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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Tulane virus protease as a structural surrogate for inhibitor screening of human norovirus proteases

Human norovirus (HuNoV) is a significant cause of gastroenteritis worldwide, affecting people of all age groups. There are currently no vaccines or drugs available, leaving susceptible populations vulnerable to severe or protracted illness. A HuNoV cultivation system is pivotal for screening norovirus antivirals. While the human intestinal enteroid cultivation system allows robust replication of multiple HuNoV strains, it presents technical and cost barriers. Tulane virus (TV), a surrogate for HuNoV, replicates well in monkey kidney cell lines and is closely related to norovirus in cellular biology. Here, we determined the structures of TV protease (TV-Pro) alone and in complex with rupintrivir, a picornavirus inhibitor that also inhibits HuNoV proteases (HuNoV-Pro). Our data validate TV as an efficient surrogate system for rapid screening of HuNoV protease inhibitors. The TV protease structure exhibits significant backbone similarity to the GI.1 HuNoV protease in the substrate-binding domain, with the BII-CII loop in an open conformation stabilized by hydrogen bonds as present in the GI.1 protease. Structural differences in the S2 pocket and two amino acid changes in the S4 pocket result in slightly altered P2 and P4 substrate and inhibitor conformations. Despite these differences, we confirm previous findings that the TV protease can cleave the GI.1 and GII HuNoV polyprotein substrates with high and moderate efficiency, respectively. We found that rupintrivir efficiently inhibits TV protease in vitro and inhibits TV replication in cell culture with similar efficacy in combination with P-glycoprotein efflux pump inhibitors. We conclude that TV is a valuable surrogate for HuNoV protease inhibitor screening and outline strategies to improve its compatibility as such.

crystal structures↗
DOE OSTI2025

Potent HIV‑1 protease inhibitors containing oxabicyclo octanol-derived P2-ligands: Design, synthesis, and X‑ray structural studies of inhibitor-HIV-1 protease complexes

Here, we describe here the design, synthesis, and X-ray structural studies of a new class of HIV-1 protease inhibitors containing 8-oxabicyclo[3.2.1]octanol-derived P2 ligands. We investigated the functional effect of these stereochemically defined fused-poly cyclic ligands on enzyme inhibition and antiviral activity in MT-2 cells. The tricyclic core of 8-oxabicyclo[3.2.1]octan-6-ol is designed to interact with the residues in the S2 subsite of HIV-1 protease. The syntheses of the ligands were carried out using the [5+2]-cycloaddition as the key step. Several inhibitors exhibited potent enzyme inhibitory activity. High resolution room-temperature X-ray structures of inhibitor-bound HIV-1 protease were determined. These structures provided important molecular insights for further design and optimization of inhibitor potency.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

GII.4 Norovirus Protease Shows pH-Sensitive Proteolysis with a Unique Arg-His Pairing in the Catalytic Site

Human noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, in this work we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substrate-binding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pH-dependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs. IMPORTANCE: Human noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Conformational flexibility is a critical factor in designing broad-spectrum human norovirus protease inhibitors

Human norovirus (HuNoV) is a leading cause of gastroenteritis worldwide and is associated with significant morbidity, mortality, and economic impact. There are currently no licensed antiviral drugs for the treatment of HuNoV-associated gastroenteritis. The HuNoV protease plays a critical role in the initiation of virus replication by cleaving the polyprotein. Thus, it is an ideal target for developing antiviral small-molecule inhibitors. While rupintrivir, a potent small-molecule inhibitor of several picornavirus proteases, effectively inhibits GI.1 protease, it is an order of magnitude less effective against GII protease. Other GI.1 protease inhibitors also tend to be less effective against GII proteases. To understand the structural basis for the potency difference, we determined the crystal structures of proteases of GI.1, pandemic GII.4 (Houston and Sydney), and GII.3 in complex with rupintrivir. These structures show that the open substrate pocket in GI protease binds rupintrivir without requiring significant conformational changes, whereas, in GII proteases, the closed pocket flexibly extends, reorienting arginine-112 in the BII-CII loop to accommodate rupintrivir. Structures of R112A protease mutants with rupintrivir, coupled with enzymatic and inhibition studies, suggest R112 is involved in displacing both substrate and ligands from the active site, implying a role in the release of cleaved products during polyprotein processing. Thus, the primary determinant for differential inhibitor potency between the GI and GII proteases is the increased flexibility in the BII-CII loop of the GII proteases caused by the H-G mutation in this loop. Therefore, the inherent flexibility of the BII-CII loop in GII proteases is a critical factor to consider when developing broad-spectrum inhibitors for HuNoV proteases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Dual Inhibitors of SARS-CoV-2 3CL Protease and Human Cathepsin L Containing Glutamine Isosteres Are Anti-CoV-2 Agents

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 P 2 position of a peptidomimetic inhibitor. At the P 1 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 P 1 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 K i = 0.6–18 nM (cathepsin L) and K i = 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 EC 50 = 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 Cys 145 , 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 P 3 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.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Characterization of molecular interactions between Zika virus protease and peptides derived from the C-terminus of NS2B

Highlights: • The TGKR residues in eZiPro protease are dynamic in solution. • Peptides derived from NS2B C-terminus exhibit different binding affinities to protease. • Free NS2B cofactor region is flexible in solution. • The eZiPro construct binds to peptides and a fragment. Zika virus (ZIKV) protease is a two-component complex in which NS3 contains the catalytic triad and NS2B cofactor region is important for protease folding and activity. A protease construct-eZiPro without the transmembrane domains of NS2B was designed. Structural study on eZiPro reveals that the Thr-Gly-Lys-Arg (TGKR) sequence at the C-terminus of NS2B binds to the active site after cleavage. The bZiPro construct only contains NS2B cofactor region and the N-terminus of NS3 without any artificial linker or protease cleavage site, giving rise to an empty pocket accessible to substrate and inhibitor binding. Herein, we demonstrate that the TGKR sequence of NS2B in eZiPro is dynamic. Peptides from NS2B with various lengths exhibit different binding affinities to bZiPro. TGKR binding to the active site in eZiPro does not affect protease binding to small-molecule compounds. Our results suggest that eZiPro will also be useful for evaluating small-molecule protease inhibitors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Mutant cytoskeletal and ECM peptides sensitive to the ST14 protease are associated with a worse outcome for glioblastoma multiforme

Highlights: • In GBM, mutant ECM proteins appear largely resistant to the ST14 protease. • Patients with resistant proteins have a better survival. • The few patients with sensitive mutants have T-cell infiltrates. We previously identified a set of the most frequently mutated cytoskeleton- and extracellular matrix-related proteins (CECMPs) in numerous cancer datasets. In this report, we used a bioinformatics approach to assess the impact of amino acid (AA) substitutions on the sensitivity of CECMPs to the ST14 protease (matriptase I), a transmembrane serine protease previously implicated in cancer development. Results indicated that AA substitutions in glioblastoma multiforme (GBM) CECMPs are skewed toward increased resistance to the ST14 protease, in comparison to the wild-type peptide sequence. Furthermore, the protease resistant AA substitutions represent relatively high binding affinities to HLA class I proteins, when assessing the binding specificities using HLA class I alleles matched to the source of the mutant AA. Moreover, samples representing AA substitutions that increased protease sensitivity also represented reduced overall and disease-free survival periods for patients with glioblastoma. To assess tumor specimen immunogenicity, we identified T-cell receptor (TCR) V(D)J recombinations in GBM exome files. The overlap between ST14 protease sensitive mutant barcodes and the TCR V(D)J recombination read positive barcodes represented significantly reduced survival.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Effect of foot-and-mouth disease virus 3C protease B 2 β-strand proline mutagenesis on expression and processing of the P1 polypeptide using a plasmid expression vector

The production of experimental molecular vaccines against foot-and-mouth disease virus utilizes the viral encoded 3C protease for processing of the P1 polyprotein. Expression of wild type 3C protease is detrimental to host cells. The molecular vaccine constructs containing the 3C protease L127P mutant significantly reduce adverse effects associated with protease expression while retaining the ability to process and assemble virus-like particles. In published 3C protease crystal structures, the L127 residue is contained within the B 2 β-strand as part of the A 2 –B 2 β-sheet. To provide insight into the mechanism by which the L127P mutant alters the properties of the 3C protease, we performed scanning proline mutagenesis of residues 123–128 of the B 2 β-strand and monitored expression and P1 processing. Simultaneously, we utilized random mutagenesis of the full 3C sequence to identify additional mutations presenting a phenotype similar to the L127P mutation. Six of the tested mutants enhanced expression over wild type, and the I22P, T100P and V124P mutations surpassed the L127P mutation in certain cell lines. These data are interpreted in conjunction with published 3C protease crystal structures to provide insight into the mechanism by which these mutations enhance expression.

3C↗