We use chemical genetic tools in Saccharomyces cerevisiae and mammalian cells to uncover targets of bioactive compounds, reveal functions of genes and identify inhibitors of Protein-Protein Interactions.
Chemogenomic profiling is a powerful tool in biology that generates mechanistic insights into how a bioactive compound interacts with a cell. This technique hinges on the principle that cellular sensitivity to a bioactive compound is altered by changing activities of their in vivo targets or interfering with components that collaborate with the target. In Chemogenomic Profiling, we determine the fitness of a library of mutants that cover the entire genome, in the presence and absence of a bioactive compound. Thus, we identify all genes in the genome that confer either resistance or sensitivity to a bioactive compound.
Chemogenomic profiling in budding yeast (Saccharomyces cerevisiae) is facilitated by the presence of 20-nucleotide barcode sequences unique for each mutant in the genome-wide yeast knockout collection. We grow the pooled collection of yeast barcoded knockout mutant cells in the presence and absence of a bioactive compound and determine the relative fitness of each mutant by quantifying the barcodes in the untreated and treated cells by PCR followed by Next Generation Sequencing (NGS). We have collaborated with several research groups from A*STAR like the Natural Product Discovery platform (BII), Molecular Engineering laboratory (MEL), Experimental Drug Development Centre (EDDC) and the p53 laboratory and elucidated the mode-of-action of their compounds by chemogenomic profiling in yeast.
In addition to yeast, we also perform chemogenomic profiling in mammalian cells (established by the Zhang laboratory at the Broad Institute) to determine Mode-of-action of bioactive compounds. Unlike in yeast where we use a commercially available barcoded knockout collection, we generate a library of mutants that span the human genome for each experiment (Figure 1). We transduce sgRNA libraries that cover the entire human genome into mammalian cells using lentiviruses. Cas9 is also encoded by the lentiviral vector. Unrepaired CRISPR/ Cas9-mediated double-strand breaks in the genome will result in deletions/insertions and inactivation of the target gene. A mammalian cell knockout library generated this way will then be grown in the presence and absence of the bioactive compound (Figure 1) and the genes that confer resistance/sensitivity of the compound will be determined by NGS methods. We performed chemogenomic profiling of vemurafenib, a BRAF kinase inhibitor in A375 cells and obtained several expected hits (Figure 2). This work is supported by A*STAR’s Innovations in Food and Chemical Safety programme and is part of the Toxicity Modes-of-Action Discovery (TOXMAD) platform that aims to uncover mode-of-action of compounds mainly used in consumer care using a multi-disciplinary approach. Apart from identifying targets/side-targets of drugs and bioactive compounds, this technique can also be used in understanding virus-host interaction. We are now seeking to collaborate with research groups and industries across the world interested in utilizing this powerful technique. Interested research groups/companies are advised to contact Dr Prakash Arumugam ( firstname.lastname@example.org ).
Protein-Protein interactions (PPI) are fundamental to growth and survival of cells and serve as excellent targets to develop inhibitors of biological processes such as host-pathogen interaction and cancer cell proliferation. However, isolation of PPI inhibitors is extremely challenging. We have established a significantly improved and thoroughly validated Yeast 2-hybrid (Y2H) assay that can be used in a high throughput manner to screen for small molecule PPI inhibitors (Wong et al BMC Biology 2017 15:108). Using the p53-Mdm2 interaction to optimize the assay, we showed that the p53-Mdm2 inhibitor nutlin-3 is a substrate for the yeast ATP-binding cassette (ABC) transporter Pdr5. By deleting nine ABC transporter-related genes, we generated a ABC9Δ yeast strain that is highly permeable to small molecules. In the ABC9Δ strain, the p53-Mdm2 interaction inhibitors like AMG232 and MI-773 completely inhibit p53-Mdm2 interaction at nanomolar concentrations in the Y2H assay. In addition, we have identified a conserved segment in the core DNA-binding domain of p53 that facilitates stable interaction with Mdm2 in yeast cells and in vitro. We are using this assay to screen for inhibitors of PPI that are of outstanding biomedical importance.
Evaluation of chemical sensory irritation is an essential component of safety testing of personal care products, cosmetics, medical devices and topical medications. Current methods for sensory irritation testing include in vivo animal tests and in vitro skin cell viability-based assays. Given the worldwide efforts to minimize the use of animals in research, the ban on animal use in cosmetic testing in Europe, and interspecies discrepancies in response to irritants, the former in vivo testing option is not preferred. Although existing in vitro assays could help in screening for irritants, it lacks sensitivity in detecting sensory irritation at lower doses of test compound. In a project funded by IFCS, we are establishing a three-pronged Mechanism-based Irritant Screening Platform that includes a Receptor-activity assay, Neuronal culture- based assays and a human sensory assay. By integrating the data from the three independent assays, we will develop a tool that will reliably predict whether a molecule will have irritant activity.
Prakash Arumugam joined the Translational Research division of BII in January 2015. He completed his doctoral work at the Centre for Cellular and Molecular Biology in India in 2001. During his PhD, he showed that the human lamin B receptor has sterol biosynthetic activity. He joined Prof. Kim Nasmyth’s laboratory at the Institute of Molecular Pathology (IMP) in Vienna as a postdoctoral fellow in 2002 and elucidated the role of cohesin’s ATPase activity in sister chromatid cohesion in budding yeast. He joined the University of Warwick in the UK as a lecturer in 2007 and established his independent laboratory to study meiotic cell cycle and chromosome segregation in budding yeast. His group identified the first binding site for monopolins at the kinetochore, and a conserved signalling module (Rim15-PP2ACdc55-Endosulfine) that regulates entry into gametogenesis, in budding yeast.
My laboratory uses chemical genetic tools in yeast and mammalian cells to uncover mode-of action of bioactive compounds, map cellular pathways of toxicity, determine functions of conserved protein complexes and screen for inhibitors of Protein-Protein interaction.