Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response.

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

Pseudomonas aeruginosa quorum sensing and biofilm attenuation by a di-hydroxy derivative of piperlongumine (PL-18).

Bacterial communication, Quorum Sensing (QS), is a target against virulence and prevention of antibiotic-resistant infections. 16 derivatives of Piperlongumine (PL), an amide alkaloid from Piper longum L., were screened for QS inhibition. PL-18 had the best QSI activity. PL-18 inhibited the lasR-lasI, rhlR-rhlI, and pqs QS systems of Pseudomonas aeruginosa. PL-18 inhibited pyocyanin and rhamnolipids that are QS-controlled virulence elements. Iron is an essential element for pathogenicity, biofilm formation and resilience in harsh environments, its uptake was inhibited by PL-18. Pl-18 significantly reduced the biofilm biovolume including in established biofilms. PL-18-coated silicon tubes significantly inhibited biofilm formation. The transcriptome study of treated P. aeruginosa showed that PL-18 indeed reduced the expression of QS and iron homeostasis related genes, and up regulated sulfur metabolism related genes. Altogether, PL-18 inhibits QS, virulence, iron uptake, and biofilm formation. Thus, PL-18 should be further developed against bacterial infection, antibiotic resistance, and biofilm formation.

The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products.

Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 1''-2' and 1''-3' glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert ~0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1-induced programmed axonal death in animals.

The differential regulation of placenta trophoblast bisphosphoglycerate mutase in fetal growth restriction: preclinical study in mice and observational histological study of human placenta.

Background: Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown. Methods: Pregnant mice were subjected to acute or chronic hypoxia (12.5% O2) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry. Results: We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1α levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation. Conclusions: This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR. Funding: This work was supported by the Weizmann Krenter Foundation and the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.

Lung dendritic-cell metabolism underlies susceptibility to viral infection in diabetes.

People with diabetes feature a life-risking susceptibility to respiratory viral infection, including influenza and SARS-CoV-2 (ref. 1), whose mechanism remains unknown. In acquired and genetic mouse models of diabetes, induced with an acute pulmonary viral infection, we demonstrate that hyperglycaemia leads to impaired costimulatory molecule expression, antigen transport and T cell priming in distinct lung dendritic cell (DC) subsets, driving a defective antiviral adaptive immune response, delayed viral clearance and enhanced mortality. Mechanistically, hyperglycaemia induces an altered metabolic DC circuitry characterized by increased glucose-to-acetyl-CoA shunting and downstream histone acetylation, leading to global chromatin alterations. These, in turn, drive impaired expression of key DC effectors including central antigen presentation-related genes. Either glucose-lowering treatment or pharmacological modulation of histone acetylation rescues DC function and antiviral immunity. Collectively, we highlight a hyperglycaemia-driven metabolic-immune axis orchestrating DC dysfunction during pulmonary viral infection and identify metabolic checkpoints that may be therapeutically exploited in mitigating exacerbated disease in infected diabetics.

A conserved family of immune effectors cleaves cellular ATP upon viral infection.

During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. Here, we report a family of immune effectors in bacteria that, upon phage infection, degrade cellular adenosine triphosphate (ATP) and deoxyadenosine triphosphate (dATP) by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems, including cyclic oligonucleotide-based antiviral signaling systems (CBASS), prokaryotic argonautes, and nucleotide-binding leucine-rich repeat (NLR)-like proteins, and we show that ATP and dATP degradation during infection halts phage propagation. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a family of bacterial defense systems with a two-component phosphotransfer-signaling architecture. The immune ATP nucleosidase domain is also encoded within diverse eukaryotic proteins with immune-like architectures, and we show biochemically that eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.

N-acetylneuraminic acid links immune exhaustion and accelerated memory deficit in diet-induced obese Alzheimer's disease mouse model.

Systemic immunity supports lifelong brain function. Obesity posits a chronic burden on systemic immunity. Independently, obesity was shown as a risk factor for Alzheimer's disease (AD). Here we show that high-fat obesogenic diet accelerated recognition-memory impairment in an AD mouse model (5xFAD). In obese 5xFAD mice, hippocampal cells displayed only minor diet-related transcriptional changes, whereas the splenic immune landscape exhibited aging-like CD4+ T-cell deregulation. Following plasma metabolite profiling, we identified free N-acetylneuraminic acid (NANA), the predominant sialic acid, as the metabolite linking recognition-memory impairment to increased splenic immune-suppressive cells in mice. Single-nucleus RNA-sequencing revealed mouse visceral adipose macrophages as a potential source of NANA. In vitro, NANA reduced CD4+ T-cell proliferation, tested in both mouse and human. In vivo, NANA administration to standard diet-fed mice recapitulated high-fat diet effects on CD4+ T cells and accelerated recognition-memory impairment in 5xFAD mice. We suggest that obesity accelerates disease manifestation in a mouse model of AD via systemic immune exhaustion.

Cryo-EM structure of the RADAR supramolecular anti-phage defense complex.

RADAR is a two-protein bacterial defense system that was reported to defend against phage by "editing" messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.

Engineering of Pyroelectric Crystals Decoupled from Piezoelectricity as Illustrated by Doped α-Glycine.

Design of pyroelectric crystals decoupled from piezoelectricity is not only a topic of scientific curiosity but also demonstrates effects in principle that have the potential to be technologically advantageous. Here we report a new method for the design of such materials. Thus, the co-doping of centrosymmetric crystals with tailor-made guest molecules, as illustrated by the doping of α-glycine with different amino acids (Threonine, Alanine and Serine). The polarization of those crystals displays two distinct contributions, one arising from the difference in dipole moments between guest and host and the other from the displacement of host molecules from their symmetry-related positions. These contributions exhibit different temperature dependences and response to mechanical deformation. Thus, providing a proof of concept for the ability to design pyroelectric materials with reduced piezoelectric coefficient (d22 ) to a minimal value, below the resolution limit of the method (<0.005 pm/V).

Bacteria deplete deoxynucleotides to defend against bacteriophage infection.

DNA viruses and retroviruses consume large quantities of deoxynucleotides (dNTPs) when replicating. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral lifecycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. Here, we report that bacteria use a similar strategy to defend against bacteriophage infection. We identify a family of defensive bacterial deoxycytidine triphosphate (dCTP) deaminase proteins that convert dCTP into deoxyuracil nucleotides in response to phage infection. We also identify a family of phage resistance genes that encode deoxyguanosine triphosphatase (dGTPase) enzymes, which degrade dGTP into phosphate-free deoxyguanosine and are distant homologues of human SAMHD1. Our results suggest that bacterial defensive proteins deplete specific deoxynucleotides (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Our study shows that manipulation of the dNTP pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.

The identification and characterization of an oxalyl-CoA synthetase from grass pea (Lathyrus sativus L.).

Oxalic acid is a small metabolite found in many plants. It serves as protection from herbivores, a chelator of metal ions, a regulator of calcium levels, and additional tasks. However, it is also a strong di-carboxylic acid that can compromise plant viability by reducing cellular pH. Several metabolic pathways have evolved to control oxalate levels in plants by enzymatic degradation. Among them is the pathway that utilizes oxalyl-CoA synthetase (OCS, EC 6.2.1.8) and ATP to convert oxalate to oxalyl-CoA. Oxalyl-CoA can then be degraded to CO2 or utilized as a precursor for the synthesis of other compounds. In grass pea (Lathyrus sativus L.), a grain legume grown in Asia and Africa for human and animal consumption, the neurotoxic compound ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP) is synthesized from oxalyl-CoA and l-α,ß-diaminopropionic acid (l-DAPA). Here, we report on the identification and characterization of oxalyl CoA-synthetase from grass pea (LsOCS). The gene encoding LsOCS was amplified from grass pea, and then expressed and purified from E. coli cells as an untagged, monomeric protein of 56 kDa. Its catalytic efficiency with oxalate, K oxalate M = 71.5 ± 13.3 µM, V max = 8.2 ± 0.8 µmole min-1 mg-1, was similar to that of OCS homologs from Arabidopsis thaliana (AtAAE3) and Medicago truncatula (MtAAE3). The enzyme was crystalized in complex with AMP and is the first OCS whose structure was determined in the thioester-forming conformation. Finally, we propose that substituting LsOCS with an oxalate oxidase or decarboxylase may reduce the levels of ß-ODAP in grass pea.

Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin ß-ODAP in grass pea.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

Protocol for studying microbiome impact on host energy and reproduction in Drosophila.

Drosophila gut microbiome in flies has been shown to have a systemic influence on energy production by the host and the energetic investment in growth and reproduction. Here we describe a protocol for studying the mechanisms responsible for this remote regulation by gut bacteria. This protocol enables whole-body and ovary-specific quantification of energy-storing molecules as well as identification of host metabolites and pathways that are regulated by gut microbiome-derived factors. Similar procedures are applicable to additional treatments and genetic manipulations. For complete details on the use and execution of this protocol, please refer to Gnainsky et al. (2021).

Computationally designed dual-color MRI reporters for noninvasive imaging of transgene expression.

Imaging of gene-expression patterns in live animals is difficult to achieve with fluorescent proteins because tissues are opaque to visible light. Imaging of transgene expression with magnetic resonance imaging (MRI), which penetrates to deep tissues, has been limited by single reporter visualization capabilities. Moreover, the low-throughput capacity of MRI limits large-scale mutagenesis strategies to improve existing reporters. Here we develop an MRI system, called GeneREFORM, comprising orthogonal reporters for two-color imaging of transgene expression in deep tissues. Starting from two promiscuous deoxyribonucleoside kinases, we computationally designed highly active, orthogonal enzymes ('reporter genes') that specifically phosphorylate two MRI-detectable synthetic deoxyribonucleosides ('reporter probes'). Systemically administered reporter probes exclusively accumulate in cells expressing the designed reporter genes, and their distribution is displayed as pseudo-colored MRI maps based on dynamic proton exchange for noninvasive visualization of transgene expression. We envision that future extensions of GeneREFORM will pave the way to multiplexed deep-tissue mapping of gene expression in live animals.

Gut microbiota modulates weight gain in mice after discontinued smoke exposure.

Cigarette smoking constitutes a leading global cause of morbidity and preventable death1, and most active smokers report a desire or recent attempt to quit2. Smoking-cessation-induced weight gain (SCWG; 4.5 kg reported to be gained on average per 6-12 months, >10 kg year-1 in 13% of those who stopped smoking3) constitutes a major obstacle to smoking abstinence4, even under stable5,6 or restricted7 caloric intake. Here we use a mouse model to demonstrate that smoking and cessation induce a dysbiotic state that is driven by an intestinal influx of cigarette-smoke-related metabolites. Microbiome depletion induced by treatment with antibiotics prevents SCWG. Conversely, fecal microbiome transplantation from mice previously exposed to cigarette smoke into germ-free mice naive to smoke exposure induces excessive weight gain across diets and mouse strains. Metabolically, microbiome-induced SCWG involves a concerted host and microbiome shunting of dietary choline to dimethylglycine driving increased gut energy harvest, coupled with the depletion of a cross-regulated weight-lowering metabolite, N-acetylglycine, and possibly by the effects of other differentially abundant cigarette-smoke-related metabolites. Dimethylglycine and N-acetylglycine may also modulate weight and associated adipose-tissue immunity under non-smoking conditions. Preliminary observations in a small cross-sectional human cohort support these findings, which calls for larger human trials to establish the relevance of this mechanism in active smokers. Collectively, we uncover a microbiome-dependent orchestration of SCWG that may be exploitable to improve smoking-cessation success and to correct metabolic perturbations even in non-smoking settings.

Cyclic CMP and cyclic UMP mediate bacterial immunity against phages.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

ASL expression in ALDH1A1+ neurons in the substantia nigra metabolically contributes to neurodegenerative phenotype.

Argininosuccinate lyase (ASL) is essential for the NO-dependent regulation of tyrosine hydroxylase (TH) and thus for catecholamine production. Using a conditional mouse model with loss of ASL in catecholamine neurons, we demonstrate that ASL is expressed in dopaminergic neurons in the substantia nigra pars compacta, including the ALDH1A1 + subpopulation that is pivotal for the pathogenesis of Parkinson disease (PD). Neuronal loss of ASL results in catecholamine deficiency, in accumulation and formation of tyrosine aggregates, in elevation of α-synuclein, and phenotypically in motor and cognitive deficits. NO supplementation rescues the formation of aggregates as well as the motor deficiencies. Our data point to a potential metabolic link between accumulations of tyrosine and seeding of pathological aggregates in neurons as initiators for the pathological processes involved in neurodegeneration. Hence, interventions in tyrosine metabolism via regulation of NO levels may be therapeutic beneficial for the treatment of catecholamine-related neurodegenerative disorders.

ß-sitosterol reduces anxiety and synergizes with established anxiolytic drugs in mice.

Anxiety and stress-related conditions represent a significant health burden in modern society. Unfortunately, most anxiolytic drugs are prone to side effects, limiting their long-term usage. Here, we employ a bioinformatics screen to identify drugs for repurposing as anxiolytics. Comparison of drug-induced gene-expression profiles with the hippocampal transcriptome of an importin α5 mutant mouse model with reduced anxiety identifies the hypocholesterolemic agent ß-sitosterol as a promising candidate. ß-sitosterol activity is validated by both intraperitoneal and oral application in mice, revealing it as the only clear anxiolytic from five closely related phytosterols. ß-sitosterol injection reduces the effects of restraint stress, contextual fear memory, and c-Fos activation in the prefrontal cortex and dentate gyrus. Moreover, synergistic anxiolysis is observed when combining sub-efficacious doses of ß-sitosterol with the SSRI fluoxetine. These preclinical findings support further development of ß-sitosterol, either as a standalone anxiolytic or in combination with low-dose SSRIs.