RhoA GEF Mcf2lb regulates rosette integrity during collective cell migration.

Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of ∼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.

Pediococcus acidilactici reduces tau pathology and ameliorates behavioral deficits in models of neurodegenerative disorders.

BACKGROUND: Alzheimer's disease (AD), affecting many elders worldwide, is characterized by A-beta and tau-related cognitive decline. Accumulating evidence suggests that brain iron accumulation is an important characteristic of AD. However, the function and mechanism of the iron-mediated gut-brain axis on AD is still unclear. METHODS: A Caenorhabditis elegans model with tau-overexpression and a high-Fe diet mouse model of cognitive impairment was used for probiotic function evaluation. With the use of qPCR, and immunoblotting, the probiotic regulated differential expression of AD markers and iron related transporting genes was determined. Colorimetric kits, IHC staining, and immunofluorescence have been performed to explore the probiotic mechanism on the development of gut-brain links and brain iron accumulation. RESULTS: In the present study, a high-Fe diet mouse model was used for evaluation in which cognitive impairment, higher A-beta, tau and phosphorylated (p)-tau expression, and dysfunctional phosphate distribution were observed. Considering the close crosstalk between intestine and brain, probiotics were then employed to delay the process of cognitive impairment in the HFe mouse model. Pediococcus acidilactici (PA), but not Bacillus subtilis (BN) administration in HFe-fed mice reduced brain iron accumulation, enhanced global alkaline phosphatase (AP) activity, accelerated dephosphorylation, lowered phosphate levels and increased brain urate production. In addition, because PA regulated cognitive behavior in HFe fed mice, we used the transgenic Caenorhabditis elegans with over-expressed human p-tau for model, and then PA fed worms became more active and longer lived than E.coli fed worms, as well as p-tau was down-regulated. These results suggest that brain iron accumulation influences AD risk proteins and various metabolites. Furthermore, PA was shown to reverse tau-induced pathogenesis via iron transporters and AP-urate interaction. CONCLUSIONS: PA administration studies demonstrate that PA is an important mediator of tau protein reduction, p-tau expression and neurodegenerative behavior both in Caenorhabditis elegans and iron-overload mice. Finally, our results provide candidates for AP modulation strategies as preventive tools for promoting brain health. Video Abstract.

Design, synthesis, discovery and SAR of the fused tricyclic derivatives of indoline and imidazolidinone against DENV replication and infection.

Dengue virus, belonging to a genus Flavivirus, caused public health problem in recent years. One controversial vaccine of DENV was approved and there is no antiviral for the clinic treatment of DENV, therefore, efficient antivirals to DENV are of great medical significance. In this study, we conducted the design, synthesis, cell-based and target-based activity evaluation of 28 compounds based on indoline structural skeleton against DENV infection. Among them, 13 active compounds against DENV infection were discovered and their structure-activity relationship (SAR) was summarized. In this study, indoline carbohydrazine has derived more active compounds than indoline carboamide. It is discovered that TBS group exhibits a good pharmacophore to enhance anti-DENV activity. Further exploration indicated that post-treatment acts as effective time of addition and compound 15 targeting the post-entry stages of DENV2 viral life cycle. SPR imaging results support there are strong interaction of 13 and 15 with RdRp and compounds 13 and 15 reduce RdRp enzymatic activity, revealing that RdRp of DENV NS5 is the drug target for these series of compounds. Molecular docking deciphered the relationship of the structural feature with the putative binding mode by 13 and 15 with RdRp domain of DENV2 NS5 by hydrogen bonds and hydrophobic interactions to establish the fitted low energy conformation. Future studies will focus on designing more potent inhibitors for the treatment and prevention of dengue virus replication and infection, and understanding the more profound underlying structural features of inhibitors and drug action of the mechanism.

Simultaneous removal of fluoride, manganese and iron by manganese oxide supported activated alumina: characterization and optimization via response surface methodology.

Fluoride, iron and manganese simultaneous exceedance of standard can be observed in groundwater in northeastern China. This work aims to apply a highly efficient method combining adsorption and oxidation for the synchronous removal of the inorganic ions. An innovative adsorbent (manganese-supported activated alumina) was synthesized by the impregnation method and showed a significant adsorption capacity better than that of fresh activated alumina. The characterization (scanning electron microscope; Brunauer, Emmett and Teller; X-ray diffraction and Fourier transform infrared spectroscopy) results verified the successful introduction of MnOOH and MnO2, and the improvement of surface microstructure enhanced the removal ability. The effect of single factors, such as pH value, reaction time or dosage on the removal performance has been verified. The maximum removal efficiencies of fluoride, iron and manganese were optimized via Response surface methodology considering the independent factors in the range of MO@AA dosage (5-9 g/L), pH (4-6) and contact time (4-12 h). Noted that compared with control, MO@AA exhibited 59.4% of improved fluoride performance. At pH of 5.79, contacting time of 12 h and 8.21 g/L of MO@AA, fluoride, iron and manganese removal were found to be 91, 100 and 23%, respectively. Herein, MO@AA was distinguished as good applicability for the treatment of fluoride-, iron- and manganese-containing groundwater.

Mechanical Forces Regulate Asymmetric Vascular Cell Alignment.

Mechanical forces between cells and their microenvironment critically regulate the asymmetric morphogenesis and physiological functions in vascular systems. Here, we investigated the asymmetric cell alignment and cellular forces simultaneously in micropatterned endothelial cell ring-shaped sheets and studied how the traction and intercellular forces are involved in the asymmetric vascular morphogenesis. Tuning the traction and intercellular forces using different topographic geometries of symmetric and asymmetric ring-shaped patterns regulated the vascular asymmetric morphogenesis in vitro. Moreover, pharmacologically suppressing the cell traction force and intercellular force disturbed the force-dependent asymmetric cell alignment. We further studied this phenomenon by modeling the vascular sheets with a mechanical force-propelled active particle model and confirmed that mechanical forces synergistically drive the asymmetric endothelial cell alignments in different tissue geometries. Further study using mouse diabetic aortic endothelial cells indicated that diseased endothelial cells exhibited abnormal cell alignments, traction, and intercellular forces, indicating the importance of mechanical forces in physiological vascular morphogenesis and functions. Overall, we have established a controllable micromechanical platform to study the force-dependent vascular asymmetric morphogenesis and thus provide a direct link between single-cell mechanical processes and collective behaviors in a multicellular environment.

The Interplay Between Cell-Cell and Cell-Matrix Forces Regulates Cell Migration Dynamics.

Cells in vivo encounter and exert forces as they interact with the extracellular matrix (ECM) and neighboring cells during migration. These mechanical forces play crucial roles in regulating cell migratory behaviors. Although a variety of studies have focused on describing single-cell or the collective cell migration behaviors, a fully mechanistic understanding of how the cell-cell (intercellular) and cell-ECM (extracellular) traction forces individually and cooperatively regulate single-cell migration and coordinate multicellular movement in a cellular monolayer is still lacking. Here, we developed an integrated experimental and analytical system to examine both the intercellular and extracellular traction forces acting on individual cells within an endothelial cell colony as well as their roles in guiding cell migratory behaviors (i.e., cell translation and rotation). Combined with force, multipole, and moment analysis, our results revealed that traction force dominates in regulating cell active translation, whereas intercellular force actively modulates cell rotation. Our findings advance the understanding of the intricacies of cell-cell and cell-ECM forces in regulating cellular migratory behaviors that occur during the monolayer development and may yield deeper insights into the single-cell dynamic behaviors during tissue development, embryogenesis, and wound healing.

Biophysical Phenotyping and Modulation of ALDH+ Inflammatory Breast Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) have been shown to initiate tumorigenesis and cancer metastasis in many cancer types. Although identification of CSCs through specific marker expression helps define the CSC compartment, it does not directly provide information on how or why this cancer cell subpopulation is more metastatic or tumorigenic. In this study, the functional and biophysical characteristics of aggressive and lethal inflammatory breast cancer (IBC) CSCs at the single-cell level are comprehensively profiled using multiple microengineered tools. Distinct functional (cell migration, growth, adhesion, invasion and self-renewal) and biophysical (cell deformability, adhesion strength and contractility) properties of ALDH+ SUM149 IBC CSCs are found as compared to their ALDH- non-CSC counterpart, providing biophysical insights into why CSCs has an enhanced propensity to metastasize. It is further shown that the cellular biophysical phenotype can predict and determine IBC cells' tumorigenic ability. SUM149 and SUM159 IBC cells selected and modulated through biophysical attributes-adhesion and stiffness-show characteristics of CSCs in vitro and enhance tumorigenicity in in vivo murine models of primary tumor growth. Overall, the multiparametric cellular biophysical phenotyping and modulation of IBC CSCs yields a new understanding of IBC's metastatic properties and how they might develop and be targeted for therapeutic interventions.

Nanoroughened adhesion-based capture of circulating tumor cells with heterogeneous expression and metastatic characteristics.

BACKGROUND: Circulating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population. METHODS: We developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs. RESULTS: The microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-ß-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines' metastatic ability. CONCLUSIONS: The microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further exploration of the biological potential of metastatic and presumably non-metastatic CTCs captured using the microfluidic chip will yield insights into their relevant differences and their effects on tumor progression and cancer outcomes.

Capturing Cancer: Emerging Microfluidic Technologies for the Capture and Characterization of Circulating Tumor Cells.

Circulating tumor cells (CTCs) escape from primary or metastatic lesions and enter into circulation, carrying significant information of cancer progression and metastasis. Capture of CTCs from the bloodstream and the characterization of these cells hold great significance for the detection, characterization, and monitoring of cancer. Despite the urgent need from clinics, it remains a major challenge to capture and retain these rare cells from human blood with high specificity and yield. Recent exciting advances in micro/nanotechnology, microfluidics, and materials science have enable versatile, robust, and efficient cell isolation and processing through the development of new micro/nanoengineered devices and biomaterials. This review provides a summary of recent progress along this direction, with a focus on emerging methods for CTC capture and processing, and their application in cancer research. Furthermore, classical as well as emerging cellular characterization methods are reviewed to reveal the role of CTCs in cancer progression and metastasis, and hypotheses are proposed in regard to the potential emerging research directions most desired in CTC-related cancer research.

General performance, kinetic modification, and key regulating factor recognition of microalgae-based sulfonamide removal.

Sulfonamides have been widely detected in water treatment plants. Advanced wastewater treatment for sulfonamide removal based on microalgal cultivation can reduce the ecological risk after discharge, achieve carbon fixation, and simultaneously recover bioresource. However, the general removal performance, key factors and their impacts, degradation kinetics, and potential coupling technologies have not been systematically summarized. To guide the construction and enhance the efficient performance of the purification system, this study summarizes the quantified characteristics of sulfonamide removal based on more than 100 groups of data from the literature. The biodegradation potential of sulfonamides from different subclasses and their toxicity to microalgae were statistically analyzed; therefore, a preferred option for further application was proposed. The mechanisms by which the properties of both sulfonamides and microalgae affect sulfonamide removal were comprehensively summarized. Thereafter, multiple principles for choosing optimal microalgae were proposed from the perspective of engineering applications. Considering the microalgal density and growth status, a modified antibiotic removal kinetic model was proposed with significant physical meaning, thereby resulting in an optimal fit. Based on the mechanism and regulating effect of key factors on sulfonamide removal, sensitive and feasible factors (e.g., water quality regulation, other than initial algal density) and system coupling were screened to guide engineering applications. Finally, we suggested studying the long-term removal performance of antibiotics at environmentally relevant concentrations and toxicity interactions for further research.

Pulses of RhoA signaling stimulate actin polymerization and flow in protrusions to drive collective cell migration.

In animals, cells often move as collectives to shape organs, close wounds, or-in the case of disease-metastasize. To accomplish this, cells need to generate force to propel themselves forward. The motility of singly migrating cells is driven largely by an interplay between Rho GTPase signaling and the actin network. Whether cells migrating as collectives use the same machinery for motility is unclear. Using the zebrafish posterior lateral line primordium as a model for collective cell migration, we find that active RhoA and myosin II cluster on the basal sides of the primordium cells and are required for primordium motility. Positive and negative feedbacks cause RhoA and myosin II activities to pulse. These pulses of RhoA signaling stimulate actin polymerization at the tip of the protrusions and myosin-II-dependent actin flow and protrusion retraction at the base of the protrusions and deform the basement membrane underneath the migrating primordium. This suggests that RhoA-induced actin flow on the basal sides of the cells constitutes the motor that pulls the primordium forward, a scenario that likely underlies collective migration in other contexts.

Effects of chemical oxygen demand and chloramphenicol on attached microalgae growth: Physicochemical properties and microscopic mass transfer in biofilm.

During the wastewater treatment and resource recovery process by attached microalgae, the chemical oxygen demand (COD) can cause biotic contamination in algal culture systems, which can be mitigated by adding an appropriate dosage of antibiotics. The transport of COD and additive antibiotic (chloramphenicol, CAP) in algal biofilms and their influence on algal physiology were studied. The results showed that COD (60 mg/L) affected key metabolic pathways, such as photosystem II and oxidative phosphorylation, improved biofilm autotrophic and heterotrophic metabolic intensities, increased nutrient demand, and promoted biomass accumulation by 55.9 %, which was the most suitable COD concentration for attached microalgae. CAP (5-10 mg/L) effectively stimulated photosynthetic pigment accumulation and nutrient utilization in pelagic microalgal cells. In conclusion, controlling the COD concentration (approximately 60 mg/L) in the medium and adding the appropriate CAP concentration (5-10 mg/L) are conducive to improving attached microalgal biomass production and resource recovery potential from wastewater.

Pulses of RhoA Signaling Stimulate Actin Polymerization and Flow in Protrusions to Drive Collective Cell Migration.

In animals, cells often move as collectives to shape organs, close wounds, or-in the case of disease-metastasize. To accomplish this, cells need to generate force to propel themselves forward. The motility of singly migrating cells is driven largely by an interplay between Rho GTPase signaling and the actin network (Yamada and Sixt, 2019). Whether cells migrating as collectives use the same machinery for motility is unclear. Using the zebrafish posterior lateral line primordium as a model for collective cell migration, we find that active RhoA and myosin II cluster on the basal sides of the primordium cells and are required for primordium motility. Positive and negative feedbacks cause RhoA and myosin II activities to pulse. These pulses of RhoA signaling stimulate actin polymerization at the tip of the protrusions and myosin II-dependent actin flow and protrusion retraction at the base of the protrusions, and deform the basement membrane underneath the migrating primordium. This suggests that RhoA-induced actin flow on the basal sides of the cells constitutes the motor that pulls the primordium forward, a scenario that likely underlies collective migration in other-but not all (Bastock and Strutt, 2007; Lebreton and Casanova, 2013; Matthews et al., 2008)-contexts.

Screening of microalgae species and evaluation of algal-lipid stimulation strategies for biodiesel production.

Microalgae is considered an alternative source for biodiesel production producing renewable, sustainable and carbon-neutral energy. Microalgae property changes among species, which determines the efficiency of biodiesel production. Besides the lipid content evaluation, multi-principles (including high lipid productivity, high biomass yield, pollution resistance and desired fatty acid, etc.) for superior oil-producing species screening was proposed in this review and three microalgae species (Chlorella vulgaris, Scenedesmus obliquus and Mychonastes afer) with high bio-lipid producing prospect were screened out based on big data digging and analysis. The multilateral strategies for algal-lipid stimulating were also compared, among which, nutrient restriction, temperature control, heterotrophy and chemicals addition showed high potential in enhancing lipid accumulation; while electromagnetic field showed little effect. Interestingly, it was found that the lipid accumulation was more sensitive to nitrogen (N)-limitation other than phosphorus (P). Nutrient restriction, salinity stress etc. enhanced lipid accumulation by creating a stressed environment. Hence, optimum conditions (e.g. N:15-35 mg/L and P:4-16 mg/L) should be set to balance the lipid accumulation and biomass growth, and further guarantee the algal-lipid productivity. Otherwise, two-step cultivation could be applied during all the stressed stimulation. Different from lab study, effectiveness, operability and economy should be all considered for stimulation strategy selection. Nutrient restriction, temperature control and heterotrophy were highly feasible after the multidimensional evaluation.

Antiviral effects of the fused tricyclic derivatives of indoline and imidazolidinone on ZIKV infection and RdRp activities of ZIKV and DENV.

The prevalence and ravages of Zika virus (ZIKV) seriously endanger human health, especially causing significant neurological defects in both neonates as pediatric microcephaly and adults as Guillain-Barré syndrome. In this work, we studied anti-ZIKV effects of the fused tricyclic derivatives of indoline and imidazolidinone and discovered that some of them are valuable leads for drug discovery of anti-ZIKV agents. The current results show that certain compounds are broad-spectrum inhibitors of ZIKV- and dengue virus (DENV)-infection while distinctive compounds are selective ZIKV inhibitors or selective DENV inhibitors. Compounds of 12, 17 and 28 are more active against Asian ZIKV SZ-VIV01 strain than African ZIKV MR766 strain. It is valued that silylation makes six TBS compounds of 4-nitrophenyl hydrazine series and phenyl hydrazine series more active against ZIKV infection than their phenols. Time-of-addition and withdrawal studies indicate that compound 12 majorly acts on post-infection of RNA synthesis stage of ZIKV life cycle. Moreover, compounds of 12, 17 and 18 are anti-ZIKV agents with the inhibitory activities to ZIKV NS5 RdRp while 12 doesn't inhibit DENV infection even though it is a DENV RdRp inhibitor, 17 is an active agent against DENV infection but is only a weak DENV NS5 RdRp inhibitor, and 28 is inactive against DENV infection and not a DENV NS5 RdRp inhibitor. As a result, a compound's antiviral difference between ZIKV and DENV is not always related to anti-RdRp difference between ZIKV RdRp and DENV RdRp, and structural features of a compound play important roles in executing antiviral and anti-RdRp functions. Further discovery of highly potent broad-spectrum or selective agents against infection by ZIKV and DENV will be facilitated.

Discovery of ZFD-10 of a pyridazino[4,5-b]indol-4(5H)-one derivative as an anti-ZIKV agent and a ZIKV NS5 RdRp inhibitor.

Zika virus (ZIKV) infection is associated with the birth defect microcephaly and Guillain-Barré syndrome in adults. There is no approved vaccine or specific antiviral agent against ZIKV. ZFD-10, a novel structural skeleton of 1H-pyridazino[4,5-b]indol-4(5H)-one, was firstly synthesized and discovered to be a potent anti-ZIKV inhibitor with very low cytotoxicity. ZFD-10's anti-ZIKV potency is independent of cell lines and ZFD-10 mainly targets the post-entry stages of ZIKV life cycle. Time-of-addition and time-of-withdrawal assays showed that 10 µM ZFD-10 displayed the ability to decrease mainly at the RNA level and weakly the viral progeny particle load. Furthermore, ZFD-10 could protect ZIKV NS5 from thermal unfolding and aggregation and increase the Tagg value of ZIKV NS5 protein from 44.6 to 49.3 °C, while ZFD-10 dose-dependently inhibits ZIKV NS5 RdRp activity using in vitro RNA polymerase assays. Molecular docking study suggests that ZFD-10 affects RdRp enzymatic function through interfering with the fingers and thumb subdomains. These results supported that ZFD-10's cell-based anti-ZIKV activity is related to its anti-RdRp activity of ZIKV NS5. The in vivo anti-ZIKV study shows that the middle-dose (4.77 mg/kg/d) of ZFD-10 protected mice from ZIKV infection and the viral loads of the blood, liver, kidney and brain in the middle-dose and high-dose (9.54 mg/kg/d) were significantly reduced compared to those of the ZIKV control. These results confirm that ZFD-10 has a certain antiviral effect against ZIKV infection in vivo.

Pediococcus acidilactici Promotes the Longevity of C. elegans by Regulating the Insulin/IGF-1 and JNK/MAPK Signaling, Fat Accumulation and Chloride Ion.

Probiotics are known to contribute to the anti-oxidation, immunoregulation, and aging delay. Here, we investigated the extension of lifespan by fermented pickles-origin Pediococcus acidilactici (PA) in Caenorhabditis elegans (C. elegans), and found that PA promoted a significantly extended longevity of wild-type C. elegans. The further results revealed that PA regulated the longevity via promoting the insulin/IGF-1 signaling, JNK/MAPK signaling but not TOR signaling in C. elegans, and that PA reduced the reactive oxygen species (ROS) levels and modulated expression of genes involved in fatty acids uptake and lipolysis, thus reducing the fat accumulation in C. elegans. Moreover, this study identified the nrfl-1 as the key regulator of the PA-mediated longevity, and the nrfl-1/daf-18 signaling might be activated. Further, we highlighted the roles of one chloride ion exchanger gene sulp-6 in the survival of C. elegans and other two chloride ion channel genes clh-1 and clh-4 in the prolonged lifespan by PA-feeding through the modulating expression of genes involved in inflammation. Therefore, these findings reveal the detailed and novel molecular mechanisms on the longevity of C. elegans promoted by PA.

Microskeletal stiffness promotes aortic aneurysm by sustaining pathological vascular smooth muscle cell mechanosensation via Piezo1.

Mechanical overload of the vascular wall is a pathological hallmark of life-threatening abdominal aortic aneurysms (AAA). However, how this mechanical stress resonates at the unicellular level of vascular smooth muscle cells (VSMC) is undefined. Here we show defective mechano-phenotype signatures of VSMC in AAA measured with ultrasound tweezers-based micromechanical system and single-cell RNA sequencing technique. Theoretical modelling predicts that cytoskeleton alterations fuel cell membrane tension of VSMC, thereby modulating their mechanoallostatic responses which are validated by live micromechanical measurements. Mechanistically, VSMC gradually adopt a mechanically solid-like state by upregulating cytoskeleton crosslinker, α-actinin2, in the presence of AAA-promoting signal, Netrin-1, thereby directly powering the activity of mechanosensory ion channel Piezo1. Inhibition of Piezo1 prevents mice from developing AAA by alleviating pathological vascular remodeling. Our findings demonstrate that deviations of mechanosensation behaviors of VSMC is detrimental for AAA and identifies Piezo1 as a novel culprit of mechanically fatigued aorta in AAA.

Discovery of dehydroandrographolide derivatives with C19 hindered ether as potent anti-ZIKV agents with inhibitory activities to MTase of ZIKV NS5.

Infection by Zika virus (ZIKV), a mosquito-transmitted arbovirus and a member of Flavivirus, could make pediatric microcephaly and Guillain-Barré syndrome, which remains an ongoing global threat. The efficient antivirals to ZIKV infection are of great medical need. Andrographolide and its analogues were discovered to be active against flaviviral infection. In this study, we discovered some dehydroandrographolide derivatives of 3-oximido- or 3-alcohol-19-hindered ether to be potent anti-ZIKV agents with low cytotoxicities (CC50 > 200 µM). Time of addition assay suggests that compound 5a and its analogues act on inhibition of post-entry stage of ZIKV life cycle. It is discovered by experimental and molecular docking studies that active anti-ZIKV compounds of 3a, 5a, 5b and 5c possess inhibitory activities of ZIKV NS5 MTase (methyl transferase) enzymatic activity. Preliminary SAR reveals that C19-modification with bulky groups is necessary for anti-ZIKV infection and replication, anti-ZIKV activity of 5a comes from itself bearing hindered trityl ether but not from its instability, the backbone of dehydroandrographolide is more effective against ZIKV infection than that of andrographolide, and 3-oxime derivatives are more active against ZIKV infection than 3-alcohol derivatives. To our knowledge, 5a is the first reported MTase inhibitor of andrographolide derivatives. More importantly, discovery of active compound 5b with acid-stable 19-OCHPh2 against ZIKV infection is valued and gives us a clue to design and discover generally acid-stable anti-ZIKV agents.

Discovery of 14S-(2'-chloro-4'-nitrophenoxy)-8R/S,17-epoxy andrographolide as EV-A71 infection inhibitor.

Human enterovirus A71 (EV-A71) is a major etiological agent of hand-foot-and-mouth disease (HFMD) and there is presently no internationally approved antiviral against EV-A71. In this study, it is disclosed that 14S-(2'-chloro-4'-nitrophenoxy)-8R/S,17-epoxy andrographolide (2) was discovered to be an effective inhibitor against EV-A71 infection showing significant reduction of viral titre. In addition to EV-A71, compound 2 exerts broad-spectrum antiviral effects against other enteroviruses. It is revealed that compound 2 inhibits the post-entry stages of EV-A71 viral replication cycle and significantly reduces viral protein expression of structural proteins such as VP0 and VP2 via inhibiting EV-A71 RNA replication. Moreover, the inhibitory property of compound 2 is specific to viral RNA replication. Furthermore, compound 2 is more likely to target a host factor in EV-A71 RNA replication. As a result, introduction of epoxide at positions 8 and 17 of andrographolide is effective for anti-EV-A71 infection and is a potential anti-EV-A71 strategy. Further work to discover more potent andrographolide derivatives and elucidate comprehensive SAR is under way.