September 28, 2022

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High-throughput screening methods in drug development

High-throughput screening methods in drug development


High-throughput screening methods in drug development. 

High-throughput screening plays an important role in the early stage of new drug discovery and provides a technical basis for the discovery of emerging compounds.

Some of the screening systems commonly used today can be roughly divided into two categories: biochemical screening systems and cell screening systems for discussion.


The biochemical screening system includes fluorescence polarization and anisotropy (FP/FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence lifetime analysis ( Fluorescence lifetime analysis).

Affinity-based screening methods also belong to this category, including nuclear magnetic resonance (NMR), surface plasmon resonance, SPR, mass spectrometry (MS), and thermal drift experiment (Differential scanning fluorimetry, DSF).


Cell-based screening methods include cell viability, reporter gene, second messenger, high-throughput microscopy assays, and CRISPR/Cas9 system-based screening.


 High-throughput screening methods in drug development     


The process of new drug development is long, complicated and uncertain.

High-throughput screening (HTS) is one of the strategies used to discover starting compounds in small molecule drug design. Generally speaking, when there is less research on the target,

HTS is the first choice. According to the relevance to the disease, the connection with other targets and the innovation are comprehensively considered to select the target or cell model of HTS.


The choice of screening technology is determined by the screenability of the target and its assumed chemical variability.

Different screening techniques are suitable for different targets, and the review of screening techniques has an important guiding role for the subsequent HTS.


High-throughput screening methods in drug development


There are certain prerequisites for HTS. The first is the preliminary confirmation of the target, the second is a sensitive and stable screening method, and the last is a structure-rich compound library. After HTS, further confirmation of the seed compound is needed.

The commonly used method is to re-purchase, confirm the purity, determine the affinity and concentration gradient experiment, conduct multi-method experimental detection, and then go to convolution analysis to eliminate false positives.

It is also possible to use alternatives such as homologous family proteins/mutants of the target protein for specific screening to confirm the selectivity of the compound in many ways.




Biochemical Screening System


Biochemical screening uses purified target proteins to determine ligand binding or enzyme activity inhibition in vitro.

These analyses are usually conducted in a competitive format, that is, the compound under study must replace a known ligand or substrate.


384-well plates are usually used for detection, usually with a detection volume of 20-50 μL, and read out using optical methods, such as absorbance, fluorescence, or luminescence.

The fluorescence method has largely replaced the traditional radiolabeled ligand analysis method due to its easier operation, high sensitivity and flexibility.

The commonly used fluorescence detection methods are as follows:


1. Total fluorescence analysis (Fluorescence intensity, FLINT)

Integrate the fluorescence value of the well in a fixed time, such as 100 ms.

It is commonly used for dye-containing substrates to produce fluorescent substances after enzyme-catalyzed reactions, such as protease-mediated cleavage of peptide fluorescent groups.

FLINT will be affected by many factors, such as autofluorescence and quenching (interacting with the tested substance instead of the substrate) of the tested substance, and sampling error.

Generally speaking, the longer the excitation wavelength of the dye used for labeling, the smaller the spectral interference from the screening compound.


2. Ratiometric fluorescence methods


High-throughput screening methods in drug development


Common HTS screening systems include fluorescence polarization (FP) and fluorescence anisotropy (FA) detection.

Compared with FLINT, this method is less susceptible to the influence of human factors.

By measuring the rotation change of the molecule for a certain period of time, the influence of the interaction on the measuring molecule is characterized.

In these techniques, polarized light is used to excite fluorophores, and polarizers polarized parallel and perpendicular to the incident light are used to measure fluorescence emission.

When the dye-labeled molecules flip over time, the emitted signal will depolarize. Binding with macromolecules reduces the mobility of the fluorophore, thereby increasing the polarization value or anisotropy, and allowing quantitative binding.

Because different fluoresceins have different fluorescence lifetimes, using this feature can maximize the sensitivity of the screening system.

Organic dyes such as fluorescein and rhodamine have a fluorescence lifetime of about 3-4 ns.

When they are combined with molecules of about 0.5-10 kD, the detection sensitivity is the highest.

Therefore, the fluorescence ratio method is most suitable for the determination of molecular interactions with very different molecular weights.


3. Fluorescence resonance energy transfer (FRET)

Energy from the fluorescent donor is absorbed by the acceptor through dipole-dipole interaction.

The efficiency of energy transfer depends on the spectral overlap between the donor and acceptor, their distance and relative direction, and other parameters in the Foster equation.

The common energy donor and acceptor distance is 26 nm, which is suitable for many protein-protein interactions.


4. Time-resolved fluorescence resonance energy transfer (TR-FRET)

It is a screening method derived from fluorescence resonance energy transfer.

Lanthanide compounds with long-lived fluorescence (usually 100 seconds to milliseconds) are used as fluorescence donors, and fluorescent proteins or organic fluorophores (for example, allophycocyanin or Cy5) are used as acceptors.

Then the fluorescence emission of the acceptor is gated (for example, delayed to 50 ms), so that when the signal is collected, the fluorescence emission of the short-lived organic fluorophore in the test compound has been attenuated.


The fluorescence lifetime of molecules in the typical commercial compound library is in the picosecond range. In addition, the large Stokes shift between the 350 nm donor excitation and the 670 nm acceptor emission further helps to minimize the background signal. The fluorescence emission of lanthanides also exhibits multiple narrow wavelengths, which is different from the broad wavelength emission of organic dyes, which allows multiplexing with fluorescent acceptors.

5. Fluorescence lifetime analysis (FLA)

A less common but effective detection method, in high-throughput screening, FLA uses time-domain data collection to determine the lifetime of fluorescent substances present in the sample.

The ideal dye for FLA is a ruthenium complex with a fluorescence lifetime of about 20 ns.

Compared with the fluorescence intensity measurement, the lifetime measurement is much less sensitive to the color of other materials in the sample (such as the test compound).

In addition, when analyzing data, both life cycles are generally applicable, which allows the deletion of shorter-lived components, similar to the effects achieved by TR-FRET.


6. Alphascreen

The wavelength of fluorescence emission is longer than the wavelength of excitation light. In contrast,

AlphaScreen is a non-radioactive distance-based analysis method that uses singlet oxygen sensitization to produce emission at a shorter wavelength, thereby avoiding fluorescence artifacts.

The molecules to be tested are coupled to two different hydrogel-coated beads.

The donor beads contain a photosensitizer (phthalocyanine derivative), which generates singlet oxygen when stimulated by light at 680 nm.

If the acceptor beads (including dimethylthiophene derivatives and Eu chelate) are close to the donor beads, chemiluminescence and short wavelength light emission (520-620 nm) are caused.




Screening system based on affinity


Binding-based analysis directly observes the binding of Curry compounds to the target protein without considering their effect on protein function.

The biochemical methods described above measure changes in enzyme/receptor function or inhibition of probe/protein binding.

In general, the biophysical techniques used in binding-based assays are more material and time-consuming than competition-based screening, and are therefore most commonly used for small compound libraries.

Combination-based methods have also been used in the later stages of the challenging orphan target and drug discovery process, such as seed compound verification, optimization, and lead compound optimization.


1. Fragment-based drug design (FBDD)

An application particularly suitable for biophysical methods. FBDD involves the screening of fragments.

These fragments are small molecules (<280 Da) and form weak interactions with proteins.

The typical dissociation constant is in the range of millimolar to micromolar.

FBDD uses a small compound library to cover a larger chemical space (for example, the use of 103 fragments may achieve the effect of the traditional HTS compound library in 105-106), and at the same time provides a flexible starting point for medicinal chemistry.


Fragment discovery is particularly helpful for targeting difficult-to-drug targets with limited effects on protein-protein interactions and traditional compound libraries.

The most commonly used methods in FBDD are NMR (nuclear magnetic resonance), surface plasmon resonance and X-ray crystallography.


2. Nuclear Magnetic Resonance (NMR)

NMR can be used in two modes: ligand detection and protein detection.

The NMR of ligand detection measures the change in the intensity or relaxation of the ligand proton or other nuclear magnetic resonance active nuclei in the presence of the target protein.

The magnetization transfer from the protein to the ligand is called the Austenite effect. Usually 1H-NMR is used, and the ligand detection mode is used.

There is no size restriction on the protein, only a low concentration of the protein is required, and isotope labeling is not required.


On the other hand, ligand-based NMR may not be able to distinguish between specific and non-specific binding.

Although protein detection NMR is more laborious, it provides specific information about the compound’s binding site, especially when the NMR peak is assigned to a specific protein residue.


Therefore, after the initial screening, protein detection NMR is usually used as a secondary detection.

The most commonly used methods for protein detection NMR are 1H-15N and 1H-13C heteronuclear single quantum relations (HSQC), which are very sensitive to changes in the chemical environment of the protein backbone or methyl side chains, respectively.

Recently, reverse micelle encapsulation has been proposed to expand the detection limit of NMR in FBDD, which may allow the measurement of dissociation constants in excess of 200 mM.


3. Surface Plasmon Resonance (SPR)



A very sensitive method to monitor the binding between two species in real time, and can perform high-throughput screening.

SPR measures the change in refractive index of the gold/glass/solvent interface, which is the angle at which light is lost on the surface plasmon (“SPR dip”).

In a typical SPR experiment, small molecules or proteins flow over the surface where the target or other binding substances are immobilized.

Importantly, the change in refractive index is directly proportional to the quality of the binding material; therefore, SPR can measure binding stoichiometry and binding kinetics.


Typically, the target protein is immobilized on the sensor, using lysine covalent attachment or by affinity capture methods, such as polyhistidine tags, biotin or Fc receptors.

This format allows hundreds of molecules to be measured per day, but this may be limited by protein stability.

Alternatively, a microarray immobilized compound library provides higher throughput, but the library needs to be chemically modified for immobilization.


In order to distinguish between specific and non-specific binding, competition experiments for known ligands or mutation verification of proteins can be performed. Although SPR is very sensitive, it is relatively expensive and requires strict control of buffers, temperature, and suspended particles.


4. Biolayer interferometry (BLI)

Similar to the principle of SPR, the signal interference between the fixed protein layer and the internal reference layer is measured.

This technique is easier to use, but less sensitive than SPR, so it is usually used to measure the interaction between proteins, rather than the interaction between proteins and small molecules.


5. Second-harmonic generation (Second-harmonic generation, SHG)

Another plate-based method for measuring affinity is to measure the total internal reflectance of dye-labeled proteins immobilized on a lipid bilayer in a 384-well plate.

The second harmonic component of the signal is very sensitive to the orientation of the second harmonic reactive dye (PyMPO derivative) relative to the surface (with sub-angstrom resolution).

When the ligand binds to the target and changes its conformation, the second harmonic component of the signal The harmonic components will change.

Therefore, SHG can screen allosteric or induced conformational changes in the compound library.


6. Mass spectrometry (MS)

It is widely used to screen the interaction between proteins and small molecules.

A common form is affinity selection-mass spectrometry (AS-MS), in which the protein and compound pool are incubated, and the protein ligand complex is quickly separated from the unbound compound, and the bound ligand is then resolved by LC-ESI-MS Separation and identification.

The separation of protein ligand complexes and unbound ligands can be achieved by various methods, such as ultrafiltration or size exclusion chromatography.

In addition, protein molecular adducts can also be measured by mass spectrometry under specific circumstances, such as the screening of disulfide bond compounds or covalent compounds.


7. Thermal drift experiment (DSF)

Differential scanning fluorescence (DSF) and thermal shift assay (Thermal shift assay, TSA) both refer to this method.

By detecting the increase in the fluorescence of the dye when the temperature rises, the unfolding of the protein is measured.

The hydrophobic part has affinity. When the protein unfolds, the hydrophobic part is exposed and the fluorescence increases. The compound that binds to the target stabilizes it and increases the melting temperature.


8. DNA-Encoded Library technology (DEL)

DEL is established through a method of equalization and merging.

The chemical scaffolds are mixed together and then separated into the container for the next round of chemical reactions.

Add different chemical substitutions to each container, then combine and divide them again for the next reaction.

The product of cracking pond chemistry is a complex mixture that can contain millions or even billions of compounds.


For DEL, the initial scaffold is labeled as a unique DNA sequence; in each reaction step, another DNA sequence is added as a barcode for the added chemical substituent.

Then, the mixture encoded by the DNA barcode is applied to the beads containing the protein of interest.

Unbound compounds are washed away, bound compounds are eluted, and PCR and next-generation sequencing are performed to determine which DNA coupling compounds bind to the protein.


9. X-ray crystallography

High-resolution methods for studying small molecule/protein complexes. Although X-ray crystallography requires the most protein (mg) and crystallization is time-consuming, it can greatly facilitate the structure-oriented design of ligands to improve affinity. At present, high-throughput crystallization methods have been developed, and there are corresponding high-throughput facilities for diffraction, but they mainly rely on compound soaking (Soaking).




Cell-based screening system


The discovery of chemical probes and drug leads can be based not only on interactions with specific targets, but also on their ability to induce cell/organism phenotypes.
Cell/organism-based screening is usually used in:

1) The ideal molecular target is unknown;
2) Biochemical methods cannot fully reproduce the target reaction system;
3) The ideal phenotype only exists in the context of cells, such as cell differentiation.


Functional results can also provide a lot of mechanism information, such as distinguishing full agonists, partial agonists, inverse agonists, and allosteric modulators.

Heterogeneity can also be measured, especially when cells can be visualized for high-content imaging.

Finally, cell-based methods can also report the membrane permeability and cytotoxicity of compounds, which are the main reasons for the failure of structure-based drug development.


Cell-based methods can be applied to the entire process of drug discovery: target identification and verification, preliminary screening, lead identification, lead optimization, and safety and toxicity screening.

The commonly used methods are as follows, cell proliferation, second messenger, reporter gene, protein fragment complementation, yeast two-hybrid method, and microscope-based analysis.

The choice of cell model is crucial.


In human-derived modeling, cell lines are usually modified with reporter genes or other characteristics.

The current trend is more to establish “disease-like” models, including primary/patient-derived cells and complex cultures involving 3D cell culture or multiple cell types.

There is also increasing interest in using perfusion culture and other microfluidic devices to assess the long-term effects of compounds on cells.

In addition, small animal models such as nematodes and zebrafish are increasingly used as high-throughput models for small molecule screening.


1. Cell viability

Cell proliferation is often used to identify compounds that can kill cancer cells or pathogens, and to detect potential safety issues in organs such as the liver.

A common and effective method of activity determination is to use luciferase/luciferin to emit light in an ATP-dependent reaction to determine ATP content.

The detection of luciferase is very sensitive (less than 10 cells can be detected per well) and can be applied to 1536-well plates.

Dyes such as ruthenium, Alma blue, and bisazo compounds (MTT, MTS, XTT, and WST-1) can also be used for proliferation assays.

These are usually converted to produce fluorescence or color to indicate survival or death.

Cell viability can also be assessed by detecting intracellular enzymes (protease, LDH) released in the medium or inserting DNA with non-membrane fluorescent dyes (such as propidium iodide).


2. Reporter gene


Reporter gene analysis (also called signal pathway analysis), the coding sequence of the reporter protein is introduced under the transcriptional control of the relevant pathway.

The expression of the reporter gene reflects the degree of promoter activation or inhibition by detecting luminescence or fluorescence.

The observed signal is the product of the entire pathway, and the compounds being screened can interact at any point.

Commonly used reporter genes are CAT (chloramphenicol acetyltransferase), GAL (β-galactosidase), LAC (β-lactamase),

LUC (luciferase) and GFP. One disadvantage of reporter gene methods is that they usually require a long time to be cultured for transcription and translation, thus increasing the possibility of indirect effects.

The latest development method is to use different reporter genes to simultaneously detect multiple pathways.


3. The second messenger (Secondary messenger)

The second messenger method detects the expression or transport of messenger molecules in the pathway and provides a more similar response than the reporter gene method.

This test is widely used for GPCR targets. For example, if the target activates intracellular Ca2+ storage, its activity can be directly measured using calcium-sensitive dyes (such as fluorine-3, fluorine-4, based on the luminescence of aequorin).

If GPCR cannot naturally regulate intracellular Ca2+ levels, it can be designed to use promiscuous or chimeric G proteins (also known as universal adaptors) to stimulate Ca2+ signaling.

One of the characteristics of these methods is their fast response time, which requires the entire plate to be injected with compounds and CCD detection, such as FLIPR or FDSS, in the order of seconds.

Slow readout analysis, such as arrestin or TANGO, has also been developed for GPCR screening.

Fluorescence assays have been developed to measure other messengers, such as K+, or membrane-bound dyes whose fluorescence signal changes with membrane potential.


4. Protein-fragment complementation analysis and two-hybrid screening (Protein-fragment complementation)

Protein fragment complementation analysis (PCA, also known as bimolecular fluorescence complementation) and two-hybrid screening can detect the formation/inhibition of protein-protein interactions in cells.

PCA divides a single detection protein (luciferase, GFP, GAL) into two parts and fused to the protein-interacting molecule of interest; if the partner binds, the detection protein is re-formed and the signal can be detected.

In small molecule screening, GFP cannot be used because the two fragments are irreversibly bound and cannot be dissociated by the binding of small molecules.

Recently, a reversible fluorescent complementary structure has been reported, which enhances fluorescence through a protein that binds to a small molecule dye.


Two-hybrid (2H) screening is another complementary method based on the formation of active transcription factors.

In the double hybrid, the DNA binding domain binds to the bait protein, and the transcription activation domain binds to the target protein. After the reporter gene (such as GAL or LAC) is inserted into the promoter, if the target protein binds to the bait, the activation domain is connected to the reporter gene, which activates the reporter gene and can be detected.

In the presence of compounds that inhibit protein interaction, activation is prevented.


5. High-content imaging


The HCS instrument automatically collects optical or fluorescent images of the cells growing in the multiwell plate; these images are then automatically analyzed for any measurable features selected.

A typical instrument can read four-color fluorescence 2-40 times the target. Wide field of view and confocal tools are available, and the top equipment can read 63 times the flood target.

After obtaining the image, use quantitative image analysis tools to extract features and convert them into digital data. There are many measurable characteristics.

For example, from a single DNA-binding dye, the size, shape, intensity, and texture (pixel-to-pixel variability) of the nucleus can be measured.



Target recognition


After identifying promising lead compound series through phenotypic-based methods, the corresponding targets need to be identified.

These techniques are usually used for preclinical molecules in development, but can also be applied to approved drugs with unknown targets.

Identifying the target of a compound will greatly help understand its physiological relevance, optimize its affinity, determine off-target effects, and obtain regulatory approval for new drugs.

Target deconvolution methods can be divided into three categories: chemical proteomics, chemical genetics and bioinformatics comparative methods.

Generally speaking, the results of these methods are verified by sensitive in vitro tests, such as SPR.


1. Chemical proteomics

The affinity-based method is the oldest and most direct method to elucidate small molecule targets. In a popular form, the ligand is immobilized on a carrier (such as magnetic beads) and incubated with cell lysate to allow it to interact with the proteome. The bound protein is eluted, digested with trypsin into polypeptides, separated by liquid chromatography, and then identified by mass spectrometry and bioinformatics analysis (shotgun proteomics).

Quantitative mass spectrometry proteomics allows to compare the amount of protein pulled down by biologically active ligands with those bound to inactive ligand analogs (negative controls), thereby distinguishing non-specific binding. Other forms based on affinity have also been reported, some of which involve labeling of protein arrays instead of ligands, using gel electrophoresis for protein separation, or chemical modification of the ligands so that they can be covalently attached to cells On the target in the.


One disadvantage of affinity-based separation is that immobilization and derivatization can affect the observed binding affinity.

Labeling also requires expertise in chemical synthesis, and may not be compatible with all ligands.

In activity-based protein analysis (ABPP), ligands and probes compete for binding to members of a specific target class.


The Cell Thermal Drift Test (CETSA) is derived from the Thermal Drift Test, in which compounds are added to the cells.

The cells or lysates are heated to different temperatures, and protein denaturation is monitored.

The binding of the ligand stabilizes the target protein, so that they melt at a higher temperature. CETSA can be detected by gel electrophoresis or mass spectrometry.


2. Bioinformatics analysis (In silico comparative profiling)

The effect of the studied molecule on the cell expression profile or phenotype can be compared with the molecules in the known biologically active small molecule reference database to infer the target and mechanism of action of the experimental molecule.

Phenotype profiles that can be used for comparison include genetic, transcriptome, proteome, and high-content microscopy data.


3. Chemical genetics


Chemical genetic methods determine how gene expression levels change the effects of chemical agents.

In particular, the expression of gene products can be increased or decreased in the whole genome, and the phenotypic changes induced by the ligand can be monitored.


Compared with other target deconvolution strategies, this offers unique advantages, such as not limited to soluble proteins, and allows the distinction between direct and indirect binders, which provides a deeper understanding of the drug’s mechanism of action.

Initially, chemical genetics was studied in a limited number of only viable biological models like yeast using multicopy plasmids containing gene fragments, or by comparing the effects of reducing the number of copies of genes.


Today, chemical genetic technologies include RNA interference (RNAi), barcoded open reading frame (ORF) libraries, and CRISPR screening, which can interfere with all genes in mammalian cells in a systematic and effective manner.

Both CRISPR and RNAi-based methods are suitable for pooled screening, because the expression structure of these two systems (sgRNA or shRNA) has DNA copies integrated into the genome, and they themselves serve as molecular barcodes to encode target genes.





Future and outlook

There are currently two popular approaches for drug development.

One is to select significant disease targets based on basic research, specifically design HTS screening methods, select appropriate targeted drug libraries for screening, and use multiple methods to carry out emerging compounds and Identification and verification of lead compounds.


The second is to select a suitable phenotypic model according to the characteristics of the disease, and according to the characteristics of the phenotype, select a suitable phenotypic screening system for phenotypic screening experiments.

After obtaining the lead compound, perform target deconvolution identification and verification of the target In combination with structure-based drug screening, a further round of structural optimization is carried out to improve drug efficacy, and the strengths of each drug design strategy are integrated to develop original new drugs.






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3. Romei, M.G. and Boxer, S.G. Split Green Fluorescent Proteins: Scope, Limitations, and Outlook. Annu. Rev. Biophys. 2019, 48, 19–44

4. Blay V , Tolani B , Ho S P , et al. High-Throughput Screening: today’s biochemical and cell-based approaches. Drug Discovery Today, 2020, 25( 10):1807-1821.

High-throughput screening methods in drug development

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