Wet-lab target validation
Identifying a promising therapeutic target is only the first step. Once selected, targets must be rigorously validated to demonstrate their direct involvement in disease mechanisms and confirm that their modulation is likely to deliver a therapeutic benefit. This critical phase transforms computational and biological hypotheses into actionable, decision-enabling evidence.
At Axxam, we integrate AI-enabled target identification with advanced wet lab validation to ensure that targets are not only biologically relevant but also suitable for downstream drug discovery. Our comprehensive portfolio of complementary techniques enables a multidimensional assessment of target function, mechanism of action, and pathway engagement. Embedded within our integrated DiscoveryMAXX platform, this approach ensures seamless progression from target identification through validation to hit and lead identification By generating robust, high-quality data across multiple experimental approaches, we build confidence in target selection while reducing the risk of late-stage failures, laying a solid foundation for successful drug discovery.
1. Genetic approaches and expression profiling
Establishing the biological relevance of a target begins with understanding where and how it is expressed, followed by functional perturbation to confirm its role in disease mechanisms.
Expression profiling
Expression profiling is performed to confirm the expression in relevant cellular systems using qPCR, Western blot, immunostaining, or advanced techniques such as MSD (mesoscale diagnostic).
Target expression assessment to determine its disease relevance includes:
- Expression across models: Evaluating target expressions in different cellular models (iPSCs, primary cells, or immortalized lines) including diseases ones to select the best model and ensure relevance across contexts.
- Differential expression: Comparing target expression levels in diseased and/or stimuli-treated versus healthy and/or untreated cells (e.g., tumors vs. normal samples) or at various developmental/differentiation stages.
These analyses are essential for guiding downstream validation and help link AI-predicted targets to experimentally measurable outcomes.
Genetic / pharmacological perturbation strategies
Once target expression is confirmed and its modulation in disease-relevant or stimulated conditions supports the disease hypothesis, genetic modulation can contribute to validating the functional role of the target.
Axxam can apply the following approaches in disease-relevant cellular models:
- Knock-out (KO) – via CRISPR/Cas9 to eliminate gene function
- Knock-down (KD) – using CRISPR interference (CRISPR-i), siRNAs, shRNAs, or antisense oligonucleotides (ASOs) to reduce gene expression
- Overexpression – via transfection, viral transduction, or CRISPR activation (CRISPR-a) to enhance gene activity
These tools enable a robust evaluation of how target modulation affects cellular behavior, supporting both phenotypic and mechanistic validation. The use of drugs and/or tool reagents (e.g., target-directed antibodies, others) able to modulate the expression of the target or mimicking a specific relevant pathological phenotype, e.g. stressing the cells or activating some pathways can be useful too.
2. Functional assays
Functional assays evaluate the biological activity of targets and the effects of their modulation using tool molecules, typically in-vitro:
- Cellular pathway analysis investigates compound effects on signaling pathways and biological processes in live cells, monitoring proliferation, apoptosis, gene and protein expression, receptor-ligand interactions, disease-specific biomarkers, etc.
- Biochemical assays focus on enzymatic activity and molecular interactions of the target and/or on enzymatic activities or interactions connected with the possible role of the target on the disease state under investigation.
- Pharmacological assays measure drug potency, efficacy, and mechanisms of action at molecular and cellular levels. Assessment of whether reference modulators of the disease state display additive or cooperative effects when used in combination with tool compounds directed against the target, when available.
Functional assays confirm that target modulation produces desired biological outcomes.
Assay development activities, including in-depth characterization, reproducibility assessment, scalability evaluation, and confirmation of HTS compatibility, are typically performed downstream of target validation. However, it is highly advisable to start considering orthogonal assay strategies already during the early stages of assay validation and development. Planning ahead for complementary assays helps ensure that on-target activity and the desired level of specificity can be accurately confirmed throughout the screening cascade.
In parallel, target engagement assays play an important role in quantitatively demonstrating the direct binding of reference compounds to the biological target. These approaches provide valuable evidence supporting the proposed mechanism of action and help strengthen confidence in the biological relevance of identified hits and lead compounds.
3. Phenotypic analysis
Phenotypic assays capture broad biological changes induced by modulating drug targets, providing a rich dataset for validation:
- Cell Painting: High-content imaging using multiplexed fluorescent dyes to record cellular morphology changes, offering phenotypic fingerprints and off-target effects analysis if present.
- Multi-electrode Array (MEA) and Calcium Oscillations: Measure electrical and calcium signaling changes in excitable cells, crucial for neuroscience and cardiotoxicity studies upon modulation of target expression and/or activity.
MEA and calcium oscillations
Activity map (left) and spike sorter with respective raster plot showing acitivity recorded from iCell Motor Neurons (FCDI) cocultured with iCell Astrocytes (FCDI) at 24 DIV
- Transcriptomics: Profiling gene expression changes driven by target modulation (e.g., RNA-seq) to reveal downstream pathways and mechanistic insights (services offered in partnership).
- Proteomics: Mass spectrometry-based profiling of protein abundance and modifications after target perturbation to validate specific effects (partnered services).
Data analysis integration transforms complex experimental data (imaging, omics, phenotypes) into biologically and clinically meaningful insights.
Together, these approaches provide a multi-dimensional understanding of target function and compound activity, improving confidence in target validation and supporting successful drug discovery and possibly biomarker validation.
Why choose Axxam for validating AI-driven insights?
By integrating advanced biological platforms with insights from AI and in silico modeling, Axxam offers a complete solution for AI-driven target identification and validation. Our scientific teams collaborate closely with strategic partners to move from digital prediction to wet-lab confirmation with precision, speed, and confidence.
Whether you are prioritizing early-stage targets or preparing for lead optimization, Axxam provides the expertise, tools, and flexibility to help you advance the right target – right from the start.