Accurate prediction of small-molecule bioactivity is critical for accelerating drug discovery. Here, we report MoActiPred, a multimodal AI framework that integrates 2D molecular graphs, physicochemical descriptors, and 3D E (3)-equivariant geometric representations. Using a curated ChEMBL-derived influenza virus neuraminidase (NA) inhibitor dataset (1245 compounds; IC50 threshold 20 nM), MoActiPred showed the strongest overall performance among the evaluated methods, including traditional machine-learning models (Random Forest, SVM, and XGBoost), SMILES-based language models (ChemBERTa, ChemFM, and PolyBERT), and graph-based baselines (MolCLR and SEGNN). On the public test set, MoActiPred obtained an accuracy of 0.952, MCC of 0.797, and F1 score of 0.898, while retaining high average precision, supporting the value of combining 2D and 3D representations. To evaluate its practical utility, MoActiPred was applied prospectively to 26 oseltamivir analogues designed, experimentally synthesised and evaluated against influenza A (H3N2) NA. The model correctly predicted 6 of 7 high-activity compounds (IC50 < 20 nM) and 18 of 19 inactive or weakly active derivatives, yielding 92.3

Human parainfluenza virus 3 is a highly abundant RNA virus that primarily affects young children, the elderly, and immunocompromised individuals, leading to severe lower respiratory infections and pneumonia. Despite an urgent need of treatment options for these high-risk patients, neither a vaccine nor specific antiviral are currently approved. Blocking viral entry by targeting the viral surface glycoprotein haemagglutinin-neuraminidase (HN) has shown promising results in vitro and, to some extent, in vivo. However, to further evaluate these antiviral approaches for potential human application, a detailed understanding of early hPIV-3 infection and drug treatment mechanisms in human lung tissue is needed. In this study, we established a model for early hPIV-3 infection in human precision-cut lung slices (PCLS). We demonstrate specific infection of small airway epithelial cells followed by a distinct antiviral and inflammatory response marked by expression and secretion of type I, II and III interferons, chemokines such as IP-10 and ITAC, and pro-inflammatory markers IL-6 and TNF-α, but only limited induction of cytokines associated with high clinical severity, such as IL-8. Prophylactic treatment with two viral entry HN-inhibitors significantly reduced hPIV-3 viral load and inflammatory response after infection in the human lung tissue slices, demonstrating the high usability of the PCLS infection model for pharmacological assessment of novel antiviral drugs.

Human parainfluenza virus type 3 (HPIV-3) remains a major cause of respiratory illness particularly among young children, the elderly and immunocompromised individuals. Despite significant efforts in therapeutic discovery research, there is neither an effective antiviral nor a vaccine available against HPIV-3. Host cell glycosylation is known to play a pivotal role in virus entry and replication. While some host glycan-based cellular receptors for HPIV-3 have been identified, the dynamics of the host-cell glycome upon HPIV-3 infection has never been studied. Herein, we report the first mass spectrometry-based study that provides direct insight into the remodelling of the human lung adenocarcinoma cell (A549) glycome upon HPIV-3 infection. Our results reveal significant remodelling of host-cell glycome, including increased expression of oligomannose and reduced expression of sialylated complex-type N -glycans. In addition, we observed altered O -glycan expression, with upregulation of core 1 and downregulation of core 2 type structures in infected cells compared to mock-infected controls. HPIV-3 infection also led to distinct remodelling of glycosphingolipid glycans. Together, these findings present the first evidence that HPIV-3 infection alters host-cell glycome, offering new insights into the virus’ impact on host-cellular processes. ### Competing Interest Statement The authors have declared no competing interest. * HPIV : Human parainfluenza virus HN : haemagglutinin-neuraminidase H.p.i : Hour’s post-infection CEP : Cell extracted protein SP : Secreted protein HN : N -Acetyl Glucosamine H : Hexose dH : Deoxyhexose Man : Mannose N : N -Acetyl Neuraminic Acid G : N -glycolylneuraminic Acid GSL : Glycosphingolipids PGC : Porous graphitized carbon ESI : Electrospray ionization CID : Collision induced dissociation National Health and Medical Research Council, https://ror.org/011kf5r70, GNT1196520, GNT1157150, APP1047824

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galβ1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

S. pneumoniae is the world’s foremost bacterial pathogen. S. pneumoniae encodes a phasevarion (phase-variable regulon), that results in differential expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population.

Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in nontypeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA (n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to human lung cells and denser biofilm formation.

Human metapneumovirus (HMPV) is recognized as an important cause of pneumonia in infants, in the elderly, and in immunocompromised individuals worldwide. The absence of an antiviral treatment or vaccine strategy against HMPV infection creates a high burden on the global health care system.

Human metapneumoviruses have emerged in the past decades as an important global pathogen that causes severe upper and lower respiratory tract infections. Children under the age of 2, the elderly and immunocompromised individuals are more susceptible to HMPV infection than the general population due to their suboptimal immune system. Despite the recent discovery of HMPV as a novel important respiratory virus, reports have rapidly described its epidemiology, biology, and pathogenesis. However, progress is still to be made in the development of vaccines and drugs against HMPV infection as none are currently available. Herein, we discuss the importance of HMPV and review the reported strategies for anti-HMPV drug candidates. We also present the fusion protein as a promising antiviral drug target due to its multiple roles in the HMPV lifecycle. This key viral protein has previously been targeted by a range of inhibitors, which will be discussed as they represent opportunities for future drug design.

SARS-CoV-2, the causative agent of COVID-19, has caused more than 60 million cases worldwide with almost 1.5 million deaths as of November 2020. Repurposing existing drugs is the most rapid path to clinical intervention for emerging diseases.

This paper reports the design, development, and testing of a novel, yet simple and low-cost portable device for the rapid detection of SARS-CoV-2. The device performs loop mediated isothermal amplification (LAMP) and provides visually distinguishable images of the fluorescence emitted from the samples. The device utilises an aluminium block embedded with a cartridge heater for isothermal heating of the sample and a single-board computer and camera for fluorescence detection. The device demonstrates promising results within 20 min using clinically relevant starting concentrations of the synthetic template. Time-to-signal data for this device are considerably lower compared to standard quantitative Polymerase Chain Reaction(qPCR) machine (~10–20 min vs. >38 min) for 1 × 102 starting template copy number. The device in its fully optimized and characterized state can potentially be used as simple to operate, rapid, sensitive, and inexpensive platform for population screening as well as point-of-need severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) detection and patient management.

Human parainfluenza viruses cause acute respiratory tract infections and disease predominantly in young children and immunocompromised individuals. Currently, there are no vaccines to prevent hPIV infections, nor licensed anti-hPIV drugs. There is therefore a need for specific antiviral therapies to decrease the morbidity and mortality associated with hPIV diseases. Haemagglutinin-neuraminidase (HN) is one of two hPIV surface proteins with critical roles in host receptor recognition, binding and cleavage; it has been explored as a key drug development target for the past few decades with variable success. Recent advancements in computational modelling and the availability of the X-ray crystal structure of hPIV3 HN have improved our understanding of the structural and mechanistic features of HN. This review explores structural features of the HN protein that are being exploited for structure-guided inhibitor design. We describe past and present hPIV HN inhibition strategies based on sialic acid scaffolds, together with other novel approaches that decrease hPIV infectivity. Although many HN inhibitors have been developed and evaluated as anti-hPIV agents, currently only a host-directed therapy (DAS181) has succeeded in phase II clinical drug trials. Hence, the review concludes with future considerations for targeting the specific function(s) of hPIV HN and suggestions for antiviral drug design.

Human parainfluenza virus (hPIV) infections are a major cause of respiratory tract illnesses in children, with currently no available vaccine or drug treatment. The surface glycoprotein haemagglutinin-neuraminidase (HN) of hPIV has a central role in the viral life cycle, including neuraminic acid-recognising receptor binding activity (early stage) and receptor-destroying activity (late stage), which makes it an ideal target for antiviral drug disovery. In this study, we showed that targeting the catalytic mechanism of hPIV-1 HN by a 2α,3β-difluoro derivative of the known hPIV-1 inhibitor, BCX 2798, produced more potent inhibition of the neuraminidase function which is reflected by a stronger inhibition of viral replication. The difluorosialic acid-based inhibitor efficiently blocked the neuraminidase activity of HN for a prolonged period of time relative to its unsaturated neuraminic acid (Neu2en) analogue, BCX 2798 and produced a more efficient inhibition of the HN neuraminidase activity as well as in vitro viral replication. This prolonged inhibition of the hPIV-1 HN protein suggests covalent binding of the inhibitor to a key catalytic amino acid, making this compound a new lead for a novel class of more potent hPIV-1 mechanism-based inhibitors.

A novel approach to human parainfluenza virus 3 (hPIV-3) inhibitor design has been evaluated by targeting an unexplored pocket within the active site region of the hemagglutinin-neuraminidase (HN) of the virus that is normally occluded upon ligand engagement. To explore this opportunity, we developed a highly efficient route to introduce nitrogen-based functionalities at the naturally unsubstituted C-3 position on the neuraminidase inhibitor template N-acyl-2,3-dehydro-2-deoxy-neuraminic acid ( N-acyl-Neu2en), via a regioselective 2,3-bromoazidation. Introduction of triazole substituents at C-3 on this template provided compounds with low micromolar inhibition of hPIV-3 HN neuraminidase activity, with the most potent having 48-fold improved potency over the corresponding C-3 unsubstituted analogue. However, the C-3-triazole N-acyl-Neu2en derivatives were significantly less active against the hemagglutinin function of the virus, with high micromolar IC50 values determined, and showed insignificant in vitro antiviral activity. Given the different pH optima of the HN protein's neuraminidase (acidic pH) and hemagglutinin (neutral pH) functions, the influence of pH on inhibitor binding was examined using X-ray crystallography and STD NMR spectroscopy, providing novel insights into the multifunctionality of hPIV-3 HN. While the 3-phenyltriazole- N-isobutyryl-Neu2en derivative could bind HN at pH 4.6, suitable for neuraminidase inhibition, at neutral pH binding of the inhibitor was substantially reduced. Importantly, this study clearly demonstrates for the first time that potent inhibition of HN neuraminidase activity is not necessarily directly correlated with a strong antiviral activity, and suggests that strong inhibition of the hemagglutinin function of hPIV HN is crucial for potent antiviral activity. This highlights the importance of designing hPIV inhibitors that primarily target the receptor-binding function of hPIV HN.

Human parainfluenza viruses represent a leading cause of lower respiratory tract disease in children, with currently no available approved drug or vaccine. The viral surface glycoprotein haemagglutinin-neuraminidase (HN) represents an ideal antiviral target. Herein, we describe the first structure-based study on the rearrangement of key active site amino acid residues by an induced opening of the 216-loop, through the accommodation of appropriately functionalised neuraminic acid-based inhibitors. We discovered that the rearrangement is influenced by the degree of loop opening and is controlled by the neuraminic acid's C-4 substituent's size (large or small). In this study, we found that these rearrangements induce a butterfly effect of paramount importance in HN inhibitor design and define criteria for the ideal substituent size in two different categories of HN inhibitors and provide novel structural insight into the druggable viral HN protein.

Human parainfluenza virus type-3 is a leading cause of acute respiratory infection in infants and children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for inhibitor development. In this study, we explore the structural features required for Neu2en derivatives to efficiently lock-open the 216-loop of the human parainfluenza virus type-3 hemagglutinin-neuraminidase protein.

Human parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive in vitro inhibitor of the hPIV-3 haemagglutinin-neuraminidase (HN). Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 μM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs.