Tulsi Upadhyay

All Publications

• A Small Molecule Substrate Mimic to Inhibit Bacterial Hsp70 Chaperone Function CHEMBIOCHEM Upadhyay, T., Kumari, R., Gangarde, Y. M., Potteth, U. S., Roychowdhury, R., Das, A., Saxena, D., Maitra, R., Chopra, S., Saraogi, I. 2026; 27 (17): e70518

  Abstract

  Given the rapid spread of antibiotic resistance worldwide, it is imperative to develop new strategies against pathogenic bacteria. Here, we report a small-molecule inhibitor of bacterial heat shock protein 70 (Hsp70), called DnaK, a central component of the bacterial protein chaperone network. Screening of an in-house pyridylamide-based peptidomimetic library identified M7 as an inhibitor of DnaK ATPase and substrate refolding activity with intrinsic fluorescence properties. Mechanistic studies revealed that M7 likely engages the substrate-binding pocket of DnaK. Treatment of bacterial cells with M7 led to the accumulation of σ-32, without affecting protein translation, consistent with impaired DnaK function. Microscopy images of Escherichia coli AS19 cells treated with M7 revealed filamentous growth, a phenotypic defect consistent with compromised DnaK function. Moreover, M7 effectively inhibited the growth of E. coli AS19 and some Gram-positive bacteria, including Staphylococcus aureus and Bacillus cereus. These findings highlight a promising class of peptidomimetic DnaK inhibitors as potential antibacterial agents, as well as a chemical biology tool to interrogate the broader network of chaperone-mediated protein folding.

  View details for DOI 10.1002/cbic.70518

  View details for Web of Science ID 001867085900001

  View details for PubMedID 42695548

  View details for PubMedCentralID PMC13543761

• Imaging of Staphylococcus aureus Infections and Biofilms Using a Selective Covalent Probe for the Unique Serine Hydrolase FphE. Angewandte Chemie (International ed. in English) Woods, E. C., Upadhyay, T., Park, K. W., Su, S. P., Xiao, Z., Rao, J., Valdez, T. A., Jo, J., Bogyo, M. 2026: e9575966

  Abstract

  Staphylococcus aureus is the leading cause of soft tissue infections which can often be treated with antibiotics. However, it can also cause significant mortality and morbidity from systemic infections and infections of surgical implants. Implant infections typically require invasive surgery, and treatment often necessitates removal of the implant because S. aureus biofilms are extremely difficult to eradicate with antibiotic treatment alone. Therefore, there is a significant need for improved diagnostic tools for rapid, noninvasive confirmation of S. aureus infections. We recently developed an activity-based probe containing an oxadiazolone electrophile that selectively and covalently labels the S. aureus-specific serine hydrolase, FphE. Here we describe a Cy5-labeled version of the probe, JJ-OX-012, and its characterization as an imaging agent for detecting biofilms both in vitro and in vivo. The probe labeled S. aureus biofilms in vitro, with virtually no background labeling of bacteria that lack FphE expression, such as Escherichia coli. Furthermore, using a mouse surgical implant infection model, we demonstrate that JJ-OX-012 can be used for noninvasive fluorescent imaging to detect S. aureus biofilms in vivo. Overall, these findings support the potential for using covalent probes targeting FphE as imaging agents for rapid detection and diagnosis of staphylococcal infections in vivo.

  View details for DOI 10.1002/anie.9575966

  View details for PubMedID 42057603

• Unique structural and ligand-binding properties of the Staphylococcus aureus serine hydrolase FphE. Proceedings of the National Academy of Sciences of the United States of America Jo, J., Upadhyay, T., You, X., Bennett, J. M., Lee, H., Bogyo, M., Fellner, M. 2026; 123 (13): e2532683123

  Abstract

  Staphylococcus aureus is a human pathogen capable of forming biofilms that complicate treatment and facilitate chronic infections. A family of S. aureus serine hydrolases are important regulators of virulence and biofilm formation. Among these, FphE is highly specific to S. aureus and therefore a viable target for both imaging and therapy. Here, we present bioinformatic and structural evidence that FphE may be involved in aromatic compound metabolism. In addition, 12 distinct crystal forms reveal that FphE exists as a highly unusual but stable and flexible, cross-subunit homodimer, unique to the large alpha/beta hydrolase superfamily. Substrate engagement favors retention of the dimeric state, which is a more catalytically active form of the enzyme, and small-angle X-ray scattering confirms that the dimeric architecture occurs in solution. High-resolution cocrystal structures of FphE covalently bound to two chemically distinct ligands reveal different modes of active site engagement, supporting an atypical structural plasticity of the dimer interface. Together, these findings establish FphE as a structurally unique alpha/beta hydrolase and provide a foundation for structure-guided development of S. aureus-specific inhibitors and imaging probes.

  View details for DOI 10.1073/pnas.2532683123

  View details for PubMedID 41875159

• Identification of covalent inhibitors of Staphylococcus aureus serine hydrolases important for virulence and biofilm formation. Nature communications Upadhyay, T., Woods, E. C., Dela Ahator, S., Julin, K., Faucher, F. F., Uddin, M. J., Hollander, M. J., Pedowitz, N. J., Abegg, D., Hammond, I., Eke, I. E., Wang, S., Chen, S., Bennett, J. M., Jo, J., Lentz, C. S., Adibekian, A., Fellner, M., Bogyo, M. 2025; 16 (1): 5046

  Abstract

  Staphylococcus aureus is a leading cause of bacteria-associated mortality worldwide. New tools are needed to both image and treat this pathogen. We previously identified a group of S. aureus serine hydrolases (Fphs), which regulate aspects of virulence and lipid metabolism. However, due to high structural and functional similarities, it remains challenging to distinguish the specific roles of members of this family. Here, we apply a high-throughput screening approach using a library of covalent electrophiles to identify inhibitors for FphB, FphE, and FphH. We identify selective covalent inhibitors for each target without the need for extensive medicinal chemistry optimization. Structural and biochemical analysis identify novel binding modes for several of the inhibitors. Functional studies using the inhibitors suggest that all three hydrolases likely play distinct functional roles in biofilm formation and virulence. This approach has the potential to be applied to target hydrolases in other diverse pathogens or higher eukaryotes.

  View details for DOI 10.1038/s41467-025-60367-3

  View details for PubMedID 40447595

  View details for PubMedCentralID 9882747

• 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

• An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor. Journal of the American Chemical Society Wang, S., Woods, E. C., Jo, J., Zhu, J., Hansel-Harris, A., Holcomb, M., Llanos, 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. 2025

  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 toward the active site serine. 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 10.1021/jacs.4c15713

  View details for PubMedID 40013487

• 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

• Investigating the functional role of a buried interchain aromatic cluster in <i>Escherichia coli</i> GrpE dimer PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Upadhyay, T., Karekar, V. V., Potteth, U. S., Saraogi, I. 2023; 91 (1): 108-120

  Abstract

  Aromatic clusters in the core of proteins are often involved in imparting structural stability to proteins. However, their functional importance is not always clear. In this study, we investigate the thermosensing role of a phenylalanine cluster present in the GrpE homodimer. GrpE, which acts as a nucleotide exchange factor for the molecular chaperone DnaK, is well known for its thermosensing activity resulting from temperature-dependent structural changes that allow control of chaperone function. Using mutational analysis, we show that an interchain phenylalanine cluster in a four-helix bundle of the GrpE homodimer assists in the thermosensing ability of the co-chaperone. Substitution of aromatic residues with hydrophobic ones in the core of the four-helix bundle reduces the thermal stability of the bundle and that of a connected coiled-coil domain, which impacts thermosensing. Cell growth assays and SEM images of the mutants show filamentous growth of Escherichia coli cells at 42°C, which corroborates with the defect in thermosensing. Our work suggests that the interchain edge-to-face aromatic cluster is important for the propagation of the structural signal from the coiled-coil domain to the four-helical bundle of GrpE, thus facilitating GrpE-mediated thermosensing in bacteria.

  View details for DOI 10.1002/prot.26414

  View details for Web of Science ID 000848510600001

  View details for PubMedID 35988048

• Novel Antibacterial Targets in Protein Biogenesis Pathways CHEMBIOCHEM Potteth, U. S., Upadhyay, T., Saini, S., Saraogi, I. 2022; 23 (4): e202100459

  Abstract

  Antibiotic resistance has emerged as a global threat due to the ability of bacteria to quickly evolve in response to the selection pressure induced by anti-infective drugs. Thus, there is an urgent need to develop new antibiotics against resistant bacteria. In this review, we discuss pathways involving bacterial protein biogenesis as attractive antibacterial targets since many of them are essential for bacterial survival and virulence. We discuss the structural understanding of various components associated with bacterial protein biogenesis, which in turn can be utilized for rational antibiotic design. We highlight efforts made towards developing inhibitors of these pathways with insights into future possibilities and challenges. We also briefly discuss other potential targets related to protein biogenesis.

  View details for DOI 10.1002/cbic.202100459

  View details for Web of Science ID 000711024800001

  View details for PubMedID 34643994

• A Stutter in the Coiled-Coil Domain of <i>Escherichia coli</i> Co-chaperone GrpE Connects Structure with Function BIOCHEMISTRY Upadhyay, T., Potteth, U. S., Karekar, V. V., Saraogi, I. 2021; 60 (17): 1356-1367

  Abstract

  In bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a non-ideal coiled coil. Partial unfolding of this region during heat stress results in reduced nucleotide exchange and disrupts protein folding by DnaK. In this study, we elucidate the role of non-ideality in the coiled-coil domain of Escherichia coli GrpE in controlling its co-chaperone activity. The presence of a four-residue stutter introduces nonheptad periodicity in the GrpE coiled coil, resulting in global structural changes in GrpE and regulating its interaction with DnaK. Introduction of hydrophobic residues at the stutter core increased the structural stability of the protein. Using an in vitro FRET assay, we show that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, these mutants were unable to support bacterial growth at 42°C in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in GrpE in regulating the DnaK-chaperone cycle during heat stress. More generally, our findings illustrate how stutters in a coiled-coil domain regulate structure-function trade-off in proteins.

  View details for DOI 10.1021/acs.biochem.1c00110

  View details for Web of Science ID 000648509400005

  View details for PubMedID 33881310

• Small-Molecule Inhibitor Prevents Insulin Fibrillogenesis and Preserves Activity MOLECULAR PHARMACEUTICS Das, A., Gangarde, Y. M., Tomar, V., Shinde, O., Upadhyay, T., Alam, S., Ghosh, S., Chaudhary, V., Saraogi, I. 2020; 17 (6): 1827-1834

  Abstract

  Amyloidosis is a well-known but poorly understood phenomenon caused by the aggregation of proteins, often leading to pathological conditions. For example, the aggregation of insulin poses significant challenges during the preparation of pharmaceutical insulin formulations commonly used to treat diabetic patients. Therefore, it is essential to develop inhibitors of insulin aggregation for potential biomedical applications and for important mechanistic insights into amyloidogenic pathways. Here, we have identified a small molecule M1, which causes a dose-dependent reduction in insulin fibril formation. Biophysical analyses and docking results suggest that M1 likely binds to partially unfolded insulin intermediates. Further, M1-treated insulin had lower cytotoxicity and remained functionally active in regulating cell proliferation in cultured Drosophila wing epithelium. Thus, M1 is of great interest as a novel agent for inhibiting insulin aggregation during biopharmaceutical manufacturing.

  View details for DOI 10.1021/acs.molpharmaceut.9b01080

  View details for Web of Science ID 000538337600005

  View details for PubMedID 32347728