Dapeng Dai

Oxidative stress is a hallmark of aging and a key driver of neurodegenerative diseases. While much attention has been given to oxidative damage of macromolecules, the functional consequences of oxidizing the abundant nucleotide triphosphate (NTP) pool remain largely unknown. Here, we identify oxidized GTP (8-oxoGTP)—a major product of NTP oxidation—not as a mere marker of damage, but as an endogenous signaling molecule that drives microglial activation and neuroinflammation. 8-oxoGTP accumulated in the aging mouse brain, and its elevation was sufficient to induce synaptic dysfunction, tau hyperphosphorylation, neuronal loss, and cognitive decline. Mechanistically, 8-oxoGTP functioned as a potent activator of heterotrimeric G proteins, binding more strongly to Gα subunits than GTP itself, triggering Src kinase/MAPK/Akt/NF-κB signaling in microglia, leading to pro-inflammatory cytokine production, reactive oxygen species generation, and neurotoxicity. Pharmacological inhibition of Src family kinases rescued the neuroinflammatory and cognitive phenotypes induced by 8-oxoGTP. Our findings reveal a previously unrecognized mechanism linking nucleotide oxidation to age-associated neurodegenerative processes.

1. Pharmacotherapy remains a cornerstone of modern clinical practice, yet interindividual variability in drug response poses significant challenges to therapeutic efficacy and patient safety. Genet...

Large language models (LLMs) have demonstrated their limitations in addressing the design of active proteins that rely on intricate intramolecular interactions, particularly in the engineering of biocatalysts. Conducting real-world studies from targeted laboratory assays has become the de facto standard for artificial intelligence (AI) research in complex biological tasks. In this study, we present a standardized strategy using function-targeted models to decode the subtle effect of sequence variations on the function. Unlike affinity-oriented protein-protein interaction studies using LLMs, our model targets the specific functional interpretation, thereby guiding enzyme evolution. We established the VERnet model using deep mutation scanning data that underwent self-distillation, achieving an optimal accuracy of 93.5% for interpreting CYP2C9 variants. Through directed evolution at conserved positions enhanced by generative AI, we identified multiple CYP2C9 variants exhibiting a broad range of functional alterations. Additionally, a fine-tuned model optimized by AlphaFold3 significantly improved the prediction of variants involving the substitution of two amino acids. Molecular dynamics simulations revealed the structural and dynamic features of the catalytic alterations in evolved variants. The in vitro validation of metabolic activity strongly corroborated the in silico predictions, highlighting the substantial potential of AI models in predicting functional evolution.

Tofacitinib is a widely used medication for the treatment of arthritis. It has been reported that some patients experience abnormal cholesterol levels following treatment, leading to recommendations for the coadministration of lipid‐lowering drugs such as statins. In this study, we investigated the potential drug–drug interactions between tofacitinib and the statins simvastatin and lovastatin. In the in vitro experiments, rat liver microsomes were employed to evaluate the inhibitory effects of lipid‐lowering agents on the metabolism of tofacitinib, with the primary metabolite M8 analyzed using ultra‐performance liquid chromatography–tandem mass spectrometry. The results showed that simvastatin and lovastatin significantly inhibited the metabolism of tofacitinib, with IC₅₀ values of 5.837 and 10.68 μM, respectively. For the in vivo pharmacokinetic studies, Sprague–Dawley rats were pretreated with simvastatin or lovastatin via oral gavage for 7 days before the oral gavage of tofacitinib. This pretreatment led to an increased area under the concentration–time curve of tofacitinib, suggesting that a potential reduction in the first metabolism and/or systemic clearance takes place. These findings demonstrate significant interactions between tofacitinib and certain lipid‐lowering agents in the rat model, particularly simvastatin and lovastatin, both in vitro and in vivo.

Cytochrome P450 3A4 (CYP3A4), a key enzyme, is pivotal in metabolizing approximately half of the drugs used clinically. The genetic polymorphism of the CYP3A4 gene significantly influences individual variations in drug metabolism, potentially leading to the severe adverse drug reactions (ADRs). In this study, we conducted a genetic analysis on CYP3A4 gene in 1,163 Chinese Han individuals to identify the genetic variations that might affect their drug metabolism capabilities. For this purpose, a multiplex PCR amplicon sequencing technique was developed, enabling us to perform the genotyping of CYP3A4 gene efficiently and economically on a large scale. As a result, a total of 14 CYP3A4 allelic variants were identified, comprising 6 previously reported alleles and 8 new non-synonymous variants that were nominated as new allelic variants *39-*46 by the PharmVar Association. Further, functional assessments of these novel CYP3A4 variants were undertaken by co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis indicated that with the exception of CYP3A4.40 and CYP3A4.45, the protein expression levels of most new variants were similar to that of the wild-type CYP3A4.1 in yeast cells. To evaluate their catalytic activities, midazolam was utilized as a probe drug. The results showed that variant CYP3A4.45 had almost no catalytic activity, while the other variants exhibited significantly reduced drug metabolism abilities. This suggests that the majority of the CYP3A4 variants identified in the Chinese population possess markedly altered capacities for drug metabolism. Significance Statement In this study, we established a multiplex PCR amplicon sequencing method and detected the maximum number of new CYP3A4 variants in a single ethnic population. Additionally, we performed the functional characterizations of these 8 novel CYP3A4 allele variants in vitro. This study not only contributes to the understanding of CYP3A4 genetic polymorphism in the Chinese Han population but also holds substantial reference value for their potential clinical applications in personalized medicine.

Macitentan was approved by the United States Food and Drug Administration (FDA) in 2013 for the treatment of pulmonary arterial hypertension (PAH). Bergapten is a furanocoumarin that is abundant in Umbelliferae and Rutaceae plants and is widely used in many Chinese medicine prescriptions. Considering the possible combination of these two compounds, this study is aimed to investigate the effects of bergapten on the pharmacokinetics of macitentan both in vitro and in vivo. Rat liver microsomes (RLMs), human liver microsomes (HLMs), and recombinant human CYP3A4 (rCYP3A4) were used to investigate the inhibitory effects and mechanisms of bergapten on macitentan in vitro. In addition, pharmacokinetic parameters were also studied in vivo. Rats were randomly divided into two groups (six rats per group), with or without bergapten (10 mg/kg), and pretreated for 7 days. An oral dose of 20 mg/kg macitentan was administered to each group 30 min after bergapten or 0.5% CMC-Na administration on day 7. Blood was collected from the tail veins, and the plasma concentrations of macitentan and its metabolites were assessed by ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS). Finally, we analyzed the binding force of the enzyme and two small ligands by in silico molecular docking to verify the inhibitory effects of bergapten on macitentan. The in vitro results revealed that the IC₅₀ values for RLMs, HLMs, and rCYP3A4 were 3.84, 17.82 and 12.81 μM, respectively. In vivo pharmacokinetic experiments showed that the AUC_(0-t), AUC_(0-∞), and C_max of macitentan in the experimental group (20,263.67 μg/L*h, 20,378.31 μg/L*h and 2,999.69 μg/L, respectively) increased significantly compared with the control group (7,873.97 μg/L*h, 7,897.83 μg/L*h and 1,339.44 μg/L, respectively), while the CL_z/F (1.07 L/h/kg) of macitentan and the metabolite-parent ratio (MR) displayed a significant decrease. Bergapten competitively inhibited macitentan metabolism in vitro and altered its pharmacokinetic characteristics in vivo. Further molecular docking analysis was also consistent with the experimental results. This study provides a reference for the combined use of bergapten and macitentan in clinical practice.

Genetic polymorphism of the cytochrome P450 (CYP) gene can significantly influence the metabolism of endogenous and xenobiotic compounds. However, few studies have focused on the polymorphism of CYP2J2 and its impact on drug catalytic activity, especially in the Chinese Han population. In this study, we sequenced the promoter and exon regions of CYP2J2 in 1,163 unrelated healthy Chinese Han individuals using the multiplex PCR amplicon sequencing method. Then, the catalytic activities of the detected CYP2J2 variants were evaluated after recombinant expression in S. cerevisiae microsomes. As a result, CYP2J2*7, CYP2J2*8, 13 variations in the promoter region and 15 CYP2J2 nonsynonymous variants were detected, of which V15A, G24R, V68A, L166F and A391T were novel missense variations. Immunoblotting results showed that 11 of 15 CYP2J2 variants exhibited lower protein expression than wild-type CYP2J2.1. In vitro functional analysis results revealed that the amino acid changes of 14 variants could significantly influence the drug metabolic activity of CYP2J2 toward ebastine or terfenadine. Specifically, 4 variants with relatively higher allele frequencies, CYP2J2.8, 173_173del, K267fs and R446W, exhibited extremely low protein expression and defective catalytic activities for both substrates. Our results indicated that a high genetic polymorphism of CYP2J2 could be detected in the Chinese Han population, and most genetic variations in CYP2J2 could influence the expression and catalytic activity of CYP2J2. Our data significantly enrich the knowledge of genetic polymorphisms in CYP2J2 and provide new theoretical information for corresponding individualized medication in Chinese and other Asian populations.

Background: As a pan-HER tyrosine kinase inhibitor with a promising application prospect, poziotinib is likely to be coadministered with Schisandrins in clinical treatment due to its anticancer activities. Methods: Eighteen Sprague-Dawley rats were randomly divided into three groups: Schisandrin A group and Schisandrin B group (20 mg/kg daily for 1 week), and control group (vehicle). On day 8, poziotinib (2 mg/kg) was administered by oral gavage 30 min later. An in vitro study was developed to identify the possible mechanisms of Schisandrins on poziotinib metabolism. All analytes were detected by UPLC/MS-MS, and molecular docking was performed by AutoDock Tools. Results: When rats were preadministered with Schisandrin A, AUC((0-infinity)) and Cmax of poziotinib were obviously increased by 0.79- and 1.17-fold, whereas the Vz/F and CLz/F values were dramatically decreased. The results in Schisandrin B group presented similarly. Both Schisandrin A and Schisandrin B were mixed inhibitors of poziotinib in RLMs, and Schisandrin B showed stronger inhibitory activity with IC50 values of 2.55 mu M for M1 and 6.97 mu M for M2. Molecular docking analysis demonstrated that Schisandrin A and Schisandrin B exhibited a strong binding ability towards CYP2D6 as compared to CYP3A4. Conclusion: All results provided the direct evidence of the pharmacokinetic drug-drug interactions (DDIs) between Schisandrin and poziotinib. Thus, particular attention should be paid when poziotinib is taken together with Schisandrins in clinical practice.