Tulsi Upadhyay

All Publications

• A robust fluorogenic substrate for chikungunya virus protease (nsP2) activity. Protein science : a publication of the Protein Society Makhaik, S., Rut, W., Choudhary, S., Upadhyay, T., Hao, C., Westberg, M., Bobst, C., Yoo, E., Fejzo, J., Lin, M. Z., Bogyo, M., Thompson, P., Drag, M., Hardy, J. A. 2025; 34 (3): e70069

  Abstract

  Chikungunya virus (CHIKV) is an emerging pathogen with pandemic potential. CHIKV infection in humans is transmitted by mosquitoes and induces common symptoms of high fever, arthralgia and myalgia. Because no specific antiviral drugs for treatment of CHIKV infection are available, drug development remains a central goal. The chikungunya virus protease from nsP2 (CHIKVP) has emerged as a key drug target due to its indispensable role in viral replication via cleavage of the viral polyprotein. To date, effective tools for screening for CHIKVP inhibitors that reflect the most critical polyprotein cleavage sites have been lacking, hampering drug-development efforts. We found that the recognition ability of CHIKVP is sensitive to the length of peptide substrates. In this study, we report a robust fluorogenic substrate comprising a 15-mer peptide derived from the nsP3/4 junction from the CHIKV polyprotein. This peptide is flanked by an ACC-Lys(dnp) donor-quencher pair. Our new substrate acc-CHIK15-dnp shows a 30-fold improved signal-to-noise ratio as compared to the previously reported edab8 substrate, which is also based on the nsP3/4 junction. We found acc-CHIK15-dnp is recognized only by CHIKVP but not by other alphavirus proteases. This is surprising due to the high level of sequence conservation in the alpha virus polyprotein junctions and indicates that the P-side residues are more important than the P'-side sequence for effective CHIKVP cleavage. The robust signal-to-noise ratio obtained using acc-CHIK15-dnp derived from the nsP3/4 cleavage site enabled much improved small molecule HTS on CHIKV relative to other fluorogenic reporters.

  View details for DOI 10.1002/pro.70069

  View details for PubMedID 39981948

• Covalent-fragment screening identifies selective inhibitors of multiple Staphylococcus aureus serine hydrolases important for growth and biofilm formation. Research square Bogyo, M., Upadhyay, T., Woods, E., Ahator, S., Julin, K., Faucher, F., Hollander, M., Pedowitz, N., Abegg, D., Hammond, I., Eke, I., Wang, S., Chen, S., Bennett, J., Jo, J., Lentz, C., Adibekian, A., Fellner, M. 2024

  Abstract

  Staphylococcus aureus is a leading cause of bacteria-associated mortality worldwide. This is largely because infection sites are often difficult to localize and the bacteria forms biofilms which are not effectively cleared using classical antibiotics. Therefore, there is a need for new tools to both image and treat S. aureus infections. We previously identified a group of S. aureus serine hydrolases known as fluorophosphonate-binding hydrolases (Fphs), which regulate aspects of virulence and lipid metabolism. However, because their structures are similar and their functions overlap, it remains challenging to distinguish the specific roles of individual members of this family. In this study, we applied a high-throughput screening approach using a library of covalent electrophiles to identify inhibitors for FphB, FphE, and FphH. We identified inhibitors that irreversibly bind to the active-site serine residue of each enzyme with high potency and selectivity without requiring extensive medicinal chemistry optimization. Structural and biochemical analysis identified novel binding modes for several of the inhibitors. Selective inhibitors of FphH impaired both bacterial growth and biofilm formation while Inhibitors of FphB and FphE had no impact on cell growth and only limited impact on biofilm formation. These results suggest that all three hydrolases likely play functional, but non-equivalent roles in biofilm formation and FphH is a potential target for development of therapeutics that have both antibiotic and anti-biofilm activity. Overall, we demonstrate that focused covalent fragment screening can be used to rapidly identify highly potent and selective electrophiles targeting bacterial serine hydrolases. This approach could be applied to other classes of lipid hydrolases in diverse pathogens or higher eukaryotes.

  View details for DOI 10.21203/rs.3.rs-5494070/v1

  View details for PubMedID 39711551

  View details for PubMedCentralID PMC11661381

• An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor. bioRxiv : the preprint server for biology Wang, S., Woods, E. C., Jo, J., Zhu, J., Hansel-Harris, A., Holcomb, M., Pedowitz, N. J., Upadhyay, T., Bennett, J., Fellner, M., Park, K. W., Zhang, A., Valdez, T. A., Forli, S., Chan, A. I., Cunningham, C. N., Bogyo, M. 2024

  Abstract

  Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

  View details for DOI 10.1101/2024.11.06.622387

  View details for PubMedID 39574702

  View details for PubMedCentralID PMC11581011

• Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections. Journal of the American Chemical Society Jo, J., Upadhyay, T., Woods, E. C., Park, K. W., Pedowitz, N. J., Jaworek-Korjakowska, J., Wang, S., Valdez, T. A., Fellner, M., Bogyo, M. 2024

  Abstract

  Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.

  View details for DOI 10.1021/jacs.3c13974

  View details for PubMedID 38411555