DRUG DISCOVERY

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Drug Discovery

• Kalyani Rajalingham

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• Goal Screen as many Drugs as possible
• How are Drugs generated?
• Combinatorial Chemistry

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HTS implies

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

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Requirements

• 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.
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Detection Systems

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

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Radiometric Detection
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Fluorescence Assays

• 4 Types
• Time-resolved fluorescence (TRF)
• Fluorescence resonance energy transfer (FRET)
• Fluorescence polarization (FP)
• Fluorescence correlation spectroscopy (FCS).

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

• 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..)

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.
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.
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Time Resolved Fluorescence
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).
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Time Resolved Fluorescence
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.
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Time Resolved Fluorescence
emission of light at 665 nm over a prolonged time
allophycocyanin acceptor molecule
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.
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Time Resolved Fluorescence
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.
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.
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Resonance Energy Transfer
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Fluorescence Polarization

• Small Molecules ? Faster Rotation ? Small FP
• Large Molecules ? Slower Rotation ? Large FP
• Concept If a molecule (eg antibody) binds a
  fluorescently tagged molecule (eg Protein A) ?
  Slower Rotation
• We get a reading on the polarization of the
  unbound molecule ? if it binds another molecule,
  polarization changes
• Commonly used to detect if molecule A interacts
  with molecule B

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

• 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 latters 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.

Rogers, M. (1997). Light on high-throughput
screening fluorescence-based assay technologies.
Drug Discovery Today, 2(4), pp.156-160.
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Fluorescence Correlation Spectroscopy
Rogers, M. (1997). Light on high-throughput
screening fluorescence-based assay technologies.
Drug Discovery Today, 2(4), pp.156-160.
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Fluorescence Correlation Spectroscopy
Rogers, M. (1997). Light on high-throughput
screening fluorescence-based assay technologies.
Drug Discovery Today, 2(4), pp.156-160.
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In Vitro Versus In Vivo

• In Vitro Screens
• Straightforward but requires the production of
  uncontaminated samples of protein, RNA, or DNA
• In Vivo Cell Based Screens

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Cell 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.
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Cell 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.
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Cell 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.
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Calcium Mediated Signal Monitoring
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.
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Reporter 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.
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BRET for Protein-Protein Interactions 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.
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Split Protein Complementation 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.
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Cell Proliferation Assay
For anti-cancer drug discovery
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.
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Sample Case CBA in Yeast 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.
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Cell 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.
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Markers
Riss, T. (2005). Selecting cell-based
assays for drug discovery screening. Cell Notes,
(13), pp.16-21.
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• 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.
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References

• 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.

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Calcium Mediated Signal Monitoring

• 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

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Calcium Mediated Signal Monitoring

• 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.

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Cell Proliferation Assay

• 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