Longlong Si

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.

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.

Neoechinulin B and its analogues as potential entry inhibitors of influenza viruses, targeting viral hemagglutinin

A class of prenylated indole diketopiperazine alkaloids including 15 new compounds namely rubrumlines A-O obtained from marine-derived fungus Eurotium rubrum, were tested against influenza A/WSN/33 virus. Neoechinulin B (18) exerted potent inhibition against H1N1 virus infected in MDCK cells, and is able to inhibit a panel of influenza virus strains including amantadine- and oseltamivir-resistant clinical isolates. Mechanism of action studies indicated that neoechinulin B binds to influenza envelope hemagglutinin, disrupting its interaction with the sialic acid receptor and the attachment of viruses to host cells. In addition, neoechinulin B was still efficient in inhibiting influenza A/WSN/33 virus propagation even after a fifth passage. The high potency and broad-spectrum activities against influenza viruses with less drug resistance make neoechinulin B as a new lead for the development of potential inhibitor of influenza viruses.

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.

Nitrosporeusines A and B, unprecedented thioester-bearing alkaloids from the Arctic Streptomyces nitrosporeus.

Chemical examination of an arctic actinomycete Streptomyces nitrosporeus resulted in the isolation of two new alkaloids named nitrosporeusines A (1) and B (2), an unprecedented skeleton containing benzenecarbothioc cyclopenta[c]pyrrole-1,3-dione. Their structures were determined through extensive spectroscopic analyses in association with X-ray single crystal diffraction. Both 1 and 2 exhibited inhibitory activities against the H1N1 virus in MDCK cells.

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.

Development of next generation of therapeutic IFN-α2b via genetic code expansion.

With the aim to overcome the heterogeneity associated with marketed IFN-α2b PEGylates and optimize the size of the PEG moiety and the site of PEGylation, we develop a viable and facile platform through genetic code expansion for PEGylation of IFN-α2b at any chosen site(s). This approach includes site-specific incorporation of an azide-bearing amino acid into IFN-α2b followed by orthogonal and stoichiometric conjugation of a variety of PEGs via a copper-free click reaction. By this approach, only the chosen site(s) within IFN-α2b is consistently PEGylated under mild conditions, leading to a single and homogenous conjugate. Furthermore, it makes the structure-activity relationship study of IFN-α2b possible by which the opposite effects of PEGylation on the biological and pharmacological properties are optimized. Upon re-examination of the PEGylated IFN-α2b isomers carrying different sizes of PEG at different sites, we find mono-PEGylates at H34, A74 and E107 with a 20-, 10- and 10-kDa PEG moiety, respectively, have both higher biological activities and better PK profiles than others. These might represent the direction for development of the next generation of PEGylated IFN-α2b.

Re-exploration of the Codon Context Effect on Amber Codon-Guided Incorporation of Noncanonical Amino Acids in Escherichia coli by the Blue–White Screening Assay

The effect of codon context on amber codon‐guided incorporation of noncanonical amino acids (NAAs) has been previously examined by antibiotic selection. Here, we re‐explored this effect by screening a library in which three nucleotides upstream and downstream of the amber codon were randomised, and inserted within the lacZ‐α gene. Thousands of clones were obtained and distinguished by the depth of blue colour upon exposure to X‐gal. Large‐scale sequencing revealed remarkable preferences in nucleotides downstream of the amber codon, and moderate preferences for upstream nucleotides. Nucleotide preference was quantified by a dual‐luciferase assay, which verified that the optimum context for NAA incorporation, AATTAGACT, was applicable to different proteins. Our work provides a general guide for engineering amber codons into genes of interest in bacteria.

Design, synthesis and biological activity evaluation of novel conjugated sialic acid and pentacyclic triterpene derivatives as anti-influenza entry inhibitors

Influenza virus is a major human pathogen that causes annual epidemics and occasional pandemics. Recently, plant-derived pentacyclic triterpenes have been shown to act as highly potent anti-viral agents by efficiently preventing the attachment of the virion to the host cells. In this report, we conjugated sialic acid with oleanolic acid (OA), a natural product with broad antiviral entry activity, as well as three other analogs echinocystic acid (EA), ursolic acid (UA) and betulinic acid (BA). A total of 24 conjugated sialic acid and pentacyclic triterpene derivatives with different linkers were synthesized and evaluated for antiviral activity against influenza A/WSN/33 (H1N1) virus in MDCK cell culture. The most potent compound had an IC50 of 41.2 μM. Time-of-addition, hemagglutination inhibition (HI), surface plasmon resonance (SPR) and molecular docking assays demonstrated that compound 20a acted as an influenza virus entry inhibitor by preventing the binding of influenza virus hemagglutinin (HA) protein to host cells.

Recent progress in the antiviral activity and mechanism study of pentacyclic triterpenoids and their derivatives

Viral infections cause many serious human diseases with high mortality rates. New drug‐resistant strains are continually emerging due to the high viral mutation rate, which makes it necessary to develop new antiviral agents. Compounds of plant origin are particularly interesting. The pentacyclic triterpenoids (PTs) are a diverse class of natural products from plants composed of three terpene units. They exhibit antitumor, anti‐inflammatory, and antiviral activities. Oleanolic, betulinic, and ursolic acids are representative PTs widely present in nature with a broad antiviral spectrum. This review focuses on the recent literatures in the antiviral efficacy of this class of phytochemicals and their derivatives. In addition, their modes of action are also summarized.