm

32 Direct Peptide Measurements Histone Demethylases Lysine Demethylase 1 (LSD-1) uses Flavin Adenine Dinucleotide (FAD) Jumanji Domain 2a (JMJD2a) uses Fe+2 mediated oxidative chemistry Histone Deacetylases Histone Deacetylase 1 (HDAC-1) uses a metal dependent hydrolysis

33 SIRT1 - Enzyme Titration Timecourse Reaction Conditions: Deacetylated Peptide 180 minutes 120 minutes 240 minutes 50 mm Tris ph mm NaCl 2.7 mm KCl 1 mm MgCl % BSA 5 mm DTT 100 mm NAD + 10 mm p53 peptide (Anaspec cat # 62121) 30 minutes Enzyme 15 minutes 0 minutes Acetylated Peptide 45 minutes 60 minutes ~12.5 minute analysis time

34 Percent Conversion Initial Velocity (V 0 ) SIRT1 - Enzyme Linearity Enzyme Titration / Timecourse Enzyme Linearity x enzyme x enzyme x enzyme R² = Time (minutes) Enzyme Dilution

35 SIRT1 - Enzymatic Parameters (K m, IC 50, etc ) K m = 25 ± 6 mm K m = 38 ± 4 mm Deacetylated peptide IC 50 = 62 ± 1 mm Acetylated peptide Literature IC 50 value ~ 50 mm Bitterman et. al., J. Biol. Chem. (2002) 277: Marcotte et. al., Anal. Biochem. (2005) 332:90-99 Nicotinamide ~ 3 minute analysis time (8-point log-dilution IC 50 curve, n=3)

36 Initial Velocity SIRT2 - Enzymatic Parameters (Linearity, K m & IC 50 ) y = x R² = Enzyme Concentration (x stock) K m = 8 ± 2 mm K m = 46 ± 11 mm IC 50 = 9.6 ± 0.1 mm

37 Initial Velocity SIRT3 - Enzymatic Parameters (Linearity, K m & IC 50 ) y = x R² = Enzyme Concentration (x K m = 4.3 ± 1.0 mm K m = 118 ± 44 mm IC 50 = 31 mm

38 Labeled vs. Un-labeled Sirtuin Assay Free fluorophore Protein Labeled Acetylated Peptide Protein Unlabeled Acetylated Peptide H N CH C CH 2 CH 2 CH 2 CH 2 HN H N CH C CH 2 CH 2 CH 2 CH 2 HN Protein Protein SIRT1/Chymotrypsin Resveratrol Howitz et. al., Nature (2003) 425: SIRT1/Chymotrypsin Resveratrol Kaeberlein et. al., J. Biol. Chem. (2005) 280: Beher et. al., Chem. Biol. Rug Des. (2009) 74: Pacholec et. al., J. Biol. Chem. (2010) 285: Protein H 2 N CH C CH 2 CH 2 CH 2 CH 2 NH 2 Protein De-Acetylated Peptide H N CH C CH 2 CH 2 CH 2 CH 2 NH 2 Protein De-Acetylated Peptide Reaction Activation X No Reaction Activation

39 SIRT1 - Substrate Dependant Activation by Resveratrol Labeled Peptide Unlabeled Peptide Rye et. al., J. Biomol. Screen. (2011) 16: Milne et. al., Nature (2007) 450:

40 Multiple Modification Events on a Single Peptide potential acetylation site Preferred acetylation sites potential acetylation site -Human p53 ( ) -19-mer peptide - Six potential acetylation sites Rye et. al., J. Biomol. Screen. (2011) 16: Prives et. al., Nature (2007) 450:

41 Percent of Control Direct Measurement of Modification Events on Whole Histone Proteins Protein (Q-TF) AcCoA/CoA (QqQ) [Garcinol] (mm) Incorporation of a high-resolution (Q-TF) mass spectrometer into the RapidFire workflow allows the substitution of whole proteins for representative peptide based sequences in a high-throughput screening compatible mode. Rye et. al., J. Biomol. Screen. (2011) 16:

42 Screening Histone Demethylases in a Pharmaceutical Drug Discovery Setting Kruidenier et. al., Nature (2012) 488: Plant et. al., J. Anal. Biochem. (2011) 419: Hutchinson et. al., J. Biomol. Screen. (2012) 17: Melanie Leveridge, MipTec 2011, oral presentation

43 Conclusions Mass spectrometry is an excellent tool for epigenetic target based drug discovery because if it s ability to: - Directly measure native, label-free peptides and generic reaction co-products - Directly and independently measure multiple modification events on single substrates - Directly measure modifications to whole protein substrates

44 Fragment Based Drug Discovery

45 Fragment Based Drug Discovery

46 BACE-1 Assay: Fragment Based Screen Cary Eclipse Fluorescence Spectrophotometer

47 Assay Setup Fluorescent Peptide Fluorescence Plate Reader X Unlabeled Peptide Fluorescence Plate Reader Fluorescent Peptide Mass Spectrometer Unlabeled Peptide Mass Spectrometer

48 Assay System Characterization Unlabeled Substrate By MS (UMS) Fluorescently Labeled Substrate by FS (FS) Fluorescently Labeled Substrate by MS (LMS)

49 UMS LMS FS all 3 Initial Screening Results Hits by Assay Format Hits by Assay Format UMS UMS LMS FS all LMS FS

50 Hits bserved by MS nly Follow-up of selected hits confirmed that compound autofluorescence (AF) obscured several hits in the FS data, including the most potent analyte. Titration of that compound revealed a concentration-dependent increase in signal in the FS assay, suggesting AF, while the MS data were consistent with a traditional inhibition curve.

51 Hits bserved with the Labeled Peptide nly A second class of compounds was uncovered consisting of those molecules that appear as hits when the labeled peptide is employed (as in the FS and LMS assays), but do not show inhibition when the more native substrate is used (UMS). These results suggest that perhaps compounds exist that interfere with the enzyme s ability to bind the peptide carrying the bulky label but not with the tighter binding exhibited by the enzyme for the unlabeled substrate, raising the possibility of misleading data being produced when modified substrates are employed.

52 Hits bserved with the Unlabeled Peptide nly Yet another class of inhibitors was detected in the unlabeled assay (UMS) but was not found with the fluorescent peptide (FS or LMS). Because MS eliminates the need for unnatural modification of substrates, it allows the study of more biologically relevant molecules. These more realistic substrates could reveal activities that are lost with modified peptides, possibly due to altered binding, which in this case was clearly revealed by the K m experiments. Unlabeled Substrate By MS (UMS) Fluorescently Labeled Substrate by MS (LMS)

53 Conclusions Robust assays were developed for both a labeled and an unlabeled substrate of the BACE-1 enzyme. Screening a fragment library against both substrates using both detection methods produced three disparate hit sets. FS and MS produced different hit sets when used as complementary detection methods on the same samples. While some MS hits (including the most potent) were obscured by autofluorescence in the FS assay, this did not account for all of the differences between the methods. MS generated different hit sets for the labeled and the unlabeled peptide, underscoring the importance of substrate selection. Label-free screening by high-throughput MS has proven to be a valid method for conducting activity-based screens of fragment libraries that enables the study of more native molecules and is less susceptible to confounding factors, such as AF.

54 Target Binding Assays

55 Mass Spectrometry Approaches to Binding Assays

56 Questions? Agilent Technologies William A. LaMarr, Ph.D. Senior R&D Manager, RapidFire