Identification of Small Molecule Inhibitors of Pseudomonas aeruginosa Exoenzyme S Using a Yeast Phenotypic Screen

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens

Sphingolipid accumulation causes mitochondrial dysregulation and cell death

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways

Noxa in rheumatic diseases: present understanding and future impact

Impaired programmed cell death is an important contributing mechanism in the development of chronic inflammatory and autoimmune diseases. Overexpression of Bcl-2 family proteins in such diseases has led to the concept of targeted suppression of these proteins as a primary therapeutic strategy. However, limited success with this approach has prompted pharmacologists to look at the other side of the coin, with the aim of reactivating jeopardized pro-apoptotic proteins that may neutralize Bcl-2 or other anti-apoptotic molecules. In this effort, BH3-only proteins have gained recent attention as endogenous molecules for the sensitization of resistant cells to undergo apoptosis. Among the BH3-only family, Noxa stands out as exceptional for its specificity to bind Mcl-1 and Bcl-2 and blunt their biological properties. Noxa is now being tested as a promising therapeutic target in cancer biology. Nonetheless, its role and clinical application still lack validation in autoimmune diseases, including rheumatic conditions. This is partly attributed to the significant gap in our understanding of its regulatory role and how either overexpression of Noxa or delivery of BH3 mimetics could be therapeutically exploited. In this review we highlight some recent studies in RA, OA, SLE and SS suggesting that Noxa may be used as a potential therapeutic target to circumvent invasive and tissue destructive processes in these rheumatic diseases

Noxa in rheumatic diseases: present understanding and future impact

Impaired programmed cell death is an important contributing mechanism in the development of chronic inflammatory and autoimmune diseases. Overexpression of Bcl-2 family proteins in such diseases has led to the concept of targeted suppression of these proteins as a primary therapeutic strategy. However, limited success with this approach has prompted pharmacologists to look at the other side of the coin, with the aim of reactivating jeopardized pro-apoptotic proteins that may neutralize Bcl-2 or other anti-apoptotic molecules. In this effort, BH3-only proteins have gained recent attention as endogenous molecules for the sensitization of resistant cells to undergo apoptosis. Among the BH3-only family, Noxa stands out as exceptional for its specificity to bind Mcl-1 and Bcl-2 and blunt their biological properties. Noxa is now being tested as a promising therapeutic target in cancer biology. Nonetheless, its role and clinical application still lack validation in autoimmune diseases, including rheumatic conditions. This is partly attributed to the significant gap in our understanding of its regulatory role and how either overexpression of Noxa or delivery of BH3 mimetics could be therapeutically exploited. In this review we highlight some recent studies in RA, OA, SLE and SS suggesting that Noxa may be used as a potential therapeutic target to circumvent invasive and tissue destructive processes in these rheumatic diseases