Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate.

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Expedient synthesis of imino-C-nucleoside fleximers featuring a one-pot procedure to prepare aryl triazoles.

Nucleoside analogues such as the antiviral agents galidesivir and ribavirin are of synthetic interest. This work reports a "one-pot" preparation of similar fleximers using a bifunctional copper catalyst that generates the aryl azide in situ, which is captured by a terminal alkyne to effect triazole formation.

Synthesis of 12ß-methyl-18-nor-avicholic acid analogues as potential TGR5 agonists.

In the quest for new modulators of the Farnesoid-X (FXR) and Takeda G-protein-coupled (TGR5) receptors, bile acids are a popular candidate for drug development. Recently, bile acids endowed with a C16-hydroxy group emerged as ligands of FXR and TGR5 with remarkable agonistic efficacies. Inspired by these findings, we synthesised a series of C16-hydroxylated 12ß-methyl-18-nor-bile acid analogues from a Δ13(17)-12ß-methyl-18-nor-chenodeoxycholic acid intermediate (16), the synthesis of which we reported previously. The preparation of these aptly named 12ß-methyl-18-nor-avicholic acids (17, 18, 41 and 42) was accomplished via allylic oxidation at C16, hydrogenation of the C13→C17 double bond and selective reduction of the C16-carbonyl group. Described also are various side products which were isolated during the evaluation of methods to affect the initial allylic oxidation. In addition, C23-methyl modified 12ß-methyl-18-nor-bile acids with (48, 49, 51 and 52) and without a C16-hydroxy group (45, 46 and 55), were synthesized to enable comparison of biological activities between these compounds and their un-methylated counterparts. As a result of our investigations we identified (23R)-12ß,23-dimethyl-18-nor-chenodeoxycholic acid (46) and 12ß-methyl-17-epi-18-nor-chenodeoxycholic acid 53 as TGR5 ligands with EC50 values of 25 µM.

Synthesis of Novel C/D Ring Modified Bile Acids.

Bile acid receptors have been identified as important targets for the development of new therapeutics to treat various metabolic and inflammatory diseases. The synthesis of new bile acid analogues can help elucidate structure-activity relationships and define compounds that activate these receptors selectively. Towards this, access to large quantities of a chenodeoxycholic acid derivative bearing a C-12 methyl and a C-13 to C-14 double bond provided an interesting scaffold to investigate the chemical manipulation of the C/D ring junction in bile acids. The reactivity of this alkene substrate with various zinc carbenoid species showed that those generated using the Furukawa methodology achieved selective α-cyclopropanation, whereas those generated using the Shi methodology reacted in an unexpected manner giving rise to a rearranged skeleton whereby the C ring has undergone contraction to form a novel spiro-furan ring system. Further derivatization of the cyclopropanated steroid included O-7 oxidation and epimerization to afford new bile acid derivatives for biological evaluation.

Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.

Mechanism-Based Inactivation of Mycobacterium tuberculosis Isocitrate Lyase 1 by (2R,3S)-2-Hydroxy-3-(nitromethyl)succinic acid.

The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retro-aldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (kinact/KI = (1.3 ± 0.1) × 103 M-1 s-1) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys191 thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.

Chemical Synthesis of the Antiviral Nucleotide Analogue ddhCTP.

3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin as part of the innate immune response. ddhCTP has been shown to act as an obligate chain terminator of flavivirus and SARS-CoV-2 RNA-dependent RNA polymerases; however, further biophysical studies have been precluded by limited access to this promising antiviral. Herein, we report a robust and scalable synthesis of ddhCTP as well as the mono- and diphosphates ddhCMP and ddhCDP, respectively. Identification of a 2'-silyl ether protection strategy enabled selective synthesis and facile purification of the 5'-triphosphate, culminating in the preparation of ddhCTP on a gram scale.

Narrow-Spectrum Antibiotic Targeting of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis.

Antibiotic resistance continues to spread at an alarming rate, outpacing the introduction of new therapeutics and threatening to globally undermine health care. There is a crucial need for new strategies that selectively target specific pathogens while leaving the majority of the microbiome untouched, thus averting the debilitating and sometimes fatal occurrences of opportunistic infections. To address these challenges, we have adopted a unique strategy that focuses on oxygen-sensitive proteins, an untapped set of therapeutic targets. MqnE is a member of the radical S-adenosyl-l-methionine (RS) superfamily, all of which rely on an oxygen-sensitive [4Fe-4S] cluster for catalytic activity. MqnE catalyzes the conversion of didehydrochorismate to aminofutalosine in the essential menaquinone biosynthetic pathway present in a limited set of species, including the gut pathogen Helicobacter pylori (Hp), making it an attractive target for narrow-spectrum antibiotic development. Indeed, we show that MqnE is inhibited by the mechanism-derived 2-fluoro analogue of didehydrochorismate (2F-DHC) due to accumulation of a radical intermediate under turnover conditions. Structures of MqnE in the apo and product-bound states afford insight into its catalytic mechanism, and electron paramagnetic resonance approaches provide direct spectroscopic evidence consistent with the predicted structure of the radical intermediate. In addition, we demonstrate the essentiality of the menaquinone biosynthetic pathway and unambiguously validate 2F-DHC as a selective inhibitor of Hp growth that exclusively targets MqnE. These data provide the foundation for designing effective Hp therapies and demonstrate proof of principle that radical SAM proteins can be effectively leveraged as therapeutic targets.

Mechanism-based inactivator of isocitrate lyases 1 and 2 from Mycobacterium tuberculosis.

Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys191.

E-cadherin-deficient cells have synthetic lethal vulnerabilities in plasma membrane organisation, dynamics and function.

BACKGROUND: The E-cadherin gene (CDH1) is frequently mutated in diffuse gastric cancer and lobular breast cancer, and germline mutations predispose to the cancer syndrome Hereditary Diffuse Gastric Cancer. We are taking a synthetic lethal approach to identify druggable vulnerabilities in CDH1-mutant cancers. METHODS: Density distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1-/- cells were used to identify protein classes affected by CDH1 mutation. The synthetic lethal relationship between selected protein classes and E-cadherin was characterised by drug sensitivity assays in both the isogenic breast MCF10A cells and CDH1-isogenic gastric NCI-N87. Endocytosis efficiency was quantified using cholera toxin B uptake. Pathway metagene expression of 415 TCGA gastric tumours was statistically correlated with CDH1 expression. RESULTS: MCF10A-CDH1-/- cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10A-CDH1-/- and NCI-N87-CDH1-/- cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. The MCF10A-CDH1-/- cell lines showed reduced capacity to endocytose cholera toxin B. Pathway metagene analysis identified 20 Reactome pathways that were potentially synthetic lethal in tumours. Genes involved in GPCR signalling, vesicle transport and the metabolism of PI3K and membrane lipids were strongly represented amongst the candidate synthetic lethal genes. CONCLUSIONS: E-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities.

A Designed Approach to Enantiodivergent Enamine Catalysis.

The rational design and implementation of enantiodivergent enamine catalysis is reported. A simple secondary amine catalyst, 2-methyl-l-proline, and its tetrabutylammonium salt function as an enantiodivergent catalyst pair delivering the enantiomers of α-functionalized aldehyde products in excellent enantioselectivities. This novel concept of designed enantiodivergence is applied to the enantioselective α-amination, aldol, and α-aminoxylation/α-hydroxyamination reactions of aldehydes.

Uncatalysed diaryldiazo cyclopropanations on bicyclic lactams: access to annulated prolines.

The uncatalysed cycloaddition of substituted diaryldiazo compounds onto bicyclic unsaturated lactams derived from pyroglutamic acid efficiently leads to highly functionalised azatricyclononanes. The products are readily elaborated to deprotected pyroglutamate derivatives, providing rapid access to conformationally constrained amino acids and their analogues. Preliminary assessment of antibacterial activity against one Gram positive and one Gram negative organism indicated high levels of efficacy in some cases.

Transition state analogues of Plasmodium falciparum and human orotate phosphoribosyltransferases.

The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (Kd) as low as 80 nM. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5'-phosphate bound to OPRTs with Kd values near 40 nM. Analogues designed with a C5-pyrimidine carbon-carbon bond to ribocation mimics gave Kd values in the range of 80-500 nM. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5'-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum.

An efficient regioconvergent synthesis of 3-aza-obeticholic acid.

Bile acids (BAs) are steroidal molecules that play important roles in nutrient absorption, distribution, and excretion. They also act on specific receptors implicated in various metabolic and inflammatory diseases demonstrating their importance as potential drug candidates. Accordingly, there has been a concerted effort to develop new BA derivatives to probe structure-activity relationships with the goal of discovering BA analogues with enhanced pharmacological properties. Among the many steroidal derivatisations reported, the formation of endocyclic azasteroids appeals due to their potential to deliver altered biological responses with minimal change to the steroidal superstructure. Here, we report the synthesis of 3-aza-obeticholic acid (6) via a regioconvergent route. Ammoniolysis of lactones, formed from an m-CPBA-mediated Baeyer-Villiger reaction on a 3-keto-OCA derivative, furnished protected intermediate amido-alcohols which were separately elaborated to amino-alcohols via Hofmann degradation with BAIB. Upon individual N-Boc-protection, these underwent annulation to the 3-aza-A-ring when subjected to either mesylation or a Dess-Martin oxidation/hydrogenation sequence. Global deprotection of the 3-aza-intermediate delivered 3-aza-OCA in ten steps and an overall yield of up to 19 %.

Galidesivir Triphosphate Promotes Stalling of Dengue-2 Virus Polymerase Immediately Prior to Incorporation.

Millions of people are infected by the dengue and Zika viruses each year, resulting in significant morbidity and mortality. Galidesivir is an adenosine nucleoside analog that can attenuate flavivirus replication in cell-based assays and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases and subsequently incorporated into viral RNA by viral RNA polymerases. This has been proposed to lead to the delayed termination of RNA synthesis. Here, we report direct in vitro testing of the effects of Galidesivir triphosphate on dengue-2 and Zika virus polymerase activity. Galidesivir triphosphate was chemically synthesized, and inhibition of RNA synthesis followed using a dinucleotide-primed assay with a homopolymeric poly(U) template. Galidesivir triphosphate was equipotent against dengue-2 and Zika polymerases, with IC50 values of 42 ± 12 µM and 47 ± 5 µM, respectively, at an ATP concentration of 20 µM. RNA primer extension assays show that the dengue-2 polymerase stalls while attempting to add a Galidesivir nucleotide to the nascent RNA chain, evidenced by the accumulation of RNA products truncated immediately upstream of Galidesivir incorporation sites. Nevertheless, Galidesivir is incorporated at isolated sites with low efficiency, leading to the subsequent synthesis of full-length RNA with no evidence of delayed chain termination. The incorporation of Galidesivir at consecutive sites is strongly disfavored, highlighting the potential for modulation of inhibitory effects of nucleoside analogs by the template sequence. Our results suggest that attenuation of dengue replication by Galidesivir may not derive from the early termination of RNA synthesis following Galidesivir incorporation.

Chemoenzymatic Synthesis of 3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP).

3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin during the early stages of the innate immune response. ddhCTP has been shown to act as a chain terminator of flavivirus RNA-dependent RNA polymerases. To date, synthesis of ddhCTP requires complicated synthetic protocols or isolation of the enzyme viperin to catalyze the production of ddhCTP from CTP. Recombinant viperin approaches preclude the production of highly pure ddhCTP (free of contaminants such as CTP), whereas the chemical synthesis involves techniques or equipment not readily available to most laboratories. Herein, we describe the chemoenzymatic synthesis of ddhCTP, starting from commercially available ddhC. We utilize these methods to produce milligram quantities of ddhCTP, ddhCDP, and ddhCMP. Using purified semisynthetic ddhCTP and fully synthetic ddhCTP, we also show ddhCTP does not inhibit NAD+-dependent enzymes such as glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, or lactate dehydrogenase, contrary to a recent report.

The discovery of 12ß-methyl-17-epi-18-nor-bile acids as potent and selective TGR5 agonists.

Recent discoveries have demonstrated that the physiological function of bile acids extends to the regulation of diverse signaling processes through interactions with nuclear and G protein-coupled receptors, most notably the Farnesoid-X nuclear receptor (FXR) and the G protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5). Targeting such signaling pathways pharmacologically, i.e. with bile acid-derived therapeutics, presents great potential for the treatment of various metabolic, inflammatory immune, liver, and neurodegenerative diseases. Here we report the discovery of two potent and selective TGR5 agonists (NZP196 and 917). These compounds are the taurine conjugates of 6α-ethyl-substituted 12ß-methyl-18-nor-bile acids with the side chain being located on the α-face of the steroid scaffold. The compounds emerged from a screening effort of a diverse library of 12ß-methyl-18-nor-bile acids that were synthesized from 12ß-methyl-18-nor-chenodeoxycholic acid and its C17-epimer. Upon testing for FXR activity, both compounds were found to be inactive, thus revealing selectivity for TGR5.