Jiyun Zhu

All Publications

• A caspase-1-cathepsin AND-gate probe for selective imaging of inflammasome activation. bioRxiv : the preprint server for biology Chen, S., Goncin, U., Zhu, J., Su, S. P., Czyzyk, T. A., Miller, C. O., Sadabad, R. K., Bogyo, M. 2025

  Abstract

  Caspase-1 is a key mediator of the inflammasome pathway, which is associated with several inflammatory disorders including obesity, diabetes mellitus (DM), cardiovascular diseases (CVDs), cancers and chronic respiratory diseases. Although substrate-based probes can be used to visualize the activity of caspase-1, none are selective enough for use as imaging agents. Here, we report the design and synthesis of a AND-gate substrate probe (Cas1-Cat-Cy7) that requires processing by both caspase-1 and cathepsins to produce a signal. Because both enzymes are only found together and active at the site of inflammasome activation, the resulting probe can be used to image caspase-1 mediated inflammation. We demonstrate that the probe produces selective signals in ex vivo biochemical and cellular assays and in a mouse model of acute inflammation.

  View details for DOI 10.1101/2025.05.27.656501

  View details for PubMedID 40501809

  View details for PubMedCentralID PMC12154666

• 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

• Dual Inhibitors of SARS-CoV-2 3CL Protease and Human Cathepsin L Containing Glutamine Isosteres Are Anti-CoV-2 Agents JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kumar, V., Zhu, J., Chenna, B. C., Hoffpauir, Z. A., Rademacher, A., Rogers, A. M., Tseng, C., Drelich, A., Farzandh, S., Lamb, A. L., Meek, T. D. 2025

  Abstract

  SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P2 position of a peptidomimetic inhibitor. At the P1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir, and PF-835321. Our inhibitors contain glutamine isosteres at the P1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from Ki = 0.6-18 nM (cathepsin L) and Ki = 2.6-124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC50 = 0.47-15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys145, and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.

  View details for DOI 10.1021/jacs.4c11620

  View details for Web of Science ID 001388636000001

  View details for PubMedID 39746101

• Catalytic Mechanism of SARS-CoV-2 3-Chymotrypsin-Like Protease as Determined by Steady-State and Pre-Steady-State Kinetics ACS CATALYSIS Zhu, J., Kemp, A. M., Chenna, B. C., Kumar, V., Rademacher, A., Yun, S., Laganowsky, A., Meek, T. D. 2024; 14 (24): 18292-18309

  Abstract

  The 3-chymotrypsin-like protease (3CL-PR; also known as Main protease) of SARS-CoV-2 is a cysteine protease that is the target of the COVID-19 drug, Paxlovid. Here, we report for 3CL-PR, the pH-rate profiles of a substrate, an inhibitor, affinity agents, and solvent kinetic isotope effects (sKIEs) obtained under both steady-state and pre-steady-state conditions. "Bell-shaped" plots of log(k cat/K a) vs pH for the substrate (Abz)SAVLQ*SGFRK(Dnp)-NH2 and pK i vs pH for a peptide aldehyde inhibitor demonstrated that essential acidic and basic groups of pK 2 = 8.2 ± 0.4 and pK 1 = 6.2 ± 0.3, respectively, are required for catalysis, and the pH-dependence of inactivation of 3CL-PR by iodoacetamide and diethylpyrocarbonate identified enzymatic groups of pK 2 = 7.8 ± 0.1 and pK 1 = 6.05 ± 0.07, which must be unprotonated for maximal inactivation. These data are most consistent with the presence of a neutral catalytic dyad (Cys-SH-His) in the 3CL-PR free enzyme, with respective pK values for the cysteine and histidine groups of pK 2 = 8.0 and pK 1 = 6.5. The steady-state sKIEs were D2O(k cat/K a) = 0.56 ± 0.05 and D2O k cat = 1.0 ± 0.1, and sKIEs indicated that the Cys-S--HisH+ tautomer was enriched in D2O. Presteady-state kinetic analysis of (Abz)SAVLQ*SGFRK(Dnp)-NH2 exhibited transient lags preceding steady-state rates, which were considerably faster in D2O than in H2O. The transient rates encompass steps that include substrate binding and acylation, and are faster in D2O wherein the more active Cys-S--HisH+ tautomer predominates. A full catalytic mechanism for 3CL-PR is proposed.

  View details for DOI 10.1021/acscatal.4c04695

  View details for Web of Science ID 001364997700001

  View details for PubMedID 39722883

  View details for PubMedCentralID PMC11667672

• 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

• Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases JOURNAL OF MEDICINAL CHEMISTRY Li, L., Chenna, B. C., Yang, K. S., Cole, T. R., Goodall, Z. T., Giardini, M., Moghadamchargari, Z., Hernandez, E. A., Gomez, J., Calvet, C. M., Bernatchez, J. A., Mellott, D. M., Zhu, J., Rademacher, A., Thomas, D., Blankenship, L. R., Drelich, A., Laganowsky, A., Tseng, C. K., Liu, W. R., Wand, A., Cruz-Reyes, J., Siqueira-Neto, J. L., Meek, T. D. 2021; 64 (15): 11267-11287

  Abstract

  Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

  View details for DOI 10.1021/acs.jmedchem.1c00628

  View details for Web of Science ID 000685644300042

  View details for PubMedID 34288674

  View details for PubMedCentralID PMC10504874

• Dinitrosyl iron complexes (DNICs) as inhibitors of the SARS-CoV-2 main protease CHEMICAL COMMUNICATIONS Pectol, D., DeLaney, C. R., Zhu, J., Mellott, D. M., Katzfuss, A., Taylor, Z. W., Meek, T. D., Darensbourg, M. Y. 2021; 57 (67): 8352-8355

  Abstract

  By repurposing DNICs designed for other medicinal purposes, the possibility of protease inhibition was investigated in silico using AutoDock 4.2.6 (AD4) and in vitro via a FRET protease assay. AD4 was validated as a predictive computational tool for coordinatively unsaturated DNIC binding using the only known crystal structure of a protein-bound DNIC, PDB- (calculation RMSD = 1.77). From the in silico data the dimeric DNICs TGTA-RRE, [(μ-S-TGTA)Fe(NO)2]2 (TGTA = 1-thio-β-d-glucose tetraacetate) and TG-RRE, [(μ-S-TG)Fe(NO)2]2 (TG = 1-thio-β-d-glucose) were identified as promising leads for inhibition via coordinative inhibition at Cys-145 of the SARS-CoV-2 Main Protease (SC2Mpro). In vitro studies indicate inhibition of protease activity upon DNIC treatment, with an IC50 of 38 ± 2 μM for TGTA-RRE and 33 ± 2 μM for TG-RRE. This study presents a simple computational method for predicting DNIC-protein interactions; the in vitro study is consistent with in silico leads.

  View details for DOI 10.1039/d1cc03103a

  View details for Web of Science ID 000679988200001

  View details for PubMedID 34337637