Area 3: Chemical Genomics in Yeast
The current projects within the laboratory focus on four critical areas:
The laboratory uses a combination of molecular, cellular, genetic, genome, and proteome approaches to study the function of many yeast and human membrane proteins as well as proteins involved in the maintenance of genome stability in humans.
(i) Identification of inhibitors of P. aeruginosa using yeast-based phenotypic screens
Pseudomonas aeruginosa, an opportunistic human pathogen, is responsible for the majority of deaths in Cystic Fibrosis (CF) patients. The extensive tissue damage caused by P. aeruginosa infections is due to the synthesis of a number of toxins by the invading microorganism, among which exoenzyme S (ExoS), exoenzyme T (ExoT), exoenzyme Y (ExoY), and exoenzyme U (ExoU), collectively called Type III effectors, play a crucial role. These effectors kill their target human cells by inhibiting several essential cellular processes.
We recently developed a novel approach that uses a yeast assay to identify specific inhibitors against one of the type III effectors, ExoS. We showed that one of these compounds, Exosin (Figure 1), significantly inhibited Pseudomonal infections in human cells.
Figure 1: Chemical structure of Exosin, the first ExoS-inhibitor
Based on the success of this approach, we are currently testing the remaining three effectors, ExoT, ExoU, and ExoY, in a similar manner. Furthermore, to advance any of these new inhibitors to the clinic, we are testing the efficacy of Exosin and its derivatives in a mouse CF model. It is anticipated that these inhibitors will graduate from mouse models to human patients to provide a means to neutralize the toxic effect of P. aeruginosa colonization within infected individuals and provide sufficient time for antibiotic treatment protocols.
(ii) Discovery of novel anti-malaria drugs using a low-cost yeast model system
Malaria is the world’s most deadly tropical parasitic disease and is transmitted through the bite of female mosquitoes. There are four species of the parasite that cause malaria in humans. One of these, Plasmodium falciparum, causes the majority of infections and can lead to death if left untreated. There are few available vaccines against P. falciparum, and, strikingly, this pathogen shows increasing drug resistance making drug treatments of malaria patients extremely difficult. Therefore new therapies developed by innovative and unconventional approaches are needed to treat this devastating human infectious disease.
Figure 2: The life cycle of P. falciparum, the causative agent of malaria.
We are applying a yeast-based approach to select potential P. falciparum drug targets and to identify their specific inhibitors. Briefly, bakers yeast Saccharomyces cerevisiae is used to identify P. falciparum open reading frames (ORFs) that inhibit yeast growth and these P. falciparum ORFs are prioritized based on biological relevance. The pathogen ORFs of interest are then screened for inhibitors by overexpressing the P. falciparum ORFs and assaying for restoration of yeast growth in the presence of small molecules. Finally, in vitro and in vivo experiments are performed to demonstrate that the inhibitors directly modulate the parasite protein biological activity.
This approach for malaria drug discovery research represents a significant departure from current approaches for identification of anti-malaria drugs since it utilizes for the first time the bakers yeast S. cerevisiae as a whole-cell tool to isolate inhibitors against the products of toxic P. falciparum ORFs.
Selected Publications
Curak, J., Rohde, J.R., and Stagljar, I. (2009) Using the baker's yeast Saccharomyces cerevisiae to study bacterial effector proteins, Curr Opin Microbiol 12, 18-23.
Arnoldo, A., Curak, J., Kittanakom, S., Chevelev, I., Lee, V.T., Sahebol-Amri, M,, Koscik, B., Ljuma, L., Roy, P.J., Bedalov, A., Giaever, G., Nislow, C., A. Merrill, R., Lory, S., and Stagljar, I. (2008)
Isolating small molecule inhibitors of Pseudomonas aeruginosa ExoS toxin using a yeast phenotypic screen,
PLoS Genet 4, e1000005.
