Our research group aims to understand the role that sulfur redox chemistry plays in physiological and pathological processes and to use this knowledge to identify novel therapeutic targets for the treatment of human disease. To achieve these goals, we develop and apply new technologies that bridge the fields of chemistry and biology. A major focus of our research is the development of chemical approaches to probe oxidative post-translational modifications in cell-based systems, and the application of these tools to studies of oxidation biology. We complement these efforts in technology development with focused studies on individual enzymes. With particular interests in microbial sulfur metabolism, we select proteins such as the sulfate-reducing enzyme APS reductase, for detailed investigation using a range of chemical, biochemical, genetic and pharmacological techniques.
Reactive oxygen species are critical mediators of cellular signal transduction pathways, where they can post-translationally modify proteins via chemoselective oxidation of redox sensitive cysteine residues that can be re-reduced in the cell. Despite important advances in recent years, many fundamental issues remain in the field including which proteins, and which cysteines within them, are most reactive, the identity of the cysteine modifications themselves, and mechanisms of specificity in redox signaling. The importance of addressing such questions becomes clear when one considers that oxidative stress and cysteine modifications are prominent features of many acute and chronic diseases as well as the normal aging process. Cell-permeable chemical probes that detect oxidative cysteine modifications with molecular specificity offer a potentially powerful approach to identify oxidation sites and map redox signaling networks in living systems. To this end, we have developed the first chemical approach for selective detection of sulfenic acid-modified proteins in living cells. Application of this new method to the discovery of protein targets of oxidation in mammalian cells has yielded identification of ~200 proteins. A small subset of these proteins are known to be oxidized in cells, but the majority of the proteins have not been previously characterized as oxidation targets. In addition, we have used this method to gain mechanistic insight into the yeast peroxide sensing system comprising the oxidase Gpx3 and the transcription factor Yap1. From this work, we have identified a functional role for sulfenic acid modifications in this redox signaling pathway. The ability to monitor cysteine oxidation in living cells opens new avenues for the rapidly expanding field of oxidation biology and we continue to develop these reagents for further study and application.
In bacteria, de novo biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for protein synthesis, but also a reduced thiol as a component of the oxidative defense mechanisms that are particularly vital for the survival of intracellular pathogens such as Mycobacterium tuberculosis. Consequently, enzymes in the cysteine biosynthetic pathway are potential targets for the development of novel antibacterial agents. The enzyme APS reductase (APSr) catalyzes the reduction of adenosine 5’-phosphosulfate (APS) to sulfite, the first committed step in sulfate assimilation. APSr is an excellent new target for antibiotic development because it is essential for bacterial survival and humans lack an analogous enzyme. However, many fundamental questions concerning the molecular determinants of binding and specificity and the catalytic mechanism remain unknown. The focus of our research in this area is on further elucidating the catalytic mechanism of this bacterial enzyme and on the development of inhibitors as valuable tools for functional studies as well as possible leads for drug development. To gain insight into substrate recognition, we have examined the relative contributions of individual portions of ligand molecules to the enzyme-binding interaction. The adenine moiety of the substrate contributes significantly to recognition by APSr, suggesting that this region can be targeted in drug development. APSr is an iron-sulfur protein and the metallocenter has been demonstrated to be essential for enzyme activity. We have developed a method to reduce APSr to the [4Fe-4S]1+ state, opening the door to EPR studies to probe the function of the iron-sulfur cluster. Several potent APSr inhibitors have been discovered through virtual ligand screening and, as an alternative strategy to identify compounds that are selective for APSr, we have developed a synthetic approach to bifunctional compounds designed to interact with the APS binding site and the adjacent iron-sulfur cluster.
