3-Oxidopyridinium Ions Are Versatile Bioorthogonal Dipoles for Use in Cycloadditions with Cyclooctynes.

3-oxidopyridinium ions are water stable and soluble heteroaromatic betaines that behave as latent dipoles and undergo a wide variety of cycloadditions. Research into the cycloaddition reactions of 3-oxidopyridiniums was spearheaded by Alan R. Katritzky and collaborators from the early 1970s until the late 1980s, but they have yet to be used for bioorthogonal applications. Herein we report that 3-oxidopyridiniums can readily react with 4-dibenzocyclooctynol (DIBO), a common bioorthogonal handle, in a [3+2] cycloaddition. The mechanism was investigated by altering the electronics of the reaction by changing the substituent on the 5 position of the pyridinium. Electron-donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.3×10-4 s-1 with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07 M-1 s-1 with 5-amino-N-methyl-3-oxidopyridinium. 3-oxidopyridiniums' appreciable cycloaddition rates and compatibility with bioorthogonally relevant environments give them the potential to be used in a variety of bioconjugation applications.

microRNA-27b regulates hepatic lipase enzyme LIPC and reduces triglyceride degradation during hepatitis C virus infection.

miRNAs are short, noncoding RNAs that negatively and specifically regulate protein expression, the cumulative effects of which can result in broad changes to cell systems and architecture. The miRNA miR-27b is known to regulate lipid regulatory pathways in the human liver and is also induced by the hepatitis C virus (HCV). However, the functional targets of miR-27b are not well established. Herein, an activity-based protein profiling method using a serine hydrolase probe, coupled with stable isotope labeling and mass spectrometry identified direct and indirect targets of miR-27b. The hepatic lipase C (LIPC) stood out as both highly dependent on miR-27b and as a major modulator of lipid pathway misregulation. Modulation of miR-27b using both exogenous miRNA mimics and inhibitors demonstrated that transcription factors Jun, PPARα, and HNF4α, all of which also influence LIPC levels and activity, are regulated by miR-27b. LIPC was furthermore shown to affect the progress of the life cycle of HCV and to decrease levels of intracellular triglycerides, upon which HCV is known to depend. In summary, this work has demonstrated that miR-27b mediates HCV infection by downregulating LIPC, thereby reducing triglyceride degradation, which in turn increases cellular lipid levels.

Bioorthogonal Reactions Utilizing Nitrones as Versatile Dipoles in Cycloaddition Reactions.

Bioorthogonal chemical reactions have emerged as convenient and rapid methods for incorporating unnatural functionality into living systems. Different prototype reactions have been optimized for use in biological settings. Optimization of 3 + 2 dipolar cycloadditions involving nitrones has resulted in highly efficient reaction conditions for bioorthogonal chemistry. Through substitution at the nitrone carbon or nitrogen atom, stereoelectronic tuning of the reactivity of the dipole has assisted in optimizing reactivity. Nitrones have been shown to react rapidly with cyclooctynes with bimolecular rate constants approaching k2 = 102 M-1 s-1, which are among the fastest bioorthogonal reactions reported (McKay et al. Org. Biomol. Chem. 2012, 10, 3066-3070). Nitrones have also been shown to react with trans-cyclooctenes (TCO) in strain-promoted TCO-nitrone cycloadditions reactions. Copper catalyzed reactions involving alkynes and nitrones have also been optimized for applications in biology. This review provides a comprehensive accounting of the different bioorthogonal reactions that have been developed using nitrones as versatile reactants, and provides some recent examples of applications for probing biological systems.

A conserved miRNA-183 cluster regulates the innate antiviral response.

Upon immune recognition of viruses, the mammalian innate immune response activates a complex signal transduction network to combat infection. This activation requires phosphorylation of key transcription factors regulating IFN production and signaling, including IFN regulatory factor 3 (IRF3) and STAT1. The mechanisms regulating these STAT1 and IRF3 phosphorylation events remain unclear. Here, using human and mouse cell lines along with gene microarrays, quantitative RT-PCR, viral infection and plaque assays, and reporter gene assays, we demonstrate that a microRNA cluster conserved among bilaterian animals, encoding miR-96, miR-182, and miR-183, regulates IFN signaling. In particular, we observed that the miR-183 cluster promotes IFN production and signaling, mediated by enhancing IRF3 and STAT1 phosphorylation. We also found that the miR-183 cluster activates the IFN pathway and inhibits vesicular stomatitis virus infection by directly targeting several negative regulators of IRF3 and STAT1 activities, including protein phosphatase 2A (PPP2CA) and tripartite motif-containing 27 (TRIM27). Overall, our work reveals an important role of the evolutionarily conserved miR-183 cluster in the regulation of mammalian innate immunity.

Cycloadditions of Trans-Cyclooctenes and Nitrones as Tools for Bioorthogonal Labelling.

Trans-cyclooctenes (TCOs) represent interesting and highly reactive dipolarophiles for organic transformations including bioorthogonal chemistry. Herein we show that TCOs react rapidly with nitrones and that these reactions are bioorthogonal. Kinetic analysis of acyclic and cyclic nitrones with strained-trans-cyclooctene (s-TCO) shows fast reactivity and demonstrates the utility of this cycloaddition reaction for bioorthogonal labelling. Labelling of the bacterial peptidoglycan layer with unnatural d-amino acids tagged with nitrones and s-TCO-Alexa488 is demonstrated. These new findings expand the bioorthogonal toolbox, and allow TCO reagents to be used in bioorthogonal applications beyond tetrazine ligations for the first time and open up new avenues for bioorthogonal ligations with diverse nitrone reactants.

A Bifunctional Nucleoside Probe for the Inhibition of the Human Immunodeficiency Virus-Type 1 Reverse Transcriptase.

Nucleoside analogs have proven effective for the inhibition of viral polymerases and are the foundation of many antiviral therapies. In this work, the antiretroviral potential of 6-azauracil analogs was assessed using activity-based protein profiling techniques and functional assays. Probes based on the 6-azauracil scaffold were examined and found to bind to HCV polymerase and HIV-1 reverse transcriptase through covalent modification of residues near the active site. The modified sites on the HIV-1 RT were examined using a mass spectrometry approach, and it was discovered that the azauracil moieties modified the enzyme in proximity to its active site. However, these scaffolds gave little or no inhibition of enzyme activity. Instead, a bifunctional inhibitor was prepared using click chemistry to link the 6-azauracil moiety to azidothymidine (AzT) and the corresponding triphosphate (AzTTP). These bifunctional inhibitors were found to have potent inhibitory function through a mode of action that includes both alkylation and chain termination. An in vitro assay demonstrated that the bifunctional inhibitor was 23-fold more effective in inhibiting HIV-1 RT activity than the parent AzTTP. The bifunctional inhibitor was also tested in HIV-1 permissive T cells where it decreased Gag expression similarly to the front-line drug Efavirenz with no evidence of cytotoxicity. This new bifunctional scaffold represents an interesting tool for inhibiting HIV-1 by covalently anchoring a chain-terminating nucleoside analog in the active site of the reverse transcriptase, preventing its removal and abolishing enzymatic activity, and represents a novel mode of action for inhibiting polymerases including reverse transcriptases.

ABPP and Host-Virus Interactions.

Successful viral infection, as well as any resultant antiviral response, relies on numerous sequential interactions between host and viral factors. These interactions can take the form of affinity-based interactions between viral and host macromolecules or active, enzyme-based interactions, consisting both of direct enzyme activity performed by viral enzymes and indirect modulation of the activity of the host cell's enzymes via viral interference. This activity has the potential to transform the local microenvironment to the benefit or detriment of both the virus and the host, favouring either the continuation of the viral life cycle or the host's antiviral response. Comprehensive characterisation of enzymatic activity during viral infection is therefore necessary for the understanding of virally induced diseases. Activity-based protein profiling techniques have been established as effective and practicable tools with which to interrogate the regulation of enzymes' catalytic activity and the roles played by these enzymes in various cell processes. This paper will review the contributions of these techniques in characterising the roles of both host and viral enzymes during viral infection in humans.

Site-Specific Cross-Linking of a p19 Viral Suppressor of RNA Silencing Protein and Its RNA Targets Using an Expanded Genetic Code.

The p19 viral suppressor of RNA silencing protein has useful applications in biotechnology due to its high affinity for binding to small RNAs such as small interfering RNAs (siRNAs). Also, its applications for the study and modulation of microRNAs are actively expanding. Here we demonstrate the successful site-specific incorporation of a photoactivatable unnatural amino acid, p-azido-l-phenylalanine (AzF), for cross-linking to RNA substrates into the p19 sequence. Incorporation of AzF was performed at three positions in the protein near the RNA binding site: K67, R115, and T111. Incorporation of AzF at position T111 of p19 did not affect the binding affinity of p19 for siRNAs and also showed nanomolar affinity for human microRNA miR-122. The affinity was less favorable with AzF incorporation at two other positions, suggesting the sensitivity of placement of the unnatural amino acid. Exposure of the T111AzF in complex with either siRNA or miRNA to ultraviolet light resulted in cross-linking of the protein with the RNA, but no cross-linking could be detected with the wild-type protein. Our results demonstrate that p19-T111AzF can be used for detection of small RNAs, including human miR-122, with high sensitivity and to irreversibly sequester these RNAs through covalent photo-cross-linking.

Visualization of the Delivery and Release of Small RNAs Using Genetic Code Expansion and Unnatural RNA-Binding Proteins.

Endogenously expressed noncoding RNAs are regulators of mRNA translation and affect diverse biological pathways spanning embryogenesis to cholesterol and fatty acid metabolism. Recently, microRNAs have become an important therapeutic target with strategies that employ oligonucleotides as both mimics and inhibitors of target microRNAs, successfully altering gene expression and cellular pathways in relevant contexts. However, delivery of these exogenous effectors remains a major challenge. Here, we present a method for evaluating noncoding RNA delivery using the viral suppressor of RNA silencing (VSRS) protein p19, optimized for cellular delivery of small RNAs. Using genetic code expansion technology, p-azidophenylalanine (AzF) was incorporated into a recombinant p19 protein and used to develop a fluorescence resonance energy transfer (FRET) sensor. AzF was used to attach FRET acceptor moieties using bioorthogonal chemistry. We show that this strategy not only gives rise to FRET signals that report on small RNA binding, but also allows for fluorescence quenching as well, convenient for measuring RNA release. We demonstrate the successful use of a modified version of the probe to track the delivery and release of small RNAs into mammalian cells. The results provide a basis for a further development of vehicles for small RNA delivery and release for intervening in noncoding RNA biology.

MicroRNAs regulate the immunometabolic response to viral infection in the liver.

Immune regulation of cellular metabolism can be responsible for successful responses to invading pathogens. Viruses alter their hosts' cellular metabolism to facilitate infection. Conversely, the innate antiviral responses of mammalian cells target these metabolic pathways to restrict viral propagation. We identified miR-130b and miR-185 as hepatic microRNAs (miRNAs) whose expression is stimulated by 25-hydroxycholesterol (25-HC), an antiviral oxysterol secreted by interferon-stimulated macrophages and dendritic cells, during hepatitis C virus (HCV) infection. However, 25-HC only directly stimulated miR-185 expression, whereas HCV regulated miR-130b expression. Independently, miR-130b and miR-185 inhibited HCV infection. In particular, miR-185 significantly restricted host metabolic pathways crucial to the HCV life cycle. Interestingly, HCV infection decreased miR-185 and miR-130b levels to promote lipid accumulation and counteract 25-HC's antiviral effect. Furthermore, miR-185 can inhibit other viruses through the regulation of immunometabolic pathways. These data establish these microRNAs as a key link between innate defenses and metabolism in the liver.

Activity-based profiling of the proteasome pathway during hepatitis C virus infection.

Hepatitis C virus (HCV) infection often leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The stability of the HCV proteins is controlled by ubiquitin-dependent and ubiquitin-independent proteasome pathways. Many viruses modulate proteasome function for their propagation. To examine the interrelationship between HCV and the proteasome pathways we employed a quantitative activity-based protein profiling method. Using this approach we were able to quantify the changes in the activity of several proteasome subunits and found that proteasome activity is drastically reduced by HCV replication. The results imply a link between the direct downregulation of the activity of this pathway and chronic HCV infection.

Hepatitis C virus induced up-regulation of microRNA-27: a novel mechanism for hepatic steatosis.

UNLABELLED: MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice. CONCLUSION: Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis.

Armand-Frappier Outstanding Student Award--The emerging role of 25-hydroxycholesterol in innate immunity.

The metabolic interplay between hosts and viruses plays a crucial role in determining the outcome of viral infection. Viruses reorchestrate the host's primary metabolic gene networks, including genes associated with mevalonate and isoprenoid synthesis, to acquire the necessary energy and structural components for their viral life cycles. Recent work has demonstrated that the interferon-mediated antiviral response suppresses the sterol pathway through production of a signalling molecule, 25-hydroxycholesterol (25HC). This oxysterol has been shown to exert multiple effects, both through incorporation into host cellular membranes as well as through transcriptional control. Herein, we summarize our current understanding of the multifunctional roles of 25HC in the mammalian innate antiviral response.

Correction: Chigrinova, M., et al. Kinugasa reactions in water: from green chemistry to bioorthogonal labelling. Molecules 2015, 20, 6959-6969.

The authors wish to make the following correction to this paper [1]: The author name "Paul Pezacki" should be "John Paul Pezacki". [...].

Kinugasa reactions in water: from green chemistry to bioorthogonal labelling.

The Kinugasa reaction has become an efficient method for the direct synthesis of ß-lactams from substituted nitrones and copper(I) acetylides. In recent years, the reaction scope has been expanded to include the use of water as the solvent, and with micelle-promoted [3+2] cycloadditions followed by rearrangement furnishing high yields of ß-lactams. The high yields of stable products under aqueous conditions render the modified Kinugasa reaction amenable to metabolic labelling and bioorthogonal applications. Herein, the development of methods for use of the Kinugasa reaction in aqueous media is reviewed, with emphasis on its potential use as a bioorthogonal coupling strategy.

A new chemical probe for phosphatidylinositol kinase activity.

Phosphatidylinositol kinases (PIKs) are key enzymatic regulators of membrane phospholipids and membrane environments that control many aspects of cellular function, from signal transduction to secretion, through the Golgi apparatus. Here, we have developed a photoreactive "clickable" probe, PIK-BPyne, to report the activity of PIKs. We investigated the selectivity and efficiency of the probe to both inhibit and label PIKs, and we compared PIK-BPyne to a wortmannin activity-based probe also known to target PIKs. We found that PIK-BPyne can act as an effective in situ activity-based probe, and for the first time, report changes in PI4K-IIIß activity induced by the hepatitis C virus. These results establish the utility of PIK-BPyne for activity-based protein profiling studies of PIK function in native biological systems.

SARS-CoV-2 Protein Nsp9 Is Involved in Viral Evasion through Interactions with Innate Immune Pathways.

The suppression of the host's innate antiviral immune response by SARS-CoV-2, a contributing factor to the severity of disease, has been considerably studied in recent years. Many of these studies have focused on the actions of the structural proteins of the virus because of their accessibility to host immunological components. However, less is known about SARS-CoV-2 nonstructural and accessory proteins in relation to viral evasion. Herein, we study SARS-CoV-2 nonstructural proteins Orf3a, Orf6, and Nsp9 in a mimicked virus-infected state using poly(I:C), a synthetic analog of viral dsRNA, that elicits the antiviral immune response. Through genome-wide expression profiling, we determined that Orf3a, Orf6, and Nsp9 all modulate the host antiviral signaling transcriptome to varying extents, uniquely suppressing aspects of innate immune signaling. Our data suggest that SARS-CoV-2 Nsp9 hinders viral detection through suppression of RIG-I expression and antagonizes the interferon antiviral cascade by downregulating NF-kB and TBK1. Our data point to unique molecular mechanisms through which the different SARS-CoV-2 proteins suppress immune signaling and promote viral evasion. Nsp9 in particular acts on major elements of the host antiviral pathways to impair the antiviral immune response.

Three-mode electrochemical sensing of ultralow microRNA levels.

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown great promise for cancer classification and prognosis. In this work, we developed a three-mode electrochemical sensor for detection and quantitation of ultralow levels of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modalities based on hybridization (H-SENS), p19 protein binding (P-SENS), and protein displacement (D-SENS). The combined three-mode sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30 µL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 µM. The HPD sensor is made on a commercially available gold nanoparticles-modified electrode and is suitable for analyzing multiple miRNAs on a single electrode. This three-mode sensor exhibits high selectivity and specificity and was used for sequential analysis of miR-32 and miR-122 on one electrode. In addition, the H-SENS can recognize miRNAs with different A/U and G/C content and distinguish between a fully matched miRNA and a miRNA comprising either a terminal or a middle single base mutation. Furthermore, the H- and P-SENS were successfully employed for direct detection and profiling of three endogenous miRNAs, including hsa-miR-21, hsa-miR-32, and hsa-miR-122 in human serum, and the sensor results were validated by qPCR.

Chemical contrast for imaging living systems: molecular vibrations drive CARS microscopy.

Cellular biomolecules contain unique molecular vibrations that can be visualized by coherent anti-Stokes Raman scattering (CARS) microscopy without the need for labels. Here we review the application of CARS microscopy for label-free imaging of cells and tissues using the natural vibrational contrast that arises from biomolecules like lipids as well as for imaging of exogenously added probes or drugs. High-resolution CARS microscopy combined with multimodal imaging has allowed for dynamic monitoring of cellular processes such as lipid metabolism and storage, the movement of organelles, adipogenesis and host-pathogen interactions and can also be used to track molecules within cells and tissues. The CARS imaging modality provides a unique tool for biological chemists to elucidate the state of a cellular environment without perturbing it and to perceive the functional effects of added molecules.

Rearrangements and addition reactions of biarylazacyclooctynones and the implications to copper-free click chemistry.

Highly strained biarylazacyclooctynone (BARAC) and analogous bioconjugation reagents were shown to undergo novel rearrangement and addition reactions leading to tetracyclic products. This may limit their practical applicability as bioorthogonal reporters for imaging biomolecules within living systems.