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PubMed2026

Molecular docking, molecular dynamics simulation, and enzyme inhibitory studies of vitamin K family members on aldose reductase.

Aldose reductase (AR) is a key enzyme in the polyol pathway and plays a major role in the progression of secondary complications of diabetes. Despite extensive efforts to develop natural and synthetic aldose reductase inhibitors (ARIs), most candidates have shown limited clinical efficacy, highlighting the need for more potent and selective inhibitors. In this study, we have systematically evaluated the inhibitory potential of vitamin K family members (vitamin K1, vitamin K2, and vitamin K3) using molecular docking, protein-ligand interaction analysis, molecular dynamics simulations, and enzyme kinetics. Docking analysis predicted that vitamin K2 has the highest binding affinity for AR. Subsequent molecular dynamics simulations revealed that both vitamin K1 and vitamin K2 formed stable complexes with the protein, exhibiting comparable RMSD (∼0.5 Å difference), similar RMSF profiles, and reduced radius of gyration, indicating compact and stable binding. Interaction analysis demonstrated that ligand binding is predominantly driven by hydrophobic interactions, with vitamin K2 forming a higher number of hydrophobic contacts, while vitamin K1 exhibited slightly more hydrogen bonding. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) results further supports stronger binding of vitamin K2 (-56 kcal/mol) compared to vitaminK1 (-51 kcal/mol). Consistent with these findings, enzyme kinetics showed a slightly lower Ki value for vitamin K2 than vitamin K1. In contrast, vitamin K3 failed to maintain stable binding and moved out of the active site during simulation. Overall, the study highlights that hydrophobic interaction-driven stabilization plays a key role in ligand binding, and identifies vitamin K1 and vitamin K2 as promising inhibitors against AR, with vitamin K2 exhibiting more favourable hydrophobic interactions and binding stability.

Aldose Reductase↗
PubMed2025

A functional enzymatic assay as potential readout for a clinical trial of a schistosomiasis vaccine.

An estimated 200 million people are currently infected with schistosomiasis and an additional 800 million reside in high transmission-risk areas in 78 endemic countries. In this report we describe a functional enzymatic assay based on the core calpain antigen (Sm-p80) of the schistosomiasis vaccine, SchistoShield®. A 44 kDa soluble variant of the core Sm-p80 antigen (B7), was assessed for its enzymatic activity using a fluorescent synthetic substrate. Inhibition of the B7 enzymatic activity by Sm-p80-specific antibodies obtained from pre-clinical trials in rodents, non-human primates as well as from participants of the human clinical trials was measured. The B7 enzyme activity followed a Michaelis-Menten-like kinetic behavior. Statistically significant inhibition of the B7 activity was observed by Sm-p80-specific antibodies produced by immunized mice, non-human primates and humans. This quantitative serological assay could be of value in assessing the effectiveness of the SchistoShield® vaccine in human trials in Africa.

Journal Article↗
PubMed2025

Noncompetitive Inhibition of DNA Polymerase β by a Nonnative Nucleotide.

Base excision repair (BER) is a DNA repair pathway responsible for protecting the genome against modified nucleotides. DNA polymerase β (Pol β) participates in this process by removing the remnants of a damaged nucleotide and filling in the resulting gap. Pol β is overexpressed in some cancers and is synthetic lethal in cells deficient in BRCA1/2, providing additional impetus for identifying inhibitors of this enzyme. We report noncovalent Pol β inhibitors that are nonnative nucleotides. The inhibitors were identified via a combination of structural and biochemical analysis, as well as serendipity, from an initial library of covalent inhibitor candidates in which diversity was introduced sequentially at the C3'- and C5-positions of pyrimidine nucleotides. The molecules are among the most potent Pol β inhibitors (Ki ≤ 70 nM) of the enzyme's polymerase and lyase activities. Kinetic analyses reveal that the molecules inhibit Pol β noncompetitively. Fluorescence anisotropy and kinetic experiments reveal that the more potent inhibitor binds in the lyase domain and does not prevent DNA binding. Neither the more potent noncompetitive inhibitor nor a neutral protide exhibits cytotoxic synergism with the DNA damaging agent methyl methanesulfonate in HeLa cells. Cell permeability experiments suggest that micromolar levels of the more potent noncompetitive inhibitor and corresponding protide are taken up by HeLa cells following 24 h incubation (25 μM). However, based upon a comparison with other molecules, it is possible that they are membrane bound. The molecules identified could be useful tools in biochemical studies and provide a starting point for creating new Pol β inhibitors that function in cells.

DNA Polymerase beta↗
PubMed2026

Dual plasmepsin IX and X inhibitors are refractory to development of resistance.

Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment, but emerging resistance threatens their efficacy. The potential for the development of drug resistance against plasmepsin X (PMX)-selective inhibitors and dual plasmepsin IX/X (PMIX/X) inhibitors was investigated in Plasmodium falciparum. A series of PMX-selective (WM4, WM76, WM92) and PMIX/X dual inhibitors (WM382, WM09, WM42) were characterised for potency against parasite growth and enzyme inhibition. In vitro selection experiments showed that all compounds had a high barrier to resistance, although parasites with reduced sensitivity to PMX‑selective inhibitors could still be selected. Resistance mechanisms involved pmx gene amplification and point mutations (D245N, S315P, S359P, I363L) that alter inhibitor binding. Recombinant expression and Michaelis-Menten kinetics demonstrated that these mutations impair drug binding whilst preserving PMX catalytic function. Reverse genetics confirmed that introducing these mutations into the pmx gene resulted in decreased potency of the inhibitors. In this study, resistance to the PMIX/X dual inhibitors evaluated here could not be selected, despite prolonged selection pressure. Antimalarial Resistome Barcoding (AReBar) assays confirmed the absence of pre-existing resistance to either inhibitor class. Critically, PMIX/X dual inhibitors maintained efficacy against parasites with decreased sensitivity to PMX-selective compounds. These findings demonstrate that dual PMIX/X inhibitors present a substantially higher barrier to resistance than PMX-selective inhibitors, informing antimalarial drug development strategies and highlighting dual-target inhibition as a promising approach to mitigate resistance risks.

Aspartic Acid Endopeptidases↗
PubMed2026

Inactivation of Aspergillus flavus spores by dielectric barrier discharge cold plasma: Kinetics, physiological properties and proteomic analysis.

A. flavus, as a pathogen, poses a grave threat to both human and livestock health, significantly influencing agricultural production as well. This study aimed to investigate the inactivation effect and mechanism of dielectric barrier discharge cold plasma (DBD-CP) on A. flavus spores. The results exhibited that DBD-CP effectively inactivated A. flavus spores by the Weibull + Tail model. Furthermore, the physiological and proteomic analysis revealed that DBD-CP destructed cell wall and membrane integrity, causing cellular protein leakage and increasing membrane penetration of ROS generated from DBD-CP. Although intracellular ROS was excessively accumulated, the protein levels and activities of SOD and CAT were decreased, indicating that intracellular redox homeostasis was disrupted by DBD-CP. Subsequently, DBD-CP treatment induced cellular protein oxidation and changed protein structures, resulting in unstable protein structures. Meanwhile, protein synthesis and degradation in A. flavus spores were disturbed by inhibiting ribosome biogenesis, initiation process and NEDD8-mediated UPS, which did not compensate for the loss of protein caused by oxidative damage and leakage, leading to A. flavus spore inactivation. Besides, DBD-CP could attenuate A. flavus virulence by downregulating hydrolytic enzymes and CFEM-related proteins. This study provides novel insight into the inactivation mechanism of DBD-CP against A. flavus spores, which establishes a basis for the application of DBD-CP in controlling pathogenic fungi contamination in grains and crops, promoting the development of DBD-CP in food and agricultural decontamination.

Spores, Fungal↗
PubMed2024

Polymorphic positions 349 and 725 of the autoimmunity-protective allotype 10 of ER aminopeptidase 1 are key in determining its unique enzymatic properties.

INTRODUCTION: ER aminopeptidase 1 (ERAP1) is a polymorphic intracellular aminopeptidase with key roles in antigen presentation and adaptive immune responses. ERAP1 allotype 10 is highly protective toward developing some forms of autoimmunity and displays unusual functional properties, including very low activity versus some substrates. METHODS: To understand the molecular mechanisms that underlie the biology of allotype 10, we studied its enzymatic and biophysical properties focusing on its unique polymorphisms V349M and Q725R. RESULTS: Compared to ancestral allotype 1, allotype 10 is much less effective in trimming small substrates but presents allosteric kinetics that ameliorate activity differences at high substrate concentrations. Furthermore, it is inhibited by a transition-state analogue via a non-competitive mechanism and is much less responsive to an allosteric small-molecule modulator. It also presents opposite enthalpy, entropy, and heat capacity of activation compared to allotype 1, and its catalytic rate is highly dependent on viscosity. Polymorphisms V349M and Q725R significantly contribute to the lower enzymatic activity of allotype 10 for small substrates, especially at high substrate concentrations, influence the cooperation between the regulatory and active sites, and regulate viscosity dependence, likely by limiting product release. CONCLUSIONS: Overall, our results suggest that allotype 10 is not just an inactive variant of ERAP1 but rather carries distinct enzymatic properties that largely stem from changes at positions 349 and 725. These changes affect kinetic and thermodynamic parameters that likely control rate-limiting steps in the catalytic cycle, resulting in an enzyme optimized for sparing small substrates and contributing to the homeostasis of antigenic epitopes in the ER.

Aminopeptidases↗
PubMed2026

Emergence of ceftazidime-avibactam resistance mediated by KPC variants KPC-71 and KPC-78 in ST463 Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is a well-recognized opportunistic pathogen and a leading cause of healthcare-associated infections. The shrinking effectiveness of available antimicrobial therapies has intensified the global threat posed by carbapenem-resistant P. aeruginosa (CRPA). Here, we elucidate the mechanisms of ceftazidime-avibactam (CZA) resistance mediated by the rare KPC variants, KPC-71 and KPC-78, identified during the treatment of CRPA infections. Two CZA-resistant P. aeruginosa strains, SY-206885 and HZ-231016032, were isolated from critically ill male patients with severe pneumonia. Whole-genome sequencing assigned both isolates to the high-risk sequence type 463 (ST463). Isolate SY-206885 harbors the blaKPC-71 gene, while HZ-231016032 carries blaKPC-78. Cloning and expression of these genes in P. aeruginosa PAO1 conferred a marked increase in the CZA minimum inhibitory concentration. Notably, expression of KPC-71 or KPC-78 conferred CZA resistance while simultaneously reducing carbapenem hydrolytic activity, a trade-off previously described for some KPC variants but still rarely documented in P. aeruginosa. Structural analysis and kinetic profiling showed that, relative to wild-type KPC-2, both KPC-71 and KPC-78 exhibited reduced catalytic turnover but increased substrate affinity for ceftazidime, together with significantly weakened binding to avibactam. In addition, elevated expression of MexAB-OprM and AmpC-related determinants in the clinical isolates likely further enhanced the high-level CZA resistance phenotype. These findings highlight the capacity of the ST463 CRPA lineage to evolve CZA resistance through KPC structural diversification under antimicrobial pressure and underscore the need for close surveillance during therapy. IMPORTANCE: In this study, we report the detection of the uncommon KPC variants KPC-71 and KPC-78 in clinical sequence type 463 (ST463) carbapenem-resistant Pseudomonas aeruginosa isolates exhibiting resistance to ceftazidime-avibactam (CZA). We demonstrate that CZA resistance is driven by specific structural alterations-a serine insertion between residues 182 and 183 or a D179A substitution within the Ω-loop-that reshape the functional balance of the KPC enzyme. These changes appear to create an evolutionary trade-off by improving ceftazidime recognition while weakening avibactam-mediated inhibition. Given the widespread dissemination of the ST463 lineage in China, the emergence of these variants highlights the urgent need for clinicians to monitor for CZA resistance development during therapy. CLINICAL TRIALS: This study is registered with ClinicalTrials.gov as ChiCTR2500105846.

Ceftazidime↗