“Drug discovery engines”

1. “Drug discovery engines” Using membrane receptor microarrays to streamline the initial stages of drug discovery

2. The present-day drug discovery process is slow and painful • Potential targets are identified • Large number of potential drug candidates are synthesized, and target binding assessed • Scale-up synthesis, do target/non-target analysis • Scale-up synthesis, do ADMET studies, flunk nearly all candidates • Few survivors go to whole cell/in-vivo studies

3. A better drug discovery platform: • Would analyze micro-quantities of candidate drugs • Would have high throughput • Would perform multiple differential target/non target binding studies + ADMET analysis • Would be simple and low-cost to run. • Would automatically send “hits” to analysis

4. Membrane microarrays could transform the drug discovery process Identify targets Synthesize multiple candidate drugs “Drug discovery engine” Combined membrane microarray target, non-target, ADMET test Big improvement if can put membrane targets, non-targets, and ADMET proteins into a unified flow-cell or microarray test device HIT!

5. Today’s talk: • Will discuss new technical approaches for membrane microarrays that might enable such high-performance “drug discovery engines” • Will give examples using serotonin receptors, Cytochrome P450, and P-glycoprotein membrane proteins

6. Quick membrane review: Membrane components are laterally mobile in the plane of the membrane

7. GPCR receptors • Transmembrane proteins • Hundreds of variants • Targets for 60% of all known drugs • Good drugs react specifically with only their target GPCR receptor

8. How to make better membrane assay systems? Reusable membrane receptor microarray flow-cell would be ideal Membrane receptors need to move freely, yet large numbers of different receptors must be bound Device must detect binding of drug candidate ligands, and then be regenerated Microarray

9. New approach to membrane microarrays: bound “evanescent liposomes” • Incorporate fluorescently tagged receptors into phospholipid vesicles (liposomes) • Bind liposomes onto a solid surface. • Expose the surface to evanescent wave illumination, and observe the fluorescent signal Liposome

10. http://www.olympusmicro.com/primer/java/tirf/penetration/ What are evanescent waves? • Produced when a beam of light bounces back from a boundary with a different index of refraction. • Part of the light penetrates a few hundred nanometers into the other medium, and rapidly decays to zero • Interesting fact: Phospholipid vesicles are a few hundred to a few thousand nanometers in diameter…

11. Liposomal quadrant (2 nm liposomes) Average distance Evanescent intensity Upper quarter 1750 nm 0.18% Upper middle quarter 1250 nm 1.10 % Lower-middle quarter 750 nm 6.67% Lower quarter 250 nm 40.56% Microarray bound base 0 nm 100% Evanescent wave intensity Microarray

12. Membrane microarray production • A: Incorporate targets into liposomes • B: Bind liposomes to microarray surface • C: Hold targets to surface using “bridge ligands” • D: Probe target distribution with evanescent illumination • E: Candidate drugs compete with “bridge ligands” for target binding

13. Label target with fluorescent moiety Biotin label anchor group A: Incorporate targets into phospholipid vesicles (liposomes) Incorporate target and anchor into phospholipid vesicle

14. B: Bind vesicles onto general-purpose evanescent wave detector surface General purpose evanescent wave microarray surface

15. GPCR binding ligand Haptein C: Hold targets onto microarray surface using “bridge ligands” Fluorescent label GPCR Liposome Anti-haptein antibody Tether

16. D1: Expose to evanescent/normal illumination and probe with test analytes Case A: receptor binding is not perturbed by external analytes

17. D2: Expose to evanescent/normal illumination and probe with test analytes Case B: receptor binding is perturbed by external analytes

18. Evanescent/Fluorescent ratio • Evanescent signal will vary as a function of receptor distance from the microarray surface • By contrast, conventional fluorescent epi-illumination signal will remain constant • Utilize Evanescent/Fluorescent signal to normalize results, and correct for errors

19. Digital camera Reference light source Band pass filter Fluorescent microscope (used for reference signal) Band pass mirror Microscope optics Microarray Collimator Mirror 488 nm Argon laser Evanescent light source Instrumentation:

20. GPCR binding ligand Haptein Drug discovery applications Fluorescent label GPCR Liposome Anti-haptein antibody To make a drug assay: • Label GPCR receptor • Bind receptor to microarray with haptein-coupled ligand Tether

21. GPCR GPCR Effect of drug binding Candidate drug Liposome Tether Anti-haptein antibody Add unlabeled drug Bond between GPCR receptor and microarray is disrupted Evanescent signal Tether

22. 5-HT2B 5-HT1A 5-HT6 5-HT2C Example: serotonin (5-HT) receptor membrane microarray • Multiple types of 5-HT serotonin receptors involved in depression, appetite, other disorders • Need high-specificity drugs with minimal cross-talk • Microarray enables rapid screening of candidate drugs

23. 5-HT2B 5-HT1A 5-HT6 5-HT2C Example: serotonin (5-HT) receptor membrane microarray • The assay in action: here a ligand against the 5-HT6 receptor has disrupted the bond between 5-HT6 and the microarray surface, quenching the evanescent signal.

24. But finding a good target is just the beginning… • Most candidates fail ADMET testing. Huge advantage to catch these failures as quickly as possible. • With liposome microarray technology, ADMET testing can be performed a few seconds after the initial target binding tests.

25. Membrane proteins in ADMET • ADMET: (Absorbtion, Distribution, Metabolism, Excretion, Toxicity) studies fundamental to modern pharmacology • AD: Many drugs are transported by the ABC membrane transporter family • MET: Most drugs are broken down by the CYP450 family of membrane proteins

26. http://www.ibcp.fr/IBCP/baggetto/LGB_FieldResearch.html ABC drug transporter family • Involved in active transport of drugs across cell membranes • P-glycoprotein (p-gp) transports many different drugs, key to the AD part of ADMET analysis • Chemotherapy resistant cells overexpress P-gp

27. http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG14_02.JPGhttp://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG14_02.JPG CYP450 drug metabolizer family • Six types of CYP450 membrane proteins metabolize 90% of all drugs (CYP3A4 >50%) • Responsible for MET part of ADMET • Bad (variant) CYP450 performance can kill a drug candidate

28. Pharmokinetics ~ P-gp + CYP3A4 P-gp transports drugs from the bloodstream into liver microsomes, where the drugs are metabolized by CYP3A4 This transport-metabolism reaction controls pharmokinetics http://www.liv.ac.uk/hivgroup/learning/basicpk/img018.gif

29. Instant micro-ADMET? • Check binding to P-gp and other ABC family transport membane proteins • Check binding to CYP34A and other CYP450 metabolism membrane proteins • Using same sample, immediately see if a drug candidate has potential ADMET problems

30. Targets CYP450s ABCs ADMET The goaL: a “drug discovery engine” flow-cell Sample

31. Bead 2 A “drug discovery engine” using a membrane flow-cell system Target ADMET Analyze single microbead quantities of candidate drugs Waste hits Bead 1 Perform multiple differential target/non target binding studies + ADMET analysis No hits Analyze Send good samples to Mass Spec. for analysis

32. Membrane flow-cell technology + microfluidic switches • Can analyze nanoscale quantities of candidate drugs • Performs multiple differential target/non target binding studies + ADMET analysis • Regenerates reagents to keep costs down • Sends good samples to Mass Spec. for analysis

33. The net benefit: a quicker and simpler drug discovery process • Identify targets • Synthesize huge number of leads, and run through a “drug discovery engine”. • Identify hits, scale-up synthesis • Go to whole cell/in-vivo studies