The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity.

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-ß production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Dual Action of Eeyarestatin 24 on Sec-Dependent Protein Secretion and Bacterial DNA.

Eeyarestatin 24 (ES24) is a promising new antibiotic with broad-spectrum activity. It shares structural similarity with nitrofurantoin (NFT), yet appears to have a distinct and novel mechanism: ES24 was found to inhibit SecYEG-mediated protein transport and membrane insertion in Gram-negative bacteria. However, possible additional targets have not yet been explored. Moreover, its activity was notably better against Gram-positive bacteria, for which its mechanism of action had not yet been investigated. We have used transcriptomic stress response profiling, phenotypic assays, and protein secretion analyses to investigate the mode of action of ES24 in comparison with NFT using the Gram-positive model bacterium Bacillus subtilis and have compared our findings to Gram-negative Escherichia coli. Here, we show the inhibition of Sec-dependent protein secretion in B. subtilis and additionally provide evidence for DNA damage, probably caused by the generation of reactive derivatives of ES24. Interestingly, ES24 caused a gradual dissipation of the membrane potential, which led to delocalization of cytokinetic proteins and subsequent cell elongation in E. coli. However, none of those effects were observed in B. subtilis, thereby suggesting that ES24 displays distinct mechanistic differences with respect to Gram-positive and Gram-negative bacteria. Despite its structural similarity to NFT, ES24 profoundly differed in our phenotypic analysis, which implies that it does not share the NFT mechanism of generalized macromolecule and structural damage. Importantly, ES24 outperformed NFT in vivo in a zebrafish embryo pneumococcal infection model. Our results suggest that ES24 not only inhibits the Sec translocon, but also targets bacterial DNA and, in Gram-negative bacteria, the cell membrane.

Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting.

Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals.

Synthesis, Biological Evaluation and Docking Studies of Ring-Opened Analogues of Ipomoeassin F.

The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61α and significantly disrupts multiple aspects of Sec61-mediated protein biogenesis at the endoplasmic reticulum, ultimately leading to cell death. However, extensive assessment of Ipom-F as a molecular tool and a therapeutic lead is hampered by its limited production scale, largely caused by intramolecular assembly of the macrocyclic ring. Here, using in vitro and/or in cellula biological assays to explore the first series of ring-opened analogues for the ipomoeassins, and indeed all resin glycosides, we provide clear evidence that macrocyclic integrity is not required for the cytotoxic inhibition of Sec61-dependent protein translocation by Ipom-F. Furthermore, our modeling suggests that open-chain analogues of Ipom-F can interact with multiple sites on the Sec61α subunit, most likely located at a previously identified binding site for mycolactone and/or the so-called lateral gate. Subsequent in silico-aided design led to the discovery of the stereochemically simplified analogue 3 as a potent, alternative lead compound that could be synthesized much more efficiently than Ipom-F and will accelerate future ipomoeassin research in chemical biology and drug discovery. Our work may also inspire further exploration of ring-opened analogues of other resin glycosides.

Membrane protein biogenesis at the ER: the highways and byways.

The Sec61 complex is the major protein translocation channel of the endoplasmic reticulum (ER), where it plays a central role in the biogenesis of membrane and secretory proteins. Whilst Sec61-mediated protein translocation is typically coupled to polypeptide synthesis, suggestive of significant complexity, an obvious characteristic of this core translocation machinery is its surprising simplicity. Over thirty years after its initial discovery, we now understand that the Sec61 complex is in fact the central piece of an elaborate jigsaw puzzle, which can be partly solved using new research findings. We propose that the Sec61 complex acts as a dynamic hub for co-translational protein translocation at the ER, proactively recruiting a range of accessory complexes that enhance and regulate its function in response to different protein clients. It is now clear that the Sec61 complex does not have a monopoly on co-translational insertion, with some transmembrane proteins preferentially utilising the ER membrane complex instead. We also have a better understanding of post-insertion events, where at least one membrane-embedded chaperone complex can capture the newly inserted transmembrane domains of multi-span proteins and co-ordinate their assembly into a native structure. Having discovered this array of Sec61-associated components and competitors, our next challenge is to understand how they act together in order to expand the range and complexity of the membrane proteins that can be synthesised at the ER. Furthermore, this diversity of components and pathways may open up new opportunities for targeted therapeutic interventions designed to selectively modulate protein biogenesis at the ER.

Biochemical and Biological Assays of Mycolactone-Mediated Inhibition of Sec61.

Mycobacterium ulcerans, the causative agent of Buruli ulcer disease, is unique among human pathogens in its capacity to produce mycolactone, a diffusible macrolide with immunosuppressive and cytotoxic properties. Recent studies have shown that mycolactone operates by inhibiting the host membrane translocation complex (Sec61), with an unprecedented potency compared to previously identified Sec61 blockers. Mycolactone binding to the pore-forming subunit of Sec61 inhibits its capacity to transport nascent secretory and membrane proteins into the endoplasmic reticulum, leading to their cytosolic degradation by the ubiquitin:proteasome system. In T lymphocytes, Sec61 blockade by mycolactone manifests as a sharp decrease in the cell's ability to express homing receptors and release cytokines following activation. Sustained exposure of human cells to mycolactone typically generates proteotoxic stress responses in their cytosol and endoplasmic reticulum (ER), ultimately inducing apoptosis. Here we describe cell-free systems for studying Sec61-mediated protein translocation that allow the impact of mycolactone on the biogenesis of secretory and membrane proteins to be probed. We also describe biological assays of mycolactone-driven inhibition of Sec61 providing rapid and sensitive means to quantitatively assess the presence of the toxin in biological samples.

An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum.

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.

Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis.

The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor.

In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2, the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation and/or insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum antiviral agents.This article has an associated First Person interview with the first author of the paper.

Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogens.

Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) associated protein degradation, Sec61-dependent Ca2+ homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61-translocon, and captures it in an open conformation that is translocation-incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG-translocon. Transcriptomic analysis suggested that ES24 has a complex mode of action, probably involving multiple targets. Interestingly, ES24 shows antibacterial activity toward clinically relevant strains. Furthermore, the antibacterial activity of ES24 is equivalent to or better than that of nitrofurantoin, a known antibiotic that, although structurally similar to ES24, does not interfere with SecYEG-dependent protein trafficking. Like nitrofurantoin, we find that ES24 requires activation by the NfsA and NfsB nitroreductases, suggesting that the formation of highly reactive nitroso intermediates is essential for target inactivation in vivo.

Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor.

In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2 the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation/insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum, antiviral agents.

Ring Expansion Leads to a More Potent Analogue of Ipomoeassin F.

Two new ring-size-varying analogues (2 and 3) of ipomoeassin F were synthesized and evaluated. Improved cytotoxicity (IC50: from 1.8 nM) and in vitro protein translocation inhibition (IC50: 35 nM) derived from ring expansion imply that the binding pocket of Sec61α (isoform 1) can accommodate further structural modifications, likely in the fatty acid portion. Streamlined preparation of the key diol intermediate 5 enabled gram-scale production, allowing us to establish that ipomoeassin F is biologically active in vivo (MTD: ∼3 mg/kg).

Membrane translocation at the ER: with a little help from my friends.

The Sec61 complex is the proteinaceous pore through which one-third of mammalian polypeptides access the lumen of the endoplasmic reticulum (ER) during their translocation across, or insertion into, the ER membrane. N-terminal ER signal peptides mediate polypeptide targeting to, and opening of, the Sec61 channel in a substrate-specific manner. Here, we discuss the recently defined features of ER signal peptides which necessitate the use of the accessory components Sec62 and Sec63 during the Sec61-mediated cotranslational translocation of newly synthesized secretory proteins.

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation.

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

Characterizing the selectivity of ER α-glucosidase inhibitors.

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.

Eeyarestatin Compounds Selectively Enhance Sec61-Mediated Ca2+ Leakage from the Endoplasmic Reticulum.

Eeyarestatin 1 (ES1) inhibits p97-dependent protein degradation, Sec61-dependent protein translocation into the endoplasmic reticulum (ER), and vesicular transport within the endomembrane system. Here, we show that ES1 impairs Ca2+ homeostasis by enhancing the Ca2+ leakage from mammalian ER. A comparison of various ES1 analogs suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analog ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca2+ leakage of ES1 analogs studied and induced Ca2+-dependent cell death. Using small interfering RNA-mediated knockdown of Sec61α, we identified Sec61 complexes as the targets that mediate the gain of Ca2+ leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61α, ES1 and ES24 likely capture Sec61 complexes in a Ca2+-permeable, open state, in which Sec61 complexes allow Ca2+ leakage but are translocation incompetent.

A randomized, double-blind, positive-controlled, 3-way cross-over human experimental pain study of a TRPV1 antagonist (V116517) in healthy volunteers and comparison with preclinical profile.

This experimental, translational, experimental pain, single-center, randomized, double-blind, single-dose, 3-treatment, 3-period cross-over proof-of-concept volunteer trial studied the efficacy of a novel TRPV1 antagonist (V116517) on capsaicin- and UV-B-induced hyperalgesia. Heat and pressure pain thresholds, von Frey stimulus-response functions, and neurogenic inflammation were assessed together with safety. Each treatment period was 4 days. The 3 single oral treatments were 300 mg V116517, 400 mg celecoxib (a COX-2 inhibitor), and placebo. The heat pain detection and tolerance thresholds were increased significantly (P < 0.0001) by V116517. Heat pain detection and tolerance thresholds showed significantly less capsaicin hyperalgesia after V116517 (P = 0.004 and P < 0.0001, respectively). Celecoxib reduced UV-B-provoked pressure pain sensitization (P = 0.01). Laser Doppler flowmetry and erythema index after UV-B were significantly (P < 0.0001) reduced by celecoxib. Stimulus-response function in capsaicin-treated areas showed significant differences between both celecoxib and placebo and between V116517 and placebo. The body temperature showed no change, and no side effects were reported for any of the treatments. The TRPV1 antagonists and the COX-2 inhibitor showed different antihyperalgesic profiles indicating different clinical targets. In addition, the preclinical profile of V116517 in rat models of UV-B and capsaicin-induced hypersensitivity was compared with the human experimental data and overall demonstrated an alignment between 2 of the 3 end points tested. The TRPV1 antagonist showed a potent antihyperalgesic action without changing the body temperature but heat analgesia may be a potential safety issue.

Effect of ketoconazole on the pharmacokinetic profile of buprenorphine following administration of a once-weekly buprenorphine transdermal system.

BACKGROUND AND OBJECTIVE: Buprenorphine is extensively metabolized by cytochrome P450 (CYP) 3A4. This study evaluated the effect of ketoconazole, a CYP3A4 inhibitor, on the metabolism of buprenorphine following the administration of a buprenorphine transdermal system 10 µg/hour (BTDS 10). METHODS: This single-centre study enrolled 20 healthy subjects who had demonstrated ketoconazole-mediated CYP3A4 inhibition via an erythromycin breath test. Subjects were randomized into a placebo-controlled, two-treatment, two-period crossover study. Subjects participated in a 7- to 14-day screening period, two baseline evaluations (day 0 [period 1] and day 16 [period 2]), two 12-day treatment periods (periods 1 and 2) separated by a 4-day washout period, and a study completion visit. Subjects received one BTDS 10 for 7 days per treatment period, administered concomitantly with either ketoconazole 200 mg twice daily or matching placebo. The main outcome measures were the ratios of geometric means for area under the plasma drug concentration versus time curve (AUC) from time zero to time of last measurable concentration (AUC(last)), AUC from time zero to infinity (AUC(∞)), and maximum plasma drug concentration (C(max)). RESULTS: The ratio of geometric means (BTDS 10 with ketoconazole/BTDS 10 with placebo) was 99.4 (90% confidence interval [CI] 87.2, 113.3) for AUC(last) and 97.8 (90% CI 87.7, 109.1) for C(max). The ratio of geometric means for AUC(∞) was 86.7 (90% CI 70.7, 106.2). The plasma concentrations of the metabolites norbuprenorphine and norbuprenorphine-3ß-glucuronide were slightly elevated following ketoconazole administration. BTDS 10 with ketoconazole was well tolerated and no apparent safety concerns were noted. CONCLUSION: The lack of a clinically significant CYP3A4 interaction with ketoconazole following transdermal delivery of buprenorphine is consistent with the parenteral administration of a high clearance drug bypassing exposure to gut wall and hepatic CYP3A4 first-pass effects. Metabolism of buprenorphine during therapy with BTDS is also not expected to be affected by co-administration of other CYP3A4 inhibitors.

Congress, courts, and commerce: upholding the individual mandate to protect the public's health.

Among multiple legal challenges to the Patient Protection and Affordable Care Act (PPACA) is the premise that PPACA's "individual mandate" (requiring all individuals to obtain health insurance by 2014 or face civil penalties) is inviolate of Congress' interstate commerce powers because Congress lacks the power to regulate commercial "inactivity." Several courts initially considering this argument have rejected it, but federal district courts in Virginia and Florida have concurred, leading to numerous appeals and prospective review of the United States Supreme Court. Despite creative arguments, the dispositive constitutional question is not whether Congress' interstate commerce power extends to commercial inactivity. Rather, it is whether Congress may regulate individual decisions with significant economic ramifications in the interests of protecting and promoting the public's health. This article offers a counter-interpretation of the scope of Congress' interstate commerce power to regulate in furtherance of the public's health.