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Nanofiltration is useful to concentrate propolis extract. During the selection of membrane, both compound rejection and permeate flux are important indicators of process economy. Brazilian green propolis extract was studied to evaluate the separation performance of Startmen 122 and NF270 membranes. Compared to Starmen 122, NF270 membrane showed better rejection of bioactive compounds. The flux decline patterns were further studied using Hermia's model. Cake formation is the major fouling mechanism on the hydrophobic surface of Starmen 122. While the fouling mechanism for NF270 is pore blocking. The fouled membranes were further characterized using SEM and FT-IR to confirm on the predicted fouling mechanisms.

The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo . The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC 50 ], 10 to 80 nM) but not DENV-1 and -4 (EC 50 , >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo . Our results validate, for the first time, that NS4B inhibitors could potentially be developed for antiviral therapy for treatment of DENV infection in humans.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is a common practice in antiviral treatment and a potential approach to search for new treatments for infectious pathogens. In this study, we performed a combination treatment in cell culture by using three distinct classes of inhibitors, including ribavirin (a guanosine analog with several antiviral mechanisms), brequinar (a pyrimidine biosynthesis inhibitor), and INX-08189 (a guanosine analog). The compound pairs were evaluated for antiviral activity by use of a DENV-2 luciferase replicon assay. Our result indicated that the combination of ribavirin and INX-08189 exhibited strong antiviral synergy. This result suggests that synergy can be achieved with compound pairs in which one compound suppresses the synthesis of the nucleoside for which the other compound is a corresponding nucleoside analog. In addition, we found that treatment of cells with brequinar alone could activate interferon-stimulated response elements (ISREs); furthermore, brequinar and NITD-982 (another pyrimidine biosynthesis inhibitor) potentiated interferon-induced ISRE activation. Compared to treatment with brequinar, treatment of cells with ribavirin alone could also induce ISRE activation, but to a lesser extent; however, when cells were cotreated with ribavirin and beta interferon, ribavirin did not augment the interferon-induced ISRE activation.