Improving endothelial cell junction integrity by diphenylmethanone derivatives at oxidative stress: A dual-action directly targeting caveolar caveolin-1.

Directly targeting caveolar caveolin-1 is a potential mechanism to regulate endothelial permeability, especially during oxidative stress, but little evidence on the topic limits therapeutics discoveries. In this study, we investigated the pharmacological effect of an antioxidant LM49 (5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanoe) and its five diphenylmethanone derivatives on endothelial permeability and establish two distinct mechanisms of action. Multiplex molecular assays with theoretical modeling indicate that diphenylmethanone molecules, including LM49, directly bind the caveolin-1 steric pocket of ASN53/ARG54, ILE49/ASP50, ILE18, LEU59, ASN60, GLU48 and ARG19 residues. They also indicated dynamic binding-affinity for diphenylmethanone derivatives. First, this molecular interaction at caveolin-1 pocket inhibits its phosphorylation at TYR14 residue in H2O2-injured endothelial cell. A positive correlation was established between diphenylmethanone derivative binding-affinity and caveolin-1 phosphorylation inhibition. Inhibition of caveolin-1 phosphorylation, however, was independent of the LM49-mediated variation of protein tyrosine kinase activity, suggesting a direct blockage of adenosine triphosphate substrate diffusion into cavelion-1 structure. Second, LM49 increases the expression of cellular adhesive and tight junction proteins, VE-cadherin and occludin, in H2O2-injured cell, in a dose dependent manner. A leakage assay of fluorescein isothiocyanate-labeled dextran 40 across cell monolayer suggested improvement in endothelial barrier integrity with diphenylmethanone treatments. Our results demonstrate a direct targeting effect of caveolin-1 on endothelial permeability, and should guide the diphenylmethanone therapy against oxidative stress-induced junction dysfunction, especially at caveolar membrane invagination.

A novel marine halophenol derivative attenuates lipopolysaccharide-induced inflammation in RAW264.7 cells via activating phosphoinositide 3-kinase/Akt pathway.

BACKGROUND: 2,4',5'-Trihydroxyl-5,2'-dibromo diphenylmethanone (LM49), a novel active halophenol derivative synthesized by our group from marine plants, exhibits strong anti-inflammatory activities. However, molecular machineries involved in its effect have not been fully identified. The study was aimed to investigate the anti-inflammatory effect of LM49 on lipopolysaccharide (LPS)-stimulated RAW264.7 cells and its underlying mechanism. METHODS: RAW264.7 cells were treated with LPS (10 µg/mL) and then exposed to different concentrations of LM49 (i.e., 5, 10, and 15 µM) for 24 h. Cytokine release in culture medium of RAW264.7 cells was measured by enzyme-linked immunosorbent assay (ELISA). Phagocytic capacity (FITC-dextran uptake) was determined by flow cytometry. The protein level of phosphoinositide 3-kinase (PI3K), AKT and p-AKT was measured by western blot analysis. RESULTS: Our findings revealed that LM49 reduced the production and mRNA levels of cytokines related to inflammation such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α), and increased the level of IL-10, an anti-inflammatory cytokine. In addition, LM49 decreased the production of nitric oxide and reactive oxygen species. Moreover, flow cytometry showed that LM49 significantly enhanced the phagocytic capacity (FITC-dextran uptake) of macrophages. The effects of LM49 were significantly inhibited by the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. In particular, LY294002 attenuated the phagocytic capacity of RAW264.7 cells induced by LM49 and prevented the effects on cytokines. CONCLUSION: These findings suggest that LM49 possesses anti-inflammatory activity on LPS-stimulated RAW264.7 cells, in which the PI3K/Akt pathway plays an essential role. LM49 may have clinical utility as an anti-inflammatory agent. In this study, we demonstrated that a halophenol derivative (LM49) could possess anti-inflammatory activity on LPS-stimulated RAW264.7 cells by reducing pro-inflammatory cytokines and enhancing the phagocytic capacity, in which the PI3K/Akt pathway plays an essential role. LM49 may have clinical utility as an anti-inflammatory agent.

Process Development and Synthesis of Process-Related Impurities of an Efficient Scale-Up Preparation of 5,2'-Dibromo-2,4',5'-Trihydroxy Diphenylmethanone as a New Acute Pyelonephritis Candidate Drug.

Based on a foregoing gram-scale laboratory process, an efficient scale-up preparation process of 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49-API), a new acute pyelonephritis candidate drug, was developed and validated aiming to reduce by-products and achieve better impurity profiles. Meanwhile, the polymorph of LM49-API and process-related impurities were also investigated. Ultimately, the optimal reaction conditions were verified by evaluating the impurity profiles and their formation during the synthesis. Six process-related impurities were synthesized and identified, being useful for the quality control of LM49-API. Its finalized preparation process was further validated at 329-410 g scale-up production in 53.4-57.1% overall yield with 99.95-99.98% high-performance liquid chromatography (HPLC) purity, and it is currently viable for commercial production. LM49-API-imC and LM49-API-imX were identified as the main single impurities in LM49-API, with the content controlled to be less than 0.03%.

5,2'-Dibromo-2,4,5-trihydroxydiphenylmethanone, a novel immunomodulator of T lymphocytes by regulating the CD4+ T cell subset balance via activating the mitogen-activated protein kinase pathway.

5,2'-Dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49) exerted therapeutic effects against rat acute pyelonephritis by regulating immune responses, especially affecting T lymphocytes. However, its underlying action mechanism remains unclear. T lymphocytes play an irreplaceable role in immune responses. Therefore, we sought to understand whether LM49 is an immunomodulator of T lymphocytes. The results showed that LM49 promoted T lymphocyte proliferation, increased the number of CD4+ T cells, and increased the CD4+/CD8+ T cell ratio. LM49 regulated the CD4+ T cell subset balance by increasing the production of CD4+IL-2+, CD4+IL-4+, and CD4+IL-10+, and reducing the production of CD4+IL-17+, without changing the production of interferon-γ. LM49 had a significant effect on the mRNA expression of the transcription factors T-bet, GATA3, Foxp3, and RORγt. Furthermore, LM49 raised the phospho (p)-extracellular signal-regulated protein kinase 1/2, p-p38, and p-c-Jun N-terminal kinase expression levels. T cell proliferation, and the production of CD4+IL-2+, CD4+IL-4+, and CD4+IL-10+ induced by LM49, were decreased by inhibitors of mitogen-activated protein kinases (MAPKs). These results revealed that LM49 possesses immunomodulatory activity on T lymphocytes, in which the MAPK pathway plays an essential role.

5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone attenuates LPS-induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction.

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (TDD), possess anti-atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD-treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS-induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose-dependently inhibited the production or expression of IL-6, IL-1ß, MCP-1, TNF-α, VCAM-1, ICAM-1 and E-selectin as well as ROS in LPS-stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti-inflammatory and anti-oxidative effects of TDD. Furthermore, TDD inhibited LPS-induced NF-κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS-induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox-sensitive NF-κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.

Therapeutic effects of 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49) in an experimental rat model of acute pyelonephritis by immunomodulation and anti-inflammation.

Antibiotics are still the primary therapy for acute pyelonephritis (APN); rarely, natural polyphenols are also used. LM49 is a novel marine bromophenol derivative displaying strong anti-inflammatory effects. We investigated the therapeutic efficacy of LM49 in an experimental rat model of APN. The model was established by injecting 0.5 mL Escherichia coli (ATCC 25922, 108 CFU/mL) into the urinary bladders of Sprague Dawley rats. This model showed increased kidney viscera indices and renal bacterial growth scores, as well as pathological changes in kidneys. We also performed a broth microdilution antimicrobial susceptibility test of the E. coli strain. Both norfloxacin and LM49 treatment reduced kidney viscera indices and decreased microbial counts in urine cultures and kidney homogenates in APN rats. However, in vitro experiments showed that LM49 did not directly inhibit bacteria. Rather, LM49 treatment inhibited inflammatory cell infiltration or abscess and improved tissue lesions in the renal medullary junction, renal pelvis, and calyx, and high-dose LM49 treatment inhibited the production of inflammatory interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in serum. CD4+ T cells were higher in the LM49 groups treated with high, medium, and low doses than in the model group, whereas only high-dose LM49 treatment increased the number of CD8+ T cells, as compared with that in the model group. However, LM49 treatment did not influence hematological parameters. Our results show that LM49 therapeutic effects in an APN animal model may be achieved by regulating immune responses and inhibiting inflammatory mediators, suggesting it as a candidate APN treatment.

Synthesis of Novel Nitrogen-Containing Heterocycle Bromophenols and Their Interaction with Keap1 Protein by Molecular Docking.

We previously reported 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanoe (LM49), a bromophenol analogue that shows strong protection from oxidative stress injury owing to its superior anti-inflammatory, antioxidant, and anti-apoptotic properties. A series of novel nitrogen-containing heterocycle bromophenols were herein synthesized by introducing substituted piperidine, piperazine, and imidazole to modify 2-position of the lead compound LM49. By further evaluating their cytoprotective activity against H2O2 induced injury in EA.hy926 cells, 14 target bromophenols showed moderate-to-potent activity with EC50 values in the range of 0.9-6.3 µM, which were stronger than that of quercetin (EC50: 18.0 µM), a positive reference compound. Of these, the most potent compound 22b is a piperazine bromophenol with an EC50 value of 0.9 µM equivalent to the LM49. Molecular docking studies were subsequently performed to deduce the affinity and binding mode of derived halophenols toward the Keap1 Kelch domain, the docking results exhibited that the small molecule 22b is well accommodated by the bound region of Keap1-Kelch and Nrf2 through stable hydrogen bonds and hydrophobic interaction, which contributed to the enhancement of affinity and stability between the ligand and receptor. The above facts suggest that 22b is a promising pharmacological candidate for further cardiovascular drug development. Moreover, the targeting Keap1-Nrf2 protein-protein interaction may be an emerging strategy for halophenols to selectively and effectively activate Nrf2 triggering downstream protective genes defending against injury.

Heme Oxygenase-1, a Key Enzyme for the Cytoprotective Actions of Halophenols by Upregulating Nrf2 Expression via Activating Erk1/2 and PI3K/Akt in EA.hy926 Cells.

Increasing evidence has demonstrated that heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, exhibiting cytoprotective, antioxidant, and anti-inflammatory abilities. Previously, we prepared a series of novel active halophenols possessing strong antioxidant activities in vitro and in vivo. In the present study, we demonstrated that these halophenols exhibited significant protective effects against H2O2-induced injury in EA.hy926 cells by inhibition of apoptosis and ROS and TNF-α production, as well as induction of the upregulation of HO-1, the magnitude of which correlated with their cytoprotective actions. Further experiments which aimed to determine the mechanistic basis of these actions indicated that the halophenols induced the activation of Nrf2, Erk1/2, and PI3K/Akt without obvious effects on the phosphorylation of p38, JNK, or the expression of PKC-δ. This was validated with the use of PD98059 and Wortmannin, specific inhibitors of Erk1/2 and PI3K, respectively. Overall, our study is the first to demonstrate that the cytoprotective actions of halophenols involve their antiapoptotic, antioxidant, and anti-inflammatory abilities, which are mediated by the upregulation of Nrf2-dependent HO-1 expression and reductions in ROS and TNF-α generation via the activation of Erk1/2 and PI3K/Akt in EA.hy926 cells. HO-1 may thus be an important potential target for further research into the cytoprotective actions of halophenols.

Synthesis and biological activity of 4-substituted benzoxazolone derivatives as a new class of sEH inhibitors with high anti-inflammatory activity in vivo.

A series of novel 4-substituted benzoxazolone derivatives was synthesized, characterized and evaluated as human soluble epoxide hydrolase (sEH) inhibitors and anti-inflammatory agents. Some compounds showed moderate sEH inhibitory activities in vitro, and two novel compounds, 3g and 4j, exhibited the highest activities with IC50 values of 1.72 and 1.07 µM, respectively. Structure-activity relationships (SARs) revealed that introduction of a lipophilic amino acid resulted in an obvious increase in the sEH inhibitory activity, especially for derivatives containing a phenyl (3d, IC(50) = 2.67 µM), pyrrolidine (3g, IC(50) = 1.72 µM), or sulfhydryl group (3e, IC(50)=3.02 µM). Several compounds (3a-3g) were tested in vivo using a xylene-induced ear edema mouse model. Three compounds (3d, 3f, and 3g) showed strong anti-inflammatory activities in vivo which were higher than that of Chlorzoxazone, a reference drug widely used in the clinic. Our investigation provided a novel type of sEH inhibitor and anti-inflammatory agent that may lead to the discovery of a potential candidate for clinical use.

Structure-activity relationship of halophenols as a new class of protein tyrosine kinase inhibitors.

A series of new benzophenone and diphenylmethane halophenol derivatives were prepared. Their structures were established based on (1)H NMR, (13)C NMR and HRMS data. All prepared compounds were screened for their in vitro protein tyrosine kinase (PTK) inhibitory activities. The effects of modification of the linker, functional groups and substituted positions at the phenyl ring on PTK inhibitory activity were investigated. Twelve halophenols showed significant PTK inhibitory activity. Among them, compounds 6c, 6d, 7d, 9d, 10d, 11d and 13d exhibited stronger activities than that of genistein, the positive reference compound. The results gave a relatively full and definite description of the structure-activity relationship and provided a foundation for further design and structure optimization of the halophenols.

Synthesis and in vitro protein tyrosine kinase inhibitory activity of furan-2-yl(phenyl)methanone derivatives.

A series of novel furan-2-yl(phenyl)methanone derivatives were synthesized, and their structures were established on the basis of ¹H-NMR, ¹³C-NMR and mass spectral data. All the prepared compounds were screened for their in vitro protein tyrosine kinase inhibitory activity and several new derivatives exhibited promising activity, which, in some cases, was identical to, or even better than that of genistein, a positive reference compound. The preliminary structure-activity relationships of these compounds were investigated and are discussed.

Synthesis and biological activity of flavanone derivatives.

A series of new flavanone derivatives of farrerol was synthesized by a convenient method. The in vitro anti-tumor activity of these compounds was evaluated against human Bel-7402, HL-60, BGC-823 and KB cell lines, the protein tyrosine kinase (PTK) inhibitor activity was also tested. Their cytoprotective activity was tested using hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells. Their in vitro anti-atherosclerosis activity was tested on vascular smooth muscle cells by the MTT method using tetrandrine as a positive contrast drug. The structures of all compounds synthesized were confirmed by 1H, 13C NMR and ESI-MS. Most of the compounds exhibited good pharmacological activity and the preliminary structure-activity relationships were described.