Sulong Xiao

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.

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.

Elucidation of the pharmacophore of echinocystic acid, a new lead for blocking HCV entry

To elucidate the pharmacophore of echinocystic acid (EA), an oleanane-type triterpene displaying substantial inhibitory activity on HCV entry, two microbial strains, Rhizopus chinensis CICC 3043 and Alternaria alternata AS 3.4578, were utilized to modify the chemical structure of EA. Eight new metabolites with regio- and stereo-selective introduction of hydroxyl and lactone groups at various inert carbon positions were obtained. The anti-HCV entry activity of the metabolites 2-13, along with their parental compound EA and other analogs 14-15, were evaluated. Most of the metabolites showed no improvement but detrimental effect on potency except compound 5 and 6, which showed similar and even a litter higher anti-HCV entry activity than that of EA. The results demonstrated that ring A, B, C and the left side of ring E of EA are highly conserved, while ring D and the right side of ring E of EA are flexible. Introduction of a hydroxyl group at C-16 enhanced the triterpene potency. Further analysis indicated that the hemolytic effect of EA disappeared upon such modifications.

Generation of influenza A viruses as live but replication-incompetent virus vaccines

Genetic code expansion and orthogonal translation machinery are used to generate live, attenuated viral vaccines. Protecting by changing the code Live attenuated vaccines can be very potent, but their potential to revert to their pathogenic form limits their use. In an attempt to get around this, Si et al. expanded the genetic code of influenza A viruses. They propagated viruses that were mutated to encode premature termination codons (PTCs) in a cell line engineered to be able to express these flu proteins. Despite not being able to replicate in conventional cells, PTC-containing viruses were highly immunogenic and protected mice, guinea pigs, and ferrets against influenza challenge. Science, this issue p. 1170 The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)–harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells. In mouse, ferret, and guinea pig models, vaccination with PTC viruses elicited robust humoral, mucosal, and T cell–mediated immunity against antigenically distinct influenza viruses and even neutralized existing infecting strains. The methods presented here may become a general approach for generating live virus vaccines that can be adapted to almost any virus.

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.

Synthesis and Anti‐HCV Entry Activity Studies of β‐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 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.

Conjugation of cyclodextrin with fullerene as a new class of HCV entry inhibitors.

An α-cyclodextrin-[60]fullerene conjugate with a flexible linker at the secondary face of α-cyclodextrin has been prepared, which displays significant water solubility and, more importantly, acts as a new class of HCV entry inhibitor with IC(50) at 0.17 μM level.

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.

Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion

With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure–function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus–host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

Synthesis and biological evaluation of ring A and/or C expansion and opening echinocystic acid derivatives for anti-HCV entry inhibitors.

Echinocystic acid (EA), a naturally occurring oleanane-type triterpene isolated from Dipsacus asperoides, was found to have anti-HCV entry activity in our previous study. Expansion of triterpene structural diversity, including the ring A and/or C expansion and opening, was performed. To elucidate the pharmacophore of EA, seven lactones (8, 16, 17, 24, 26, 35 and 41), three 3,28-dioic acids (9, 36 and 42) and two pentols (10 and 27) were synthesized. The anti-HCV entry activities of those derivatives, along with their parental compound EA and analogs α,β-unsaturated ketone (18), were evaluated. All the products showed no improvement but detrimental effect on potency of EA. The results demonstrated that ring A and C of EA are highly conserved, indicating the steric effects of the rigid skeleton have a profound effect on the potency.