Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors.

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Exploring the Potential of Iminosugars as Antivirals for Crimean-Congo Haemorrhagic Fever Virus, Using the Surrogate Hazara Virus: Liquid-Chromatography-Based Mapping of Viral N-Glycosylation and In Vitro Antiviral Assays.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a pathogen of increasing public health concern, being a widely distributed arbovirus and the causative agent of the potentially fatal Crimean-Congo haemorrhagic fever. Hazara virus (HAZV) is a genetically and serologically related virus that has been proposed as a surrogate for antiviral and vaccine testing for CCHFV. Glycosylation analysis of HAZV has been limited; first, we confirmed for the first time the occupation of two N-glycosylation sites in the HAZV glycoprotein. Despite this, there was no apparent antiviral efficacy of a panel of iminosugars against HAZV, as determined by quantification of the total secretion and infectious virus titres produced following infection of SW13 and Vero cells. This lack of efficacy was not due to an inability of deoxynojirimycin (DNJ)-derivative iminosugars to access and inhibit endoplasmic reticulum α-glucosidases, as demonstrated by free oligosaccharide analysis in uninfected and infected SW13 and uninfected Vero cells. Even so, iminosugars may yet have potential as antivirals for CCHFV since the positions and importance of N-linked glycans may differ between the viruses, a hypothesis requiring further evaluation.

Antiviral effects of deoxynojirimycin (DNJ)-based iminosugars in dengue virus-infected primary dendritic cells.

Dendritic cells (DCs) are important targets for dengue virus (DENV) infection and play a significant role in the early immune response. Antiviral effects of iminosugars against DENV in primary cells have been demonstrated previously in monocyte-derived macrophages (MDMΦs). Given the important role played by DCs in innate immune defense against DENV, the antiviral effects of three deoxynojirimycin (DNJ) derivatives (NN-DNJ, EOO-DNJ and 2THO-DNJ) and a deoxygalactonojirimycin (DGJ) negative control were evaluated in DENV-infected primary human monocyte-derived immature DCs (imDCs). DNJ- but not DGJ-derivatives elicited antiviral activity in DENV-infected imDCs, similar to that observed in MDMΦs. The DNJ-derivatives inhibited DENV secretion in a dose-dependent manner. Endoplasmic reticulum (ER) α-glucosidase I inhibition by DNJ-derived iminosugars, at concentrations of 3.16 µM, correlated with a reduction in the specific infectivity of virions that were still secreted, as well as a reduction in DENV-induced tumour necrosis factor alpha secretion. This suggests iminosugar-mediated ER α-glucosidase I inhibition may give rise to further benefits during DENV infection, beyond the reduction in viral secretion associated with ER α-glucosidase II inhibition.

Iminosugar C-Glycosides Work as Pharmacological Chaperones of NAGLU, a Glycosidase Involved in MPS IIIB Rare Disease*.

Mucopolysaccharidosis type IIIB is a devastating neurological disease caused by a lack of the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU), leading to a toxic accumulation of heparan sulfate. Herein we explored a pharmacological chaperone approach to enhance the residual activity of NAGLU in patient fibroblasts. Capitalizing on the three-dimensional structures of two modest homoiminosugar-based NAGLU inhibitors in complex with bacterial homolog of NAGLU, CpGH89, we have synthesized a library of 17 iminosugar C-glycosides mimicking N-acetyl-D-glucosamine and bearing various pseudo-anomeric substituents of both α- and ß-configuration. Elaboration of the aglycon moiety results in low micromolar selective inhibitors of human recombinant NAGLU, but surprisingly it is the non-functionalized and wrongly configured ß-homoiminosugar that was proved to act as the most promising pharmacological chaperone, promoting a 2.4 fold activity enhancement of mutant NAGLU at its optimal concentration.

Assessing Antigen Structural Integrity through Glycosylation Analysis of the SARS-CoV-2 Viral Spike.

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of March 29, 2021, has claimed 2 776 175 lives worldwide. Vaccine development efforts focus on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralization, and antigen efficacy is influenced by spike glycoprotein production in vivo. Therefore, immunogen integrity is important for glycoprotein-based vaccine candidates. Here, we show how site-specific glycosylation differs between virus-derived spikes, wild-type, non-stabilized spikes expressed from a plasmid with a CMV promoter and tPA signal sequence, and commonly used recombinant, engineered spike glycoproteins. Furthermore, we show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, including shedding of spike monomeric subunits for the non-stabilized wild-type spike tested, which may have implications for the resulting immune response and vaccine design.

Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus-neutralizing activity in vaccinated mice.

Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the full-length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.

Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name "Parodi limit" the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70-80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a "one-size-fits-all adjustable spanner" UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target.

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.

Targeting Endoplasmic Reticulum α-Glucosidase I with a Single-Dose Iminosugar Treatment Protects against Lethal Influenza and Dengue Virus Infections.

Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.

Structural Insights into the Broad-Spectrum Antiviral Target Endoplasmic Reticulum Alpha-Glucosidase II.

Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme's α- and ß-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins.

ToP-DNJ, a Selective Inhibitor of Endoplasmic Reticulum α-Glucosidase II Exhibiting Antiflaviviral Activity.

Iminosugars have therapeutic potential against a range of diseases, due to their efficacy as glycosidase inhibitors. A major challenge in the development of iminosugar drugs lies in making a compound that is selective for the glycosidase associated with a given disease. We report the synthesis of ToP-DNJ, an antiviral iminosugar-tocopherol conjugate. Tocopherol was incorporated into the design of the iminosugar in order to direct the drug to the liver and immune cells, specific tissues of interest for antiviral therapy. ToP-DNJ inhibits ER α-glucosidase II at low micromolar concentrations and selectively accumulates in the liver in vivo. In cellular assays, the drug showed efficacy exclusively in immune cells of the myeloid lineage. Taken together, these data demonstrate that inclusion of a native metabolite into an iminosugar provides selectivity with respect to target enzyme, target cell, and target tissue.

Interdomain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint.

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two ß-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first ß-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.

Iminosugar antivirals: the therapeutic sweet spot.

Many viruses require the host endoplasmic reticulum protein-folding machinery in order to correctly fold one or more of their glycoproteins. Iminosugars with glucose stereochemistry target the glucosidases which are key for entry into the glycoprotein folding cycle. Viral glycoproteins are thus prevented from interacting with the protein-folding machinery leading to misfolding and an antiviral effect against a wide range of different viral families. As iminosugars target host enzymes, they should be refractory to mutations in the virus. Iminosugars therefore have great potential for development as broad-spectrum antiviral therapeutics. We outline the mechanism giving rise to the antiviral activity of iminosugars, the current progress in the development of iminosugar antivirals and future prospects for this field.

Minimal In Vivo Efficacy of Iminosugars in a Lethal Ebola Virus Guinea Pig Model.

The antiviral properties of iminosugars have been reported previously in vitro and in small animal models against Ebola virus (EBOV); however, their effects have not been tested in larger animal models such as guinea pigs. We tested the iminosugars N-butyl-deoxynojirimycin (NB-DNJ) and N-(9-methoxynonyl)-1deoxynojirimycin (MON-DNJ) for safety in uninfected animals, and for antiviral efficacy in animals infected with a lethal dose of guinea pig adapted EBOV. 1850 mg/kg/day NB-DNJ and 120 mg/kg/day MON-DNJ administered intravenously, three times daily, caused no adverse effects and were well tolerated. A pilot study treating infected animals three times within an 8 hour period was promising with 1 of 4 infected NB-DNJ treated animals surviving and the remaining three showing improved clinical signs. MON-DNJ showed no protective effects when EBOV-infected guinea pigs were treated. On histopathological examination, animals treated with NB-DNJ had reduced lesion severity in liver and spleen. However, a second study, in which NB-DNJ was administered at equally-spaced 8 hour intervals, could not confirm drug-associated benefits. Neither was any antiviral effect of iminosugars detected in an EBOV glycoprotein pseudotyped virus assay. Overall, this study provides evidence that NB-DNJ and MON-DNJ do not protect guinea pigs from a lethal EBOV-infection at the dose levels and regimens tested. However, the one surviving animal and signs of improvements in three animals of the NB-DNJ treated cohort could indicate that NB-DNJ at these levels may have a marginal beneficial effect. Future work could be focused on the development of more potent iminosugars.

Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals.

The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.

Iminosugars Inhibit Dengue Virus Production via Inhibition of ER Alpha-Glucosidases--Not Glycolipid Processing Enzymes.

It has long been thought that iminosugar antiviral activity is a function of inhibition of endoplasmic reticulum-resident α-glucosidases, and on this basis, many iminosugars have been investigated as therapeutic agents for treatment of infection by a diverse spectrum of viruses, including dengue virus (DENV). However, iminosugars are glycomimetics possessing a nitrogen atom in place of the endocyclic oxygen atom, and the ubiquity of glycans in host metabolism suggests that multiple pathways can be targeted via iminosugar treatment. Successful treatment of patients with glycolipid processing defects using iminosugars highlights the clinical exploitation of iminosugar inhibition of enzymes other than ER α-glucosidases. Evidence correlating antiviral activity with successful inhibition of ER glucosidases together with the exclusion of alternative mechanisms of action of iminosugars in the context of DENV infection is limited. Celgosivir, a bicyclic iminosugar evaluated in phase Ib clinical trials as a therapeutic for the treatment of DENV infection, was confirmed to be antiviral in a lethal mouse model of antibody-enhanced DENV infection. In this study we provide the first evidence of the antiviral activity of celgosivir in primary human macrophages in vitro, in which it inhibits DENV secretion with an EC50 of 5 µM. We further demonstrate that monocyclic glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. By comparison to bicyclic glucose-mimicking iminosugars which inhibit glycoprotein processing but do not inhibit glycolipid processing and galactose-mimicking iminosugars which do not inhibit glycoprotein processing but do inhibit glycolipid processing, we demonstrate that inhibition of endoplasmic reticulum-resident α-glucosidases, not glycolipid processing, is responsible for iminosugar antiviral activity against DENV. Our data suggest that inhibition of ER α-glucosidases prevents release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.

Inhibition of endoplasmic reticulum glucosidases is required for in vitro and in vivo dengue antiviral activity by the iminosugar UV-4.

The antiviral activity of UV-4 was previously demonstrated against dengue virus serotype 2 (DENV2) in multiple mouse models. Herein, step-wise minimal effective dose and therapeutic window of efficacy studies of UV-4B (UV-4 hydrochloride salt) were conducted in an antibody-dependent enhancement (ADE) mouse model of severe DENV2 infection in AG129 mice lacking types I and II interferon receptors. Significant survival benefit was demonstrated with 10-20 mg/kg of UV-4B administered thrice daily (TID) for seven days with initiation of treatment up to 48 h after infection. UV-4B also reduced infectious virus production in in vitro antiviral activity assays against all four DENV serotypes, including clinical isolates. A set of purified enzyme, in vitro, and in vivo studies demonstrated that inhibition of endoplasmic reticulum (ER) α-glucosidases and not the glycosphingolipid pathway appears to be responsible for the antiviral activity of UV-4B against DENV. Along with a comprehensive safety package, these and previously published data provided support for an Investigational New Drug (IND) filing and Phases 1 and 2 clinical trials for UV-4B with an indication of acute dengue disease.

A Novel Iminosugar UV-12 with Activity against the Diverse Viruses Influenza and Dengue (Novel Iminosugar Antiviral for Influenza and Dengue).

Iminosugars are capable of targeting the life cycles of multiple viruses by blocking host endoplasmic reticulum α-glucosidase enzymes that are required for competent replication of a variety of enveloped, glycosylated viruses. Iminosugars as a class are approved for use in humans with diseases such as diabetes and Gaucher's disease, providing evidence for safety of this class of compounds. The in vitro antiviral activity of iminosugars has been described in several publications with a subset of these demonstrating in vivo activity against flaviviruses, herpesviruses, retroviruses and filoviruses. Although there is compelling non-clinical in vivo evidence of antiviral efficacy, the efficacy of iminosugars as antivirals has yet to be demonstrated in humans. In the current study, we report a novel iminosugar, UV-12, which has efficacy against dengue and influenza in mouse models. UV-12 exhibits drug-like properties including oral bioavailability and good safety profile in mice and guinea pigs. UV-12 is an example of an iminosugar with activity against multiple virus families that should be investigated in further safety and efficacy studies and demonstrates potential value of this drug class as antiviral therapeutics.

Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease.

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

Non-specific accumulation of glycosphingolipids in GNE myopathy.

BACKGROUND: UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) is a bifunctional enzyme responsible for the first committed steps in the synthesis of sialic acid, a common terminal monosaccharide in both protein and lipid glycosylation. GNE mutations are responsible for a rare autosomal recessive neuromuscular disorder, GNE myopathy (also called hereditary inclusion body myopathy). The connection between the impairment of sialic acid synthesis and muscle pathology in GNE myopathy remains poorly understood. METHODS: Glycosphingolipid (GSL) analysis was performed by HPLC in multiple models of GNE myopathy, including patients' fibroblasts and plasma, control fibroblasts with inhibited GNE epimerase activity through a novel imino sugar, and tissues of Gne(M712T/M712T) knock-in mice. RESULTS: Not only neutral GSLs, but also sialylated GSLs, were significantly increased compared to controls in all tested models of GNE myopathy. Treatment of GNE myopathy fibroblasts with N-acetylmannosamine (ManNAc), a sialic acid precursor downstream of GNE epimerase activity, ameliorated the increased total GSL concentrations. CONCLUSION: GNE myopathy models have increased total GSL concentrations. ManNAc supplementation results in decrease of GSL levels, linking abnormal increase of total GSLs in GNE myopathy to defects in the sialic acid biosynthetic pathway. These data advocate for further exploring GSL concentrations as an informative biomarker, not only for GNE myopathy, but also for other disorders of sialic acid metabolism.