drug discovery

Drug Drug DiscoveryDiscovery

Kalyani Rajalingham

• Goal: Screen as many Drugs as possible

• How are Drugs generated?o Combinatorial Chemistry

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

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.

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

Radiometric DetectionRadiometric Detection

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).

Problems with Problems 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.

Time Resolved Time 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).”


“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.”



allophycocyanin acceptor moleculeallophycocyanin

acceptor molecule

emission oflight at 665 nm

over a prolonged time

emission oflight at 665 nm

over a prolonged time



“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.”

Resonance Energy Resonance Energy TransferTransfer

Fluorescence Fluorescence PolarizationPolarization

Not best detection system for Cell based

assays

Not best detection system for Cell based

assays


o Small Molecules Faster Rotation Small FPo 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

Fluorescence Fluorescence PolarizationPolarization


Fluorescence Fluorescence Correlation Correlation

SpectroscopySpectroscopy


“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.”

Fluorescence Fluorescence Correlation Correlation

SpectroscopySpectroscopy


In VitroIn Vitro Versus Versus In VivoIn Vivo

• In Vitro Screens• Straightforward but requires the production of uncontaminated

samples of protein, RNA, or DNA

• In Vivo – Cell Based Screens

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.

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.

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.

Calcium Mediated Calcium Mediated Signal MonitoringSignal Monitoring


Reporter Gene AssayReporter Gene Assay


BRET for Protein-BRET for Protein-Protein Interactions Protein Interactions

with CBAwith CBA


Split Protein Split Protein Complementation Complementation

AssayAssay


Cell Proliferation Cell Proliferation AssayAssay


For anti-cancer drug discovery

For anti-cancer drug discovery

Sample Case: CBA in Sample 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.

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.

MarkersMarkers


• 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


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.

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


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.


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 Cell Proliferation AssayAssay

drug discovery

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