Title: DRUG DISCOVERY
1Drug Discovery
2- Goal Screen as many Drugs as possible
- How are Drugs generated?
- Combinatorial Chemistry
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4HTS implies
- Automation
- Stable reagents, and signals
- Small signal to noise ratio (S/N)
- Performed in 96, 384, or 1536 wells
5Requirements
- 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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8Detection Systems
- Radiometric Detection
- Non-Isotopic Detection Method
- Luminescence, colorimetry, resonance energy
transfer, time resolved fluorescence, cell based
fluorescence assays, fluorescence polarization,
fluorescence correlation spectroscopy
9Radiometric Detection
10Fluorescence 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.
11Problems 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.
12Time 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).
13Time 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.
14Time 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.
15Time 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.
16Resonance Energy Transfer
17Fluorescence 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.
18Fluorescence Polarization
Rogers, M. (1997). Light on high-throughput
screening fluorescence-based assay technologies.
Drug Discovery Today, 2(4), pp.156-160.
19Fluorescence 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.
20Fluorescence Correlation Spectroscopy
Rogers, M. (1997). Light on high-throughput
screening fluorescence-based assay technologies.
Drug Discovery Today, 2(4), pp.156-160.
21Fluorescence 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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23In 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
24Cell 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.
25Cell 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.
26Cell 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.
27Calcium 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.
28Reporter 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.
29BRET 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.
30Split 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.
31Cell 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.
32Sample 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.
33Cell 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.
34Markers
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.
36References
- 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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38Calcium 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
39Calcium 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.
40Cell 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