High-Throughput Screening

In small molecule drug discovery, chance is one driver of hit identification in High-Throughput Screening (HTS). However, it is wrong to reduce the process to chance only. Louis Pasteur’s famous “Chance favors the prepared mind” most appropriately captures the notion that the purposeful design of all elements of the HTS experiment has equal weight: first, a suitable screening assay, combining sensitivity and robustness; second, a well thought through screening cascade to discriminate specific hits from unwanted bycatch; and third, a compound collection assembled based on a multitude of medicinal chemistry quality criteria and a smart coverage of chemical space.

To run an HTS campaign, clients can access our assay development services, select one of our ready-to-use assays or transfer their own assays to us.

We carry out hit-discovery programs using either Axxam’s comprehensive compound collections or our clients’ preferred libraries, which are handled professionally at our facility.

State-of-the-art laboratory automation platforms allow the experimental testing in HTS using either 384- (40 μl) or 1536-well (5-10 μl) microplates. The integration of multi-modality plate readers, liquid handling devices and robotic plate handling enable the realization of a large spectrum of diverse assay formats.

Besides small molecules, we also offer screenings on chemical mixtures, natural product extracts, oligonucleotides, and antibodies.

HTS platforms & technologies

We provide to our clients access to our fully automated, state-of-the-art screening platforms designed to run cell-free and cell-based assays in miniaturized microplate format using a variety of detection technologies such as luminescence, fluorescence (intensity polarization, time-resolved such as HTRF or LANCE, calcium flux, membrane potential, thallium flux), absorbance, flashplate assays as well as transporter assays.

Cell-based screenings can also be performed using electrophysiological, quantitative genetic expression, and phenotypic imaging-based readouts.

We routinely run automated screening campaigns with either cell-free or cell-based assays (homogeneous and non-homogeneous) in both high-throughput screening (384 well plate) and ultra-high-throughput screening (1536 well plate) format:

For projects targeting ion channels or electrogenic proteins, our clients can rely on high quality patch-clamp screenings run on the SyncroPatch instrument (Nanion) integrated in a fully automated robotic platform. Beyond primary screenings on a large compound collection, this instrumentation can be used for:

Clone selection
Compound profiling
Hit validation/structure–activity relationship (SAR) studies

The integration of TaqMan^TM RT-qPCR readers in automated systems enables us to study drug-induced changes in gene expression directly in relevant cells types.

Suitable for any cell type or target gene
Close to physiological conditions
Multiplexing: up to 3 different genes/labels can be monitored simultaneously (including housekeeping genes as reference)
Appropriate primer selection allows to quantitatively follow RNA splicing

The imaging of cellular structures and function in live or fixed cells using automated fluorescence microscopy offers another option to study the impact of drugs on relevant phenotypes. Our experienced and skilled High-Content Screening team integrates our imaging capabilities (Operetta^® and Opera Phenix^®, PerkinElmer) with customized assay designs and state-of-the-art data analysis to visualize drug effects on cell morphology and/or function. Our offerings include:

Immortalized cell lines, iPSC-derived cells and primary cells
Cell painting approach
Live imaging or staining of fixed cells
Cell line generation (genetically encoded labels and reporter)

Hit identification

The ingredients for a typical discovery program encompass various essential elements – compound collections, a primary target assay and secondary assay for specificity testing, primary counter screening, secondary orthogonal assays utilized during the screening campaign, and a meticulously planned screening approach.

A test compound in HTS is typically tested only once. For this reason, assays for primary screening need to comply to highest quality criteria combining sensitivity towards the desired mode-of-action with a low-noise signal and high reproducibility. “HTS-grade” assays perform robustly most importantly within the fully-automated setup. At Axxam, we use internal controls and consequent tracking of assay signal statistics to ensure and document test performance from early assay development throughout the entire HTS and follow-up process.

Discover more about assay development at Axxam

Compound management is crucial to supply test compounds in microplate format to be processed in automated HTS or subsequent follow up experiments. Our compound management unit consists of dedicated specialists responsible for secure long-term compound storage, and suitable logistics for picking, plating and processing (replication, dilutions, etc.). Our systems operate using either microliter tip-based or nanoliter acoustic dispensing (ECHO) technology. Consequent barcode and process tracking finally generates a seamless record of each plate and corresponding compounds in our screening database.

Discover more about compound management at Axxam

Microplate-based data a generally processed and analyzed using GeneData Screener software. Assay performance related to signal-to-noise and integrated experimental controls is rigorously assessed and documented in our central database. All experiments need to comply with predefined quality criteria. Data analysis is handled by a specialized team of data scientists tailoring analysis and reporting to the individual project needs and providing an instance of peer review in our workflow. The group also integrates chemoinformatic expertise applied at multiple steps in our hit identification process to minimize the risk of ‘false negatives’, to capture features of emerging structure-activity relationships, and/or to extend and enrich screening results also into the space of commercially available analogues.

Hit validation

Following the elimination of false positives, i.e. statistical events originating from random biological noise and/or technical errors, through simple retesting, screening hit lists still contain large numbers of compounds acting through off-target or otherwise undesired mechanisms. To discriminate desired from unwanted hit compounds, hit compounds are typically tested in a set of secondary assays to probe for target specificity, selectivity and mode-of-action (MoA). The design of these secondary assays (or screening cascade) is dictated by the target, the assay technology used for primary screening and the desired MoA.
At Axxam, the structural integrity of hit compounds is generally tested by LC/MS. Upon request, some basic physico-chemical properties, like lipophilicity or solubility, and selected baseline ADME characteristics, e.g. plasma and hepatocyte stability or membrane permeability, can be measured to facilitate hit prioritization in the downstream process.