Background: Berberine (BBR) is a drug with multiple effects on cellular energy metabolism. The present study explored answers to the question of which CYP450 (Cytochrome P450) isoenzymes execute the phase-I transformation for BBR, and what are the bioactivities of its metabolites on energy pathways.Methods: BBR metabolites were detected using LC-MS/MS. Computer-assistant docking technology as well as bioassays with recombinant CYP450s were employed to identify CYP450 isoenzymes responsible for BBR phase-I transformation. Bioactivities of BBR metabolites in liver cells were examined with real time RT-PCR and kinase phosphorylation assay.Results: In rat experiments, 4 major metabolites of BBR, berberrubine (M1), thalifendine (M2), demethyleneberberine (M3) and jatrorrhizine (M4) were identified in rat's livers using LC-MS/MS (liquid chromatography-tandem mass spectrometry). In the cell-free transformation reactions, M2 and M3 were detectable after incubating BBR with rCYP450s or human liver microsomes; however, M1 and M4 were below detective level. CYP2D6 and CYP1A2 played a major role in transforming BBR into M2; CYP2D6, CYP1A2 and CYP3A4 were for M3 production. The hepatocyte culture showed that BBR was active in enhancing the expression of insulin receptor (InsR) and low-density-lipoprotein receptor (LDLR) mRNA, as well as in activating AMP-activated protein kinase (AMPK). BBR's metabolites, M1-M4, remained to be active in up-regulating InsR expression with a potency reduced by 50-70%; LDLR mRNA was increased only by M1 or M2 (but not M3 and M4) with an activity level 35% or 26% of that of BBR, respectively. Similarly, AMPK-α phosphorylation was enhanced by M1 and M2 only, with a degree less than that of BBR.Conclusions: Four major BBR metabolites (M1-M4) were identified after phase-I transformation in rat liver. Cell-free reactions showed that CYP2D6, CYP1A2 and CYP3A4 seemed to be the dominant CYP450 isoenzymes transforming BBR into its metabolites M2 and M3. BBR's metabolites remained to be active on BBR's targets (InsR, LDLR, and AMPK) but with reduced potency.

Using chemoproteomic techniques, we first identified EIF2AK2, eEF1A1, PRDX3 and VPS4B as direct targets of berberine (BBR) for its synergistically anti-inflammatory effects. Of them, BBR has the strongest affinity with EIF2AK2 via two ionic bonds, and regulates several key inflammatory pathways through EIF2AK2, indicating the dominant role of EIF2AK2. Also, BBR could subtly inhibit the dimerization of EIF2AK2, rather than its enzyme activity, to selectively modulate its downstream pathways including JNK, NF-κB, AKT and NLRP3, with an advantage of good safety profile. In EIF2AK2 gene knockdown mice, the inhibitory IL-1β, IL-6, IL-18 and TNF-α secretion of BBR was obviously attenuated, confirming an EIF2AK2-dependent anti-inflammatory efficacy. The results highlight the BBR's network mechanism on anti-inflammatory effects in which EIF2AK2 is a key target, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders.

Programmed death-ligand 1 (PD-L1) is a T-cell inhibitory checkpoint molecule that suppresses antitumor immunity. Anti-PD-L1 antibodies have shown remarkable promise in treating tumors, but the patient response rate is low. Therefore, small-molecule checkpoint inhibitors blocking PD-L1 function are urgently needed. Changes of protein expression and phosphorylation levels were determined by immunoblotting. The level of Membrane PD-L1 was examined by flow cytometer. Cytotoxicity of T cells and NK cells toward tumor cells were detected using LDH and cell index assays. Lysosome function was investigated by NAG assay. Changes in lysosomal-related genes were measured by RT-PCR. In vivo anti-NSCLC cancer effects were assessed using C57BL/6 mice bearing Lewis tumor xenografts. We identified SA-49 as a new regulator of PD-L1 expression from a series of novel aloperine derivatives. SA-49 decreased the expression of PD-L1 in NSCLC cells and enhanced the cytotoxicity of co-cultured T and NK cells toward tumor cells. Importantly, lysosomal pathway contributed to SA-49-mediated down-regulation of PD-L1. SA-49 increased the biogenesis of lysosome and promoted translocation of PD-L1 to lysosome for proteolysis, which was associated with nuclear translocation of MITF. SA-49-induced MITF translocation acted through activation of PKCα and subsequently suppression of GSK3β activity. Furthermore, SA-49 suppressed Lewis tumor xenograft growth by activating immune microenvironment in C57BL/6 mice. Our data demonstrate that SA-49 can be used to regulate PD-L1 in cancer cells and trigger its degradation by activating lysosome function.

To discover small-molecule cancer immunotherapy candidates through targeting Indoleamine 2,3-dioxygenase 1 (IDO1), twenty-five new berberine (BBR) derivatives defined with substituents on position 3 or 9 were synthesized and examined for repression of IFN-γ-induced IDO1 promoter activities. Structure-activity relationship (SAR) indicated that large volume groups at the 9-position might be beneficial for potency. Among them, compounds 2f, 2i, 2n, 2o and 8b exhibited increased activities, with inhibition rate of 71-90% compared with BBR. Their effects on IDO1 expression were further confirmed by protein level as well. Furthermore, compounds 2i and 2n exhibited anticancer activity by enhancing the specific lysis of NK cells to A549 through IDO1, but not cytotoxicity. Preliminary mechanism revealed that both of them inhibited IFN-γ-induced IDO1 expression through activating AMPK and subsequent inhibition of STAT1 phosphorylation. Therefore, compounds 2i and 2n have been selected as IDO1 modulators for small-molecule cancer immunotherapy for next investigation.