By Kalyani Rajalingham

1. Drug DiscoveryDrug Discovery
Kalyani Rajalingham

2. • Goal: Screen as many Drugs as possible
• How are Drugs generated?
o Combinatorial Chemistry

4. HTS impliesHTS implies
• Automation
• Stable reagents, and signals
• Small signal to noise ratio (S/N)
• Performed in 96, 384, or 1536 wells

5. RequirementsRequirements
•Drug Target Sample - enzymes, cell surface receptors,
nuclear receptors, ion channels, and signal
transduction proteins
•Test Drug Sample – Combinatorial Chemistry
•A Detection System
Sittampalam, G., Kahl, S. and Janzen, W. (1997). High-throughput screening: advances in assay technologies. Current Opinion
in Chemical Biology, 1(3), pp.384-391.

8. Detection SystemsDetection Systems
• Radiometric Detection
• Non-Isotopic Detection Method
• Luminescence, colorimetry, resonance energy transfer, time resolved
fluorescence, cell based fluorescence assays, fluorescence
polarization, fluorescence correlation spectroscopy

9. Radiometric DetectionRadiometric Detection

10. Fluorescence AssaysFluorescence Assays
Rogers, M. (1997). Light on high-throughput screening: fluorescence-based assay technologies. Drug Discovery Today, 2(4),
pp.156-160.
• 4 Types:
o Time-resolved fluorescence (TRF)
o Fluorescence resonance energy transfer (FRET)
o Fluorescence polarization (FP)
o Fluorescence correlation spectroscopy (FCS).

11. Problems withProblems with
FluorescenceFluorescence
• Quenchers in reaction can interfere with detection of signal
• Quenching by media or plastic
• Background fluorescence - Autofluorescence by free
probes/contaminants
• (eg: flavins, porphyrins, elastin, collagen, etc..)
Autofluorescence: Causes and Cures. (n.d.). 1st ed. [ebook] Toronto: Wright Cell Imaging Facility. Available at:
http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf [Accessed 7 Feb. 2015].
Grepin, C. and Pernelle, C. (2000). High-throughput screening Evolution of Homogeneous Time Resolved Fluorescence
(HTRF) technology for HTS. Drug Discovery Today, 5(5), pp.212-214.

12. Time ResolvedTime Resolved
FluorescenceFluorescence
“HTRF uses a europium (III) ion caged in a proprietary macropolycyclic ligand,
containing 2,2’-bipyridines as light absorbers (Eu-cryptate). Energy is nonradiatively
transferred from Eu-cryptate excited at 337 nm to a fluorescence acceptor molecule, a
proprietary chemically modified allophycocyanin, termed XL665. In the presence of
pulsed laser light, energy is transferred from the Eu-cryptate to the XL665 resulting in
emission of light at 665 nm over a prolonged timescale (microseconds).”
“HTRF uses a europium (III) ion caged in a proprietary macropolycyclic ligand,
containing 2,2’-bipyridines as light absorbers (Eu-cryptate). Energy is nonradiatively
transferred from Eu-cryptate excited at 337 nm to a fluorescence acceptor molecule, a
proprietary chemically modified allophycocyanin, termed XL665. In the presence of
pulsed laser light, energy is transferred from the Eu-cryptate to the XL665 resulting in
emission of light at 665 nm over a prolonged timescale (microseconds).”

13. Time ResolvedTime Resolved
FluorescenceFluorescence
“europium emission peaks (620 nm) has been used as an internal control, as the signal
at 620 nm is proportional to the concentration of free Eu-cryptate.”
“europium emission peaks (620 nm) has been used as an internal control, as the signal
at 620 nm is proportional to the concentration of free Eu-cryptate.”

14. Time ResolvedTime Resolved
FluorescenceFluorescence
Grepin, C. and Pernelle, C. (2000). High-throughput screening Evolution of Homogeneous Time Resolved Fluorescence (HTRF)
technology for HTS. Drug Discovery Today, 5(5), pp.212-214.
allophycocyanin acceptor
molecule
allophycocyanin acceptor
molecule
emission of
light at 665 nm over a
prolonged time
emission of
light at 665 nm over a
prolonged time

15. Time ResolvedTime Resolved
FluorescenceFluorescence
Grepin, C. and Pernelle, C. (2000). High-throughput screening Evolution of Homogeneous Time Resolved Fluorescence (HTRF)
technology for HTS. Drug Discovery Today, 5(5), pp.212-214.
“This light emission
is recorded in a time-
resolved fashion over
a 400 ␮s period,
starting 50 ␮s after the
excitation pulse so
that the auto-
fluorescence from the
media and the short-
lived fluorescence of
the free APC are not
recorded.”
“This light emission
is recorded in a time-
resolved fashion over
a 400 ␮s period,
starting 50 ␮s after the
excitation pulse so
that the auto-
fluorescence from the
media and the short-
lived fluorescence of
the free APC are not
recorded.”

16. Resonance EnergyResonance Energy
TransferTransfer

17. Fluorescence PolarizationFluorescence Polarization
Not best detection system for
Cell based assays
Not best detection system for
Cell based assays
Rogers, M. (1997). Light on high-throughput screening: fluorescence-based assay technologies. Drug Discovery Today, 2(4),
pp.156-160.
o Small Molecules  Faster Rotation  Small FP
o Large Molecules  Slower Rotation  Large FP
• Concept: If a molecule (eg: antibody) binds a
fluorescently tagged molecule (eg: Protein A) 
Slower Rotation
o We get a reading on the polarization of the unbound molecule  if it
binds another molecule, polarization changes
o Commonly used to detect if molecule A interacts with molecule B

18. Fluorescence PolarizationFluorescence Polarization
Rogers, M. (1997). Light on high-throughput screening: fluorescence-based assay technologies. Drug Discovery Today, 2(4),
pp.156-160.

19. Fluorescence CorrelationFluorescence Correlation
SpectroscopySpectroscopy
Rogers, M. (1997). Light on high-throughput screening: fluorescence-based assay technologies. Drug Discovery Today, 2(4),
pp.156-160.
“In FCS, single molecules are measured as they diffuse
through the extremely small measurement volume of 1
fl (the size of an E.coli cell). Free ligands diffuse more
rapidly than ligand-receptor complexes because of
the latter’s greater molecular mass. Statistics
associated with these diffusion events are recorded
and automatically processed in real time during an
FCS measurement. The entire task of measurement
and data processing takes only a few seconds.”

20. Fluorescence CorrelationFluorescence Correlation
SpectroscopySpectroscopy
Rogers, M. (1997). Light on high-throughput screening: fluorescence-based assay technologies. Drug Discovery Today, 2(4),
pp.156-160.

21. Fluorescence CorrelationFluorescence Correlation
SpectroscopySpectroscopy
Rogers, M. (1997). Light on high-throughput screening: fluorescence-based assay technologies. Drug Discovery Today, 2(4),
pp.156-160.

23. In VitroIn Vitro VersusVersus In VivoIn Vivo
• In Vitro Screens
• Straightforward but requires the production of uncontaminated
samples of protein, RNA, or DNA
• In Vivo – Cell Based Screens

24. Cell Based AssaysCell Based Assays
• in vivo
• Used to measure cell
proliferation, toxicity,
production of markers,
motility, activation of specific
signalling pathways, and
changes in morphology
Sundberg, S. (2000). High-throughput and ultra-high-throughput screening: solution- and cell-based approaches. Current
Opinion in Biotechnology, 11(1), pp.47-53.

25. Cell Based AssaysCell Based Assays
• Use of Immortalized Human
Cells or Rodent Cell Lines
• Recombinant DNA
technology required in many
cases (not required for cell
proliferation assay)
• Low supply of cells is a
problem  Use fewer cells
Zaman, G. (2008). Editorial [Hot Topic: Cell-Based Screening (Guest Editor: Guido J.R. Zaman) ]. Combinatorial Chemistry &
High Throughput Screening, 11(7), pp.494-494.

26. Cell Based AssayCell Based Assay
• 1 – Second Messenger Assay
• 2 - Reporter Gene Assay
• 3 – Cell Proliferation Assay
Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A. and Roda, A. (2010). Cell-based assays: fuelling drug discovery. Analytical
and Bioanalytical Chemistry, 398(1), pp.227-238.

27. Calcium Mediated SignalCalcium Mediated Signal
MonitoringMonitoring
Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A. and Roda, A. (2010). Cell-based assays: fuelling drug discovery. Analytical
and Bioanalytical Chemistry, 398(1), pp.227-238.

28. Reporter Gene AssayReporter Gene Assay
Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A. and Roda, A. (2010). Cell-based assays: fuelling drug discovery. Analytical
and Bioanalytical Chemistry, 398(1), pp.227-238.

29. BRET for Protein-ProteinBRET for Protein-Protein
Interactions with CBAInteractions with CBA
Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A. and Roda, A. (2010). Cell-based assays: fuelling drug discovery. Analytical
and Bioanalytical Chemistry, 398(1), pp.227-238.

30. Split ProteinSplit Protein
Complementation AssayComplementation Assay
Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A. and Roda, A. (2010). Cell-based assays: fuelling drug discovery. Analytical
and Bioanalytical Chemistry, 398(1), pp.227-238.

31. Cell Proliferation AssayCell Proliferation Assay
Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A. and Roda, A. (2010). Cell-based assays: fuelling drug discovery. Analytical
and Bioanalytical Chemistry, 398(1), pp.227-238.
For anti-cancer
drug discovery
For anti-cancer
drug discovery

32. Sample Case: CBA inSample Case: CBA in
Yeast CellsYeast Cells
If Drug prevents interaction between Protein X, and Y, then cell lives.
Tucker, C. (2002). High-throughput cell-based assays in yeast. Drug Discovery Today, 7(18), pp.S125-S130.

33. Cell Based AssayCell Based Assay
• 1 – Select Cell Lines to be Screened
• 2 – Select traits you want to measure
• ex: cell viability – is the drug toxic? (Apoptosis/Necrosis)
• There are Markers to detect dead cells, live cells, number of cells,
etc…
• 3 – Immobilization of Cells
Riss, T. (2005). Selecting cell-based assays for drug discovery screening. Cell Notes, (13), pp.16-21.

34. MarkersMarkers
Riss, T. (2005). Selecting cell-based assays for drug discovery screening. Cell Notes, (13), pp.16-21.

35. • 4 – Choose Markers or Detection system
• 5 - Select Dosage of Drug, and Exposure Time
• Response to a Drug can occur within minutes – days
• 6 - Experiment
Riss, T. (2005). Selecting cell-based assays for drug discovery screening. Cell Notes, (13), pp.16-21.

36. ReferencesReferences
• Sittampalam, G., Kahl, S. and Janzen, W. (1997). High-throughput screening: advances in assay
technologies. Current Opinion in Chemical Biology, 1(3), pp.384-391.
• Riss, T. (2005). Selecing cell-based assays for drug discovery screening. Cell Notes, (13), pp.16-21.
• Sundberg, S. (2000). High-throughput and ultra-high-throughput screening: solution- and cell-based
approaches. Current Opinion in Biotechnology, 11(1), pp.47-53.
• Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A. and Roda, A. (2010). Cell-based assays: fuelling
drug discovery. Analytical and Bioanalytical Chemistry, 398(1), pp.227-238.
• Tucker, C. (2002). High-throughput cell-based assays in yeast. Drug Discovery Today, 7(18), pp.S125-S130.
• Zaman, G. (2008). Editorial [Hot Topic: Cell-Based Screening (Guest Editor: Guido J.R. Zaman) ].
Combinatorial Chemistry & High Throughput Screening, 11(7), pp.494-494.
• Autofluorescence: Causes and Cures. (n.d.). 1st ed. [ebook] Toronto: Wright Cell Imaging Facility.
Available at: http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf [Accessed 7 Feb.
2015].
• Grepin, C. and Pernelle, C. (2000). High-throughput screening Evolution of Homogeneous Time Resolved
Fluorescence (HTRF) technology for HTS. Drug Discovery Today, 5(5), pp.212-214.
• Rogers, M. (1997). Light on high-throughput screening: fluorescence-based assay technologies. Drug
Discovery Today, 2(4), pp.156-160.

38. Schematic representation of a cell-based assay for
calcium mediated signalling pathway monitoring
using the calcium-sensitive bioluminescent
photoprotein aequorin. The cells are stably
transfected with a gene construct for expression of
the apoprotein aequorin that is reconstituted by
addition of its prosthetic group coelenterazine. The
presence of an agonist triggers an intracellular
signalling pathway that increases intracellular calcium
concentration causing the aequorin to emit light
Calcium Mediated SignalCalcium Mediated Signal
MonitoringMonitoring

39. Aequorin is a photoprotein, originally isolated from the
jellyfish Aequorea victoria, which needs an organic
imidopyrazine substrate, coelenterazine, and the
presence of Ca2+ for emission of bioluminescence.
Calcium Mediated SignalCalcium Mediated Signal
MonitoringMonitoring

40. Schematic representation of a cell-based impedance
sensing system. The cells grown on the electrode act
as insulators, impeding the flow of current, thus
increasing the resistance of the system. Addition of
compounds able to alter the cell morphology or to
disrupt the cell monolayer produce openings
between the cells causing a rapid drop of resistance
Cell Proliferation AssayCell Proliferation Assay