Affinity selection of double-click triazole libraries for rapid discovery of allosteric modulators for GLP-1 receptor.

The recently developed double-click reaction sequence [G. Meng et al., Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.

A Metabolic-Tag-Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria.

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.

KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export.

CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. Previous studies have revealed the feasibility of modulating Cas9-based genome- and base-editing tools using protein or small-molecule CRISPR inhibitors. Here we screened a set of small molecule compounds with irreversible warhead, aiming to identifying small-molecule modulators of CRISPR-Cas9. It was found that selective inhibitors of nuclear export (SINEs) could efficiently inhibit the cellular activity of Cas9 in the form of genome-, base- and prime-editing tools. Interestingly, SINEs did not function as direct inhibitors to Cas9, but modulated Cas9 activities by interfering with the nuclear export process of Cas9 mRNA. Thus, to the best of our knowledge, SINEs represent the first reported indirect, irreversible inhibitors of CRISPR-Cas9. Most importantly, an FDA-approved anticancer drug KPT330, along with other examined SINEs, could improve the specificities of CRISPR-Cas9-based genome- and base editing tools in human cells. Our study expands the toolbox of CRISPR modulating elements and provides a feasible approach to improving the specificity of CRISPR-Cas9-based genome engineering tools.

Metal-Free Synthesis of Functional 1-Substituted-1,2,3-Triazoles from Ethenesulfonyl Fluoride and Organic Azides.

The boom in growth of 1,4-disubstituted triazole products, in particular, since the early 2000's, can be largely attributed to the birth of click chemistry and the discovery of the CuI -catalyzed azide-alkyne cycloaddition (CuAAC). Yet the synthesis of relatively simple, albeit important, 1-substituted-1,2,3-triazoles has been surprisingly more challenging. Reported here is a straightforward and scalable click-inspired protocol for the synthesis of 1-substituted-1,2,3-triazoles from organic azides and the bench stable acetylene surrogate ethenesulfonyl fluoride (ESF). The new transformation tolerates a wide selection of substrates and proceeds smoothly under metal-free conditions to give the products in excellent yield. Under controlled acidic conditions, the 1-substituted-1,2,3-triazole products undergo a Michael addition reaction with a second equivalent of ESF to give the unprecedented 1-substituted triazolium sulfonyl fluoride salts.

Modular click chemistry libraries for functional screens using a diazotizing reagent.

Click chemistry is a concept in which modular synthesis is used to rapidly find new molecules with desirable properties1. Copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) triazole annulation and sulfur(VI) fluoride exchange (SuFEx) catalysis are widely regarded as click reactions2-4, providing rapid access to their products in yields approaching 100% while being largely orthogonal to other reactions. However, in the case of CuAAC reactions, the availability of azide reagents is limited owing to their potential toxicity and the risk of explosion involved in their preparation. Here we report another reaction to add to the click reaction family: the formation of azides from primary amines, one of the most abundant functional groups5. The reaction uses just one equivalent of a simple diazotizing species, fluorosulfuryl azide6-11 (FSO2N3), and enables the preparation of over 1,200 azides on 96-well plates in a safe and practical manner. This reliable transformation is a powerful tool for the CuAAC triazole annulation, the most widely used click reaction at present. This method greatly expands the number of accessible azides and 1,2,3-triazoles and, given the ubiquity of the CuAAC reaction, it should find application in organic synthesis, medicinal chemistry, chemical biology and materials science.

Novel Approaches to Access Arylfluorosulfates and Sulfamoyl Fluorides Based on Sulfur (VI) Fluoride Exchange.

Sulfur (VI) fluoride exchange (SuFEx) is a new family of click chemistry reactions that relies on readily available sulfuryl fluoride (SO2 F2 ) and ethenesulfonyl fluoride to build diverse chemical structures bearing the SVI -F motif, such as fluorosulfate (-OSO2 F) and sulfonyl fluoride (-SO2 F). These motifs could be useful functional groups and connective linkers in organic synthesis. This unit describes two protocols for performing SuFEx. The first protocol describes an in situ method for rapid generation of arylfluorosulfates in 96-well plates for high-throughput screening. The second protocol outlines use of a shelf-stable fluorosulfuryl imidazolium salt for generating arylfluorosulfates and sulfamoyl fluorides. © 2019 by John Wiley & Sons, Inc.

Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon.

Porcine epidemic diarrhea virus (PEDV) causes acute and devastating enteric disease in suckling piglets and results in huge economic losses in the pig industry worldwide. To establish productive infection, viruses must first circumvent the host innate immune response. In this study, we found that PEDV infection stimulated epidermal growth factor receptor (EGFR) activation, which has been linked to not only anticancer therapeutics, but also antiviral signaling. Therefore, we determined whether EGFR activation affected PEDV infection by using an activator or overexpression assay. The data showed that EGFR activation enhanced virus replication in both cases. We also found that specific inhibition of EGFR by either inhibitors or small interfering RNA (siRNA) led to a decrease in virus yields. Further analysis revealed that inhibition of EGFR produced augmentation of type I interferon genes. We next observed that the EGFR downstream cascade STAT3 was also activated upon PEDV infection. Similar to the case of EGFR, specific inhibition of STAT3 by either inhibitor or siRNA increased the antiviral activity of interferon and resulted in decreased PEDV RNA levels, and vice versa. The data on STAT3 depletion in combination with EGFR activation suggest that the attenuation of antiviral activity by EGFR activation requires activation of the STAT3 signaling pathway. Taken together, these data demonstrate that PEDV-induced EGFR activation serves as a negative regulator of the type I interferon response and provides a novel therapeutic target for virus infection.IMPORTANCE EGFR is a transmembrane tyrosine receptor that mediates various cellular events, as well as several types of human cancers. In this study, we investigated for the first time the role of EGFR in PEDV infection. We observed that PEDV infection induced EGFR activation. The role of EGFR activation is to impair the antiviral activity of type I interferon, which requires the involvement of the EGFR downstream signaling cascade STAT3. Our findings reveal a new mechanism evolved by PEDV to circumvent the host antiviral response, which might serve as a therapeutic target against virus infection.

A New Portal to SuFEx Click Chemistry: A Stable Fluorosulfuryl Imidazolium Salt Emerging as an "F-SO2 +" Donor of Unprecedented Reactivity, Selectivity, and Scope.

Sulfuryl fluoride, SO2 F2 , has been found to derivatize phenols in all kinds of environments, even those in highly functional molecules. We now report that a solid fluorosulfuryl imidazolium triflate salt delivers the same "F-SO2 +" fragment to Nu-H acceptor groups in the substrates. However, this triflate salt is a far more reactive fluorosulfurylating agent than SO2 F2 and displays selectivity preferences of its own. Moreover, the new azolium triflate reagent reacts once with primary amines and anilines before the reaction stops. On the other hand, with triethylamine and two equivalents of the "F-SO2 +" donor present, it proceeds on to the bis(fluorosulfuryl)imides in good yield-two important conversions that we have never seen with sulfuryl fluoride as the electrophile.