Tianjin Institute of Pharmaceutical Research Co., Ltd.

Currently, the majorty of metabolomics studies rely on the use of a single analytical platform, either NMR or MS, which hampers the ability to conduct an unbiased and comprehensive analysis. However, the use of multiple platforms poses an additional challenge: the limited sample material. Consequently, it is highly desirable to develop comprehensive, effective and sample-conserving procedures for metabolite extraction. In this study, we presented pretreatment strategies enabling sequential NMR and multi-LC-MS (UHPLC-Q-Orbitrap MS, and UHPLC-QqQ MS) platform analysis using a single plasma and liver tissue sample. For the plasma sample, the biphasic CHCl₃/MeOH/H₂O method was suitable for polar and lipid extraction after NMR-based metabolomics analysis, in terms of the number of annotated metabolites, reproducibility, and the amount of sample needed. While for the liver sample, the two-step extraction involving CHCl₃/MeOH followed by MeOH/H₂O was recommended. In this method, resuspension of dried lipid extracts was used for lipidomics, and polar extracts were transferred for further untargeted metabolic profiles by UHPLC-Q-Orbitrap MS following an NMR-based metabolomics study. Finally, the proposed preparation protocols were evaluated for robustness, and the identification data were used to generate a comprehensive metabolic map for plasma and liver tissue. In summary, this study provides a unique sample preparation procedure for two biological specimens, allowing for multi-platform analysis using a single sample. By adopting this approach, comprehensive metabolic profiling can be conducted to detect metabolic alterations under different physiological or pathological conditions.

Yuanhuzhitong prescription (YZP), composed by Corydalis yanhusuo and Angelica dahurica, is mainly used to treat stomach pain, rib pain, headache. However, there is limited information on the effects of YZP on the activity of CYP450 enzymes, and studies on the compatibility mechanism of YZP in view of CYP-mediated herb-herb interactions are rarely conducted. This study, utilizing probe substrates, human liver microsomes, recombinant CYP metabolic enzymes, CYP inhibitors, and molecular docking, focuses on the metabolic stability and inhibitory activity of YZP components on P450 isozymes, and how imperatorin (IPT) and isoimperatorin (IIP) affect the elimination pathway of tetrahydropalmatine (THP) by inhibiting the activity of the CYP450 enzyme to enhance the analgesic efficacy of CYH. The results showed that THP shows weak inhibition of CYP2C9, CYP2D6, and 3A4; while IPT and IIP are potent inhibitors of CYP1A2, moderate inhibitors of CYP2C19, CYP2C9, CYP2D6, and 3A4. In addition, CYP2B6 was strongly inhibited by IPT and moderately inhibited by IIP. THP is primarily metabolized by CYP1A2, CYP2C9, CYP2D6, and CYP3A4. IPT and IIP undergo metabolism mainly through CYP1A2, CYP2B6, CYP2C19, and 3A4. Molecular docking studies confirmed that THP, IPT and IIP occupy the active sites of CYP1A2, CYP2C9, CYP2D6, and CYP3A4. In vitro and in vivo experiments have shown a significant reduction in metabolism of THP in combination with IPT or IIP. IPT and IIP mediate the metabolism of THP in the liver by inhibiting P450 enzymes, thereby enhancing the bioavailability of THP, reducing the elimination rate of THP, and consequently improving the analgesic efficacy.

A series of novel potent HIV-1 protease inhibitors featuring diverse hydroxyaromatic acetanilide derivatives as P2 ligands and 4-substituted phenyl sulfonamides as P2' ligands were designed, synthesized, and biologically evaluated. The majority of the target compounds demonstrated potent enzyme inhibitory activity with IC₅₀ values below100 nM. Notably, compound 18h, incorporating a 2-(4-hydroxypyrimidin-5-yl) acetamide P2 ligand and a 4-methoxybenzenesulfonamide as the P2' ligand, exhibited exceptional potency with an enzyme IC₅₀ of 0.46 nM and antiviral EC₅₀ of 0.26 μM against HIV-1_NL4-3 strain. In addition, 18h displayed activity with EC₅₀ value of 0.25 μM against the subtype C HIV-1 Indie strain. The extensive hydrogen-bonding interactions with the protease active site revealed in the molecular docking analysis of 18h-bound HIV-1 protease provided valuable structural insights for the rational design of next-generation HIV-1 protease inhibitors.