finding better compounds: label-free assays in compound identification profiling

1. Finding Better Compounds: Label-free Assays in Compound Identification & Profiling

3. 3 Development of automation strategies improve quality & speed of screening campaigns

5. 5 Label-free technology can overcome limits / expand reach of classic biochemistry Definition: Ligand used for screening assay is presented to target free of external, unnatural label (fluorophore, etc) Native ligand can be used Detection does not impact interaction between ligand and target Strategies: Use biophysical properties of substrate / product / system to measure biochemistry Use biology of cell to measure ligand / target interaction

6. 6 Strategies for no-label screening Biochemistry altered surface optical property with hit binding surface plasmon resonance (BiaCore) resonance waveguide grating (Corning EPIC) biolayer interferometry (BioForte) altered protein confirmation with hit binding Surface Enhanced Raman Spectroscopy ligand effect on protein thermal stability ThermoFluor® dye binding high throughput microcalorimetry (Vivactis) Cell-based Cell-substrate sensing resonance waveguide grating (Corning EPIC) peak wavelength value modulation (SRU BIND) cellular dielectric spectroscopy (MDS Sciex) electric impedance sensing (ACEA) Pathway screens using biosensor target activation utilizes native ligand, detection separate

7. 7 Mass spectrometry as no-label detection platform Biologic molecules can be ionized, providing a form that can be detected by appropriate detector. The mass spectrometer sorts ions according to their mass/charge ratios with high accuracy – each mass/charge signal specific for specific biomolecule. The detector converts the ion signal to a proportional electrical current. Quantitating the magnitude of these electrical signals as a function of m/z identifies the specific compound/compounds in question.

8. 8 MS to identify bound library compound(s)

9. 9 J Biomol Screen. 2006 Mar;11(2):194-207. Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry. Whitehurst CE, Nazef N, Annis DA, Hou Y, Murphy DM, Spacciapoli P, Yao Z, Ziebell MR, Cheng CC, Shipps GW Jr, Felsch JS, Lau D, Nash HM. NeoGenesis Pharmaceuticals, Inc., Cambridge, MA 02139 Example of Affinity selection- mass spectrometry

10. 10 Characteristics of affinity selection-mass spectrometry screening Identify compounds which bind target Platform independent of target bioactivity Binding to novel sites on target protein e.g., allosteric sites, etc. Biologic efficacy of hits determined by 2o analysis Binders with no bioactivity common Requires specialized, mass encoded compound library Requires purified, soluble protein target Compounds that destabilize target conformation may represent false positives

12. 12 Technique for rapid sampling / purification / analysis of MS samples

13. BioTrove RapidFireTM technology accurately measures enzymatic activity / kinetics.

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15. 15 “No-label” thermodenaturation method to confirm ligand binding to lipid deacylase Bound ligand stabilizes protein, increasing denaturation temperature exposing hydrophobic dye binding sites

19. The evasive nature of drug efficacy: implications for drug discovery “…ligands that behave as agonists toward a given receptor can act, through the same receptor, as antagonists or even inverse agonists on a different pathway in the same cell. These observations… have important implications for the molecular definition of efficacy and the process of drug discovery…” 19

20. Cannabinoid CB2 receptor-specific agonists & inverse agonists modulate chemotaxis - HOW?

22. Cannabinoid CB2 receptor-specific agonists & inverse agonists modulate chemotaxis - HOW?

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26. 26 Operating Principal: Cell-based Assays Measures changes in local index of refraction resulting from ligand-induced mass redistribution within bottom region (~150 nm) of the cell monolayer Change in index measured as shift in resonance wavelength Mass changes at interface detected using CORNING Epic resonance waveguide grating technology

27. 27 Effect G-protein coupling on EPIC signal

28. 28 Profiling biology of b2 adrenoceptor ligands using optical biosensor

30. 30 CONCLUSION: Roles for no-label technologies in drug discovery effort Identification of chemotypes that might be missed using “non-biological” ligands Intermediate throughout screening tool for refractory targets Targets with complex ligands Targets where fluorophore alters ligand binding Compound profiling Identification of unknown properties of chemotype Characterizing efficacy within primary cell preparations

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