Enabling Lead Discovery at Epigenetics Targets with RapidFire Mass Spectrometry

Transcription

1 Enabling Lead Discovery at Epigenetics Targets with RapidFire Mass Spectrometry Stuart Baddeley Director, Screening and Compound Profiling GlaxoSmithKline R&D Stevenage, UK

2 Overview 1. Introduction to epigenetics 2. Introduction to the RapidFire high throughput mass spectrometry system 3. Compare and contrast various technologies; does RapidFire offer advantages for lead discovery at epigenetic targets? 4. Summary and conclusions

3 Molecular Basis of Epigenetics Epigenetics (epi: over, above) refers to change in phenotype through mechanisms other than changes in DNA sequence E.g cellular differentiation (activation/repression genes) N-terminal tail of histones can carry posttranslational modifications which play an important role in regulation of gene expression modification events can be activating or repressive

4 Molecular Basis of Epigenetics

5 Histone Methylation and Demethylation Two families of histone lysine demethylases have been identified: Lysine specific demethylase (amine oxidases) (Shi et al, 2004) Jumonji C (JmjC) domain family proteins. (Tsukada et al, 2006) Jmj Demethylase + 2-OG + FeII + formaldehyde + succinate

6 The Challenges of Screening for Inhibitors of Histone Demethylase Enzymes A number of assays have been developed in house to study inhibition of histone demethylases but each have their limitations: Analytical Biochemistry 404 (1):86-93, 2010.

7 Mass Spectrometry as a Label Free Technology for the Study of Histone Demethylases Enzyme activity can be measured directly via MS in a label free environment : Substrate (MW1) Enzyme Product (MW2) % Conversion = MW2 (product) * 100 MW1 (substrate) + MW2 (product) Highly sensitive technique, ideal for low turnover enzymes Direct quantification of substrate and product (label free) Eliminates need to develop specific antibodies or fluorescent labels Decreased risk of interference No coupling enzymes Fast assay development

8 Agilent RapidFire Technology

9 Case Study; Hit Identification for a Jmj domain family demethylase using RapidFire Assay development, determination of kinetic parameters Pharmacological validation Jmj Demethylase Hit Id Campaign: + 2-OG 100,000 compound screening set + FeII + formaldehyde + succinate 5µl 5µl Stop reaction with 30ul 0.5% TFA

10 Example RapidFire Data; Demethylase Assay Me 3 Substrate Me 2 Product

11 % Conversion Me3 to Me2 Assay Development Titration of (NH4)2Fe(SO4)2 6 K m = 5.66µM +/ K act = 9.59µM +/ [(NH4)2Fe(SO4)2] (µm) Assay allows determination of kinetic parameters e.g. K m

12 MTS Assay validation Replicate Rob Mean Rob SD Rob Cut-off N hits HR(%) n= n=

13 MTS Screening Statistics Total of 104K compounds screened in 10 days on 2 instruments 6500 compounds/18 x 384 well plates per instrument per day

14 IC 50 Follow-up 2028 compounds progressed to IC 50 : All compounds with >40% inhibition at 10µM Compounds with % inhibition between 20 and 40% at 10µM but highly developable.

15 % Inhibition % Inhibition % Inhibition Example IC 50 Data [GSK A] µm [GSK887162A] µm [GSK558331A] µm Exemplars from a known series of Jmj demthylase inhibitors were successfully identified in this campaign. Quantitative High-Throughput Screening Identifies 8-Hydroxyquinolines as Cell-Active Histone Demethylase Inhibitors Oliver N. F. King., Xuan Shirley Li., Masaaki Sakurai, Akane Kawamura, Nathan R. Rose,Stanley S. Ng, Amy M. Quinn,G anesha Rai, Bryan T. Mott, Paul Beswick, Robert J. Klose, Udo Oppermann, Ajit Jadhav, Tom D. Heightman, David J. Maloney, Christopher J. Schofield, Anton Simeonov PLoS ONE 12 November 2010 Volume 5 Issue 11

16 Hit Confirmation Jmj Demethylase Assay: RapidFire vs. TR-FRET Data TR-FRET RapidFire HTMS TR-FRET Interference

17 Multiplexing Possibilities RapidFire for True HTS? Challenge to bring RapidFire throughput into same league as conventional fluorescent based HTS techniques Single process flow the limiting step. Option 1: Pool compounds in well Dilution effect reduces effective screening concentration, issue with these assays as hit rate is already low. GSK experience suggests poorer follow-up rates for pooled compound screening. Option 2: Can we exploit the advantages of mass spectrometry? Direct measurement of substrate and product dependent on mass Gives the ability to multiplex

18 Multiplexing Possibilities RapidFire for True HTS? Enzyme reaction -1 Enzyme reaction -2 Pooling MS IT Enzyme reaction -3 Enzyme reaction -4 Liquid handling Method Creation Template/Macro Creation Peptide design Peptide Peptide Peptide Peptide

19 K m, k cat Determination for Multiplex Peptides K cat = V max /[E] = sec -1 All K m are within 2 fold of original peptide K m

20 Multiplex Pharmacology Multiplex Individual

21 Multiplex Spiking Experiment 4 DMSO plates were Spiked with four Jumanji inhibitors at either 1uM or 10uM final assay concentration in random locations on the plate and plates were screened either in the standard single peptide assay or as a multiplex: Both single peptide and multiplex assays gave Z of >0.7 and S:B > 3:1 and similar statistical hit cut-offs The single peptide assay identified all 14 wells containing compound, but had a false positive rate of 0.85% The multiplex assay detected 13 out of the 14 of the compound wells at 1uM as a hit Samples Control_1 Control_2/3 Cut-off Multiplex Single Robust Cut off Hit rate % of spiked cpds identified 93% 100% % false -ve 7% 0 % false +ve 0.10% 0.85% Single Peptide

22 Features RapidFire MALDI-TOF AlphaScreen TR-FRET Coupled Assays Direct Measurement Kinetic Characterisation YES YES NO NO NO YES YES NO NO YES Potential for Fluorescent Interference NO NO YES YES YES Deconvolution of Data Required NO NO NO NO YES Cost LOW (<5p/well) HIGH (>15p/well) HIGH (>15p/well) MEDIUM (5-15p/well) MEDIUM (5-15p/well) Protein Consumption MEDIUM (< 500nM) MEDIUM (<500nM) LOW (< 50nM) MEDIUM ( < 500nM) HIGH ( > 500nM) Throughput MEDIUM LOW HIGH HIGH MEDIUM

23 Summary and Conclusions Modification of histones plays an important role in chromatin regulation, and as such, enzymes which catalyse these modifications represent a novel and expanding class of drug targets. Multiple fluorescent assays have been developed to study small molecule modulators of these enzymes but suffer from interference or are limited in their scope. Mass spectrometry offers a label free alternative to these assays, with direct measurement of demethylation events and the ability to generate tight data from a small assay window by calculating % conversion. RapidFire HTMS has the capacity and robustness to be used for high throughput screening, focus screening and SAR support

24 Acknowledgements Specific Acknowledgements To: Melanie Leveridge Laura Williams Archie Argyrou Sue Hutchinson Roy Katso Laurie Gordon Laurens Kruidenier Alice Mandryk Jack Brown Michelle Gee David Wilson Margaret Martin Michelle Heathcote Isabel Marilia Steve Rees Laura Vela Jon Hutchinson David Calvo Penny Smee Angela Bridges Anthony Shillings Rachel Grimley Mike Woodrow Jordi Munoz-Muriedas

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26 % Inhibition % Inhibition Pharmacological validation Compound IC 50 AlphaScreen (µm) IC 50 FDH (µm) IC 50 MALDI TOF (µm) IC 50 RapidFire (µm) 2,4-PDCA GSK A (NOG analogue) HO O N O OH [GSK A] µm [2,4-PDCA] µm IC 50 Values Taken From: Analytical Biochemistry 404 (1):86-93, 2010.