Reactive oxygen species are associated with the inhibitory effect of N-(4-hydroxyphenyl)-retinamide on the entry of the severe acute respiratory syndrome-coronavirus 2.

N-(4-hydroxyphenyl)-retinamide (4-HPR) inhibits the dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity. We previously reported that 4-HPR suppresses the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein-mediated membrane fusion through a decrease in membrane fluidity in a DEGS1-independent manner. However, the precise mechanism underlying the inhibition of viral entry by 4-HPR remains unclear. In this study, we examined the role of reactive oxygen species (ROS) in the inhibition of membrane fusion by 4-HPR because 4-HPR is a well-known ROS-inducing agent. Intracellular ROS generation was found to be increased in the target cells in a cell-cell fusion assay after 4-HPR treatment, which was attenuated by the addition of the antioxidant, α-tocopherol (TCP). The reduction in membrane fusion susceptibility by 4-HPR treatment in the cell-cell fusion assay was alleviated by TCP addition. Furthermore, fluorescence recovery after photobleaching analysis showed that the lateral diffusion of glycosylphosphatidylinositol-anchored protein and SARS CoV-2 receptor was reduced by 4-HPR treatment and restored by TCP addition. These results indicate that the decrease in SARS-CoV-2 spike protein-mediated membrane fusion and membrane fluidity by 4-HPR was due to ROS generation. Taken together, these results demonstrate that ROS production is associated with the 4-HPR inhibitory effect on SARS-CoV-2 entry.

Antimitochondrial Antibodies: from Bench to Bedside.

Anti-mitochondrial antibodies (AMA) are directed against the E2 subunits of the 2-oxo acid dehydrogenase complexes (PDC-E2) and are the typical biomarkers of primary biliary cholangitis (PBC), being present in 90-95% of patients, with increasing sensitivity at increasing titers. Albeit being highly specific for PBC diagnosis, AMA can be detected in less than 1% of healthy subjects, and thus the management subjects with no sign or symptom of liver disease is still a challenge and data concerning clinical risk of developing PBC in this subgroup of patients are controversial. Moreover, AMA can also be detected in patients affected by overlap syndrome, as well as hepatic diseases (i.e., NASH and viral hepatitis), while the association with autoimmune diseases, in particular Sjögren's syndrome, systemic sclerosis, and systemic lupus erythematosus, is well established. Furthermore, new associations are being identified with inflammatory myositis and heart disease. AMA are directed towards the pyruvate dehydrogenase multi enzyme complex (PDC-E2) subunit, which represents an epithelial specific autoantigen for PBC. This review focuses on the main characteristics of AMA, their association with autoimmune diseases and liver diseases.

Dihydroceramide Δ4-Desaturase 1 Is Not Involved in SARS-CoV-2 Infection.

Dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity is inhibited with N-(4-hydroxyphenyl)-retinamide (4-HPR). We reported previously that 4-HPR suppresses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry through a DEGS1-independent mechanism. However, it remains unclear whether DEGS1 is involved in other SARS-CoV-2 infection processes, such as virus replication and release. Here we established DEGS1 knockout (KO) in VeroE6TMPRSS2 cells. No significant difference was observed in virus production in the culture supernatant between wild-type (WT) cells and DEGS1-KO cells, although the levels of dihydroceramide (DHCer), a DEGS1 substrate, were significantly higher in DEGS1-KO cells than WT cells. Furthermore, the virus-induced cytopathic effect was also observed in DEGS1-KO cells. Importantly, the EC50 value of 4-HPR in DEGS1-KO cells was almost identical to the value reported previously in WT cells. Our results indicated the lack of involvement of DEGS1 in SARS-CoV-2 infection.

Prognostic performance of an index based on lactic dehydrogenase and transaminases for patients with liver steatosis and COVID-19.

BACKGROUND: Metabolic associated fatty liver disease (MAFLD) is associated with complications and mortality in patients with coronavirus disease 2019 (COVID-19). However, there are no prognostic scores aimed to evaluate the risk of severe disease specifically in patients with MAFLD, despite its high prevalence. Lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase have been used as markers of liver damage. Therefore, we propose an index based on lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase for the prediction of complications and mortality in patients with MAFLD and COVID-19. AIM: To evaluate the prognostic performance of an index based on lactate dehydrogenase and transaminases (aspartate aminotransferase/alanine aminotransferase) in patients with COVID-19 and MAFLD [liver fibrosis and nutrition (LNF)-COVID-19 index]. METHODS: In this retrospective cohort study, two cohorts from two different tertiary centers were included. The first was the derivation cohort to obtain the score cutoffs, and the second was the validation cohort. We included hospitalized patients with severe COVID-19 and MAFLD. Liver steatosis was evaluated by computed tomography scan. Area under the receiver operating characteristic (ROC) curve analysis and survival analysis were used. RESULTS: In the derivation cohort, 44.6% had MAFLD; ROC curve analysis yielded a LFN-COVID-19 index > 1.67 as the best cutoff, with a sensitivity of 78%, specificity of 63%, negative predictive value of 91% and an area under the ROC curve of 0.77. In the multivariate analysis, the LFN-COVID-19 index > 1.67 was independently associated with the development of acute kidney injury (odds ratio: 1.8, 95% confidence interval: 1.3-2.5, P < 0.001), orotracheal intubation (odds ratio: 1.9, 95% confidence interval: 1.4-2.4, P < 0.001), and death (odds ratio: 2.86, 95% confidence interval: 1.6-4.5, P < 0.001) in both cohorts. CONCLUSION: LFN-COVID-19 index has a good performance to predict prognosis in patients with MAFLD and COVID-19, which could be useful for the MAFLD population.

SARS-CoV-2 Nsp14 protein associates with IMPDH2 and activates NF-κB signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and identified that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14- mediated NF-κB activation and cytokine induction. Furthermore, IMPDH2 inhibitors (RIB, MPA) or NF-κB inhibitors (bortezomib, BAY 11-7082) restricted SARS-CoV-2 infection, indicating that IMPDH2-mediated activation of NF-κB signaling is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in inducing NF-κB activation through IMPDH2 to promote viral infection.

SARS-CoV-2-induced hypomethylation of the ferritin heavy chain (FTH1) gene underlies serum hyperferritinemia in severe COVID-19 patients.

High serum ferritin (hyperferritinemia), a reliable hallmark of severe COVID-19 often associates with a moderate decrease in serum iron (hypoferremia) and a moderate increase in serum hepcidin. This suggests that hyperferritinemia in severe COVID-19 is reflective of inflammation rather than iron overload. To test this possibility, the expression status of ferritin heavy chain (FTH1), transferrin receptor 1 (TFRC), hepcidin (HAMP), and ferroportin (SLC40A1) genes and promoter methylation status of FTH1 and TFRC genes were examined in blood samples obtained from COVID-19 patients showing no, mild or severe symptoms and in healthy-donor monocytes stimulated with SARS-CoV-2-derived peptides. Severe COVID-19 samples showed a significant increase in FTH1 expression and hypomethylation relative to mild or asymptomatic COVID-19 samples. S-peptide treated monocytes also showed a significant increase in FTH1 expression and hypomethylation relative to that in controls; treatment with ECD or NP did not change FTH1 expression nor its methylation status. In silico and in vitro analysis showed a significant increase in the expression of the TET3 demethylase in S peptide-treated monocytes. Findings presented here suggest that S peptide-driven hypomethylation of the FTH1 gene promoter underlies hyperferritinemia in severe COVID-19 disease.

Association of the Transmembrane Serine Protease-2 (TMPRSS2) Polymorphisms with COVID-19.

SARS-CoV-2 uses the ACE2 receptor and the cellular protease TMPRSS2 for entry into target cells. The present study aimed to establish if the TMPRSS2 polymorphisms are associated with COVID-19 disease. The study included 609 patients with COVID-19 confirmed by RT-PCR test and 291 individuals negative for the SARS-CoV-2 infection confirmed by RT-PCR test and without antibodies anti-SARS-CoV-2. Four TMPRSS2 polymorphisms (rs12329760, rs2298659, rs456298, and rs462574) were determined using the 5'exonuclease TaqMan assays. Under different inheritance models, the rs2298659 (pcodominant2 = 0.018, precessive = 0.006, padditive = 0.019), rs456298 (pcodominant1 = 0.014, pcodominant2 = 0.004; pdominant = 0.009, precessive = 0.004, padditive = 0.0009), and rs462574 (pcodominant1 = 0.017, pcodominant2 = 0.004, pdominant = 0.041, precessive = 0.002, padditive = 0.003) polymorphisms were associated with high risk of developing COVID-19. Two risks (ATGC and GAAC) and two protectives (GAGC and GAGT) haplotypes were detected. High levels of lactic acid dehydrogenase (LDH) were observed in patients with the rs462574AA and rs456298TT genotypes (p = 0.005 and p = 0.020, respectively), whereas, high heart rate was present in patients with the rs462574AA genotype (p = 0.028). Our data suggest that the rs2298659, rs456298, and rs462574 polymorphisms independently and as haplotypes are associated with the risk of COVID-19. The rs456298 and rs462574 genotypes are related to high levels of LDH and heart rate.

SARS-CoV-2 ORF8 reshapes the ER through forming mixed disulfides with ER oxidoreductases.

The replication machinery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely associated with the endoplasmic reticulum (ER) in host cells. Activation of the unfolded protein response (UPR) is a strategy hijacked by coronavirus to facilitate its replication and suppress host innate immunity. Here, we have found that SARS-CoV-2 ORF8 protein accumulates in the ER and escapes the degradation system by forming mixed disulfide complexes with ER oxidoreductases. ORF8 induces the activation of three UPR pathways through targeting key UPR components, remodels ER morphology and accelerates protein trafficking. Moreover, small molecule reducing agents release ORF8 from the mixed disulfide complexes and facilitate its degradation, therefore mitigate ER stress. Our study reveals a unique mechanism by which SARS-CoV-2 ORF8 escapes degradation by host cells and regulates ER reshaping. Targeting ORF8-involved mixed disulfide complexes could be a new strategy to alleviate SARS-CoV-2 induced ER stress and related diseases.

Disengaging the COVID-19 Clutch as a Discerning Eye Over the Inflammatory Circuit During SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the cytokine release syndrome (CRS) and leads to multiorgan dysfunction. Mitochondrial dynamics are fundamental to protect against environmental insults, but they are highly susceptible to viral infections. Defective mitochondria are potential sources of reactive oxygen species (ROS). Infection with SARS-CoV-2 damages mitochondria, alters autophagy, reduces nitric oxide (NO), and increases both nicotinamide adenine dinucleotide phosphate oxidases (NOX) and ROS. Patients with coronavirus disease 2019 (COVID-19) exhibited activated toll-like receptors (TLRs) and the Nucleotide-binding and oligomerization domain (NOD-), leucine-rich repeat (LRR-), pyrin domain-containing protein 3 (NLRP3) inflammasome. The activation of TLRs and NLRP3 by SARS-CoV-2 induces interleukin 6 (IL-6), IL-1ß, IL-18, and lactate dehydrogenase (LDH). Herein, we outline the inflammatory circuit of COVID-19 and what occurs behind the scene, the interplay of NOX/ROS and their role in hypoxia and thrombosis, and the important role of ROS scavengers to reduce COVID-19-related inflammation.

Evaluation of clinical and laboratory findings in severe group COVID-19 pregnants without comorbidity.

BACKGROUND: COVID - 19 disease may be seen with different clinical presentations in pregnant women. Comorbid diseases are important factors affecting the progression of this disease. In this study, we aimed to evaluate the clinical and laboratory findings in pregnant women with COVID - 19 who had no comorbid disease. METHODS: This retrospective designed study included 217 patients with Covid PCR positive in typically COVID - 19 clinic. The patients were classified into asymptomatic, nonsevere, and severe disease groups. The symptoms, laboratory results, hospital followups and intensive care records of the patients and the findings of new borns are presented. RESULTS: Most of the patients (78%) were in the third trimester of pregnancy, and 103 patients in the study group had severe disease. Fever in the non-severe group and respiratory distress in the severe group were the most common symptoms in the patients. The severe clinical manifestations were specifically observed in the third trimester patients. In the severe group, neutrophil, lactat dehydrogenase, ferritin, CK - MB, IL - 6, and hospital stay were statistically higher than those in other groups (p < 0.05). Increase in BUN and creatine were the most predictive parameters in intensive care admission. While the intensive care unit (ICU) requirement was higher in patients in the severe group, premature birth was observed more frequently in the severe group (p < 0.05) .

N-(4-Hydroxyphenyl) Retinamide Suppresses SARS-CoV-2 Spike Protein-Mediated Cell-Cell Fusion by a Dihydroceramide Δ4-Desaturase 1-Independent Mechanism.

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

A method for intuitively extracting macromolecular dynamics from structural disorder.

Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an analytical approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.

From Plant Survival Under Severe Stress to Anti-Viral Human Defense - A Perspective That Calls for Common Efforts.

Reprogramming of primary virus-infected cells is the critical step that turns viral attacks harmful to humans by initiating super-spreading at cell, organism and population levels. To develop early anti-viral therapies and proactive administration, it is important to understand the very first steps of this process. Plant somatic embryogenesis (SE) is the earliest and most studied model for de novo programming upon severe stress that, in contrast to virus attacks, promotes individual cell and organism survival. We argued that transcript level profiles of target genes established from in vitro SE induction as reference compared to virus-induced profiles can identify differential virus traits that link to harmful reprogramming. To validate this hypothesis, we selected a standard set of genes named 'ReprogVirus'. This approach was recently applied and published. It resulted in identifying 'CoV-MAC-TED', a complex trait that is promising to support combating SARS-CoV-2-induced cell reprogramming in primary infected nose and mouth cells. In this perspective, we aim to explain the rationale of our scientific approach. We are highlighting relevant background knowledge on SE, emphasize the role of alternative oxidase in plant reprogramming and resilience as a learning tool for designing human virus-defense strategies and, present the list of selected genes. As an outlook, we announce wider data collection in a 'ReprogVirus Platform' to support anti-viral strategy design through common efforts.

Laccase isoform diversity on basal medium in Cyathus bulleri and role in decolorization/detoxification of textile dyes and effluent.

Laccases (EC 1.10.3.2) are multi-copper oxidases that can degrade several xenobiotics, including textile dyes. Present study investigated the nature of laccase isoforms induced by 2,6-dimethylaniline in Cyathus bulleri cultivated on basal salt medium. Two isoforms, LacI and LacII were identified and purified by a combination of ultrafiltration and ion-exchange chromatography. The MS spectrum of the two proteins displayed a number of non-identical and identical molecular peaks (m/z), and, the latter were mapped to protein originating from the previously reported Laccase (Lcc) 1 gene. The LacI isoform exhibited higher catalytic efficiency (Kcat/Km) towards 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol and pyrogallol and was tolerant to high levels of chloride ions and resistant to EDTA. Higher decolorization of several dyes such as Direct Scarlet B (67%), Reactive Brilliant blue-R (96%), Direct Orange 34 (50%) and Reactive Red198 (95%) by the LacI isoform makes it a good candidate for degradation of synthetic dyes. The decolorization of Direct Orange 34 by laccases is being reported for the first time. Many of the properties exhibited by this isoform make it a good candidate for large scale production and applications for use in the dyeing industry.

ROS/RNS Balancing, Aerobic Fermentation Regulation and Cell Cycle Control - a Complex Early Trait ('CoV-MAC-TED') for Combating SARS-CoV-2-Induced Cell Reprogramming.

In a perspective entitled 'From plant survival under severe stress to anti-viral human defense' we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named 'ReprogVirus' was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the 'ReprogVirus platform' was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to 'RegroVirus' complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called 'CoV-MAC-TED'. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target 'CoV-MAC-TED' in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that 'de-stressing' disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.

Kinetic characterisation and inhibitor sensitivity of Candida albicans and Candida auris recombinant AOX expressed in a self-assembled proteoliposome system.

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose-response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.

Secretion of von Willebrand Factor and Suppression of ADAMTS-13 Activity by Markedly High Concentration of Ferritin.

Hyperferritinemia is associated with poor outcomes in critically ill patients with sepsis, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndromes (MAS) and coronavirus disease 19 (COVID-19). Autopsies of hyperferritinemic patients that succumbed to either sepsis, HLH, MAS or COVID-19 have revealed disseminated microvascular thromboses with von Willebrand factor (VWF)-, platelets-, and/or fibrin-rich microthrombi. It is unknown whether high plasma ferritin concentration actively promotes microvascular thrombosis, or merely serves as a prognostic biomarker in these patients. Here, we show that secretion of VWF from human umbilical vein endothelial cells (HUVEC) is significantly enhanced by 100,000 ng/ml of recombinant ferritin heavy chain protein (FHC). Ferritin fraction that was isolated by size exclusion chromatography from the plasma of critically ill HLH patients promoted VWF secretion from HUVEC, compared to similar fraction from non-critically ill control plasma. Furthermore, recombinant FHC moderately suppressed the activity of VWF cleaving metalloprotease ADAMTS-13. These observations suggest that a state of marked hyperferritinemia could promote thrombosis and organ injury by inducing endothelial VWF secretion and reducing the ADAMTS-13 activity.

Biosynthesis of medicinal tropane alkaloids in yeast.

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.

Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2.

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.