Biology subjects

Upadhyay, T.

Publications and source records attributed to Upadhyay, T..

9 recordsLinked to original sources

bioRxiv2026

A covalent irreversible inhibitor binds in two mutually exclusive conformations to the active-site cysteine residue of human aldehyde dehydrogenase 1A3

Aldehyde dehydrogenases (ALDH) are enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes into carboxylic acids, playing roles in detoxification, biosynthesis, and regulatory functions. Dysfunction of ALDH is associated with serious conditions such as alcohol intolerance, cancer, cardiovascular problems, and neurological disorders. In humans, ALDH1A1 and ALDH1A3 isoforms act as retinaldehyde dehydrogenases and are overexpressed in various cancers, where high levels are associated with increased tumor malignancy, cancer stem cell traits, and therapeutic resistance. ALDH1A3 is recognized as a promising target for anticancer therapies, with several inhibitors, mainly reversible, developed to specifically target it or the enzyme family. Since ALDH enzymes can also display esterase activity, we used this property to develop an in vitro assay specifically targeting the esterase function of ALDH1A3. A highly conserved active-site cysteine in ALDH1A3 is located at the bottom of two converging channels, which define the substrate- and cofactor-binding pockets. To target this catalytic cysteine, we screened a library of 3,200 cysteine-focused covalent fragments. This led to the identification of Z3405279217 (Z34), an acrylamide-based covalent compound that inhibits both ALDH1A1 and ALDH1A3 at sub-micromolar levels. Biochemical and biophysical tests confirmed that Z34 acts as a time-dependent, covalent, and irreversible binder to the active-site cysteine. In this work, we determined the Cryo-EM structure of the ALDH1A3-Z34 complex at 2.26 [A] resolution, confirming the covalent attachment to the catalytic cysteine of Z34. Notably, two mutually exclusive covalent binding modes were observed: one occupying the substrate-binding pocket and the other the cofactor-binding region. Z34 displayed unexpected binding modes within the active site and holds promise as a lead compound for future drug development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/738401v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1d9ebd2org.highwire.dtl.DTLVardef@96470org.highwire.dtl.DTLVardef@a110c7org.highwire.dtl.DTLVardef@548a93_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Imaging of Staphylococcus aureus infections and biofilms using a selective covalent probe for the unique serine hydrolase FphE

Staphylococcus aureus is the leading cause of soft tissue infections that can be treated with antibiotics. However, it can also cause significant mortality and morbidity due to 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, non-invasive confirmation of S. aureus infections. We recently developed an activity-based probe containing an oxadiazolone electrophile that selectively labels the S. aureus-specific serine hydrolase, FphE, by covalent binding to its active site serine residue. 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. Furthermore, we demonstrate that JJ-OX-012 can be used for non-invasive fluorescent imaging as a way 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.

microbiology↗

bioRxiv2025

Unique structural and ligand binding properties of the Staphylococcus aureus serine hydrolase FphE

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 therefor 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, twelve 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 (SAXS) confirms the dimeric architecture occurs in solution. High-resolution co-crystal 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.

microbiology↗

bioRxiv2025

Coaxially Electrospun Myocardial dECM-based Nanofibrous Scaffolds Demonstrate Enhanced Cardiomyocyte Adhesion and Function

Abstract textLimited regenerative capability of mature cardiomyocytes (CMs) makes myocardial repair more challenging, requiring effective and viable alternatives to conventional heart transplants. Cardiac tissue engineering is a substitutionary approach combining cells, scaffolds, and growth factors to develop functional heart tissues in vitro. Induced pluripotent stem cells (iPSCs) represent a significant advancement in cardiac regenerative medicine, offering a continuous supply of CMs, however, the limited understanding of their microenvironment hinders translational research. Decellularized extracellular matrix (dECM) derived from myocardium is a highly promising natural scaffold for CTE, given its tissue-specific composition, mechanical properties, and biochemical cues that promote cellular regeneration. This study investigates myocardial dECM-based fibrous scaffolds for iPSC-derived CM use. Coaxially electrospun nanofibers comprising a polyurethane core and a blend of polycaprolactone and myocardial dECM as the sheath were optimized. Morphological analysis confirms the resemblance of the nanofibers to fibrillar collagen in the native dECM. ATR-FTIR and immunostaining results confirm the presence of dECM which enhanced their hydrophilicity and enzymatic degradation. Biocompatibility results show higher phenotypic retention of iPSC-CMs due to microenvironments enriched with native proteins. On the contrary, the scaffolds without myocardial proteins exhibit higher dedifferentiation of iPSC-CMs, proving that ECM proteins provide a suitable microenvironment for iPSC-CMs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/673432v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@31a497org.highwire.dtl.DTLVardef@12cee05org.highwire.dtl.DTLVardef@beca77org.highwire.dtl.DTLVardef@1de5337_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

A Robust Fluorogenic Substrate for Chikungunya Virus Protease (nsP2) Activity

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 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 20-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.

biochemistry↗

bioRxiv2024

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor

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.

microbiology↗

bioRxiv2024

Activity-Based Protein Profiling Identifies Klebsiella pneumoniae Serine Hydrolases with Potential Roles in Host-Pathogen Interactions

Klebsiella pneumoniae is a normal resident of the human gastro-intestinal tract and an opportunistic, critical priority pathogen that can cause a variety of severe systemic infections. Due to emerging multi-drug resistance of this pathogen, the discovery and validation of novel targets for the development of new treatment options is an urgent priority. Here, we explored the family of serine hydrolases, a highly druggable and functionally diverse enzyme family which is uncharacterized in K. pneumoniae. Using functionalized covalent fluorophosphonate inhibitors as activity-based probes we identified 10 serine hydrolases by mass spectrometry-based activity-based protein profiling, 7 of which were previously uncharacterized. Functional validation using transposon mutants deficient in either of the putative lysophospholipase PldB, esterase YjfP and patatin-like phospholipase YchK revealed severe growth defects in human colonic organoid co-culture models and reduced virulence during Galleria mellonella infection. Mutants deficient in the PldB and YjfP, but not YchK show increased susceptibility to killing by complement and the antimicrobial peptide antibiotic polymyxin B, suggesting a role in maintaining cell envelope integrity. Biochemical characterization and structural analysis of recombinant YjfP suggest this protein is a deacetylase. This study gives important insights into the molecular mechanisms underlying virulence and cell physiology of K. pneumoniae at the host-pathogen interface and it positions PldB, YjfP and YchK as potential antimicrobial or anti-virulence target candidates, inhibition of which might synergize with existing antibiotics and human immune defenses.

microbiology↗

bioRxiv2023

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of S. aureus Infections

Staphylococcus aureus is a major human pathogen 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 homologs 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 (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

microbiology↗

bioRxiv2020

A stutter in the coiled coil domain of E.coli co-chaperone GrpE connects structure with function

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 long non-ideal coiled-coil. During heat stress, this region partially unfolds and prevents DnaK nucleotide exchange, ultimately ceasing the chaperone cycle. In this study, we elucidate the role of thermal unfolding of the coiled-coil domain of E. coli GrpE in regulating its co-chaperonic activity. The presence of a stutter disrupts the regular heptad arrangement typically found in an ideal coiled coil resulting in structural distortion. Introduction of hydrophobic residues at the stutter altered the structural stability of the coiled-coil. Using an in vitro FRET assay, we show for the first time that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, the mutants were defective in in vitro functional assays, and were unable to support bacterial growth at heat shock temperature in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in the GrpE coiled-coil, and its role in regulating the DnaK-chaperone cycle for bacterial survival during heat stress. More generally, our findings illustrate how a sequence specific stutter in a coiled-coil domain regulates the structure function trade-off in proteins.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗