Live cell imaging: Visualization of [Ca 2+ ] i fluctuation Dual-wavelength methods –Alternating excitation wavelength with fixed emission observation (Fura) –Looking at different emission bands with constant excitation (Indo, GFP- FRET) Single-wavelength methods –Fast sequential acquisition of fluorescent calcium indicators (Fluo 3, Fluo 4) Hit to lead

Dual-wavelength ratiometric dyes vs single-wavelength dyes for HTS Dual-wavelength Pros –Correction for variation in cell number –Correction for efficiency loading Cons –Require UV excitation that could result in significant autofluorescence –Photolysis of photosensitive caged compounds –Difficult to measure small variation in calcium –More complex technology Single-wavelength Pros –Dissociation constant ideal for measuring intracellular calcium variation (200 – 400 nM) –Instrument more simple –Excited by wavelength in the visible region. Cons –Cannot compensate for variability in the sample –Cannot correct for efficiency loading Hit to lead

Some examples: fluorescence based Ca 2+ measurement G q PCR IP 3   qq PIP 2 DAG PKC PLC Ca 2+ Dye FLUO4 Dye loading Hit to lead

FLIPR ® system Hit to lead

ss GDP ss GTP ii ii GDP Ligand 1 Ligand 2 Some examples: cAMP measurement Hit to lead

Enzyme fragment complementation (EFC) technology -galattosidase (-gal) protein from E. coli is split in two fragments: a bigger one (acceptor enzyme, EA), and a smaller one (donor enzyme, ED). These fragments are inactive but when put together interact rapidly and form the active enzyme that hydrolyzes the substrates, producing a detectable signal. The cAMP from the cell lysates competes with the marked cAMP for the antibody (conjugated ED-cAMP). The non-linked ED-cAMP is free to complement the EA fragment, creating the active enzyme that will hydrolyze the luminescent substrate Some examples: cAMP measurement Hit to lead

Questions?

Reporter gene are DNA sequence coding for exogenous proteins which are either easy to detect (fluorescent) or produce products that are easy to detect. Reporter gene technology Examples: GAL (-galactosidase) - hydrolize a substrate that become colored - colorimetric assay GFP (green fluorescent protein) - produce fluorescence under UV light - fluorescence assay LUC (luciferase) - by oxidazing luciferine produces photons - luminometric assay Hit to lead

Screening on cells I. Cell Production II. Cell treatment &/or compound exposure III. Signal development/capture Hit to lead

Screening steps Creation of a cell line containing the gene of interest Standardization of the assay First screening of the library and identification of the putative hits Confirmation of the hits by performing a dose response Hit to lead

SP1999 construct for FLIPR Screen Control Co-transfection Chimera PLC Ca++ GiGqCotransfection GiGq PLC Fused receptor/Gi/Gq allows for transfection of a single molecule and increases signal.Chimera Hit to lead

EC500.0 nM1.5950e nM1.9350e nM1.0580e nM2.8510e nM5.2240e-008 Assay standardization Hit to lead 2-MeS-ADP dose response MeS-ADP Concentration [log M] Relative Fluorscence Units (max-min) EC 50 =1.59 nM Dose response of standard agonist Parameter to be standardized -cell number per well -solvent concentration -dye loading time -incubation time -instrument parameter setting -Etc, etc

Example of screening results Screening resulted in 2926 hits of which 1310 were confirmed after 5-points IC 50 run These compouds were clustered in 200 groups By combining the most active cluster members and the top 200 compounds (sorted by average IC 50 ) were identified 338 compounds to be tested Hit to lead

Example of hits Ki 278 nM Ki 848 nM Ki 902 nM Ki 592 nMKi 796 nM Ki 775 nM Hit to lead Evaluation of compounds’ chemical tractability Evaluation of patent position Identification of lead compound/series

Identification of the pathology of interest What would be better between an: –Antithrombotic? –Antibleeding? –Analgesic? Knowles and Gromo, Nature Rev Drug Disc 2002 Anticoagulants are useful in primary and secondary prevention of deep vein thrombosis, pulmonary embolism, myocardial infarctions, and strokes

Reference standard for in vitro and in vivo In vitro: –to compare efficacy and potency at the receptor In vivo: –to compare efficacy in disease models –to evaluate side effects Hit to lead

Commercially available drugs acting on platelets’ activation cascade Aspirin X Resting platelet Thrombin Collagen ADP TxA 2 PAFEpinephrine GP IIb/IIIa Activated platelet X Your drug Orbofiban Integrilin Abciximab X Hit to lead Clopidogrel: unknown site of action

Surprise, surprise!! Clopidogrel is acting on P2Y12!!! Hit to lead

Effect of clopidogrel on platelets aggregrations Hit to lead (Foster et al 2001) Clopidogrel blocks ADP and Collagen-induced aggregration in wild type but not in P2Y12 KO mice

And now what we do? Hit to lead

A close look to the drug profile Clopidogrel profile: -Clopidogrel is an effective antithrombotic drug that provides significant protection against heart attack and stroke. - It is a prodrug that must be metabolized to an active species. - Responses in patients are variable. - It binds covalently to the receptor. - The drug must be given for several days before the maximum clinical effect is observed. - Elimination of the drug is slow. Hit to lead

There is an unmet medical need!! Improvement of available therapy To discovery an antithrombotic agents with improved efficacy, that binds reversibly to the receptor and has a better pharmacokinetic profile. Hit to lead