High-Throughput Screening(1) by pptfiles



            What is HTS ?

Identification of one or more positive candidates

            extracted from a pool of

         103 to 108 possible candidates

           based on specific criteria

          High Throughput Screening

   Library design (Library – Nature)

   Assay technology (screening assay)

   Laboratory Automation (high throughput)

   The HTS work flow (target selection -> hit)

   Data analysis of screening results

                What do you use HTS for?

To screen for all kind of novel biological active compounds (libraries):
 Natural products
 Combinatorial Libraries (peptides, chemicals…)
 Biological libraries

To screen MicroArrays such as:
 DNA chips
 RNA chips
 Protein chips

                 Screening for novel compounds

                       Natural product extracts

                      Screening for novel biological
                           active compounds

           Combinatorial libraries           biological libraries
            (organic chemistry)              (proteins, Virus,…)

Future ?

             Screening for novel compounds

Screening of natural product extracts: -> Yes or No ???

In the past -> major source of diversity

Originated mainly from plants and microorgansims

Today -> 0.5% of microbes in soil tested
      -> same percentage of plants and microbes in ocean tested

              -> still a big potential of diversity !!!!!

Difficulties: -> time and resource-intensive

              -> frequently not accessible by chemical synthesis
                 (no derivates possible)

              -> chronic lack of success

                   Screening for novel compounds
Screening of natural product extracts: -> Yes or No ???

Problems with screening different purity of extracts:

 -> crude extract: - many substances (complexity of function)
                   - follow up isolation of active compound time consuming
                   - many screens necessary to evaluate for tolerance in the screen
                     (many different substances at different concentrations in the

 -> partially purified: - still some substances (decreased complexity factor)
                        - follow up isolation shortened
                        - active substance lost partially (problem of concentration) or
                          completely during purification process
                        - substance not active any more – cofactor missing

 -> highly purified: - purification of individual substances from extract time consuming
                     - high amounts of “start up” material necessary
                       (problem with “unculturable” microbes)
                     - wrong substances purified -> none of the candidates give a signal
                     - active substance originally at too level to be purified
                       (-> lost during purification process)
                     - substance not active any more – cofactor missing

-> Culture conditions determine what substances are produced !!!!
          Screening for novel compounds

Screening of Libraries:

1. What is a library?
    A pool of many related/similar substances (chemical compounds,
   proteins,…..) -> not natural occurring -> man made !!!

2. What libraries can we make – what are they used for?

   -> Combinatorial libraries (chemical synthesis)
      all kind of chemical compounds (including peptides) that can
      be synthesized, used for ligand discovery, drug discovery,
      interaction analysis, ….

   -> Biological libraries (DNA libraries, RNA libraries, gene-
      encoded libraries, protein libraries)
      Microarrays, chip technology, screening for and engineering of
      enzymes, antibodies, pathways, viruses, ligand discovery,
      interaction analysis, …
              Screening for novel compounds

Screening of Microorganisms -> for novel enzymes

-> by using gene probes       -> cultivating
-> metagenomic screening      -> non-cultivating

1. Gene probe technology -> hybridization technique

    -> detection of presents and location of specific genes in organism
    -> evaluate distribution of a gene among different species
    -> taxonomic markers
    -> search for novel enzymes in microbial isolates

Limitation: microbial strains can just be screened -> if cultivation possible !!!

             Screening for novel compounds

Screening of Microorganisms -> for novel enzymes

2. Metagenome -> screening of uncultivated microorganisms

   -> Fluorescence in situ hybridisation (FISH)
   -> 16S rRNA clone library
   -> metagenome

->16S rRNA clone library     -> used to identify uncultivated microorganisms
                           -> map their phylogenetic relationships (taxonomy)

              Screening for novel compounds

Screening of Microorganisms -> for novel enzymes

-> Fluorescence in situ hybridization (FISH)
   -> can be used to detect and localize the presence or absence of specific DNA
   sequences on chromosomes.
   FISH can also be used to compare the genomes of two biological species, to deduce
   evolutionary relationships. Bacterial FISH probes are often primers for the 16s
   rRNA region.

             Screening for novel compounds

Screening of Microorganisms -> for novel enzymes

-> Metagenome
  -> extraction of DNA from all microorganisms present in isolate -> generate
    library or use as template for PCR

   Library -> BAC vector based (20-500 kbp insert)

                                                     Can be screened by Microarray system:

                                                     Target (immobilized) -> BAC clones

                                                     Probe (labled) -> oligos directed to
                                                     specific gene

Screening for novel compounds
                 Screening of Microorganisms ->
                    for novel enzymes

                 Comparison of culturing and
                    metagenome strategy

                 -> easier to clone complete pathways
                 -> more difficult to make library

              Screening for novel compounds

Screening of Libraries:

Library design:

! Size and Diversity -> direct impact on successful screening !

-> Targeted libraries -> derive from information gained on biomedical
                        programs (find new lead structures)
-> Focused libraries -> preselected subset of compounds based on
                        virtual screening of a targeted library
-> Discovery libraries -> randomly assembled (find new lead structures)

3 aspects of library design:    -> combinatorial synthesis
                                -> statistical considerations
                                -> molecular recognition models

              Screening for novel compounds
Screening of Libraries:

Library design:

! Size and Diversity -> direct impact on successful screening !

Limiting factors for library expansion: -> costs
                                        -> technical feasibility
                                           (storage, logistics)

   !!!Screening success -> frequently due to unexpected findings !!!
                           (predictive capabilities still limited)

                 Screening for novel compounds
What is a “lead structure” ?

-> displays desired biological activity
-> but does not yet combine all properties needed for therapeutic use

Iterative cycles -> synthesis of structural analogs -> pharmacological
                    testing -> suitable candidate for therapeutic use

Properties a lead structure must have to become a pharmaceutical product:
-   Functional in vitro and in vivo (animal tests)
-   Reduced side-effects (understanding the structure-activity-relationship of the interaction
    between lead structure and receptor)
-   Chemical structure related to biological active molecule (optimal binding to receptor)
-   Accessible to chemical synthesis -> derivatisation
-   Substance and metabolic products not toxic
-   Novelty

                  Screening for novel compounds
What is a “lead structure” ?

Statins -> inhibits HMG-CoA
reductase -> cholesterol

Many companies have generated
optimized products

        Cholesterol Biosynthesis Blocker

Potent blocker of HMG CoA reductase -> reduce level of synthesized cholesterol

        Screening for novel compounds
Assay Technology in HTS

                       Screening for novel compounds
        Assay Technology in HTS

A -> Cell growth tests (cell-based assays) -> Phenotypic assays

-> have problems to discriminate between pharmaceutical meaningful and toxic
-> cellular response time can be long (min, hours, days)
-> source of unspecific hits (compounds interfering with energy metabolism -> complexity!!!)

B -> Tissue response -> targeted functional cell-based assay

-> sensitivity and specificity towards receptor crucial for meaningful assay (Worse in A)
-> recombinant cell lines (nowadays) make it possible to monitor functional activity
-> response time (sec, hours) -> worse in A
-> distinguishes between agonist and inhibitor

C -> Enzyme test -> biochemical test

-> best sensitivity
-> can be run at high compound/solvent concentration
-> discriminates between inhibitors and stimulators
-> require recombinant gene expression and reconstitution of biological activity of protein
   (time consuming)
-> great experimental freedom for varying conditions
-> best for automated screening
-> “fragment screening” (structural fragments used to identify low-affinity binding
                          -> design larger compounds -> enhance affinity)

                    Screening for novel compounds
       Assay Technology in HTS

A -> Cell growth tests (cell-based assays) -> Phenotypic assays

-> mechanism of action + relevance to disease -> complex targets
-> necessary substrate and co-factor available at physiological conditions
-> target difficult or expensive to express and purify
-> sometime the fastest and least expensive approach

Formats used:
-> Reporter assay -> detect transcriptional regulation (luciferase, GFP, beta-galactosidase, …)
-> Proximity assay -> screening for transporter targets (carrier)
-> proliferation assay -> screens for targets that stimulate/inhibit cell growth
-> assays to measure changes in gene expression (past: antibodies) -> today with microarray systems

Implementation challenges:
-> growth and adherence properties of cells
-> solvent tolerance
-> cytotoxicity
-> stability of cellular phenotype

Possible Future -> cell-based screenings on chips !!!

       Requirements for screening assays
     -> High Throughput Screening Assay

   High sensitivity of assay (single molecule detection)
   High speed of assay (automation)
   Minimization of assay (microtiter plate assay)
   Low background signal
   Clear message (best: Yes/No answer)
   Low complexity of assay (specific interaction)
   Reproducibility
   Fast data processing of results
   Acceptable costs !!!

             Screening for novel compounds

    Laboratory Automation

Beginning in late 1980s ->    - microtiter plate assays
                              - liquid handling automats
                              - plate readers

Speeded up by -> miniaturization and parallelization of assays (96-1536 well plates)

Assays can be run:

-> semi manual (batch mode)
   small differences in timing of the steps -> additional assay noise (time jitter)

-> continuous (robotic integration of processing automats)
   since cycle time are constant -> easier to identify systematic trends and errors
   more complex logistic

      Screening for novel compounds

Laboratory Automation – Assay automation

         Screening for novel compounds
Laboratory Automation

                                         1 cycle -> 230s

                                         Overnight run (16h):

                                         -> 250 microtiter

                                         -> 384 000 tests

                Screening for novel compounds
   From target selection to confirmed hits

Identification of
drug targets

Choice of assay technology:                   Primary test:
-> expertise                                  -> final assay concentration cannot be controlled
-> compatibility with existing HTS hardware      -> signal scattering
-> sensitivity                                -> liquid handling errors -> signal scattering
-> profile of drug
-> susceptibility to artifacts                -> false negative (cannot be excluded)
-> unspecific interference                      -> active compounds not detected (5-15%)
-> cost of assay
-> reproducibility                            -> false positive (-> cross-contamination,
                                                assay noise) -> identified by repetition

                                              -> controls are very important!!!!
                  Screening for novel compounds
    From target selection to confirmed hits

Screening inhibitors of
a serine protease

-> Completed in 2 weeks
9 robots testing
170 000 a day

A -> compounds with 20%
And more inhibition were

B -> activity in re-testing
Compared to primary test
Degree of reproducibility
-> false positive

C -> evaluation ->
conformation rate
correlates positive with

           Screening for novel compounds
Specific <-> unspecific hits (assay artifacts)

 In HTS -> small number of lead candidates

         -> large number of unreproducible hits (false positives)

         -> compounds acting unspecific

 Unspecific acting -> same effect on assay as the desired drug

 !!! Interference increases -> complexity of assay !!!! -> cell-based assays

 -> reference assays -> probing for specificity and selectivity (challenging)

                Screening for novel compounds
   Specific <-> unspecific hits (assay artifacts)

Discrimination between
Target molecule and unspecific
Acting molecules.

-> cell-based assay
Inhibitor for G-protein coupled
Receptor (GPCR)

Activity monitored by release of
Ca2+ -> Ca2+ concentration was
visualized by Ca2+ sensitive
photoprotein -> connected to
stimulation of ATP

Library: 1 000 000 compounds
-> 10 000 hits (too many)

ATP signal as reference to find
unspecific hits
Specific hits -> enhancing
response to ATP -> 656 hits
              Screening for novel compounds
Data analysis and screening results

Data analysis combines -> biological activity + chemoinformatic tools

                     evaluate + interpreting experimental observations

Grouping active compounds into structural classes (clusters) -> enhances resolution
looking at scattered data

               Screening for novel compounds
Data analysis and screening results

Chemoinformatics in HTS:

-> Elimination of unspecific compounds from hit sets -> detection of false negative
   + borderline activity

 Search library for:
 - Overall similarity
 - shared substructure
 - known bio-isosteric replacements

Computer tool:
-> identify molecular fragment contributing
   to biological activity

Possible to extract information that is not
detectable by cluster methods

     Screening for novel compounds

!!! Experimental testing remains the major route for lead discovery !!!

Prerequisite for success:

Convert knowledge of mechanism + molecular recognition principle

-> robust + sensitive assay

-> HTS should deliver more than one candidate !!!

           Detection Methods in HTS:

   Spectroscopy
   Mass Spectrometry
   Chromatography
   Calorimetry
   X-ray diffraction
   Surface plasmon resonance
   Microscopy
   Radioactive methods

             Spectroscopy in HTS:
   Fluorescence Spectroscopy
   Total internal reflection fluorescence
   Nuclear magnetic resonance (NMR)
   Absorption and luminescence sp.
   Circular dicroism sp. (CD)
   Fourier transformed infrared sp. (FTIR)
   Light scattering

     Fluorescence Spectroscopy in HTS:

   Fluorometric Imaging Plate Reader
   Fluorescence polarization spectroscopy (FP)
   Time-resolved fluorescence spectroscopy (TRF)
   Fluorescence resonance energy transfer spectroscopy
   Fluorescence correlation spectroscopy (FCS)
   Fluorescence correlation microscopy (FCM)
   Confocal fluorescence coincidence analysis (CFCA)

Fluorometric Imaging Plate Reader

   384 samples can be measured simultaneously in using a FLIPR
   (Fluorometric Imaging Plate Reader). The fluorescent species measured
   is a sensitive dye. Using 384-well microplates with a 5-min turnaround
   time, nearly 40,000 samples can be screened in an 8-h period.
              Fluorescence polarisation (FP):
                                                                   FP (Fluorescence polarization) can
                                                                   measure the binding of a small
                                                                    to a much larger protein. According
                                                                   to FP theory, unbound fluorescent
                                                                   tracers will exhibit lower
                                                                   polarization, because the unbound
                                                                   molecules tumble
                                                                   more rapidly than bound tracers.

-> molecule's rate of tumbling (more formally known as the rotational relaxation
time) is directly proportional to its molecular volume

-> measuring the extent of fluorescence polarization, this method can determine
binding equilibrium and the competition for binding at a site

-> well suited for assays that rely on small ligands binding to large biomolecules, including
enzyme assays (kinase assays, protease assays), inhibitor assays, and immunoassays
(pesticides, metal ions)…

-> Used for diagnostic assays (disease detection), drug discovery, signal transduction discovery, single
nucleotide polymorphism genotyping (identification of genetic variations in the human genome)…
Time-resolved fluorescence spectroscopy
                         Time-resolved fluorescence
                         (TRF) -> closely related to
                         fluorescence intensity techniques.
                         The detector is gated for a short
                         period of time (e.g., 10 ns) -> the
                         initial burst of fluorescence (most
                         of the background fluorescence)
                         not measured.
                         After the gating period -> the
                         longer lasting fluorescence in the
                         sample is measured.
                         TRF techniques can be used to
                         substantially enhance sensitivity

      Time-resolved fluorescence (TRF):

   For compounds with a longer fluorescent lifetime. -> e.g. rare earth
    lanthanides (Eu).

   TRF further enhanced by developing of a class of cage compounds called
    cryptates. Cryptates are macropolycyclic compounds that can serve as cages ->
    trapping an ion and protecting it from solvent. The cryptate cage enhances
    fluorescence by acting as an antenna for the trapped lanthanide ion (i.e.,
    absorbing excitation light and transferring the energy to the ion) and by
    protecting it from quenching by water.
    (These compounds were discovered by Jean Marie Lehn, who received the 1987
    Nobel Prize in Chemistry for this work)
   Eu cryptate, Eu(K), can be combined with fluorescence resonance energy
    transfer (FRET) -> time-resolved, fluorescence resonance energy transfer, or

Fluorescence resonance energy transfer
         spectroscopy (FRET) :
   In HTRF (Homogeneous Time-Resolved Fluorescence) -> fluorescence depends on bringing
   the donor and acceptor fluorophores together.
   In an HTRF assay, the transfer of resonance energy between a donor and acceptor
   fluorophore provides a way to measure ligand binding to a biological target.

                                           Two conditions must pertain for an effective FRET assay
                                           1) The donor fluorescence must be significantly quenched
                                           2) The starting material and product need to differ
                                           significantly in extent of quenching (Forster radius ca. 50 A)

FRET -> common tool for studying biochemical reaction kinetics and molecular motors.
    Fluorescence resonance energy transfer
             spectroscopy (FRET)
   FRET is based on the principle of time-resoved fluorescence (TRF).
   FRET -> energy transfer from a donor fluorophore to an acceptor
    fluorophore. The efficiency of energy transfer is a function of the distance
    (1/d<SUP6< sup>) between the donor and acceptor.
   Possible FRET pair: Donor is (Eu)K (molecular weight ~1000), and the acceptor is
    XL665 (a modified allophycocyanine, a phycobiliprotein from red algae, molecular
    weight ~105,000). This donor- acceptor pair has a 50% energy transfer
    efficiency at a distance of 9 nm and a 75% energy transfer efficiency at 7.5 nm.
    The transfer distance is long enough to be compatible with biomolecular
    interactions of interest, yet short enough to minimize nonspecific energy
   The most popular FRET pair for biological use is a cyan fluorescent protein
    (CFP)-yellow fluorescent protein (YFP) pair (variants of GFP)
   Limitation of FRET -> requirement for external illumination to initiate the
    fluorescence transfer -> can lead to direct excitation of the acceptor or to
   Avoided by Bioluminescence Resonance Energy Transfer (or BRET)
    Donor: luciferase (typically the luciferase from Renilla reniformis)
    Acceptor: GFP or variant of it.
           Application of TRF and FRET:

   based on the principle of dissociative fluorescence enhancement:
    immunoassays, DNA hybridization assays, cytotoxicity assays, protein-
    protein binding, receptor binding, enzyme assays,…

   Various reagents pre-labeled with TRF donors and acceptors, including
    epitope tag antibodies, streptavidin, biotin, and several antispecies
    antibodies, are available and can be adapted to many assays.

   Fluorescence correlation spectroscopy

                                            -> laser beam illuminates a tiny volume
                                            element on the femtoliter scale (10-15 fL)

                                            -> Single fluorescent molecules that diffuse
                                            through this volume are excited and undergo
                                            fluorescence transmission -> emitted photons
                                            are recorded in a time-resolved manner with a
                                            single-photon detection system

Fluorescence correlation spectroscopy (FCS) allows molecular interactions to be
studied at the single-molecule level. This technique can be applied to assays in which
the molecular weight of a fluorescently labelled molecule changes as the result of
the interaction.

Fluorescence correlation spectroscopy
                   ->    Based on the fluctuations in the
                         fluorescence signal which are caused
                         by single molecules diffusing

                       Investigation of biomolecules at ultralow
                        concentrations (single molecular level) on
                        surfaces, in solution, and in living cells
                       can be used in binding assays and enzymatic
                       Structural and molecular dynamics of
                        fluorescent proteins in vivo and in vitro
                       drug discovery
                       Protein transport

    Fluorescence correlation microscopy

   Fluorescence correlation microscopy (FCM) is a combination of FCS with
    powerful light microscopy, e. g., confocal laser scanning microscopy

   This allows measurements inside living cells or on cell membranes.

                  Application for FCM:

   Provides direct quantitative information on molecular
    concentrations in different subcellular compartments

   Provides complementary information on molecular interactions
    and molecular distribution within the context of an intact cell

   Gives absolute numbers of mobile molecules inside the cell and
    information of their diffusion

   Can follow protein trafficking inside the cell

       Confocal fluorescence coincidence
               analysis (CFCA):

   Confocal fluorescence coincidence analysis (CFCA) extracts fluorescence
    fluctuations that occur coincidently in two different spectral ranges from a
    tiny observation volume of below 1 femto litre.

   It possible to monitor whether an association between molecular fragments
    that are labeled with different fluorophores is established or broken.
    -> For characterization of a variety of cleavage and ligation reactions in
    biochemistry -> enzymatic assays, binding assays

   Confocal fluorescence coincidence analysis -> very sensitive and ultrafast
    technique -> readout times of 100 ms and below.
    -> Allows throughput rates as high as 106 samples per day with using only small
    amounts of sample substance.

          Chromatography in HTS:

   Gas chromatography (GC)
   Thin layer chromatrography
   Liquide chromatography (HPLC)
   Ion Exchange chromatography
   Reverse phase chromatography
   Gel filtration chromatography
   Hydrophobic interaction chrom.
   Affinity chromatography

              Calorimetry in HTS:

   Isothermal titration calorimetry (ITC)

   Differential scanning calorimetry (DSC)

      Surface Plasmon Resonance (SPR):
                                                                Kinetic data of macromulecular
                                                                interations in real time at
                                                                almost single molecular level
                                                                (high sensitivity)

                                                                RNA-protein, DNA-protein
                                                                complexes, Protein-Protein
                                                                interaction, Protein-Ligand

Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from
a conducting film at the interface between two media of different refractive index. The media
are the sample and the glass of the sensor chip, and the conducting film is a thin layer of gold
on the chip surface. SPR causes a reduction in the intensity of reflected light at a specific
angle of reflection. This angle varies with the refractive index close to the surface on the side
opposite from the reflected light.

When molecules in the sample bind to the sensor surface, the concentration and therefore the
refractive index at the surface changes and an SPR response is detected.

              Microscopy in HTS:

   Scanning Tunnelling Microscopy

   Atomic Force Microscopy

   Confocal Microscopy


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