Science Spyglass

From Gene to Validated and Qualified Hits
High-Throughput Screening at Axxam

Despite a constantly expanding repertoire of therapeutic modalities, small molecules continue to account for the majority of new drug approvals and continue to attract attention and investigation, especially with the expansion of the knowledge on different mechanisms of action, including pharmacological chaperones, targeted protein degraders, covalent protein modification, etc.

The small molecule drug discovery research is a complex endeavor that involves multiple stages, each with its own set of hurdles and uncertainties. The hit identification phase is a foundational step in the whole process; by providing the initial set of compounds that have the potential to be developed, it lays the groundwork for a successful drug development. The efficiency, quality, and diversity of hits identified during this phase significantly influence the success and progress of the entire drug development process.

Adaptation to Automation: adapt experimental protocols for automated testing

Hit Confirmation: re-test of cherry-picked hits from primary screening in triplicates at screening concentrations

Activity Determination: full concentration-response in triplicates for confirmed hits

At Axxam we are passionate about small molecules and eager to translate innovative target and disease biology into new therapies to patients in need. We offer clients access to a broad cutting-edge infrastructure for automated high-throughput screening, a comprehensive high-quality compound library with modern compound management logistics, a leading expertise in assay development and integrated discovery for hit selection. We believe that leveraging on Axxam’s knowledge and experience into clients’ drug discovery programs will facilitate the translation from Gene to Qualified Hit along the discovery workflow.

Assay development

High-Throughput Screening (HTS) assays come in many different flavours monitoring target binding or function in either biochemical or cell-based assay systems. Similarly, assays employ a large variety of (mostly optical) readouts.
Independent of the many diverse technologies available, HTS assays share 3 main features:

  • Specificity towards a molecular target and mode-of-action (MoA)
  • Robustness combining reproducible low-noise signals with experimental protocols amenable to automation
  • Sensitivity towards the desired molecular MoA

Our assay development aims to optimally combine all features in the HTS assay. In-depth assay optimization to probe and fine-tune the assay parameters and protocols ensures assay performance and sensitivity also throughout the adaptation to automation, which is finally assessed by testing a limited compound subset in a pilot run under HTS conditions.

Hit validation and qualification

Definitions of hit validation and hit qualification differ significantly in various organizations. Very often, no distinction is made at all. At Axxam we refer to hit qualification as an additional post-HTS service used to increase the value delivered with a hit list report.

The single HTS assay is typically not sufficient to ensure discrimination of the desired pharmacological modulators from the inevitable “by-catch” in bioassay testing, i.e. compounds acting through off-target or unspecific interference mechanisms. Therefore, well-designed screening cascades with additional tests tailored towards the individual project needs are required for a diligent hit qualification ensuring specific target interaction via the desired molecular MoA. These include:

  • Orthogonal assays: same target, but different assay format – for positive selection of hits
  • Counter assays: same assay format, but different target – for hit de-seletion
  • Selectivity assays: related target, frequently same assay format

As a final step, purity of hit compounds will typically be probed by mass spectroscopy.

To further facilitate a data-driven hit prioritization process in the HTS follow-up, Axxam provides ad hoc services designed to deliver information on:

  • Physicochemical properties, i.e. solubility lipophilicity, chemical stability
  • Metabolic stability
  • Membrane permeability
  • Plasma protein binding
  • Plasma stability

The additional services also include a detailed medicinal chemistry evaluation, addressing:

  • Chemical tractability and resynthesis of hit compounds
  • Initial novelty and intellectual property check
  • Acquisition and/or synthesis of relevant and strategic analogs to probe emerging principles of the underlying structure-activity relationship

This additional hit qualification by expert medicinal chemists aims to provide valuable information on the hit compounds, facilitating the selection of the best-suited molecules and thus enhancing the probability of downstream success.

Infrastructural backbone:
HTS automation and compound & data management

While a comprehensive quality compound library and the smart design of the HTS assay and screening cascade greatly impact the result of individual screening projects, a sophisticated automation and IT infrastructure is another, equally important enabler of success. Laboratory robots and automation not only perform recurrent experimental tasks beyond regular office hours but also with superior precision and reproducibility.

Similarly, in compound management oversized high-tech refrigerators combine optimized conditions of long-term sample storage of compound plates with flexible input and retrieval function. They interface with pipettors and acoustic dispensers to flexibly generate plate copies, cherry-pick hit compounds, and generate serial dilutions in variable formats and volumes (from nl to μl). Tracking each plate’s unique barcode throughout all steps of the process serves not only to protocol the technical execution, it also avoids errors and mis-match between data pharmacological activity data and chemical compounds of the library. Beyond state-of-the-art instrumentation it requires laboratory automation specialists and data scientists with a relevant pharmacology training to operate the HTS workflow successfully.

In summary, Axxam is the perfect partner to take your target biology one step further – from gene to validated and qualified hit.

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