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 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) Develop inhibitors of the proteasome in the human parasite Plasmodium falciparum for the treatment of malaria
- 2) Identify novel serine hydrolase targets in the pathogenic bacteria Staphylococcus aureus and Mycobacterium tuberculosis
- 3) Identify proteases that regulate aspects of bacterial signaling and survival in gut commensal microbes
- 4) Develop fluorescent enzyme-activated sensors for applications in image guided cancer surgery and imaging of sites of bacterial infections.
- 5) Target viral proteases to create the next generation of drugs for use in global pandemics
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.
A current area of interest of our laboratory is identification of hydrolase enzymes used by obligate intracellular parasites and bacterial pathogens to establish infection of a human host. In particular, we study the apicomplexa parasites Plasmodium falciparum and Toxoplasma Gondii as well as the pathogenic bacteria Staphylococcus aureus and Mycobacterium tuberculosis, all of which cause serious disease conditions in humans. In each of these pathogens we have performed either phenotypic screens using small molecule inhibitors or activity-based probes to identify novel enzymes involved in various aspects of pathogenesis. In Plasmodium, we have developed selective inhibitors of the parasite proteasome as way to kill parasites without causing toxicity to the human host. In Toxoplasma, we have identified a family of serine hydrolases involved in 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 colonization of the host. We have also used activity-based probes to identify enzymes in Mycobacterium tuberculosis that represent promising new therapeutic and diagnostic targets.
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 targets for imaging 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. Our first generation fluorecent probe has been licensed and recently entered human clinical trials in the United States and Australia.
Targeting hydrolases for imaging of infectious diseases
We have used activity-based probes to identify serine hydrolase targets in Staphylococcus aureus that are active on the surface of the bacteria in biofilms. We are using these enzymes as biomarkers for sites of infection with the goal of using covalent inhibitors to target imaging contrast agents to sites of infeciton inside the body. We are currently developing highly selective inhibitors of the S. aureus hydrolases using phage display and fragment screening. We will then use these selective binding agents to target optical and ultrasound contrast agents to sites of biofilm formation. We are also using a similar approach to target a serine protease in Mycobacterium tuberculosis for rapid and low-cost diagnosis of infection. These chemiluminescent probes are activated by the M. tuberculosis serine protease HIP1 to produce a light signal that can detected using simple and inexpensive luminescent detectors. We are currently testing our FLASH probe in human clinical samples with the help of our collaborators in South Africa.
Identifying proteases that regulate aspects of bacterial signaling and survival in gut commensal microbes
We have recently initiated projects to use activity-based probes and functional screening assays to identify proteases produced by commensal bacteria found in the human gut. Specifically, we have identified a family of dipeptidyl peptidases (DPPs) that are important for regulation of bacterial cell wall integrity and that are inhibited by current FDA approved drugs desgined to target human DPP4. We have also developed screens to identify secreted proteases produced by gut microbes that are capable of processing host receptors that regulate aspects of inflammation and pain signaling. These enzymes are potential targets for treatment of inflammatory bowl diseases.
Targeting viral proteases to create new drugs for future pandemics
We have recently joined two NIH-funded Anti-Viral Drug Develoment (AViDD) teams (see this link for more info) - AI-Driven Structure-Enabled Antiviral Platform (ASAP) and Development of Outpatient Antiviral Cocktails against SARS-CoV-2 and other Potential Pandemic RNA Viruses. Both of these teams are working to validate novel viral drug targets and to advance lead molecules into clinical testing with the hope of having new drugs ready for the next major pandemic. We are focused on helping to engineer inhibitors for several different protease targets in various high-priority viruses.