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What do compound libraries do?

Compound libraries are widely used in target identification, high throughput/focused screening for new hits that might be developed into a drug, drug repositioning/repurposing, predictive toxicology, or new target discovery through the integration of small-molecule chemogenomics with genetic approaches, such as RNA-mediated interference and CRISPR–Cas9.

Each chemical in the library has associated information stored in some kind of database with information such as the chemical structure, purity, quantity, and physiochemical characteristics of the compound.

Target identification.

The compound libraries will enable biological targets and molecular mechanisms to be linked to many critical phenotypic effects in a variety of cell types. In isolation, it is unlikely that small-molecule chemical probes will directly reveal novel therapeutic targets. Therefore, small-molecule screening, when used in conjunction with other chemical and molecular biology validation techniques, such as genetic or genomic screening: knockdown or knock-in experiments, contributes to a 'body-of-evidence' approach to target identification.

High throughput/focused screening, High-content, and in vivo screening.

Compound Libraries | TargetMol
Figure 1

Advances in high-throughput, high-content and multiparametric imaging assays have proven utility in mechanistic elucidation. TargetMol compound libraries will also be appropriate to be screened in high throughput, high-content and multiparametric imaging assays with multiple features of cell morphology being measured. This allows gene-drug interactions to be effectively profiled, creating a resource termed the Pharmacogenetic Phenome Compendium. Through this work, new molecular modes of action and opportunities for drug repurposing will be discovered.

Drug repurposing

In some instances, chemogenomic screening is able to reveal new uses for existing drugs. This can be indirect, when target or pathway hits are coincidently modulated by clinical agents, or direct, when the drugs themselves are hits in the phenotypic screen. Such findings on a therapeutic target can considerably accelerate the drug discovery process, as compounds in the libraries can be directly tested in animal models or clinical efficacy experiments.

Combinatorial library screening with existing drugs may also reveal novel synergistic therapeutic opportunities. The library usually is screened in combination with a known approved drug for a target, to identify new therapeutically relevant drug combinations for the treatment of some drug-resistant disease. Usually some potential sensitizers targeting other pathway molecules will be identified to have synergistic effects with this known approved drug, and the potentially clinical utility of this combination will be demonstrated.

Predictive toxicology

Another emerging application of chemogenomic screening is the classification of the toxic mechanisms of new compounds. Usually a panel of primary human cell types (hiPSC, cardiomyocytes, hepatocytes, etc.) are used to detect a broad range of toxic mechanisms using an array of different endpoints. Screening of molecules with known mechanisms defined signatures in these assays allows new molecules to be screened and compared against the reference database using computational predictive methods. Using this approach, a pharmaceutical compound will be correctly predicted to be a potential drug with a toxicity profile suggestive of skin rash, a side effect that was actually observed in clinical trials of this compound. Therefore, profiling such libraries to provide functional 'fingerprints' can predict the mechanisms of action of new molecules discovered in phenotypic screens.

Novel hits on-target

Focused screening of a chemogenomic subset library has been shown to reveal novel pharmacological mechanisms within the same gene family. A biochemical cell-free screen (target-based assays) will be usually carried out. This work leads to the identification of new inhibitor that had been shown previously to inhibit other target, thus suggesting that the new hit will be amenable to this mode of inhibition, a hypothesis that will be subsequently confirmed by X-ray crystallography.

How do we build a compound library at TargetMol

TargetMol uniquely offers a series of focused compound libraries comprised of FDA-approved drugs, natural compounds, compounds for chemical genomics, pathway targeting, toxicity prediction, and disease-related. They cover a diverse range of biological targets, including major families such as oncology targets, kinases, GPCRs and hydrolases, but also relatively underexplored targets such as GTPases and RNA-binding proteins. To create the TargeMol compound libraries, millions of compounds and their associated screening data points (of which there were billions) were mined using bioinformatics. Key features of this selection process included a number of parameters that ensured the appropriateness of a compound for cell-based screening, such as selectivity, permeability, solubility and cytotoxicity (predictive algorithms were used if these values were not available). Compounds were not included if they had promiscuous activity that resulted from 'false-positive' pharmacology (such as highly lipophilic). Another important aspect in the design of the TargetMol compound libraries was the selection, where available, of different chemical templates with the same annotated on-target pharmacology, as the inclusion of these in the library would provide more confidence that the putative target arising from a phenotypic screen represents a real hit.

Screening instruments considerations

Automated liquid handling workstation

Liquid handling automated workstations are tools designed to do much of the sampling, mixing, and combining of liquid samples automatically. The workstations can measure out samples, add reagents, and make sure liquids are added to bioassays in a uniform fashion. They will save valuable time and reduce errors inherent in complex, repetitive liquid handling protocols for large scale screening tasks. There is such a huge difference in the scale and scope of automation for robotic liquid dispensing ranging from single-tasking workstation to multitasking integrated system. The volume of sample the liquid handling automated workstation can handle is one feature to consider when making a purchase. Other features to consider include how large a footprint the workstation makes and the ease of use of its software interface.

Multichannel Pipettes

Multichannel pipettes (manual or electronic) are a valuable tool for laboratories working with microplates for small to medium scale screening purpose, an affordable alternative to automated liquid handling workstation when restricted financially.

There are suggestions on pipettes:

  1. Find a comfortable, ergonomic fit to prevent poor pipetting technique, which affects accuracy and precision.
  2. Look for pipettes and tips that were designed together.
  3. Don't skimp on consumables: consistency in pipette tips reduces fit variability and benefits overall performance.

Multi-mode Microplate Reader

The primary goal of HTS is to identify through compound library screenings, candidates that affect the target in the desired way, so called 'hits' or 'leads'. This is usually achieved employing liquid handling devices, robotics, plate readers as detectors and dedicated software for instrumentation control and data processing. Consequently, high throughput screening should be seen as a fast scan of biological processes by which compounds with a poor or no effect can be rapidly excluded from the analysis pipeline. Specialized instrumentation, like multi-mode microplate readers, have the flexibility to perform different assays. A HTS-dedicated plate reader can measure hundreds of plates in a single day, generating a considerable amount of data points.

The following features are the gold standard for HTS-dedicated multi-mode microplate reader:

  1. Highest sensitivity on the market for fluorescence intensity and polarization;
  2. Measurement in 96-, 384-, 1536- as well as 3456-well plate formats;
  3. Simultaneous Dual Emission detection for fast and robust detection of fluorescence polarization assays, BRET, FRET and TR-FRET as well as AlphaScreen® assays;
  4. Dedicated AlphaScreen®/AlphaLISA® excitation laser;
  5. High-frequency TRF laser allows efficient measurement of 1536-well plates on the fly (1 flash), still delivering a high Z` value (> 0.8).

Automated imaging and analysis platforms

High-content screening (HCS) or high-content analysis (HCA) requires an instrument designed to extract the maximum information from your sample in a robust and reproducible manner with the speed for high-throughput analysis. With powerful imaging and analysis capabilities for a wide range of applications -- from basic research to assay development and screening – these powerful automated imaging and analysis platforms (HCS based automated microscope systems) produce the highest possible image quality to take your research further, in less time, due to their ability to study many features simultaneously in complex biology systems.

In contrast to traditional HTS, which has a single read out of activity, HCS allows a scientist to measure many properties or features of individual cells or organisms at once. The ability to study many features and multiplex simultaneously is both what gives HCS tremendous power and challenging complexity. It can enable both targeted and phenotypic assays that measure movement within a cell or between cells or allow analysis of specific sub-populations of cells in a heterogeneous mix that would be difficult or impossible to run with other techniques.

The most obvious applications of HCS are primary screens of potential leads, molecules that can be further optimized into drug candidates, for cellular activities that cannot be easily measured by a single endpoint, such as spatially localized proteins or measurements of cellular morphology. In addition, HCS increases the power of the experiment and confidence in the outcome by coupling targeted assay measurements with a visual measure together.

Molecular Devices LLC, Thermo Fisher Scientific, GE Healthcare Life Sciences, IntelliCyt Corporation, and Perkin Elmer provide many options of such instruments for you to choose.

Plate type selection

The selection of the appropriate assay plate type is important and mainly depends on the assay detection method. The light reflecting properties of the assay plate surfaces profoundly affect the final signal intensities, background noise levels and well-to-well crosstalk. Black, solid bottom, opaque-walled plates are recommended for fluorescence-based reading technologies to achieve lower background signal and minimal crosstalk, while white plates are good for luminescence signal detection to enhance light output. On the other hand, clear-bottom plates are needed for colorimetric assays, as well as for cell-based assays, where the cells need to be monitored by microscopy throughout the course of the experiment.

Despite these general selection guidelines, a suitable assay plate type should be carefully chosen in compliant with the overall project goals. For instance, in a luminescence assay with low signal window and relatively high assay volume/well, where the 'hit' compound is defined as the test sample that causes a drop in the signal intensity in comparison to the negative control, detection of the 'hits' may be impaired if white plates are used. That is because a well containing the active compound with low signal intensity would be surrounded by several inactive wells with high signal intensities, and the crosstalk from the surrounding wells would greatly alter the original signal magnitude in the active well leading to increased false negative rates. In such luminescence assays, where the scientist is aiming to detect a signal decrease, black plates would be more appropriate to conduct the experiment.

High content assays commonly require specially designed microtiter plates to attain the maximal scan performances, when high content imagers are used. These plates are generally intended to have optically clear, very thin and uniform well bottoms to ensure high quality images. Additionally, the assays that require fixation and staining processes, and involve multiple washing steps may necessitate plates that enhance cell retention. For this purpose, plates with poly-D-lysine, poly-L-lysine and collagen-coated surfaces are available to promote cell adhesion and growth.

Many more types of plates are offered for different assay methodologies, such as low attachment plates for cell-based assays using cells in suspension, and non-specific binding surface plates for protein-binding experiments. Besides, selection of the correct plate type for multiplex assays may require extra effort and testing process, especially if luminescence and fluorescence signals are being measured within the same plate. Performing a detailed search of the available plate options for the assay of interest is a time worthy practice, which would eventually save the scientist from developing and validating the assay repeatedly.

Preparation of Compound Library

The key platform or sample carrier used in HTS is the microplate. Typical formats include 96-, 384-, 1536-, or 3456-well plates. The nature of the sample and of the detection assay may affect the choice of the microplate format and its color.

Screening facilities usually keep their compound library collections stored in so called “stock plates”. Stock plates are not directly used in experiments. Instead, when needed, compounds from a stock plate are “copied” to an assay plate through a pipetting station. In high-throughput screening (HTS), the historical practice of using a single compound concentration for the primary screen has been associated with a high proportion of false positives. To minimize the number of false positives, it is better to introduce quantitative HTS (qHTS) where concentration-response profiles, as opposed to single-concentration data points, are generated at the primary-screen level for each library compound. To enable qHTS, compound libraries have to be prepared as a dilution series. Usually the ‘vertical’ or inter-plate method is utilized to place all concentration points for a given compound on different plates. The first plate contains the highest concentration of a set of compounds, or sub-libraries, while subsequent plates contain the same compounds in the same well locations, but at successive lower concentrations. In this manner, a dilution series of plates can be generated where multiple copies of the same library are made with each copy differing only in the concentration of library members and where the number of copies is determined by the number of titration points desired for the dilution series. In vertically-developed multi-concentration compound collections, the user is free to choose between screening all concentrations and selecting dilution plates (single concentration screening) in a random-access manner.  

Compound Libraries | TargetMol
Figure 2

We suggest that the processing of an incoming compound library involve both physical and virtual components (Figure 2). Before any physical manipulation of the compounds can occur, the process of preparation of compound library should be planned carefully (map layout) and their structures should be registered at your compound management system. Additional information on compounds such as analytical data is added to the database once registration is complete. The following protocol for creation of the seven-point dilution series used in cell-based assay is based on 4 of 96-tube racks compounds, with each of these racks representing one quadrant of a 384-well plate, or a total of these racks corresponding to one 384-well plate (Figure 3). Note: Though qHTS can be performed with compounds in 384- or 1,536-well plates, 384-well format seems more suitable for labs without automated liquid dispensing handler system. You can easily scale it up to 1,536-well plate format. (Figure 2)

Compound Libraries | TargetMol
Figure 3

Compound Receipt and Initial Processing

Compounds are received either in solid state or as solutions contained in 96-well 2D-barcoded Matrix (#3791) tube racks (Figure 2 and 3). For powder (use 1 mg as example), in general, the entire sample is dissolved in DMSO (about 250 μL calculated based on M.W. of 400) to produce a 10 mM solutions. The tubes are recapped, inverted and vortexed to capture any powder that might have adhered to the cap or side of the tube, and centrifuged to 1,000 rpm (or 145×g, Eppendorf 5810R) for 1 minute. While most powder samples dissolved after brief vortexing, visual inspection of the tubes was implemented in order to identify and segregate mixtures containing undissolved material. Tubes containing such mixtures were subjected to sonication treatment for up to 10 minutes in order to complete the dissolution process.

Sample Compression into 384-well Plates

Compounds in 96-tube racks are compressed into 384-well Greiner Bio-One or Matrix polypropylene plates via interleaved quadrant transfer using automated liquid dispensing handler or manually by multichannel pipettes. For each 96-tube rack, column 1 is left empty for control placement resulting in blank columns 1 and 2 of the final 384-well plate. The sequence of the transfer is from rack 1 to Q1-2 (column 1,3,5,…,23), rack 2 to Q1-2 (column 2,4,6,…24), rack 3 to Q3-4 (column 1,3,5,…,23), and rack 4 to Q3-4 (column 2,4,6,…,24) of 384-well plate. The process for compression from 96-tube racks to 384-well plates involves the following steps (Figure 3). First, four 96-tube racks are centrifuged at 1,000 rpm for 1 minute. The samples in the first tube rack are mixed 3 times by aspirating and dispensing solution with a 12-channel pipette. This is followed by a 30 μL transfer into quadrant 1 (Q1-2: column 1,3,5,…,23) of the 384-well plate. A fresh set of tips is loaded for the next rack (into Q1-2: column 2,4,6,…,24) with all the steps repeated until all the remaining racks are completed. Depending on the volume of solution contained in the tubes, multiple copies of these 384-well top-concentration plates can be made and stored (as mother plates). One of these 384-well top-concentration plates could then be used for the downstream dilution.

Inter-plate Titration Series Preparation

10 mM stock solutions in DMSO from one of 384-well top-concentration plates made above are served as the highest concentration point in preparing libraries for qHTS. Usually the final highest compound concentration in a cell-based assay should approach or exceed 100 μM (required to answer the biological question), and the final concentration of DMSO should be controlled by not greater than 1% (minimize the effect on cell growth). Our chosen dilution series that spans a range of over four logs, consisting of seven points separated by five-fold dilution steps, cover a meaningful range from the highest concentration 100 μM to the lowest concentration 6.4 nM, applicable to a broad range of assay detection formats and target types. The creation of 5 × working plates (0.5 mM compound highest concentration) is the core of this step.

1. To make the 5 × working plates that meet the above criteria, 10 copies of 384-well top-concentration plate will be made first with 2.5 μL of solution contained in each copy.
Note: Thus, the preparation of 10 copies of plates containing 2.5 μL of solution per well consumes 25 μL, leaving approximately 5 μL in the well. This volume serves three purposes. First, it represents an amount needed to ensure that the 25 μL aspirated does not contain air bubbles due to minor variations in meniscus. Second, this sample can be used for retrospective QC analysis to investigate compound stability and to resolve library plating or registration issues. Third, it represents an archive amount which can be accessed to perform limited follow-up tests should a re-supply of the compound become difficult.

2. Then the solution in these top-concentration plates is mixed with 47.5 μL of culture medium (now the concentration of DMSO becomes 5% and the highest concentration of compound would be 0.5 mM after 20-fold dilution). The 60 plates for creation of the 10 identical copies of the seven-point dilution series that will contain the lower concentrations are pre-filled with 40 μL culture medium containing 5% DMSO. Then 10 μL of solution is aspirated from the higher concentration plate and dispensed into the 40 μL culture medium containing 5% DMSO contained in the next-concentration plate to achieve a 5-fold dilution. The steps are then repeated until all seven 384-well plates are prepared. Tip changing occurs only once (after dispensing into the fourth-concentration plate) for each seven-plate dilution set to minimize the carryover of highest-concentration solutions to the lowest-concentration plates. Upon conclusion, the 384-well plates are centrifuged at 1,000 rpm for 1 minute, and the 9 sets are heat-sealed or sealed by aluminum sealing tape for long-term storage at −80 °C, while the remaining set is fitted with compound lids and made available for immediate use in screening (Figure 4).
Note: If you don't have time to finish all the qHTS in a short time, you can only use one top-concentration plate and create one set of the seven-point dilution series, and leave the remaining 9 top-concentration plates stored at −80 °C for future use.

Compound Libraries | TargetMol
Figure 4

Plate Registration

A unique identifier that used for registration and to enable tracking of qHTS inter-plate concentrations can be assigned for each set of plates at the time of initial reformatting. A qHTS plate job typically consists of 10 copies of an inter-plate titration series, with each plate containing the same compound layout.

All plates, starting with the initial 96- and 384-well source plates and down to the individual 384-well compound titration sets, can be registered using your processing tools. The 96-tube racks' quadrant mapping into their respective 384-well plates, information about the relationships between plates, wells compounds, and their concentration mappings, need to be saved and stored in your in-house database.

For optimal database performance, indexes are created on plate, row, column, sample batch identifier, parent compound identifier, and supplier data. Second, a separate table is updated to insert new plate and well concentration information from the compound plating jobs. During processing of screening data, these two tables are used jointly to create qHTS concentration mappings that convert plate-well driven data into titration response activities centered on individual samples.

Considerations during assay development

Pharmacological relevance of the assay

If available, studies should be performed using known ligands with activity at the target under study, to determine if the assay pharmacology is predictive of the disease state and to show that the assay is capable of identifying compounds with the desired potency and mechanism of action.

Reproducibility of the assay

Within a compound screening environment it is a requirement that the assay is reproducible across assay plates, across screen days and, within a programme that may run for several years, across the duration of the entire drug discovery programme.

Assay costs

Compound screening assays are typically performed in microtitre plates. Within academia or for relatively small numbers of compounds assays are typically formatted in 96-well or 384-well microtitre plates whereas in industry or in HTS applications assays are formatted in 384-well or 1536-well microtire plates in assay volumes as small as a few microlitires. In each case assay reagents and assay volumes are selected to minimize the costs of the assay.

Assay Validation & Quality Control

A typical assay validation process consists of multiple major components such as repeating the assay of interest on multiple days with the proper experimental controls, verifying the optimum assay conditions using the high throughput instruments in subject and exploring the overall assay quality with various statistical metrics and visualization tools.

Compound Management

Because of the expense and effort involved in chemical synthesis, the chemicals must be correctly stored and banked away for later use to prevent early degradation. Each chemical has a particular shelf life and storage requirement and in a good-sized chemical library, there is a timetable by which library chemicals are disposed of and replaced on a regular basis. Most chemical libraries are managed with information technologies such as barcoding and relational databases, compound registration system, plate registration & tracking system, HTS Data QC system, report generation system, etc.  

Because a chemical library's individual entries can easily reach up into the millions of compounds, the Management of even modest-sized chemical libraries can be a full-time endeavor. Compound Management requires inventory control of small molecules and biologics needed for assays and experiments, especially in high-throughput screening.