Introduction3-Alkynyl-6-aryl-isothiazolo[4,3-b]pyridines have previously been shown to be potent inhibitors of the lipid kinase FYVE finger-containing phosphoinositide kinase (PIKfyve), displaying broad-spectrum antiviral activity.MethodsTo further study their structure–activity relationship (SAR), an efficient synthesis toward 3- bromo-5-chloro-isothiazolo[4,3-b]pyridine was established. It allowed to introduce structural modifications at positions 3 and 5 by palladium-catalyzed cross-coupling reactions and nucleophilic aromatic substitutions.Results and discussionIt led to the generation of a focused library of 3,5-disubstituted isothiazolo[4,3-b]pyridines. Several derivatives exhibited potent PIKfyve inhibition (in the low nM range) in a biochemical assay and antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (in the low μM range). To gain an insight in their binding mode, molecular modeling was applied, indicating that these 3,5- disubstituted isothiazolo[4,3-b]pyridines bind to the ATP-binding site of PIKfyve, although with a different binding mode from that of the 3,6- disubstituted isothiazolo[4,3-b]pyridines.

Various 3,5-disubstituted isothiazolo[4,3-b]pyridines were previously shown to be potent inhibitors of the lipid kinase PIKfyve, displaying broad-spectrum antiviral activity. To further study their structure-activity relationship and to discover novel skeletons as antivirally active PIKfyve inhibitors, a scaffold hopping strategy was applied yielding isothiazolo[4,5-b]pyridines, pyrazolo[4,3-b]pyridines and isothiazolo[3,4-b]pyrazines. Among the newly synthesized scaffolds, the isothiazolo[3,4-b]pyrazines were the most promising, displaying potent and selective PIKfyve inhibition in a biochemical assay, and, in addition, showing antiviral activity against SARS-CoV-2 (in the low μM range). Finally, molecular docking of the various scaffolds in the ATP-binding site of PIKfyve allowed to rationalize their differences in PIKfyve inhibitory activity.

Current mRNA-based vaccine platforms are limited by complex fabrication, stringent cold-chain dependence, and off-target in vivo delivery, restricting their global accessibility and targeting precision. Here we show a modular mRNA delivery platform; our approach integrates inverse microemulsion-derived polymeric nanogels with adsorption-based mRNA loading and the capability for ligand-directed targeting to develop targeted reductively cleavable acrylate-based inverse microemulsion nanogels (TRAINs). By decoupling nanoparticle fabrication from mRNA loading, preformed TRAINs can be stockpiled and later combined with newly designed mRNA, enabling a plug-and-play strategy for rapid adaptation to emerging pathogens, which is particularly advantageous for pandemic preparedness. This architecture supports efficient mRNA association and robust cellular uptake and translation in HEK293T cells. TRAIN also enables postsynthetic surface functionalization; as a proof of concept, CD206-targeted TRAINs promoted selective delivery to antigen-presenting cells (APCs), producing sustained and localized protein expression in BALB/c mice and preferential mRNA delivery to myeloid APCs in heterogeneous human peripheral blood mononuclear cells. Together, these results demonstrate TRAINs as a surface-customizable, adsorption-based mRNA delivery platform with potential for APC-targeted vaccination and rapid, adaptable vaccine deployment.

Discover the world’s best science and medicine | Nature.com

Current single-cell RNA atlases largely capture polyadenylated transcripts while missing critical regulatory layers from noncoding RNA. To address this, we develop a generalizable framework that adapts total RNA profiling for use in standard droplet-based platforms and captures a broad complement of coding and noncoding RNAs using a unified pipeline. Applying this approach to the developing human brain, we generate a dataset mapping diverse RNA biotypes across all neuronal and non-neuronal lineages, revealing biotype-specific expression programs with cell-type and temporal specificity. Tracking microRNA dynamics in Cajal-Retzius neurons, transient and early-born neurons in the cortex, we show the enrichment and target anticorrelation of MIR137, associated with schizophrenia and intellectual disability, suggesting tight regulatory control. We apply TotalX to human peripheral blood mononuclear cells and identify transcriptional modules combining coding and noncoding RNAs and tRNA dynamics. In addition, we analyze dengue-infected hepatocytes and capture non-adenylated viral transcripts that distinguish infection states. This expanded coverage helps with understanding cellular identity and gene regulation at the atlas scale.

Our understanding of a protective humoral immune response to dengue virus (DENV) remains limited. The envelope (E) protein is the main antibody (Ab) target. While anti-fusion loop (FL) epitope monoclonal Abs (mAbs) can induce antibody dependent enhancement (ADE) in vitro, some mAbs targeting quaternary epitopes can cross-neutralize different DENV serotypes. However, the contribution of each Ab subset to disease outcome remains poorly characterized. We defined DENV2 E epitope-specific Abs dynamics and assessed their association with disease outcome in a cohort of hospitalized and subclinical dengue patients during post-primary DENV2 infection. We quantified and isolated anti-E epitope-specific Abs and tested their enhancing and neutralizing capacity. During the critical phase, FL-targeting antibodies were increased in hospitalized patients compared to subclinical cases. Antibodies targeting quaternary epitopes were reduced in severe dengue compared to classical dengue fever patients. Functionally, quaternary epitope-targeting antibodies showed stronger neutralization and cross-neutralization properties, while FL-binding antibodies displayed stronger in vitro enhancement. These findings show that anti-DENV2 E epitope-specific Ab proportions correlate with disease susceptibility and severity, with important implications for novel vaccine design. Antibodies against dengue virus (DENV) can play both a protective and a disease enhancing role via antibody-dependent enhancement. The envelope (E) protein of the virus is the main target of the antibody (Ab) response. The composition, kinetics, and function of antibodies targeting different epitopes on the E protein, and their direct link to disease severity, remain insufficiently defined during natural infection. Antibodies against dengue virus (DENV) can play both a protective and a disease enhancing role via antibody-dependent enhancement. The envelope (E) protein of the virus is the main target of the antibody (Ab) response. The composition, kinetics, and function of antibodies targeting different epitopes on the E protein, and their direct link to disease severity, remain insufficiently defined during natural infection.

Non-coding RNAs represent a widespread and diverse layer of post-transcriptional regulation across cell types and states, yet much of their diversity remains uncharted at single-cell resolution. This gap stems from the limitations of widely used single-cell RNA-sequencing protocols, which focus on polyadenylated transcripts and miss many short or non-polyadenylated RNAs. Here, we adapted single-cell RNA-sequencing on the 10x Genomics platform to capture a broad complement of coding and non-coding RNAs-including miRNAs, tRNAs, lncRNAs, histone RNAs, and non-adenylated viral transcripts. This approach enabled the discovery of rich, dynamic non-coding RNA programs across immune cells, virally infected hepatocytes, and the developing human brain. In dengue virus-infected hepatocytes, we detect non-adenylated viral transcripts and distinguish active from transcriptionally quiescent infected states, each with distinct host regulatory signatures. In brain tissue, we identify biotype-specific, cell-type-restricted non-coding RNAs, including miRNAs whose expression anticorrelates with predicted targets, consistent with post-transcriptional regulatory relationships. We show that MIR137, one of the strongest GWAS loci associated with schizophrenia and intellectual disability, is expressed specifically in Cajal-Retzius cells, an early-born but transient population that guides subsequent cortical neuron migration. These findings demonstrate the importance of non-coding RNAs in defining cell identity and state, and show how expanded transcriptome coverage can reveal additional layers of gene control-now accessible through practical and scalable single-cell profiling.

Background/Objectives: RMC-113, a 3-alkynyl-6-aryl-disubstituted isothiazolo[4,3-b]pyridine, is a dual inhibitor of the lipid kinases PIKfyve and PIP4K2C with broad-spectrum antiviral activity. The aim was to study the structure–activity relationship (SAR) of isothiazolo[4,3-b]pyridines as dual PIKfyve/PIP4K2C inhibitors. Methods: A series of isothiazolo[4,3-b]pyridines was synthesized by introducing structural variety at positions 3 and 6 of the central scaffold. The primary assay to guide the synthetic chemistry was a biochemical PIKfyve assay, with a number of analogues also tested for PIP4K2C binding affinity. Finally, isothiazolo[4,3-b]pyridines were also evaluated for antiviral and antitumoral activity in cell-based assays. Results: PIKfyve inhibition tolerated a wide variety of substituents on the aryl ring at position 6 of the isothiazolo[4,3-b]pyridine scaffold, with the 4-carboxamide analogue emerging as the most potent (IC50 = 1 nM). The SAR at position 3 was more restricted, although the introduction of electron-donating groups (such as a methyl and methoxy) on the pyridinyl ring yielded potent PIKfyve inhibitors, with IC50 values in the low nM range. The acetylenic moiety was essential for PIKfyve inhibition, and only the saturated ethyl linker displayed potent PIKfyve inhibition, albeit less active than the acetylene counterpart. The compounds were 2- to 5-fold less potent on PIP4K2C relative to PIKfyve. These dual PIKfyve/PIP4K2C inhibitors displayed antiviral activity against both the venezuelan equine encephalitis virus (VEEV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A screening against a panel of cancer cell lines revealed antitumoral activity, although some of the potent PIKfyve/PIP5K2C inhibitors lacked antitumoral activity. Conclusions: Isothiazolo[4,3-b]pyridines are dual PIKfyve/PIP4K2C inhibitors displaying broad-spectrum antiviral, as well as antitumoral, activity.

Metabolic profiling of respiratory samples from individuals infected and uninfected with respiratory viral infections may identify biomarker signatures that complement routine clinical diagnostic testing and offer unique insights into pathophysiology. We used liquid chromatography quadrupole time-of-flight mass spectrometry to generate untargeted metabolomic profiles and identified top biomarker signatures differentiating severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) positive from negative samples via machine learning. We then adapted these signatures to liquid chromatography-tandem mass spectrometry for targeted profiling and assessed classification performance, including samples positive for other respiratory viruses and negative for viral testing. A total of 1,226 samples were tested, including 521 positive samples for SARS-CoV-2, 97 for influenza A, 96 for respiratory syncytial virus (RSV), 211 for other respiratory viruses, and 301 negative samples. The top-performing model was the Light Gradient Boosting Model, which showed an area under the receiver operating characteristic curve (AUC) of 0.99 (95% confidence interval [CI], 0.99-1.00), sensitivity of 0.96 (95% CI, 0.91-0.99), and specificity of 0.95 (95% CI, 0.90-0.97). A separate machine learning analysis investigating the performance by viral subtype showed high performance for the identification of influenza A virus with an AUC of 0.97 (95% CI, 0.94-0.99) and RSV with an AUC of 0.99 (95% CI, 0.97-1.00). The two features with the highest ranking were identified as 3-oxo-heneicosanoic acid and 2-(4-hydroxyphenyl) ethanol. These findings extend our understanding of the metabolic impact of respiratory viral infections and support the potential of metabolomics to complement routine clinical diagnostic methods.IMPORTANCEMolecular testing has greatly improved how viruses are diagnosed; however, gaps remain, including limited sensitivity directly from specimens and inability to differentiate active from resolved infection. In this study, we investigated the use of a distinct diagnostic approach, mass spectrometry for detection of metabolites (small molecules) combined with machine learning analysis, for the diagnosis of SARS-CoV-2 and other respiratory viruses. We demonstrated strong performance of this approach directly from upper respiratory swab samples to differentiate SARS-CoV-2-infected versus uninfected individuals. Extension of this approach to influenza and RSV maintained a high level of performance. This research suggests that mass spectrometry-based infectious disease diagnostic testing has clinical potential and that these metabolomic features may reveal novel host-pathogen interactions and therapeutic targets. Applying a similar approach to prospective, multisite cohorts of patients with other infectious diseases carries potential to extend our understanding of the metabolic pathways involved in the host response to infection.

In search for broad-spectrum antivirals, we discover a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrate selective inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Lipidomics analysis reveals alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and links its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We identify PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays, reveals that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced autophagic flux impairment. Promoting viral protein degradation by reversing autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual PIP4K2C and PIKfyve inhibition as a candidate strategy to combat emerging viruses.

Since the outbreak of SARS-CoV-2 in recent years, our society has become more aware that zoonotic diseases pose a real threat. Therefore, the demand for small molecules that target host proteins, essential for viral entry and replication, has increased as an interesting strategy for the development of antiviral agents, as these agents may be effective against several different pathogens. NAK kinases is one such potential target family because they are involved in a variety of cellular functions, hijacked by viruses to invade host cells, such as clathrin-mediated endocytosis. A large number of different inhibitors have already been reported targeting NAK kinases, but there are still no compounds that selectively target AAK1 over other NAK family members, in particular the closely related family member BIKE. Here, we developed a series of pyrazolo[1,5-a]pyrimidine-based macrocyclic NAK inhibitors, starting from the acyclic AAK1 inhibitor LP-935509. Through a structure-guided activity relationship study within the NAK family, we identified potent AAK1 inhibitors 16, 18 and 27, which show promising selectivity within the NAK family. The inhibitors showed a potent inhibition of the phosphorylation of the AP-2 complex and the antiviral activity of the compounds was evaluated against various RNA viruses.

Non-coding RNAs represent a widespread and diverse layer of post-transcriptional regulation across cell types and states, yet much of their diversity remains uncharted at single-cell resolution. This gap stems from the limitations of widely used single-cell RNA-sequencing protocols, which focus on polyadenylated transcripts and miss many short or non-polyadenylated RNAs. Here, we adapted single-cell RNA-sequencing on the 10x Genomics platform to capture a broad complement of coding and non-coding RNAs—including miRNAs, tRNAs, lncRNAs, histone RNAs, and non-adenylated viral transcripts. This approach enabled the discovery of rich, dynamic non-coding RNA programs across immune cells, virally infected hepatocytes, and the developing human brain. In dengue virus-infected hepatocytes, we detect non-adenylated viral transcripts and distinguish active from transcriptionally quiescent infected states, each with distinct host regulatory signatures. In brain tissue, we identify biotype-specific, cell-type– restricted non-coding RNAs, including miRNAs whose expression anticorrelates with predicted targets, consistent with post-transcriptional regulatory relationships. We show that MIR137 , one of the strongest GWAS loci associated with schizophrenia and intellectual disability, is expressed specifically in Cajal-Retzius cells, an early-born but transient population that guides subsequent cortical neuron migration. These findings demonstrate the importance of non-coding RNAs in defining cell identity and state, and show how expanded transcriptome coverage can reveal additional layers of gene control—now accessible through practical and scalable single-cell profiling. ### Competing Interest Statement The authors have declared no competing interest. CZ Biohub, https://ror.org/00knt4f32 Swiss National Science Foundation, https://ror.org/00yjd3n13 National Institute of Allergy and Infectious Diseases

Adaptor protein-2 associated kinase 1 (AAK1) has been proposed as a promising host target for the development of broad-spectrum antiviral agents. Different regioisomers, as well as scaffolds that were not explored before for their AAK1 affinity, were prepared that were derived from a known and potent 7-azaindole-based AAK1 inhibitor. This effort led to the discovery of various pyrrolo[2,3-b]pyridines, pyrazolo[3,4-b]pyridines and pyrazolo[3,4-d]pyrimidines with low nanomolar AAK1 binding affinity. The AAK1 data were rationalized by molecular modeling. Selected congeners displayed antiviral activity against dengue virus and Venezuelan equine encephalitis virus.

Infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, conducted in high-containment laboratories, requires transferring samples to lower containment labs for downstream applications, mandating sample inactivation. Here, we present a stepwise protocol for chemical inactivation of SARS-CoV-2 virus in culture supernatants or within infected cells and organoids, using eight chemical reagents validated via plaque assays. Additionally, we describe steps for troubleshooting virus inactivation, titer calculation, and log reduction. This protocol offers valuable resources for the COVID-19 research community, providing essential tools to advance research on this virus.

Inhibition of AP-2-associated protein kinase 1 (AAK1) has been shown to be a promising avenue for the development of broad-spectrum antiviral agents. On a previously described AAK1 inhibitor based on a pyrrolo[2,3-b]pyridine scaffold, the concept of isosterism was applied, by replacing a carboxamide linker by various five-membered heterocycles. It led to the discovery of a novel series of AAK1 inhibitors with IC50 values in the low nM range, that also displayed antiviral activity against the dengue virus and Venezuelan equine encephalitis virus.

In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C’s roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited.This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 M pro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here we have optimized 2-aminopyrimidine-based macrocycles to use these compounds as chemical tools for the ephrin kinase family. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55, a potent inhibitor of EPHA2/A4 and GAK. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/A4 and GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells. However, further investigations are needed to determine whether this was a compound-specific effect or target-related.