High Content Screening (HCS) is a screening approach where phenotypes that can be visualized using microscopic techniques are used to develop an assay that is relevant for the screening needs. The cells of interest are imaged and analyzed in high throughput, yielding results with high statistical power. 

HCS penetrated all stages of the drug discovery pipeline, and there are numerous reasons for that, specific to each stage of the process. A common denominator is that HCS is:

• A powerful analytical technology (in statistical sense) 
• Yielding biologically relevant, statistically robust data 
• That data is amenable to high throughput 

This approach is very flexible in building new assays. It is unique in its ability to build morphological assays such as neurite outgrowth and tube formation or assays where only a sub-population of cells is targeted. Typical examples are cell cycle, infection or cellular differentiation assays. 

High Content Screening at Selvita

We offer HCS-based functional and phenotypic assay platform for drug discovery integrated with innovative AI-based analytical pipeline. Our imaging facility is equipped with advanced Leica inverted fluorescent microscope and Perkin Elmer Operetta CLS, high throughput widefield and confocal imager, with 2D, 3D and live imaging capabilities. 

Images generated by the HCS system are analyzed by human-defined or AI-based protocols, depending on project needs. Perkin Elmer Harmony and PhenoLOGIC packages or in-house developed flexible AI solutions are used. 

Highly efficient data flow solutions along with self-learning bias-free analytical pipelines allow for analysis of large datasets resulting from screening of large compound libraries. 

Assay development 

Selvita`s Team specializes in HCS assay development for particular IDD project and target needs in multiple therapeutic indications. If the relevant phenotypic change can be visualized, in most cases it can be used to build an assay. We have developed assays covering the following therapeutic areas: 

• Oncology
• Neurosciences
• Fibrosis
• Inflammation
• Infectious Diseases
• In vitro Toxicology

These assays were used at all preclinical stages of the drug development pipeline: 

• Target ID and validation – siRNA studies 

• Primary and secondary screens 
• H2L and LO – monitoring on-target, off-target activities and toxicity in one assay is often possible 

Apart from assays developed on-demand, there is a number of ready-to-use or readily adaptable assays that cover many areas of interest. 

Universal assays:

• Cellular viability (propidium iodide, GelGreen, 7-AAD, LIVE/DEAD)
• Cellular proliferation (Ki67, EdU incorporation)
• Apoptosis (TUNEL, Annexin V, casp-3/7 activation)
• DNA damage (γH2A.X, P53BP1, TUNEL)
• Protein expression levels – immunofluorescence (IF)
• Protein modification (phosphorylation, acetylation etc. levels – IF)
• Intracellular protein localization (e.g. transcription factor nuclear translocation) – GFP and IF
• Protein phosphorylation levels – proximity ligation assay (PLA)
• Protein-protein interactions (e.g. receptor dimerization) – PLA
• Wound healing, migration assay
• Multiplexing several assays for cost effectiveness and enhanced informational content

Complex assays, adapted to particular project needs:

• Particle, virus, antibody, ligand internalization
• Tubes, fibers, neurites – number, length, branching
• Multiparametric shape descriptors (including CellPaint)
• Matrix invasion of cancer cells in 3D culture
• Labelled biologics internalization or monolayer penetration

Functional live assays:

• GPCR research: calcium (Fluo4, GCaMP-expressing reporter cell lines)
• Membrane electrical potential: FluoVolt
• Autophagy assay (GFP-LC3 reporter cell line)
• Mitochondrial health (MitoTracker Red, CMXRos)

Drug discovery stage-specific advantages of HCS 

Target discovery and validation 

• Carefully designed and executed RNAi screens (off-target effects) 

• Underlying assumption that gene silencing mimics inhibition by a small compound 
• Genome-wide screens or targeted libraries 
• Specific readouts or target-agnostic morphological profiling 

Primary screening 

• Due to the multiplexing and multi-parametric characteristics of HCS, imaging is very efficient and could save both cost and time

• It is possible to devise screens that assay concomitantly several modes of action
  or toxicity, bringing secondary screening activity at the primary screen stage 
• Because phenotypes are scored with several parameters, the rate of false positives tends to be smaller, reducing attrition rates at the hit verification stage 
• When exploiting quantitative cellular phenotypes, compounds are assayed directly for the biological outcome under study and no a priori information about targets is necessary 
• Compounds identified in cellular assays are known to penetrate cells and therefore fulfill
  a minimal requirement of ADMET 

Lead optimization 

• The ability to combine several readouts in a single assay reduces the cost and time to reach decisions during LO cycles 
• A lead series can be tested simultaneously for on-target (efficiency) and off-target effects (specificity), toxicity, cell entry, stability and precipitation 
• IC50 curves can be generated on many parameters, allowing classification of compounds
  in a lead series according to several criteria 
• Capacity to score toxic compounds in high throughput immediately after a primary homogeneous screen is a great advantage. A drug having adverse effects on a subset of the population (for instance undergoing cell division) can be identified using a cell-by-cell analysis. Such effects remain completely hidden in homogeneous assay and will only become apparent at much later stages of the drug development pipeline 
• The multi-parametric subcellular resolution provided by HCS allows detailed studies of the mode of action of lead candidates. Having access to this kind of information is very important for decisions during structure activity relationship (SAR) studies of lead candidates to ensure the compound remain on target and off-target effects can be avoided