Chemical Genomics

Prakash ARUMUGAM
Principal Investigator

Mohammad ALFATAH
Post Doc Research Fellow

WONG Jin Huei
Senior Research Officer

GOH Jie Hui, Corinna
Research Officer

We employ the Forward and Reverse Chemical Genetic tools in the unicellular yeast Saccharomyces cerevisiae (budding yeast) to uncover mechanism of action of bioactive compounds, screen for inhibitors of Protein-Protein Interaction and reveal functions of conserved signaling complexes.

Chemogenomic profiling in yeast

Chemogenomic profiling is a powerful technique which identifies genes that confer sensitivity and resistance to a given bioactive compound, in a genome-wide scale, in a single experiment. This technique is based on the observation that cellular sensitivity to bioactive compounds is altered by changing activities of their in vivo targets (or components that collaborate with the target). In this approach, a genome-wide screen is performed by determining fitness data of a defined library of bar-coded yeast mutants in the presence of a bioactive compound (Figure 1). We are analyzing novel compounds present in the A-STAR’s Natural Product Library (NPL) and bioactive compounds from collaborators. We are a part of two exciting multidisciplinary A*STAR initiatives namely the Biotransformation Innovation Platform (BIP) and the Toxicity Modes-of-Action Discovery (TOXMAD) platform. We are also establishing chemogenomic profiling in mammalian cells using a CRISPR/Cas9-based approach.

Figure 1
Figure 1: Chemogenomic profiling in yeast

Using Yeast 2-hybrid assay to screen for small molecule inhibitors of Protein-Protein Interaction

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 ABC9∆ yeast strain that is highly permeable to small molecules. In the ABC9∆ 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 now using this assay to screen for inhibitors of PPI that are of outstanding biomedical importance.

Cellular sensitivity to a bioactive compound is altered by changing activities of their in vivo targets (or components that collaborate with the target). In chemogenomic profiling, we determine the fitness of a library of barcoded knockout yeast strains in the presence and absence of a bioactive compound (Figure 1). In a single experiment, we can identify all the genes that confer either resistance or sensitivity to a bioactive compound. There are three major types of chemogenomic profiling namely haploinsufficiency profiling (HIP), homozygous deletion profiling (HOP) and multicopy suppression profiling (MSP). In HIP, relative sensitivities of a genomewide collection of strains heterozygous for a gene deletion (these strains contain one copy of a gene instead of two) are determined. Reducing dosage of a gene can sensitize cells to a compound that targets the gene product. HOP is similar to HIP except that a genome-wide collection of strains homozygous for gene deletions is used. HOP generates genomewide maps of synthetic growth interactions between gene deletions and a bioactive compound. Although HOP will not identify target gene(s) per se it will pinpoint pathways that act in parallel to the target gene pathway. In MSP, a collection of yeast strains in which each strain expresses one of the 6000 possible ORF’s from a strong inducible promoter is exposed to a bioactive compound. Target genes which when over-expressed confer resistance to a bioactive compound can be identified by MSP.

Figure 2
Figure 2:Yeast 2-hybrid assay for screening of small-molecule inhibitors of Protein-Protein Interaction.
Left Panel: Interacting proteins A and B are fused to the DNA-binding Domain (DBD) and Activation Domain (AD) of GAL4 resulting in its activation and reporter gene expression. Right Panel: Inhibition of interaction between Proteins A and B by a small-molecule inhibitor prevents activation of GAL4-driven reporter gene expression.

Chemical Genomics Members

Dr. Prakash ARUMUGAM
Principal Investigator
 
  Biography Details
NameTitle
Dr. ARUMUGAM PrakashPrincipal Investigator
Dr. Mohammad AlfatahPostdoctoral Fellow
Ms. WONG Jin HueiResearch Officer
Ms. GOH Jie Hui, CorinnaResearch Officer

This section is still work in progress.