Synthesis and In Vitro Anti-Influenza Virus Evaluation of Novel Sialic Acid (C-5 and C-9)-Pentacyclic Triterpene Derivatives.

The emergence of drug resistant variants of the influenza virus has led to a great need to identify novel and effective antiviral agents. In our previous study, a series of sialic acid (C-2 and C-4)-pentacyclic triterpene conjugates have been synthesized, and a five-fold more potent antiviral activity was observed when sialic acid was conjugated with pentacyclic triterpene via C-4 than C-2. It was here that we further reported the synthesis and anti-influenza activity of novel sialic acid (C-5 and C-9)-pentacyclic triterpene conjugates. Their structures were confirmed by ESI-HRMS, ¹H-NMR, and 13C-NMR spectroscopic analyses. Two conjugates (26 and 42) showed strong cytotoxicity to MDCK cells in the CellTiter-Glo assay at a concentration of 100 µM. However, they showed no significant cytotoxicity to HL-60, Hela, and A549 cell lines in MTT assay under the concentration of 10 µM (except compound 42 showed weak cytotoxicity to HL-60 cell line (10 µM, ~53%)). Compounds 20, 28, 36, and 44 displayed weak potency to influenza A/WSN/33 (H1N1) virus (100 µM, ~20-30%), and no significant anti-influenza activity was found for the other conjugates. The data suggested that both the C-5 acetylamide and C-9 hydroxy of sialic acid were important for its binding with hemagglutinin during viral entry into host cells, while C-4 and C-2 hydroxy were not critical for the binding process and could be replaced with hydrophobic moieties. The research presented herein had significant implications for the design of novel antiviral inhibitors based on a sialic acid scaffold.

Phylodynamic reconstruction of the spatiotemporal transmission and demographic history of coxsackievirus B2.

BACKGROUND: Studies regarding coxsackievirus (CV) tend to focus on epidemic outbreaks, an imbalanced topology is considered to be an indication of acute infection with partial cross-immunity. In enteroviruses, a clear understanding of the characteristics of tree topology, transmission, and its demographic dynamics in viral succession and circulation are essential for identifying prevalence trends in endemic pathogens such as coxsackievirus B2 (CV-B2). This study applied a novel Bayesian evolutionary approach to elucidate the phylodynamic characteristics of CV-B2. A dataset containing 51 VP1 sequences and a dataset containing 34 partial 3D(pol) sequencing were analyzed, where each dataset included Taiwan sequences isolated during 1988-2013. RESULTS: Four and five genotypes were determined based on the 846-nucleotide VP1 and 441-nucleotide 3D(pol) (6641-7087) regions, respectively, with spatiotemporally structured topologies in both trees. Some strains with tree discordance indicated the occurrence of recombination in the region between the VP1 and 3D(pol) genes. The similarities of VP1 and 3D(pol) gene were 80.0%-96.8% and 74.7%-91.9%, respectively. Analyses of population dynamics using VP1 dataset indicated that the endemic CV-B2 has a small effective population size. The balance indices, high similarity, and low evolutionary rate in the VP1 region indicated mild herd immunity selection in the major capsid region. CONCLUSIONS: Phylodynamic analysis can reveal demographic trends and herd immunity in endemic pathogens.

Phylodynamic analysis of the canine distemper virus hemagglutinin gene.

BACKGROUND: Canine distemper (CD) is one of the most contagious and lethal viral diseases in dogs. Despite the widespread use of vaccines, the prevalence of the CD virus (CDV) has increased at an alarming rate in recent years. In this phylodynamic study, we investigated the spatiotemporal modes of dispersal, viral demographic trends, and effectiveness of vaccines for CDV. A total of 188 full-length CDV hemagglutinin (H) gene sequences dataset were subjected to recombination analysis, including seven from modified live vaccine (MLV) strains and 12 from Taiwan specimens. After excluding the MLV strains and potential recombinant strains, alignments of 176 of 188 previous CDV strains were further used to analyze phylodynamic characteristics, and evidence of selection, and co-evolution. RESULTS: The CDV genotype consisted of MLV-associated genotypes such as America-1 and Rockborn-like strains, which were characterized by long terminal branches and no distinct geographical patterns among lineages. In contrast, wild-type isolates clustered into lineages with a spatiotemporal structure and short terminal branches. Co-circulation and extensive diversification were simultaneously observed. The sequence variation signature was shaped by both geographic diversity and host tropism. Codon 506 was identified as a multi-epistatic interacting in the H protein. CONCLUSIONS: The topological signature revealed in this study suggests different epidemic scenarios. For example, a ladder-like backbone is a hallmark of directional selection, and cladogenesis at long terminal branches indicates the emergence of a surviving lineage. The stable effective viral population of CDV indicate the effectiveness of vaccines currently used to control the virus.

Subacute infective endocarditis due to Lodderomyces elongisporus: a case report and review of the literature.

Lodderomyces elongisporus, a rare emerging pathogen, can cause fungemia often related to immunosuppression or intravenous devices. Herein, we report the case of a 58-year-old woman with subacute infective endocarditis due to Lodderomyces elongisporus identified by blood fungal culture and whole-genome sequencing, who was treated with antifungals, mitral replacement and endocardial vegetation removal surgery.

Targeted treprostinil delivery inhibits pulmonary arterial remodeling.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is a fatal disease caused by the progressive remodeling of pulmonary arteries (PAs). Treprostinil (TPS) is a tricyclic benzidine prostacyclin clinically used for PAH treatment. However, due to low bioavailability, short half-times, and severe systemic side effects, TPS efficacy remains limited. METHODS: In this study, glucuronic acid (GlcA)-modified liposomes were developed to improve the site-specific delivery of TPS to pulmonary arterial smooth muscle cells (PASMCs) by targeting the glucose transporter-1 (GLUT-1) in vitro and in vivo. RESULTS: Non-GlcA-modified and GlcA-modified liposomes encapsulating TPS were 106 ± 1.12 nm in diameter. The drug encapsulation efficiency (EE) was 92%. Data from rat PASMCs showed that GlcA-liposomes enhanced the inhibitory effects of TPS on PASMC proliferation and migration by suppressing growth factor expression, including transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), and cAMP, which was possibly mediated by the cAMP-C/EBP-α p42-p21 signaling pathway. In PAH model rats, GlcA-modified liposomes significantly improved TPS bioavailability and sustained its release over time. Most importantly, the selective inhibition of pulmonary arterial pressure, rather than systemic arterial pressure, indicated the increased pulmonary-specific accumulation of TPS. Of the three TPS formulations, TPS-loaded GlcA-modified liposomes exhibited the most potent activity by inhibiting PA remodeling and muscularization, decreasing PA medial thickening, suppressing collagen deposition in PAs, and attenuating right ventricle hypertrophy (RVH) in sugen-5416-induced PAH rats. CONCLUSIONS: The GLUT-1-targeted delivery of TPS increased pulmonary specificity and enhanced TPS anti-PAH activities in vivo and in vitro.

Azilsartan ameliorates ventricular hypertrophy in rats suffering from pressure overload-induced cardiac hypertrophy by activating the Keap1-Nrf2 signalling pathway.

OBJECTIVES: Investigate if azilsartan protects against myocardial hypertrophy by upregulating nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated pathways. METHODS: Abdominal aortic constriction (AAC)-induced cardiac hypertrophy in rats was applied. Azilsartan or vehicle was administered daily for 6 weeks in sham or AAC rats. Cardiac morphology and ventricular function were determined. Azilsartan effects upon neonatal rat cardiomyocyte (NRCM) hypertrophy and molecular mechanisms were studied in angiotensin (Ang) II-stimulated NRCMs in vitro. Nrf2-small interfering RNA (siRNA) was used to knockdown Nrf2 expression. Messenger RNA (mRNA)/protein expression of Kelch-like erythroid cell-derived protein (Keap)1 and Nrf2 and its downstream antioxidant enzymes was determined by real-time reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. KEY FINDINGS: Azilsartan treatment ameliorated cardiac hypertrophy/fibrosis significantly in AAC rats. Azilsartan increased expression of Nrf2 protein but decreased expression of Keap1 protein. Upregulation of protein expression of Nrf2's downstream antioxidant enzymes by azilsartan treatment was observed. Azilsartan inhibited Ang II-induced NRCM hypertrophy significantly and similar effects on the Keap1-Nrf2 pathway were observed in vivo. Nrf2 knockdown markedly counteracted the beneficial effects of azilsartan on NRCM hypertrophy and the Keap1-Nrf2 pathway. CONCLUSIONS: Azilsartan restrained pressure overload-induced cardiac remodelling by activating the Keap1-Nrf2 pathway and increasing expression of downstream antioxidant enzymes to alleviate oxidative stress.

Evolutionary histories of coxsackievirus B5 and swine vesicular disease virus reconstructed by phylodynamic and sequence variation analyses.

Coxsackievirus (CV)-B5 is a common human enterovirus reported worldwide; swine vesicular disease virus (SVDV) is a porcine variant of CV-B5. To clarify the transmission dynamics and molecular basis of host switching between CV-B5 and SVDV, we analysed and compared the VP1 and partial 3Dpol gene regions of these two viruses. Spatiotemporal dynamics of viral transmission were estimated using a Bayesian statistical inference framework. The detected selection events were used to analyse the key molecules associated with host switching. Analyses of VP1 sequences revealed six CV-B5 genotypes (A1-A4 and B1-B2) and three SVDV genotypes (I-III). Analyses of partial 3Dpol revealed five clusters (A-E). The genotypes evolved sequentially over different periods, albeit with some overlap. The major hub of CV-B5 transmission was in China whereas the major hubs of SVDV transmission were in Italy. Network analysis based on deduced amino acid sequences showed a diverse extension of the VP1 structural protein, whereas most sequences were clustered into two haplotypes in the partial 3Dpol region. Residue 178 of VP1 showed four epistatic interactions with residues known to play essential roles in viral host tropism, cell entry, and viral decoating.

Synthesis of novel pentacyclic triterpene-Neu5Ac2en derivatives and investigation of their in vitro anti-influenza entry activity.

Sialic acid derivatives, analogs, and their conjugates are important pharmacophores. Modification of the C-4 hydroxyl group of sialic acid can lead to derivatives, such as zanamivir, with potent anti-influenza activities. Herein, we described the synthesis of C-4-modified sialic acid derivatives via conjugation with naturally derived pentacyclic triterpenes, which are active ingredients of traditional Chinese medicine, and the evaluation of their in vitro anti-influenza virus activity in MDCK cells. Interestingly, a set of configurational isomers was obtained during the de-O-acetylation reaction of two pentacyclic triterpene-sialic acid conjugates under Zemplén conditions, and a mechanism was proposed. Owing to the attachment of the Neu5Ac2en moiety, all synthesized conjugates displayed lower hydrophobicity than their parent compounds. In comparison with ursane- and lupane-type triterpenes, oleanane-type triterpene-functionalized Neu5Ac2en conjugates were most promising. The insertion of a (1,2,3-triazol-4-yl)-methyl between the amide bond and Neu5Ac2en caused a substantial decrease in activity. Compound 15a exhibited the highest inhibitory activity (IC50 = 8.3 µM) and selectivity index (SI = 22.7). Further studies involving hemagglutination inhibition and neuraminidase inhibition suggested that compound 15a inhibited virus-induced hemagglutination with no effect on the enzymatic activity of neuraminidase, indicating that the antiviral activity appeared to be mediated via interaction with hemagglutinin at the initial stage of viral infection.

Design, synthesis and biological evaluation of novel L-ascorbic acid-conjugated pentacyclic triterpene derivatives as potential influenza virus entry inhibitors.

Since the influenza viruses can rapidly evolve, it is urgently required to develop novel anti-influenza agents possessing a novel mechanism of action. In our previous study, two pentacyclic triterpene derivatives (Q8 and Y3) have been found to have anti-influenza virus entry activities. Keeping the potential synergy of biological activity of pentacyclic triterpenes and l-ascorbic acid in mind, we synthesized a series of novel l-ascorbic acid-conjugated pentacyclic triterpene derivatives (18-26, 29-31, 35-40 and 42-43). Moreover, we evaluated these novel compounds for their anti-influenza activities against A/WSN/33 virus in MDCK cells. Among all evaluated compounds, the 2,3-O,O-dibenzyl-6-deoxy-l-ascorbic acid-betulinic acid conjugate (30) showed the most significant anti-influenza activity with an EC50 of 8.7 µM, and no cytotoxic effects on MDCK cells were observed. Time-of-addition assay indicated that compound 30 acted at an early stage of the influenza life cycle. Further analyses revealed that influenza virus-induced hemagglutination of chicken red blood cells was inhibited by treatment of compound 30, and the interaction between the influenza hemagglutinin (HA) and compound 30 was determined by surface plasmon resonance (SPR) with a dissociation constant of KD = 3.76 µM. Finally, silico docking studies indicated that compound 30 and its derivative 31 were able to occupy the binding pocket of HA for sialic acid receptor. Collectively, these results suggested that l-ascorbic acid-conjugated pentacyclic triterpenes were promising anti-influenza entry inhibitors, and HA protein associated with viral entry was a promising drug target.

Phylodynamic Characterization of an Ocular-Tropism Coxsackievirus A24 Variant.

Recent phylodynamic studies have focused on using tree topology patterns to elucidate interactions among the epidemiological, evolutionary, and demographic characteristics of infectious agents. However, because studies of viral phylodynamics tend to focus on epidemic outbreaks, tree topology signatures of tissue-tropism pathogens might not be clearly identified. Therefore, this study used a novel Bayesian evolutionary approach to analyze the A24 variant of coxsackievirus (CV-A24v), an ocular-tropism agent of acute hemorrhagic conjunctivitis. Analyses of the 915-nucleotide VP1 and 690-nt 3Dpol regions of 21 strains isolated in Taiwan and worldwide during 1985-2010 revealed a clear chronological trend in both the VP1 and 3Dpol phylogenetic trees: the emergence of a single dominant cluster in each outbreak. The VP1 sequences included three genotypes: GI (prototype), GIII (isolated 1985-1999), and GIV (isolated after 2000); no VP1 sequences from GII strains have been deposited in GenBank. Another five genotypes identified in the 3Dpol region had support values >0.9. Geographic and demographic transitions among CV-A24v clusters were clearly identified by Bayes algorithm. The transmission route was mapped from India to China and then to Taiwan, and each prevalent viral population declined before new clusters emerged. Notably, the VP1 and 3Dpol genes had high nucleotide sequence similarities (94.1% and 95.2%, respectively). The lack of co-circulating lineages and narrow tissue tropism affected the CV-A24v gene pool.

Transmission and Demographic Dynamics of Coxsackievirus B1.

The infectious activity of coxsackievirus B1 (CV-B1) in Taiwan was high from 2008 to 2010, following an alarming increase in severe neonate disease in the United States (US). To examine the relationship between CV-B1 strains isolated in Taiwan and those from other parts of the world, we performed a phylodynamic study using VP1 and partial 3Dpol (414 nt) sequences from 22 strains of CV-B1 isolated in Taiwan (1989-2010) and compared them to sequences from strains isolated worldwide. Phylogenetic trees were constructed by neighbor-joining, maximum likelihood, and Bayesian Monte Carlo Markov Chain methods. Four genotypes (GI-IV) in the VP1 region of CV-B1 and three genotypes (GA-C) in the 3Dpol region of enterovirus B were identified and had high support values. The phylogenetic analysis indicates that the GI and GIII strains in VP1 were geographically distributed in Taiwan (1993-1994) and in India (2007-2009). On the other hand, the GII and GIV strains appear to have a wider spatiotemporal distribution and ladder-like topology A stair-like phylogeny was observed in the VP1 region indicating that the phylogeny of the virus may be affected by different selection pressures in the specified regions. Further, most of the GI and GII strains in the VP1 tree were clustered together in GA in the 3D tree, while the GIV strains diverged into GB and GC. Taken together, these data provide important insights into the population dynamics of CV-B1 and indicate that incongruencies in specific gene regions may contribute to spatiotemporal patterns of epidemicity for this virus.

Analysis of circulating cholesterol levels as a mediator of an association between ABO blood group and coronary heart disease.

BACKGROUND: Non-O type of ABO blood group has been associated with a predisposition to coronary heart disease. It is thought that this association is partly mediated by increased cholesterol levels in non-O-type individuals. In this study, we sought to estimate the mediation effect size. METHODS AND RESULTS: In a group of individuals (n=6476) undergoing coronary angiography, we detected associations of non-O type with significant coronary artery disease with >50% stenosis in ≥1 coronary arteries (odds ratio, 1.24; 95% confidence interval, 1.10-1.39; P=2.6×10(-4)) and with prevalent or incident myocardial infarction (odds ratio, 1.22; 95% confidence interval, 1.09-1.37; P=1.2×10(-3)). Subjects of non-O type had higher levels of total cholesterol, low-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol (mean [SEM] in mmol/L: 4.931[0.021], 3.041 [0.018], and 3.805 [0.020] in non-O type compared with 4.778 [0.026], 2.906 [0.021], and 3.669 [0.024] in O type; P=3.8×10(-7), P=1.5×10(-7), and P=3.1×10(-7), respectively). Mediation analyses indicated that 10% of the effect of non-O type on coronary artery disease susceptibility was mediated by increased low-density lipoprotein cholesterol level (P=7.8×10(-4)) and that 11% of the effect of non-O type on myocardial infarction risk was mediated by raised low-density lipoprotein cholesterol level (P=2.0×10(-3)). CONCLUSIONS: In a model in which it is presumed that cholesterol is a mediator of the associations of ABO group with coronary artery disease and myocardial infarction, around 10% of the effect of non-O type on coronary artery disease and myocardial infarction susceptibility was mediated by its influence on low-density lipoprotein cholesterol level.

Discovery of pentacyclic triterpenoids as potential entry inhibitors of influenza viruses.

Entry inhibitors are of particular importance in current efforts to develop a new generation of anti-influenza virus drugs. Here we report certain pentacyclic triterpenes exhibiting conserved structure features and with in vitro anti-influenza virus activity comparable to and even higher than that of oseltamivir. Mechanistic studies indicated that these lead triterpenoids bind tightly to the viral envelope hemagglutinin (HA), disrupting the interaction of HA with the sialic acid receptor and thus the attachment of viruses to host cells. Docking studies suggest that the binding pocket within HA for sialic acid receptor potentially acts as a targeting domain, and this is supported by structure-activity data, sialic acid competition studies, and broad anti-influenza spectrum as well as less induction of drug resistance. Our study might establish the importance of triterpenoids for development of entry inhibitors of influenza viruses.

Synthesis and anti-HCV entry activity studies of ß-cyclodextrin-pentacyclic triterpene conjugates.

In our previous studies, oleanolic acid (OA) and echinocystic acid (EA), isolated from Dipsacus asperoides, were found to have anti-HCV entry properties. The major issue for members of this type of triterpene is their low water solubility. In this study, a series of new water-soluble triazole-bridged ß-cyclodextrin (CD)-pentacyclic triterpene conjugates were synthesized via click chemistry. Thanks to the attached ß-CD moiety, all synthesized conjugates showed lower hydrophobicity (Alog P) than their parent compounds. Several conjugates exhibited moderate anti-HCV entry activity. With the exception of per-O-methylated ß-CD-pentacyclic triterpene conjugates, all other conjugates showed no cytotoxicity based on an alamarBlue assay carried out with HeLa, HepG2, MDCK, and 293T cells. More interestingly, the hemolytic activity of these conjugates disappeared upon the introduction of ß-CDs. Easy access to such conjugates that combine the properties of ß-CD and pentacyclic triterpenes may provide a way to obtain a new class of anti-HCV entry inhibitors.

Development of bivalent oleanane-type triterpenes as potent HCV entry inhibitors.

The development of entry inhibitors is an emerging approach to the prevention and reduction of HCV infection. Starting from echinocystic acid (EA), a µM HCV entry inhibitor, we have developed a series of bivalent oleanane-type triterpenes which, upon optimization of the length, rigidity and hydrophobicity of the linker, exert dramatically potent enhancement of inhibition with IC50 values extending into the nM level. This study establishes the importance of triterpene natural products as new leads in the development of potential HCV entry inhibitors.

Accumulation of the mutations in basal core promoter of hepatitis B virus subgenotype C1 increase the risk of hepatocellular carcinoma in Southern China.

UNLABELLED: Hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, HBV subgenotype C1 is the major subgenotype in Southern China. The aim of this study was to investigate whether there was the specific mutation patterns in HBV/C1 associated with Southern Chinese patients with HCC. METHODS: Mutations in HBV basal core promoter (BCP) and their association with HCC were assessed in a matched cross-sectional control study of 102 HCC and 105 chronic hepatitis (CH) patients (from Guangdong, China) infected with HBV/C1. Functional analysis of HBx mutants was performed by the colony formation assay and the luciferase assays. RESULTS: T1762/A1764 double mutations was frequently found in patients infected with HBV/C1, regardless of clinical status (64.7% in HCC and 51.4% in CH, P>0.05). Unexpectedly, the adjacent V1753 or A1768 mutation significantly increased the risk of HCC (P<0.05). Moreover, the prevalence of triple or quadruple mutations in BCP was significantly higher in patients with HCC than those with CH, particularly for HBeAg-positive-carriers (P<0.05). Functional analysis revealed that T1762/A1764 mutation alone did not alter the transcriptional activity and the inhibitory effects on cell proliferation of HBx, but triple or quadruple mutations largely abrogated this effect. CONCLUSIONS: Accumulation of mutations involving V1753 or/and A1768 in addition to T1762/A1764 in BCP region were closely related to HCC among the patients infected with HBV/C1, particularly for HBeAg-positive-carriers. The increased risk of HCC caused by BCP variants may be attributable partially to modifying the biological functions of HBx.

Development of oleanane-type triterpenes as a new class of HCV entry inhibitors.

Development of hepatitis C virus (HCV) entry inhibitors represents an emerging approach that satisfies a tandem mechanism for use with other inhibitors in a multifaceted cocktail. By screening Chinese herbal extracts, oleanolic acid (OA) was found to display weak potency to inhibit HCV entry with an IC50 of 10 µM. Chemical exploration of this triterpene compound revealed its pharmacophore requirement for blocking HCV entry, rings A, B, and E, are conserved while ring D is tolerant of some modifications. Hydroxylation at C-16 significantly enhanced its potency for inhibiting HCV entry with IC50 at 1.4 µM. Further modification by conjugation of this new lead with a disaccharide at 28-COOH removed the undesired hemolytic effect and, more importantly, increased its potency by ~5-fold (54a, IC50 0.3 µM). Formation of a triterpene dimer via a linker bearing triazole (70) dramatically increased its potency with IC50 at ~10 nM. Mechanistically, such functional triterpenes interrupt the interaction between HCV envelope protein E2 and its receptor CD81 via binding to E2, thus blocking virus and host cell recognition. This study establishes the importance of triterpene natural products as new leads for the development of potential HCV entry inhibitors.