Mode of Action Analysis

After hit identification, the measurement of the activity on the actual target is essential to avoid chasing off-target effects. Such measurements can include:

Quantification of the target activity in cells, e.g., phosphorylation, methylation, acetylation
Quantification of a downstream effect of a target, e.g., gene expression, protein degradation, or protein localization

In a more holistic approach, the assumed mode of action of a new compound can be confirmed with our MoA Finder tool based on compound screening of cytotoxic effects on a large panel of tumor cell lines.

In most cancer drug discovery projects, the ultimate goal is the death of tumor cells. The investigation of the cell death mechanisms gives insight into the events downstream of target inhibition. We can examine various cell death mechanisms, including the accumulation of reactive oxygen species, mitochondrial outer membrane permeabilization, and apoptosis. Cell cycle arrest studies can aid the understanding of the mode of action.

Mode of Action Analysis - Example 1

In-depth Characterization of Histone Modifications

Epigenetic enzyme activity can be quantified by the detection of posttranslational modifications of histones. One challenge is probing the target-specific action of epigenetic inhibitors in intact cells on one specific amino acid instead of looking at global changes of histone modification.

Antibody-based methods including Western Blot and ELISA enable the detection of posttranslational modifications on specific amino acids. The tests comprise:

Histone Methylation
Histone Demethylation
Histone and Tubulin Acetylation
Histone and Tubulin Deacetylation
Histone Phosphorylation

Roam our cellular epigenetic assays webpage

Mode of Action Analysis - Example 2

Question

Quantification of histone H3 methylation after NSD2 knockdown

Challenge

To monitor the specific activity of our target NSD2 we needed a readout for quantification of dimethylation of histone H3 at position Lysine 36.

Achievement

We chose the human tumor cell line U-2 OS due to a relatively high expression of endogenous NSD2 protein. Three siRNA molecules were used for NSD2 knockdown and dimethylation of lysine 36 on histone 3 (H3K36me2) was quantified via a specific antibody in Western Blot analysis.

NSD2

Three siRNA molecules siWHSC1 A-C were incubated with U-2OS cells for 72 hours before cell lysis and subjection to SDS-PAGE and Western Blot analysis to quantify NSD2 expression (A), quantify Histone H3 and H3K36me2 to detect the activity of methyltransferase NSD2 (B, C).

View publication

High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2

Mode of Action Analysis - Example 3

Question

Confirmation of the mode of action of a PI3K inhibitor

Challenge

To gain insight into the global phenotypic effects of a novel inhibitor that was presumed to act through inhibition of PIK3.

Achievement

We performed a large cell panel screen, the ProliFiler assay, to determine IC50 values of the inhibitor impacting cellular proliferation on 140 human tumor cell lines. We correlated the resulting sensitivity profile of the assumed PI3K inhibitor with that of more than 1000 cancer drugs with a known mode of action. The computational analysis was performed with the MoA Finder service by our partner CRO 4HF Biotec.

MoA

MoA Table

The volcano blot shows the correlation between our PI3K inhibitor and more than 1000 cancer drugs in the 4HF Biotec Cancer Data Mining database.

The cancer drugs that resemble our compounds’ profile mostly comprise PI3K inhibitors confirming that inhibition of PI3K causes the phenotype.

View our MoA Finder webpage