Our laboratory is interested in developing and applying chemical tools to dissect the functional roles of hydrolases in a number of human health conditions. Our research group is made up of a mix of synthetic organic chemists, biologists, biochemists, and cell biologists. We are currently using synthetic chemistry to build new reagents that allow enzyme activity to be monitored in complex biological samples including cells, tissues and whole organisms. We are applying these tools to 1) functional studies of the proteasome and serine hydrolases in the human parasites Toxoplasma gondii and Plasmodium falciparum 2) Identification of novel serine hydrolase targets in the pathogenic bacteria Staphylococcus aureus and Micobacterium tuberculosis 3) development of fluorescent enzyme sensors for applications in image guided surgery.

Chemical synthesis of activity based probes and inhibitors

One of the main focuses of the laboratory is the design and synthesis of novel activity based probes for hydrolases that use serine and cysteine nucleophiles for catalysis. We have extensive experience developing probes that form irreversible covalent bonds to target hydrolases using an enzyme catalyzed chemical reaction. Probe labeling of targets therefore serves as an indirect readout of their enzymatic activity. Our fluorescent and biotinylated peptide epoxide and acyloxymethyl ketone (AOMK) probes that target the papain family of cysteine proteases have led to the identification and characterization of proteases involved in diverse biological processes. We are currently working to expand our repertoire of probes by diversification of both general scaffolds and reactive functional groups. We have recently developed a series of probes that can be used to study various CD clan cysteine proteases including the caspases involved in cell death, legumain involved in antigen presentation, and several bacterial proteases involved in virulence. We are also working on the design of probes that target diverse families of serine hydrolases. Finally, we are exploring the use of phage display methods to identify constrained peptide sequences that can be used to enhance overall selectivity of activity based probes for a given enzyme target.

Functional roles of cysteine and serine hydrolases in human pathogens

A current area of interest of our laboratory is identification of hydrolase enzumes used by obligate intracellular parasites and bacterial pathogens to establish infection of a human host. In particular, we study two apicomplexa parasites, Toxoplasma gondii and Plasmodium falciparum and the pathogenic bacteria Staphylococcus aureus and Micobacterium tubercluosis, all of which cause serious disease conditions in humans. In each of thes pathogens we have performed either phenotypic screens using small molecule hydroalse inhibitors or activity based probes to identify novel enzymes involved in various aspects of pathogenesis. In Toxoplasma, we have identified a family of serine hydrolases involved host cell invasion as well as parasite replication and are currently working to better understand their biochemical functions. In Staphylococcus aureus we have identified a previously uncharacterized family of serine hydrolases that process lipid esters and function in the establishment of productive colonozation of the host. We have also used activity based probes to identify novel essential enzymes in Micobacterium tuberculosis that represent promising new therapeutic and diagnostic targets.

Imaging of protease activity for optical surgical guidance

We have developed fluorescently quenched probes that produce a fluorescent signal when processed by a protease. These fluorescent probes allow protease activity to be monitored in real time using existing clinical instrumentation. We and others have found that a number of cysteine proteases that are produced primarily by stromal cells within the tumor microenvironment are ideal target for imaging of the location of tumors in vivo. Our current protease activated probes can be used to highlight locations of tumors, thus allowing more effective surgical resection with complete removal of tumor tissue. We are currently working to increase the overall selectivity of our imaging probes by using multiple tumor-derived enzyme signatures to trigger probe activation.