The indole ring, adamantane, and urea groups are important components of bioactive molecules. The orphan nuclear receptor Nur77 as a unique transcription factor encoded by an immediate early gene is a potential therapeutic target for cancer treatment. We synthesized a series of 1-(2-(adamantane-1-yl)-1H-indol-5-yl)-3-substituted urea/thiourea derivatives and identified which of these potential anticancer candidates could modulate the expression and activity of Nur77. The synthesized compounds were initially evaluated for their anti-proliferative activity against H460 lung cancer cells, HepG2 liver cancer cells, and MCF-7 breast cancer cells. Major compounds were found to be active against these tested cancer cell lines. The compounds with IC50 values down to 20 μM exhibited selective cytotoxicity effects on the human lung cancer cell line (H460) and the normal lung cell line (MCR-5). Compounds 7n, 7s, and 7w induced Nur77-expression in a time- and dose-dependent manner in H460 cells. Compounds 7n and 7s strongly induced Parp cleavage in H460 cells, but 7w resulted in a slight induction of apoptosis. The apoptotic effect of 7s was largely inhibited when the Nur77 was knocked down by shRNA. This indicated that Nur77 served as a critical mediator for the anticancer action of 7s. The molecular docking study between Nur77 and 7s revealed that compound 7s exhibited a promising binding affinity with Nur77. These findings will provide a direction for the developing Nur77 regulator as anticancer agents.

Human stimulator of interferon genes protein (hSTING) is an essential signaling adaptor and a cytosolic DNA sensor (CDS) that functions as a homodimer in innate immunity. Natural mutation V155M in hSTING can cause hSTING-associated vasculopathy with onset in infancy (SAVI) which is an autoinflammatory disease. However, the mechanisms between the variant and SAVI are poorly understood. To explore the mechanisms, we performed all-atom molecular dynamics (MD) simulations on the complexes of wild type (WT)/mutated (V155M) hSTING and endogenous agonist 2′3′-cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP) to investigate whether the interaction between hSTING and 2′3′-cGAMP could be affected by natural mutation V155M, which plays a crucial role in SAVI disease. Based on the MD simulations, the dynamic cross-correlation analysis and principal component analysis indicated that the single point mutation on residue 155 from valine to methionine changed some residues' motions of hSTING. Furthermore, MM/PBSA calculation, hydrogen bond analysis and energy decomposition analysis showed that this single point mutation increased the binding affinity of 2′3′-cGAMP with hSTING. Finally, residual network analysis indicated the way that the V155M mutation around 17 Å from the binding site controlled the strength of binding interaction and the conformation change of the substructure. These results will help in the understanding of the mechanism of SAVI disease induced by hSTING mutation at the molecular level, and provide new ideas to design innovative drugs targeted on hSTING.

Human peroxisome proliferator-activated receptor alpha (hPPARα) is a ligand-dependent transcription factor that mainly controls lipid metabolism in the liver. It has drawn wide attention as a significant target for developing new hypoglycaemic drugs. However, a central and largely unresolved question in finding new drugs targeted on hPPARα concerns ligand action mechanism: what makes certain molecules act as antagonists while others behave as agonists in the same binding site? To understand this, we performed a total of 600 ns all-atom molecular dynamics (MD) simulations to explore how four small molecule ligands bind to the hPPARα and play opposite effects. We characterized and compared the protein backbone fluctuation, and investigated the interaction networks and the movements of helixes and loops near binding site during MD simulations. Moreover, by free energy calculation and phylogenetic tree analysis, 11 key residues favouring binding ligands and some other residues playing important roles in inducing the active conformation changing of hPPARα were discovered. The results could help to understand the activation/deactivation of hPPARα by agonists or antagonists, and provide insightful prospective into hPPARα targeted structure-based drug designs.

•ESI-MSn, HRMS and H/D exchange were used.•The fragmentation pathways of NPAAE-BFA in ESI-MSn were described.•Fragment ions involved in phosphorus group’s rearrangement reactions were observed.•Two rearrangement mechanisms about phosphorylation–dephosphorylation were proposed.As mini-chemical models, amino acid ester isopropyl phosphoramidates of Brefeldin A (compounds 2a–2d) were synthesized and investigated by electrospray ionization tandem mass spectrometry in combination with H/D exchange. To further confirm the fragments’s structures, off-line Fourier transform resonance tandem mass spectrometry (FT-ICR-MS/MS) was also performed. The fragmentation rules of compounds 2a–2d have been summarized and the plausible schemes for the fragmentation pathways were proposed. In this study, one dephosphorylated ion and two phosphorylated ions were observed in ESI-MS2 spectra of [M + Na]+ ions for compounds 2a–2d. The possible mechanisms about phosphorylation and dephosphorylation were proposed and confirmed by H/D exchange. For the “dephosphorylation” rearrangement, a nitrogen atom was migrated from the phosphoryl group to the carbon atom of Brefeldin A’s backbone with losing a molecule of C3H7PO3 (122 Da). For the “phosphorylation” rearrangement, an oxygen atom of one phosphoryl group attacked the sideward phosphorus atom to form a nine-member ring intermediate, then two steps of CH covalent bond cleavage with consecutive migration of hydrogen atom to lose a molecule of C16H20O2 (244 Da). The two proposed rearrangement mechanisms about phosphoryl group transfer might be valuable for the structure analysis of other analogs and provide insights into elucidating the dynamic process of the phosphorylation–dephosphorylation of proteins.

•2D flow programming CCC × HPLC system was developed.•Initial exploration into preparative isolation, using online CCC × HPLC techniques.•One-step preparative separation of 12 compounds from the crude extract of toad venom.In this work, a new on-line two-dimensional chromatography coupling of flow programming counter-current chromatography and high-performance liquid chromatography (2D CCC × HPLC) was developed for preparative separation of complicated natural products. The CCC column was used as the first dimensional isolation and a preparative ODS column operated in reversed-phase (RP) mode as the second dimension. The CCC was operated at a controlled flow rate to ensure that each fraction eluted within one hour, corresponding to the isolation time of the 2nd dimensional preparative HPLC. The eluent from the 1st dimensional CCC was diluted using a makeup pump and trapped onto holding column, before been eluted and transferred to the 2nd dimensional HPLC. The performance of the holding column was evaluated, in terms of column size, dilution ratio and diameter-height ratio, as well as system pressure, for the solution to the issue of online trapping of low pressure eluent from a CCC column. Satisfactory trapping efficiency and tolerable CCC pressure can be achieved using a commercially available 15 mm × 30 mm i.d. ODS pre-column. The present integrated system was successfully applied in a one-step preparative separation of 12 compounds, from the crude methanol extract of venom of Bufo bufo gargarizans. Compounds 1–12 were isolated in overall yield of 1.0%, 0.8%, 2.0%, 1.3%, 2.0%, 1.5%, 1.9%, 3.6%, 6.1%, 4.8%, 3.5% and 4.1%, with HPLC purity of 99.9%, 99.7%, 90.6%, 99.9%, 77.0%, 99.9%, 90.4%, 99.9%, 52.0%, 99.9%, 99.3%, and 85.0%, respectively. All the results demonstrate that the flow programming CCC × HPLC method is an efficient and convenient way for the separation of compounds from toad venom and it can also be applied to isolate other complex multi-component natural products.