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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.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.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.The GLUT-1-targeted delivery of TPS increased pulmonary specificity and enhanced TPS anti-PAH activities in vivo and in vitro.

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.

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

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.

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.

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.

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 Zemplen 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.

To identify tumour and tumour-associated antigens in patients with hepatocellular carcinoma (HCC) one may find potential diagnostic markers and immunotherapeutic targets. In the current study, 30 distinct antigens reactive with serum IgG from HCC patients were identified by serological analysis of cDNA expression libraries (SEREX). The mRNA expression patterns of 14 of these 30 antigens were altered in cancer as further revealed by cDNA microarray, with upregulation for nine and downregulation for five antigens. One of the upregulated antigens was cancer-testis (CT) antigen (CAGE), which had been previously reported to be expressed exclusively in normal gametogenic tissues and aberrantly expressed in a variety of cancer cells. In our study, CAGE mRNA was expressed in 39.4% of HCC patients, 73.3% of patients with gastric cancer and 30.8% of patients with colorectal cancer. Antibodies against CAGE protein were detected in approximately 5.1% of the sera from HCC patients, 8.3% of that from gastric cancer patients and 7.3% of that from colorectal cancer patients. The relative high incidence of CAGE in cancer cells makes it a potential target for vaccine design. Another antigen of great interest is transgelin 2. The overexpression of transgelin 2 mRNA in a large per cent (69%) of HCC points to its potential as a diagnostic marker for HCC.

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 β-cyclodextrin (CD)-pentacyclic triterpene conjugates were synthesized via click chemistry. Thanks to the attached β-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 β-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 β-CDs. Easy access to such conjugates that combine the properties of β-CD and pentacyclic triterpenes may provide a way to obtain a new class of anti-HCV entry inhibitors.

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.

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.

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