Understanding the mode of action of a compound

To help clients understand the mode of action of their compounds, we use tools such as gene expression analysis, gene editing and custom (bespoke) assays.

Gene Expression Analysis

In cases where a compound’s mechanism of action is unknown, we can help elucidate it by examining differential gene expression, either on a targeted scale using qRT-PCR or on an -omics scale using RNA-seq.  When using RNA-seq, we have the computational expertise and tools to develop biologically meaningful hypotheses from differential gene expression signatures.

Figure 9.  Differential gene expression analysis.  Drug treatment caused up-regulation of genes involved in the indicated biological processes.  Color indicates which experimental conditions induced upregulation (red = 8 h treatment, yellow = 6 h and 8 h treatments, gray = 4 h, 6 h, and 8 h treatments).

Hypothesis Testing with RNAi and Gene Editing

Whereas transcriptomics is an excellent technique for hypothesis development, testing those hypotheses frequently relies on manipulations of protein expression levels through overexpression, knockdown, or knockout.  We have experience using lentiviral transduction to generate stable cells lines overexpressing wild-type or mutant proteins (Figure 10), expressing shRNAs for stable knockdown of target proteins.  We have also used the CRISPR/Cas9 system to generate cell lines with homozygous knockout of target proteins.  Such cell lines allow us to test the roles of proteins and pathways of interest in pharmaceutical function (see Figures 1, 5, and 7).

Figure 10.  Drug activity is modulated by expression level of a protein of interest.

Custom Assays

When clients want to investigate a specific mode of action for a compound, one of the tools we have used is to leverage our knowledge of the biology to design cell-based experiments to measure the relevant endpoint. For example, a client was concerned that their chemistries were causing liver toxicity through fatty acid build-up (a phenomenon) known as steatosis. We developed an assay to measure measure specifically the amount of fatty acids that is chemically-induced. The uterine assay is another example of a bespoke assay that we have developed.

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Case Study: Using Transcriptomics To Investigate MoA

Case Study:The Application Of Benchmark Dose To Toxicogenomic Data