Synthesis of Novel Pentacyclic Triterpenoid Derivatives that Induce Apoptosis in Cancer Cells through a ROS-dependent, Mitochondrial-Mediated Pathway.

Betulinic acid (BA) and oleanolic acid (OA) are plant-derived conjugates found in various medicinal plants that have emerged as potential antitumor agents. Herein, a series of novel BA and OA derivatives were synthesized by conjugation with per-O-methylated-ß-cyclodextrin (PM-ß-CD), and their anticancer properties against a panel of three human cancer cell lines were evaluated. Two OA-PM-ß-CD conjugates (48 and 50) were observed to be the most potent conjugates against the three cell lines (MCF-7, BGC-823, and HL-60), with a 15- to 20-fold decrease in the IC50 values (IC50: 6.06-8.47 µM) compared with their parental conjugate (OA). Annexin V-FITC/propidium iodide staining and Western blot analysis revealed that both conjugates induced apoptosis in HL-60 cells. Additionally, in the representative conjugate 48-treated HL-60 cells, a decrease in mitochondrial membrane potential and subsequent release of cytochrome c into the cytosol were observed, indicating the activation of the intrinsic apoptosis pathway. Furthermore, 48 dramatically induced the generation of reactive oxygen species (ROS) in HL-60 cells, and the corresponding effect could be reversed using the ROS scavenger N-acetylcysteine. Collectively, these results suggest that the novel pentacyclic triterpenoid derivatives trigger the intrinsic apoptotic pathways via the ROS-mediated activation of caspase-3 signaling, inducing cell death in human cancer cells.

Facial Synthesis and Bioevaluation of Well-Defined OEGylated Betulinic Acid-Cyclodextrin Conjugates for Inhibition of Influenza Infection.

Betulinic acid (BA) and its derivatives exhibit a variety of biological activities, especially their anti-HIV-1 activity, but generally have only modest inhibitory potency against influenza virus. The entry of influenza virus into host cells can be competitively inhibited by multivalent derivatives targeting hemagglutinin. In this study, a series of hexa-, hepta- and octavalent BA derivatives based on α-, ß- and γ-cyclodextrin scaffolds, respectively, with varying lengths of flexible oligo(ethylene glycol) linkers was designed and synthesized using a microwave-assisted copper-catalyzed 1,3-dipolar cycloaddition reaction. The generated BA-cyclodextrin conjugates were tested for their in vitro activity against influenza A/WSN/33 (H1N1) virus and cytotoxicity. Among the tested compounds, 58, 80 and 82 showed slight cytotoxicity to Madin-Darby canine kidney cells with viabilities ranging from 64 to 68% at a high concentration of 100 µM. Four conjugates 51 and 69-71 showed significant inhibitory effects on influenza infection with half maximal inhibitory concentration values of 5.20, 9.82, 7.48 and 7.59 µM, respectively. The structure-activity relationships of multivalent BA-cyclodextrin conjugates were discussed, highlighting that multivalent BA derivatives may be potential antiviral agents against influenza infection.

In vitro elimination of autoreactive B cells from rheumatoid arthritis patients by universal chimeric antigen receptor T cells.

OBJECTIVES: Autoreactive B cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA), and B cell-depleting therapies using an antibodies, such as rituximab, have been suggested to be effective in RA treatment. However, transient B cell depletion with rituximab is associated with significant safety challenges related to global suppression of the immune system and thus increases the risks of infection and cancer development. To address selective and persistent issues associated with RA therapy, we developed a customised therapeutic strategy employing universal antifluorescein isothiocyanate (FITC) chimeric antigen receptor T cells (CAR-T cells) combined with FITC-labelled antigenic peptide epitopes to eliminate autoreactive B cell subsets recognising these antigens in RA. METHODS: For a proof-of-concept study, four citrullinated peptide epitopes derived from citrullinated autoantigens, namely, citrullinated vimentin, citrullinated type II collagen, citrullinated fibrinogen and tenascin-C, and a cyclocitrulline peptide-1 were selected as ligands for targeting autoreactive B cells; Engineered T cells expressing a fixed anti-FITC CAR were constructed and applied as a universal CAR-T cell system to specifically eliminate these protein-specific autoreactive B cells via recognition of the aforementioned FITC-labelled autoantigenic peptide epitopes. RESULTS: We demonstrated that anti-FITC CAR-T cells could be specifically redirected and kill hybridoma cells generated by immunisation with antigenic peptides, and autoreactive B cell subsets from RA patients via recognition of corresponding FITC-labelled citrullinated peptide epitopes. Additionally, the cytotoxicity of the CAR-T cells was dependent on the presence of the peptides and occurred in a dose-dependent manner. CONCLUSIONS: The approach described here provides a direction for precise, customised approaches to treat RA and can likely be applied to other systemic autoimmune diseases.

Generation of a caged lentiviral vector through an unnatural amino acid for photo-switchable transduction.

Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging-uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.

Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation*.

There is an urgent need to develop antiviral drugs and alleviate the current COVID-19 pandemic. Herein we report the design and construction of chimeric oligonucleotides comprising a 2'-OMe-modified antisense oligonucleotide and a 5'-phosphorylated 2'-5' poly(A)4 (4A2-5 ) to degrade envelope and spike RNAs of SARS-CoV-2. The oligonucleotide was used for searching and recognizing target viral RNA sequence, and the conjugated 4A2-5 was used for guided RNase L activation to sequence-specifically degrade viral RNAs. Since RNase L can potently cleave single-stranded RNA during innate antiviral response, degradation efficiencies with these chimeras were twice as much as those with only antisense oligonucleotides for both SARS-CoV-2 RNA targets. In pseudovirus infection models, chimera-S4 achieved potent and broad-spectrum inhibition of SARS-CoV-2 and its N501Y and/or ΔH69/ΔV70 mutants, indicating a promising antiviral agent based on the nucleic acid-hydrolysis targeting chimera (NATAC) strategy.

Synthesis of a Hexavalent Betulinic Acid Derivative as a Hemagglutinin-Targeted Influenza Virus Entry Inhibitor.

Naturally occurring pentacyclic triterpenes, such as betulinic acid (BA) and its derivatives, exhibit various pharmaceutical activities and have been the subject of great interest, in particular for their antiviral properties. Here, we found a new anti-influenza virus conjugate, hexakis 6-deoxy-6-[4-N-(3ß-hydroxy-lup-20(29)-en-28-oate)aminomethyl-1H-1,2,3-triazol-1-yl]-2,3-di-O-acetyl-α-cyclodextrin (CYY1-11, 1), in a mini library of pentacyclic triterpene-cyclodextrin conjugates by performing a cell-based screening assay and then exploring the underlying mechanisms. Our results showed that conjugate 1 possessed a high-level activity against the influenza virus A/WSN/33 with an IC50 value of 5.20 µM (SI > 38.4). The study of the mechanism of action indicated that conjugate 1 inhibited viral replication by directly targeting the influenza hemagglutinin protein (KD = 1.50 µM), thus efficiently preventing the attachment of the virion to its receptors on host cells and subsequent infection. This study suggests that multivalent BA derivatives have possible use as a new class of influenza virus entry inhibitors.

Design, synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors.

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza virus, we designed and synthesized 32 oleanolic acid (OA) analogues in this study by conjugating different amino acids to the 28-COOH of OA. The antiviral activity of these compounds was evaluated in vitro. Some of these compounds revealed impressive anti-influenza potencies against influenza A/WSN/33 (H1N1) virus. Among them, compound 15a exhibited robust potency and broad antiviral spectrum with IC50 values at the low-micromolar level against four different influenza strains. Hemagglutination inhibition (HI) assay and docking experiment indicated that these OA analogues may act in the same way as their parent compound by interrupting the interaction between HA protein of influenza virus and the host cell sialic acid receptor via binding to HA, thus blocking viral entry.

Synthesis, Structure Activity Relationship and Anti-influenza A Virus Evaluation of Oleanolic Acid-Linear Amino Derivatives.

Oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor in our earlier studies. In the present work, 20 compounds were prepared by structural modifications of OA, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in Madin-Darby canine kidney (MDCK) cells were evaluated. Based on the biological result, structure-activity relationship (SAR) was discussed. Compound 10 with six-carbon chain and a terminal hydroxyl group showed the strongest anti-influenza activity with an IC50 of 2.98 µM, which is an order of magnitude more potent than OA. Hemagglutination inhibition and Surface plasmon resonance (SPR) assay indicated that compound 10 might interfere with influenza invasion by interacting with HA protein.

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.

Medium Rings Bearing Bitriazolyls: Easily Accessible Structures with Superior Performance as Cu Catalyst Ligands.

Benefiting from their unique properties, the development of structurally novel and easily accessible medium rings is of significant interest in the pharmaceutical industry and academic research. However, synthetic access to medium-ring scaffolds is very difficult due to their rigid skeleton and large-angle strains. In this paper, a new class of medium rings bearing bitriazolyls (MRBTs) was designed, synthesized, identified as a promising new skeleton ligand for the Cu(I)-catalyzed click reaction, and used in site-special modification of protein. One of the MRBTs, 3aa, exhibited a turnover number (TON) as high as 55 000 and dramatic accelerating effects ( kobs = 1.95 M-1 s-1) and ranked among the most efficient ligands for copper-catalyzed alkyne and azide cycloaddition. Unlike the difficult access to other known medium rings, these 7-12-membered MRBTs can be prepared in straightforward, one-step manner from structurally diverse linear terminal diynes and azides. The unique accessibility and intriguing properties therefore imply their broad application perspectives.

Construction of an inducible stable cell line for efficient incorporation of unnatural amino acids in mammalian cells.

The genetic incorporation of unnatural amino acids (Uaas) with defined properties into proteins at designated sites represents an extremely powerful tool for protein engineering. However, the efficient incorporation of Uaas in response to the amber stop codon in mammalian cells remains a substantial challenge due to the competition from release factor 1(RF1). Addressing this challenge will greatly broaden the power and scope of this technology. Here, we chose the eRF1 mutant, which can selectively enhance Uaa incorporation in response to the amber codon without increasing the readthrough of the opal and ochre codons. Then, we developed an engineered stable cell line using a tetracycline-controlled inducible lentiviral system for the conditional expression of mutant eRF1, which can minimize the potential effect on normal translation termination. Using the eRF1-engineered cells, we provided a 2-fold improvement in the yield of protein containing a Uaa incorporated at a single site, with the protein yield approaching 90% of the wild-type control without the amber codon. Moreover, we achieved the successful incorporation of Uaas at four sites in various proteins at a measured level of 20%.

Redirecting Killer T†Cells through Incorporation of Azido Sugars for Tethering Ligands.

The genetic expression of chimeric antigen receptors (CARs) on the surfaces of T cells enables the redirection of T cell specificity. To enhance the versatility of T cells as tumor-specific killers, we developed a nongenetic approach by which azide-containing sialic acids were metabolically incorporated into T cells to modify cellular sialyl glycans. After successful display of these moieties on the T cells, small-molecule ligands such as RGD and folate (as proof-of-concept, rather than supersized antibodies) were clicked orthogonally, leading to highly selective time- and dose-dependent cytotoxicity to integrin αv ß3 - and folate-receptor-positive cells, respectively. This chemical approach provides a facile platform for rational design of tumor-specific cytotoxic T cells for targeted immunotherapy.

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.

Site-Specific PEGylated Adeno-Associated Viruses with Increased Serum Stability and Reduced Immunogenicity.

Adeno-associated virus (AAV) is one of the most extensively studied and utilized viral vectors in clinical gene transfer research. However, the serum instability and immunogenicity of AAV vectors significantly limit their application. Here, we endeavored to overcome these limitations by developing a straightforward approach for site-specific PEGylation of AAV via genetic code expansion. This technique includes incorporation of the azide moiety into the AAV capsid protein followed by orthogonal and stoichiometric conjugation of a variety of polyethylene glycols (PEGs) through click chemistry. Using this approach, only the chosen site(s) was consistently PEGylated under mild conditions, preventing nonselective conjugation. Upon a series of in vitro examinations, AAVs conjugated with 20-kD PEG at sites Q325+1, S452+1, and R585+1 showed a 1.7- to 2.4-fold stability improvement in pooled human serum and a nearly twofold reduction in antibody recognition. Subsequent animal research on Sprague Dawley rats displayed a promising 20% reduction in antibody inducement and a higher virus titer in the blood. Together, our data demonstrate successful protection of an AAV vector from antibody neutralization and blood clearance, thereby increasing the efficiency of therapeutic gene delivery.

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.

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.

Synthesis of Site-Specific Radiolabeled Antibodies for Radioimmunotherapy via Genetic Code Expansion.

Radioimmunotherapy (RIT) delivers radioisotopes to antigen-expressing cells via monoantibodies for the imaging of lesions or medical therapy. The chelates are typically conjugated to the antibody through cysteine or lysine residues, resulting in heterogeneous chelate-to-antibody ratios and various conjugation sites. To overcome this heterogeneity, we have developed an approach for site-specific radiolabeling of antibodies by combination of genetic code expansion and click chemistry. As a proof-of-concept study, model systems including anti-CD20 antibody rituximab, positron-emitting isotope 64Cu, and a newly synthesized bifunctional linker (4-dibenzocyclooctynol-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid, DIBO-DOTA) were used. The approach consists of three steps: (1) site-specific incorporation of an azido group-bearing amino acid (NEAK) via the genetic code expansion technique at the defined sites of the antibody as a "chemical handle"; (2) site-specific and quantitative conjugation of bifunctional linkers with the antibodies under a mild condition; and (3) radiolabeling of the chelate-modified antibodies with the appropriate isotope. We used heavy-chain A122NEAK rituximab as proof-of-concept and obtained a homogeneous radioconjugate with precisely two chelates per antibody, incorporated only at the chosen sites. The conjugation did not alter the binding and pharmacokinetics of the rituximab, as indicated by in vitro assays and in vivo PET imaging. We believe our research is a good supplement to the genetic code expansion technique for the development of novel radioimmunoconjugates.

Bioactive Constituents of Glycyrrhiza uralensis (Licorice): Discovery of the Effective Components of a Traditional Herbal Medicine.

Traditional herbal medicines have been reported to possess significant bioactivities. In this investigation, a combined strategy using both phytochemical and biological approaches was conducted to discern the effective components of licorice, a widely used herbal medicine. Altogether, 122 compounds (1-122), including six new structures (1-6), were isolated and identified from the roots and rhizomes of Glycyrrhiza uralensis (licorice). These compounds were then screened using 11 cell- and enzyme-based bioassay methods, including Nrf2 activation, NO inhibition, NF-κB inhibition, H1N1 virus inhibition, cytotoxicity for cancer cells (HepG2, SW480, A549, MCF7), PTP1B inhibition, tyrosinase inhibition, and AChE inhibition. A number of bioactive compounds, particularly isoprenylated phenolics, were found for the first time. Echinatin (7), a potent Nrf2 activator, was selected as an example for further biological work. It attenuated CCl4-induced liver damage in mice (5 or 10 mg/kg, ip) and thus is responsible, at least in part, for the hepatoprotective activity of licorice.

Transition Metal-Free Oxidative Radical Decarboxylation/Cyclization for the Construction of 6-Alkyl/Aryl Phenanthridines.

A radical cascade decarboxylation/cyclization of 2-isocyanobiphenyls with aliphatic carboxylic acids as well as aromatic carboxylic acids under the transition metal-free conditions was reported. This process, which included formation of two new C-C bonds and cleavage of C-COOH bonds, afforded a novel and environmentally friendly approach to producing 6-alkyl/aryl phenanthridines with moderate to good yields.