Development of a one-step immunochromatographic strip test for the rapid detection of nevirapine (NVP), a commonly used antiretroviral drug for the treatment of HIV/AIDS

Currently, high-performance liquid chromatographic (HPLC) methods are mainly used to measure antiretroviral plasma concentrations in HIV-infected patients. Although the utility of routine therapeutic drug monitoring (TDM) as an additional tool to optimize long-term antiretroviral therapy is unclear, if TDM is to be widely used, the availability of simple, cheap and reliable methods for the measurement of antiretroviral drug levels are needed, particularly in resource-limited settings. In this study, an immunochromatograhic (IC) strip test to detect the presence of nevirapine (NVP) in body fluids has been developed. Antiserum to NVP was first raised in rabbits by immunization against NVP chemically conjugated with bovine serum albumin, and subsequently validated by Western immunoblotting and competitive indirect ELISA. The partially purified anti-NVP antibodies were conjugated with colloidal gold particles. The conjugation of the colloidal gold and polyclonal antibodies was monitored by UV-vis spectroscopy, while transmission electron microscopy images were used to characterize the particle size and shape of the conjugates. The resulting colloidal gold conjugates were used for the production of an IC strip test to detect nevirapine in human plasma. Preliminary assessment suggests no-cross reactivity of the NVP polyclonal antibodies but assessment of plasma samples from HIV-infected patients receiving HAART needs to be conducted. This assay could potentially be used for drug monitoring as part of the clinical care of HIV infected patients.     

Fungus-Derived 3-Hydroxyterphenyllin and Candidusin A Ameliorate Palmitic Acid-Induced Human Podocyte Injury via Anti-Oxidative and Anti-Apoptotic Mechanisms

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. An elevated fatty acid plasma concentration leads to podocyte injury and DN progression. This study aimed to identify and characterize cellular mechanisms of natural compounds that inhibit palmitic acid (PA)–induced human podocyte injury. By screening 355 natural compounds using a cell viability assay, 3-hydroxyterphenyllin (3-HT) and candidusin A (CDA), isolated from the marine-derived fungus Aspergillus candidus PSU-AMF169, were found to protect against PA-induced podocyte injury, with half-maximal inhibitory concentrations (IC₅₀) of ~16 and ~18 µM, respectively. Flow cytometry revealed that 3-HT and CDA suppressed PA-induced podocyte apoptosis. Importantly, CDA significantly prevented PA-induced podocyte barrier impairment as determined by 70 kDa dextran flux. Reactive oxygen species (ROS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) direct scavenging assays indicated that both compounds exerted an anti-oxidative effect via direct free radical–scavenging activity. Moreover, 3-HT and CDA upregulated the anti-apoptotic Bcl2 protein. In conclusion, 3-HT and CDA represent fungus-derived bioactive compounds that have a novel protective effect on PA-induced human podocyte apoptosis via mechanisms involving free radical scavenging and Bcl2 upregulation.     

Water‐Assisted Nitrile Oxide Cycloadditions: Synthesis of Isoxazoles and Stereoselective Syntheses of Isoxazolines and 1,2,4‐Oxadiazoles

Conventional methods generate nitrile oxides from oxime halides in organic solvents under basic conditions. However, the present work revealed that water‐assisted generation of nitrile oxides proceeds under mild acidic conditions (pH 4–5). Cycloadditions of nitrile oxides with alkynes and alkenes easily occurred in water without using catalysts, thus yielding isoxazoles and isoxazolines, respectively, with excellent stereoselectivity toward five‐ and six‐membered cyclic alkenes. A double stereoselective cycloaddition of two units of a nitrile oxide with cyclohexene was also achieved, thus yielding 1,2,4‐oxadiazole derivatives having a unique hybrid isoxazoline‐oxadiazole skeleton. Enantiomerically pure isoxazolines were prepared from monoterpenes with a ring strain. In one case, the isoxazoline with a butterfly‐like structure was simply prepared, and it might be used as a ligand in asymmetric catalysis.     

Crystal structure of an antiviral ankyrin targeting the HIV-1 capsid and molecular modeling of the ankyrin-capsid complex

Ankyrins are cellular repeat proteins, which can be genetically modified to randomize amino-acid residues located at defined positions in each repeat unit, and thus create a potential binding surface adaptable to macromolecular ligands. From a phage-display library of artificial ankyrins, we have isolated Ank^GAG1D4, a trimodular ankyrin which binds to the HIV-1 capsid protein N-terminal domain (NTD^CA) and has an antiviral effect at the late steps of the virus life cycle. In this study, the determinants of the Ank^GAG1D4-NTD^CA interaction were analyzed using peptide scanning in competition ELISA, capsid mutagenesis, ankyrin crystallography and molecular modeling. We determined the Ank^GAG1D4 structure at 2.2 Å resolution, and used the crystal structure in molecular docking with a homology model of HIV-1 capsid. Our results indicated that NTD^CA alpha-helices H1 and H7 could mediate the formation of the capsid-Ank^GAG1D4 binary complex, but the interaction involving H7 was predicted to be more stable than with H1. Arginine-18 (R18) in H1, and R132 and R143 in H7 were found to be the key players of the Ank^GAG1D4-NTD^CA interaction. This was confirmed by R-to-A mutagenesis of NTD^CA, and by sequence analysis of trimodular ankyrins negative for capsid binding. In Ank^GAG1D4, major interactors common to H1 and H7 were found to be S45, Y56, R89, K122 and K123. Collectively, our ankyrin-capsid binding analysis implied a significant degree of flexibility within the NTD^CA domain of the HIV-1 capsid protein, and provided some clues for the design of new antivirals targeting the capsid protein and viral assembly.     

Alpha-Glucosidase Inhibitory Assay-Screened Isolation and Molecular Docking Model from Bauhinia pulla Active Compounds

The aim of this research was to establish the constituents of Bauhinia pulla as anti-diabetic agents. A phytochemistry analysis was conducted by chromatographic and spectroscopic techniques. The alpha-glucosidase inhibitory assay screening resulted in the isolation of eight known compounds of quercetin, quercitrin, luteolin, 5-deoxyluteolin, 4-methyl ether isoliquiritigenin, 3,2′,4′-trihydroxy-4-methoxychalcone, stigmasterol and β-sitosterol. Ethanol leaf extracts showed potential effects, which led to a strong inhibitory activity of isolated quercetin at 138.95 µg/mL and 5.41 µg/mL of IC₅₀, respectively. The docking confirmed that flavonoids and chalcones had the same potential binding sites and responsibilities for their activity. This study was the first report of Bauhinia pulla chemical constituents and its alpha-glucosidase inhibition.     

Depsides and depsidones from the soil-derived fungus Aspergillus unguis PSU-RSPG204

Three new depsides, aspergisides A-C, and one new depsidone, aspergisidone, were isolated from the soil-derived fungus Aspergillus unguis PSU-RSPG204 together with one phthalide derivative and 11 depsidones including emeguisin A, aspersidone and folipastatin. The structures were determined by spectroscopic evidence. Aspersidone and emeguisin A showed remarkably antibacterial activity against Staphylococcus aureus and methicillin-resistance S. aureus with equal MIC values of 0.5?μg/mL. Emeguisin A, which displayed the highest activity with 87.06% inhibition of human colon carcinoma (HCT-116) cell viability, decreased numbers of live cells/numbers of dead cells in a dose-dependent manner with the IC₅₀ values of 34.8–84.7?μM in a 3D culture model depending on durations of incubation. In addition, folipastatin dose-dependently inhibited forskolin-stimulated chloride secretion in human intestinal epithelial (T84) cells with an IC₅₀ value of 0.5?μM. These depsidones were considered to be inactive against noncancerous Vero Cells with the IC₅₀ values of >10?μM.     

Synthesis of Molecularly Imprinted Polymers for Nevirapine by Dummy Template Imprinting Approach

Nevirapine (NVP) and its structurally related analogs including nicotinamide (NAM), benzamide (BZM) and benzophenone (BZP) were used as templates in the synthesis of molecularly imprinted polymers for NVP. Molecular modeling was used to estimate binding energy of the complex formation between methacrylic acid (MAA) monomer and the selected templates, while equilibrium binding studies were applied to evaluate the polymer binding efficiency. The data indicated that NAM is the best candidate to prepare MIPs for retaining NVP due to a relatively similar binding energy between the NVP–MAA and NAM–MAA complex. The NAM-imprinted polymer showed a high binding affinity and selectivity toward NVP. When the polymer was applied as a sorbent in solid-phase extraction of NVP from human plasma, high recovery and reproducibility were obtained.

     

Deoxyelephantopin and Its Isomer Isodeoxyelephantopin: Anti-Cancer Natural Products with Multiple Modes of Action

Cancer is a leading cause of morbidity and mortality worldwide. The development of cancer involves aberrations in multiple pathways, representing promising targets for anti-cancer drug discovery. Natural products are regarded as a rich source for developing anti-cancer therapies due to their unique structures and favorable pharmacology and toxicology profiles. Deoxyelephantopin and isodeoxyelephantopin, sesquiterpene lactone compounds, are major components of Elephantopus scaber and Elephantopus carolinianus, which have long been used as traditional medicines to treat multiple ailments, including liver diseases, diabetes, bronchitis, fever, diarrhea, dysentery, cancer, renal disorders, and inflammation-associated diseases. Recently, deoxyelephantopin and isodeoxyelephantopin have been extensively explored for their anti-cancer activities. This review summarizes and discusses the anti-cancer activities of deoxyelephantopin and isodeoxyelephantopin, with an emphasis on their modes of action and molecular targets. Both compounds disrupt several processes involved in cancer progression by targeting multiple signaling pathways deregulated in cancers, including cell cycle and proliferation, cell survival, autophagy, and invasion pathways. Future directions of research on these two compounds towards anti-cancer drug development are discussed.