ACS Infectious Diseases

In a previous screen for compounds that inhibit Plasmodium falciparum merozoite invasion of red blood cells, we identified the Medicines for Malaria Venture compound MMV006833. This compound inhibits PfSTART1, a protein implicated in the expansion of the nascent parasitophorous vacuole membrane following invasion, to accommodate the developing ring-stage parasite. Live-cell lattice light-sheet microscopy of invading merozoites revealed that mNeonGreen-tagged PfSTART1 is released from structures within the merozoite into the nascent parasitophorous vacuole approximately 109 seconds after invasion. Expansion microscopy of PfSTART1-HA merozoites further showed that these punctate PfSTART1-containing structures do not colocalise with known secretory organelles (rhoptries, micronemes and dense granules). Although analogues of MMV006833 are highly potent against P. falciparum, they were previously found to be ineffective against P. berghei parasites in the mouse malaria model. Here, we demonstrate that PbSTART1 is highly resistant to MMV006833 and its analogues when expressed in P. falciparum, indicating that structural differences between the orthologous proteins reduce inhibitor potency. The crystal structure of PfSTART1 in complex with WEHI-991 revealed the molecular basis for inhibition and provided a structural explanation for the reduced potency of this family of compounds against P. berghei. To sensitise P. berghei parasites to MMV006833 analogues, the parasites were engineered to express PfSTART1; however, these chimeric parasites remained insensitive to the compounds. This suggests that factors beyond target engagement, such as compound half-life or bioavailability, contribute to the lack of efficacy observed in the mouse malaria model.

Malaria kills {approx}600,000 people annually despite recently deployed vaccines. The parasite's blood-stage invasion of erythrocytes, driven by the essential, non-redundant interaction between Plasmodium falciparum RH5 and erythrocyte basigin, remains a leading therapeutic target. Here, we designed de novo mini-proteins (minibinders) to occlude this interface. From {approx}1,500 computationally generated designs, we synthesized 18 candidates ranging from 9.3 to 16.9 kDa which were easily expressed in E. coli as soluble, well-folded proteins. Of these, 7 minibinders blocked blood-stage replication of P. falciparum asexual stage parasites with greater potency than {approx}300nM IC50. Three potent candidate minibinders, mb-5, mb-7, and mb-21, were confirmed to bind RH5 via SPR and BLI and block the merozoite-to-ring transition in stage-synchronized parasite progression assays, with CryoEM of minibinder-RH5 complex confirming the predicted binding at RH5-basigin interface. Although pharmacological challenges remain, our data demonstrate that potent inhibitors of parasite growth, rivaling existing antimalarials, can be designed rather than discovered via extensive screening. Our data highlight the potential for artificial intelligence to change the development landscape of new antimalarial therapies.

Screening of the open-access CRESTdb small-molecule library identified the imidazothiazole carboxamide sCQG200 (1) as an initial hit, with activity against H37Rv and Erdman Mycobacterium tuberculosis strains in cholesterol-containing medium (MIC90= 6.88 uM and 4.48 uM, respectively) and against intracellular Mtb (EC50= 3.80 uM). sCQG200 retained activity against drug-resistant Mtb strains, including clinical MDR/XDR isolates. SAR optimization led to analogs 21 and 24 with substantially improved antimycobacterial potency. Mouse pharmacokinetic studies following oral and intravenous administration showed 62.1% and 29.5% oral bioavailability for 21 and 24, respectively. This structurally differentiated chemotype provides a promising starting point for further optimization as antitubercular agent.

New therapeutic approaches are needed to treat infections due to multidrug resistant (MDR) Gram-negative bacteria. The use of antibody-recruiting molecules (ARMs) is a promising approach to address the MDR problem given their potential to engage both the adaptive and innate immune systems to treat bacterial infections. Here we present two new strategies to create ARMs. First, we show that a 9-mer antimicrobial peptide (K6) can be fused to a 11-mer mimotope peptide recognized by antibodies targeting the Vibrio cholerae O1 O-antigen to produce an ARM peptide (FZ5) that can render E. coli more sensitive to complement killing in the presence of antibodies that recognize this mimotope. FZ5 can drive killing by complement in the presence of rabbit antibodies raised against O1 serogroup V. cholerae as well as antibodies present in human sera elicited by immunization with the live attenuated cholera vaccine PanChol. We also describe a strategy that combines bacterial surface binding with hapten display through the use of homodimers of the antibiotic streptomycin (Str) and anti-Str antibodies elicited by Str-protein conjugate vaccines. When combined with dimeric Str derivatives and human complement, polyclonal anti-streptomycin antisera kills E. coli by targeting the Str hapten. Dimeric forms of Str also significantly enhanced killing of a Str-resistant P. aeruginosa clinical isolate dependent on low levels of human complement and polyclonal anti-Str antibody. Together these data suggest that anti-bacterial ARM molecules based on mimotope display through simple synthetic fusion peptides, or dimeric haptens have promise as new approaches for the treatment of MDR bacterial infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is non-susceptible to beta-lactams. Blockade of cell wall biosynthesis is a potential target for beta-lactam enhancement but requires further investigation. A butyrolactone derivative enhanced beta-lactams against MRSA strains by reducing the availability of D-Ala-D-Ala. Unlike D-cycloserine, it did not inhibit D-Ala-D-Ala ligase (Ddl). Nor did it show an additive or synergistic effect when combined with cycloserine, indicating a unique mechanism for blocking cell wall precursor production that does not involve the traditional Lipid II pathway. Notably, beta-lactam potentiation by our chemical or D-cycloserine was highly dependent on the intrinsic autolytic ability of the tested MRSA strains. Strains that resisted lysis upon Triton X-100 exposure showed a minimal increase in beta-lactam susceptibility, whereas highly autolytic strains showed significant changes in their beta-lactam MICs. We have thus identified autolytic ability as the Achilles Heel in the strategy of targeting cell wall biosynthesis for beta-lactam potentiation.

We aimed to repurpose the non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI) rilpivirine (RPV) as a targeted protein degrader (TPD) of HIV-1 RT. Structure-guided modeling identified a TPD bifunctional linker exit trajectory from RPV that threads the NNRTI entrance channel. Ten RPV analogs (RPV') with varied warhead-linker attachments retained RT inhibition and antiviral activity, guiding selection of an amide-linked connector for degrader construction. We synthesized Proteolysis Targeting Chimeras (PROTACs) designed to recruit CUL4CRBN and CUL2VHL, alongside adamantyl acetic acid-based Hydrophobic Tags (HyTs). TPDs were evaluated for RT inhibition, virus inhibition, biophysical target engagement, and proteasome-dependent degradation. Some PROTACs showed minimal antiviral activity or limited solubility for our assays, whereas the HyT with a tetraethylene glycol (PEG4) linker (PEG4-Ad) exhibited low nanomolar potency to suppress HIV-1 replication and acceptable solubility. PEG4-Ad reduced RT levels in a proteasome-dependent manner without cytotoxicity but did not exhibit superior potency against RPV resistance mutations. MD simulations suggested that PEG4 linkers maximize episodic exposure of the hydrophobic tag, consistent with PEG4-Ad efficacy. These data highlight the promise - and constraints - of antiviral degraders, indicating that linker-controlled hydrophobic tag exposure and subcellular target accessibility may be critical design parameters for prospective therapeutics.

The sulfur mobilization (SUF) system is the only Fe-S cluster biosynthesis pathway in Mycobacterium tuberculosis (M. tb) and Mycolicibacterium smegmatis (M. smegmatis). In this pathway, SufC protein acts as an ATPase in the SufBC2D scaffold for Fe-S cluster biogenesis. This crucial role positions SufC as a promising target for the development of antimycobacterial therapeutics. However, the SufC ATPases from Mycobacterial species are not yet characterized. This study includes cloning, expression, purification, and characterization of SufC homologs from M. tb (Rv1463) and M. smegmatis (MSMEG_3124) using biochemical, biophysical, and computational approaches. Both SufC proteins were purified using affinity and size-exclusion chromatography and observed to exist as monomers in solution. Although both proteins have the conserved domains, they exhibit different ATP-dependent kinetic efficiencies, demonstrating differences in their local environment and catalytic behavior. Furthermore, CRISPR interference targeting MSMEG_3124 results led to diminished M. smegmatis growth, highlighting the critical role of SufC in cellular viability. Modeling and virtual screening led to the identification of three candidate compounds, of which Z2911048515 and Z66741142 were selected for experimental evaluation. Circular dichroism spectroscopy analysis revealed slight differences in the spectral features of Rv1463 in the presence of Z2911048515 and Z66741142, while significant spectral variations were observed for MSMEG_3124. Importantly, Z66741142 was able to cause changes in the fluorescence spectra of both SufC protein homologs. Surprisingly, both compounds showed only limited inhibition of SufC ATPase activity, exhibited distinct modes of action, and may serve as preliminary chemical scaffolds for optimization and testing as effective inhibitors. This study presents functional diversity of mycobacterial SufC proteins across pathogenic and non-pathogenic species and provides a basis for developing a framework for further mechanistic and structure-guided investigation within the indispensable SUF pathway.

The pathogenicity of Mycobacterium tuberculosis relies on its ability to withstand hostile host environments. Coenzyme F420 is critical for redox metabolism in Mycobacterium tuberculosis an organism distinguished by its abundant use of this low-redox-potential cofactor and a repertoire of F420-dependent enzymes. While F420-dependent mechanisms are known to confer protection against antimicrobials and oxidative stress, the specific enzymes responsible remain largely unidentified. In this study, using the F420-dependent detoxification of malachite green as a phenotypic marker, we performed a genome-wide screen in Mycobacterium tuberculosis using transposon mutagenesis and whole genome sequencing. The results of transposon mutagenesis and whole genome sequencing revealed that, among the 28 genes encoding F420-dependent enzymes, only mutations in fgd (essential for F420H2 re-generation) and Rv2061c, a gene encoding a putative F420-dependent oxidoreductase, were highly susceptible to malachite green. Subsequent experiments confirmed that Rv2061c and its homolog MT2120 are required for detoxifying malachite green and crystal violet. Furthermore, the Rv2061c mutant exhibited increased susceptibility to isoniazid and pretomanid; as well as menadione-induced oxidative stress. We also identified mutations in the transcriptional repressor Rv0678 as a key determinant of resistance to malachite green and crystal violet. This study establishes Rv2061c as a pivotal F420-dependent oxidoreductase that protects Mycobacterium tuberculosis from malachite green and other antimicrobial compounds as well as oxidative stress and further supports Rv0678 as a master regulator of multidrug resistance. These findings expand our knowledge of Mycobacterium tuberculosis defense mechanisms and highlight F420-dependent pathways as a potential target for new drug development.

The incidence of infections caused by multidrug-resistant bacterial agents has increased at an alarming rate worldwide. With the aim of identifying novel antimicrobial peptides (AMPs), we screened bacteria isolated from various environmental samples. Our hypothesis was that the sequence space of natural AMPs belonging to known AMP classes is far from saturation. We used a classical pipeline of bacterial culturing, overlay assays, extraction and fractionation to purify AMPs for identification. Using LC/MS analysis, the structure of AMPs isolated from a strain of Paenibacillus was narrowed down and a new subclass of polymyxins was uncovered. These harbor three aliphatic residues within the cyclic C-terminal, and in certain cases Ser replaces Thr at position A2. Four discrete polymyxins fitting the new class, but not identical to the natural polymyxins were synthesized and tested against 29 human pathogenic bacteria. Each displayed an antibacterial spectrum different from that of colistin, with the best candidate surpassing it in potency against ten bacterial strains, but underperforming against three others. These results demonstrate that current screening of natural bacterial isolates can still permit the design of novel antimicrobial peptide variants without the substantial threat of diminishing returns.

Staphylococcus epidermidis is a common member of the healthy skin microbiome that contributes to barrier function and protection against pathogen colonization. In contrast, Staphylococcus aureus is a major human pathogen and the leading cause of skin and soft tissue infections, owing in part to its robust biofilm-forming capacity and increasing antimicrobial resistance. We previously demonstrated that S. epidermidis cell-free conditioned media (CFCM) inhibit S. aureus biofilm formation by altering bacterial gene expression without affecting growth. Here, we identify the metabolite responsible for this activity. Bioactivity-guided fractionation of S. epidermidis CFCM revealed pyroglutamic acid (PCA) as the active antibiofilm compound. Commercially sourced PCA inhibited S. aureus biofilm formation and reduced adhesion to epithelial cells. In a murine skin infection model, PCA treatment significantly attenuated disease severity, accelerated wound healing, and reduced bacterial burden. Analysis of S. epidermidis CFCM by chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry confirmed the presence of PCA and identified it as the bioactive constituent responsible for antibiofilm activity. Together, these findings uncover a previously unrecognized mechanism by which S. epidermidis suppresses S. aureus virulence and highlight the potential of microbiome-derived metabolites as therapeutics for the prevention and treatment of S. aureus skin infections.

Heme metabolism regulates the availability of heme, an iron-containing molecule essential for oxygen transport, mitochondrial respiration, electron transfer, and enzymatic reactions. Increasing evidence implicates heme metabolism in regulating viral infection, potentially representing an important innate antiviral mechanism. Components of both heme biosynthesis and degradation pathways exhibit antiviral activity against different viruses; however, their contributions and conservation across human coronaviruses remain incompletely defined. We investigated the impact of modulating heme metabolism on infection by seasonal human coronaviruses HCoV-OC43 and HCoV-229E. Treatment with 5-aminolevulinic acid (5-ALA) produced cell line-dependent antiviral effects, with robust inhibition in H1299 cells and selective HCoV sensitivity in MRC-5 cells. These differences correlated with intracellular accumulation of protoporphyrin IX (PPIX), suggesting that cellular metabolic capacity influences antiviral efficacy. In contrast, components of the heme degradation pathway, including heme oxygenase-1 (HO-1), carbon monoxide, and biliverdin, did not exhibit antiviral effects against HCoV-OC43 or HCoV-229E. PPIX and synthetic porphyrin CoPPIX inhibited early infection through direct viral inactivation. Collectively, these findings indicate that antiviral effects of heme metabolism against seasonal human coronaviruses are primarily driven by direct inactivation by PPIX rather than cellular antiviral responses induced by the heme degradation pathway

Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen capable of adapting to diverse and challenging environments, including the oxygen- and nutrient-limited conditions encountered during chronic infections. Its ability to utilize a broad range of carbon sources and to switch from aerobic to anaerobic respiration contributes to its persistence within the host and its resilience to therapeutic interventions. In particular, denitrification enables P. aeruginosa to grow under oxygen-limited conditions by using nitrate and nitrite as alternative electron acceptors, a process particularly relevant in biofilms and chronic infections such as cystic fibrosis. Ubiquinone (UQ) is essential for respiratory metabolism in P. aeruginosa, which possesses two distinct UQ biosynthetic pathways that ensure UQ production across a broad range of oxygen concentrations. Recent studies have identified the O2-independent UQ biosynthetic pathway as a key determinant of chronic infection. Because this pathway is restricted to a limited group of bacteria, we hypothesized that its selective inhibition could represent a strategy to specifically target P. aeruginosa under anaerobic conditions. To test this hypothesis, we screened analogues of 4-hydroxybenzoic acid (4-HB), the earliest precursor of the UQ biosynthetic pathway, and identified 4-aminobenzoic acid, also known as para-aminobenzoic acid (pABA), as a potential inhibitor. pABA primarily affects the anaerobic metabolism of P. aeruginosa by acting as a competing substrate for UQ biosynthetic enzymes, thereby interfering with UQ biosynthesis. This inhibitory effect positions pABA as a valuable tool for investigating, and potentially targeting, anaerobic metabolism in P. aeruginosa.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading global health threat, especially due to multidrug-resistant strains. The first-line drug isoniazid (INH) and second-line drugs ethionamide (ETH) and prothionamide (PTH), are prodrugs that target the essential enoyl-ACP reductase InhA, a key enzyme involved in mycolic acid biosynthesis. Resistance to these drugs primarily arises from genetic mutations that inactivate the enzymatic function of their respective bioactivating enzymes. While the recent development of chemical boosters has shown promising results in restoring drug susceptibility in these mutants, genetic tools to dynamically modulate these pathways and fine-tune resistance could be extremely valuable to develop the next-generation of anti-InhA drugs. In this context, we leverage CRISPR interference (CRISPRi) to develop inducible and reversible genetic systems to fine-tune antibiotic susceptibility in Mycobacterium smegmatis (Msmeg) and Mtb. By using our CRISPRi-based approach we first generated an inducible inhA hypomorph, an essential target required for mycolic acid production and bacterial growth, therefore confirming that our method is fully functional. Then, we developed genetic tools to selectively target katG, ethA, and the transcriptional repressor ethR, in order to generate conditional hypomorphs with altered antibiotic susceptibility profiles. Antibiotic susceptibility assays confirmed that the conditional hypomorphs selectively displayed high-level resistance or hypervulnerability to INH or ETH upon CRISPRi induction. Altogether, this work establishes CRISPRi as a powerful genetic platform for selectively fine-tuning mycobacterial drug susceptibility, with potential applications in developing next-generation anti-TB therapies.

Recognition of host glycosaminoglycans (GAGs) is critical for the adhesion and colonization of Borrelia burgdorferi, but the structural requirements for these interactions remain incompletely understood. Using surface plasmon resonance (SPR), we characterized the heparin-binding properties of three Borrelia adhesins, i.e. Bgp, DbpA-A9, and DbpB-B31 and evaluated inhibitors comprising heparin oligosaccharides, selectively desulfated heparins, mammalian GAGs, marine sulfated glycans, pentosan polysulfate (PPS), and mucopolysaccharide polysulfate (MPS). All three adhesins bound directly to heparin, with equilibrium dissociation constants (KD) of 2.18 nM for Bgp and 36.4 nM for DbpB-B31. Competition assays identified N-sulfation and 6-O-sulfation as major determinants of binding, whereas 2-O-sulfation had a lower effect. Bgp showed chain-length dependence over dp4-dp20, whereas DbpB-B31 preferentially recognized longer oligosaccharides, particularly those of dp12 or greater. Chondroitin sulfate E also exhibited strong inhibitory activity, indicating that recognition was not restricted to heparin/heparan sulfate backbones. Among the tested sulfated glycans, fucosylated chondroitin sulfate (HfFucCS) from Holothuria floridana was the most potent inhibitor of Bgp, with an IC50 of approximately 4 ng/mL, whereas PPS was the strongest inhibitor of DbpB-B31, followed by MPS. These findings reveal protein-specific sulfated-glycan recognition and identify promising candidates for disrupting B. burgdorferi-host GAG interactions.

COVID-19 highlighted the need for antivirals that can be rapidly deployed against both current and future coronavirus threats. CMX990 is a clinical stage coronavirus main protease (Mpro) inhibitor bearing a unique trifluoromethoxymethyl ketone warhead. Here, we broadly evaluate its pan-coronavirus target engagement, antiviral breadth, and pharmacologic differentiation. Across a reporter panel of 24 Mpro enzymes spanning alpha-, beta-, delta-, and gamma-coronaviruses, CMX990 retained robust potent activity, including against nirmatrelvir-resistant SARS-CoV-2 variants. In direct comparison, nirmatrelvir and ensitrelvir showed diminished potency against non-SARS-CoV-2 coronaviruses. Live-virus assays corroborated this breadth, with potent, selective activity against SARS-CoV-2, SARS-CoV-1, MERS-CoV, and additional human coronaviruses. Structural studies revealed a conserved covalent binding mode centered on the catalytic Cys of coronavirus Mpro. In serial-passage studies, resistance emerged only after approximately twice as many serial passages under CMX990 selection as under nirmatrelvir selection, without selecting for the canonical nirmatrelvir-resistance substitution E166V. Extracellular washout experiments revealed superior persistence of antiviral activity relative to nirmatrelvir, consistent with durable target engagement. In a murine SARS-CoV-2 infection model, once-daily CMX990 protected against weight loss and reduced lung viral titers to near baseline levels. Collectively, these findings identify CMX990 as a structurally and functionally validated pan-coronavirus protease inhibitor supporting its potential as a pandemic-ready antiviral candidate.

The emerging neonatal bacterial pathogen Paenibacillus thiaminolyticus causes sepsis and postinfectious hydrocephalus, yet few tools are available to study or specifically detect it. We developed fluorescent probes based on cell wall-binding domains (CBDs) from bacteriophage-derived cell-wall hydrolases. CBDs can target structurally conserved cell-wall glycans, making them attractive affinity reagents for bacterial detection. None of the 11 probes we designed labeled intact cells under standard conditions, and we identified a surface layer (S-layer) that prevented access to their cell-wall targets. Brief acidic treatment (pH 2.0) released abundant high-molecular-mass proteins from the cell surface and enabled labeling by five distinct CBD probes, each containing a single C-terminal S-layer homology domain. Mass spectrometry identified the main released protein as an ortholog of the P. alvei SpaA S-layer protein, which we designate SlsA. Across 26 surveyed P. thiaminolyticus genomes, we identified four distinct SlsA variants with 58-79% pairwise amino acid identity, encoded within a conserved cluster homologous to the P. alvei S-layer-associated locus. After either the acidic treatment or a quick flame fixation, the lead probe mGL-BDPt9 labeled six P. thiaminolyticus strains, including three Ugandan clinical isolates, as well as a P. dendritiformis clinical isolate from a US infant, and three additional Paenibacillus species. mGL-BDPt9 also distinguished P. thiaminolyticus from five gram-positive sepsis pathogens in pairwise mixtures and detected it in a five-species suspension. Together, these findings establish the presence of an S-layer in P. thiaminolyticus and provide a rapid fluorescent labeling strategy for multiple Paenibacillus pathogens associated with neonatal paenibacilliosis.

Fosfomycin is increasingly used to treat multidrug-resistant Gram-negative infections, yet resistance surveillance largely relies on homology-based detection of known fos genes. Here, we identify Gfa, a previously unrecognized integron-borne fosfomycin resistance determinant uncovered through functional screening of 129 integron gene cassettes. Gfa confers high-level fosfomycin resistance across Enterobacterales and Pseudomonas aeruginosa and is resistant to phosphonoformate inhibition. Structural modelling and mutagenesis reveal a conserved cysteine-rich, zinc-organized architecture essential for activity and distinct from canonical Fos enzymes. Crude-extract assays confirm glutathione-dependent fosfomycin inactivation under metal-chelating conditions. Distant Gfa homologs also conferred resistance, although activity was restricted to a subset of proteins tested. Together, these findings establish Gfa as a previously unrecognized fosfomycin resistance family and highlight the limitations of homology-based mining for discovering novel resistance determinants and predicting which family members are functional.

Plant-derived flavonoids show measurable anti-dengue activity in cell culture, but their translational value is limited by a narrow therapeutic window: the concentrations that inhibit the virus approach or exceed those that are cytotoxic. Whether nanoparticle formulation can resolve this constraint, rather than simply add potency, remains untested for a chemically defined fraction. Here we show that biogenic silver nanoparticle (AgNP) formation using a flavonoid-enriched fraction of Carica papaya inverts an unusable selectivity profile into a viable one. The unformulated fraction was cytotoxic below the concentrations required for antiviral activity: its 50% cytotoxic concentration (CC50 = 134.9 ug/mL) lay below its 50% effective concentration against dengue virus serotype 2 (DENV-2; EC50 = 254.4 ug/mL), giving a Selectivity Index (SI) of 0.53. Using the same flavonoids as sole reducing and capping agents produced AgNPs (Z-average 128.4 nm; PDI 0.232; zeta potential -28.4 mV) that moved both parameters simultaneously. CC50 rose approximately 8.6-fold to 1,165.74 ug/mL while EC50 fell approximately 8.8-fold to 29.05 ug/mL, raising the SI to 40.12, an approximately 75-fold shift. Time-of-addition analysis localised the effect to the extracellular phase: inhibition was significant under pre-treatment and co-treatment but not after viral adsorption, identifying the AgNPs as entry inhibitors rather than replication inhibitors. Consistent with a serotype-independent physical mechanism, AgNP treatment at 30 ug/mL reduced viral RNA across all four serotypes, using inocula standardised against WHO-traceable NAAT reference reagents. These findings identify capping chemistry, rather than silver content alone, as a determinant of the therapeutic window in phytosynthesised nanoantivirals.

Protein kinase CK2 (CK2), a ubiquitously expressed serine/threonine kinase, has emerged as a key host factor exploited by several viruses, including Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we investigate the role of CK2 subunits in SARS-CoV-2 replication and evaluate the antiviral potential and broad-spectrum anti-coronavirus activity of two CK2 inhibitors: the previously described ATP-competitive inhibitor CX-4945 and a newly developed, highly selective bivalent inhibitor, AB668. The siRNA-mediated knockdown of CK2 subunits significantly reduced SARS-CoV-2 replication, highlighting its essential role in the viral lifecycle. Co-immunoprecipitation assays revealed that the SARS-CoV-2 nucleoprotein interacts with CK2. Furthermore, bio-layer interferometry assays demonstrated that the nucleoprotein specifically binds to the tetrameric CK2 holoenzyme (CK22{beta}2) but not to individual CK2 or CK2{beta} subunits. Pharmacological inhibition of CK2 with AB668 exhibited potent antiviral activity, significantly reducing viral replication by targeting a pre-replicative or RNA-replicative step of the viral life cycle. Importantly, AB668 displayed broad-spectrum antiviral activity, effectively inhibiting both HCoV-OC43 ({beta}-CoV) and HCoV-229E (-CoV). In summary, our findings establish CK2 as a critical host factor for SARS-CoV-2 replication and identify AB668 as a promising candidate for broad-spectrum antiviral therapy. With its high selectivity, minimized off-target effects, and efficacy against multiple coronaviruses, AB668 represents a valuable chemobiological and therapeutic tool.

Successful treatment of primary Clostridioides difficile infection (CDI) while preventing its recurrence remains a major clinical challenge, with recurrent CDI (R-CDI) affecting 20-30% of treated patients. Persistence of C. difficile spores within the intestinal mucosa is thought to drive R-CDI following antibiotic treatment. Here, we show that intracellular C. difficile spores remain protected from taurocholate-triggered germination, and that nystatin, an FDA-approved antifungal that blocks spore entry into host cells, reduces this protected intracellular population and redirects spores toward an extracellular, germinating population accessible to antibiotics. In a murine model of R-CDI, oral co-administration of taurocholate with nystatin and antibiotics during primary CDI reduced R-CDI incidence from 100% to 44%. When administered during an active R-CDI episode, the same formulation reduced a second R-CDI from 100% to 62%. To improve bioavailability, we encapsulated nystatin and vancomycin in chitosan nanoparticles. Oral administration of nanoparticle-encapsulated vancomycin, alone or combined with nanoparticle-encapsulated nystatin, reduced R-CDI colonization to 33% and 44%, respectively, compared with 100% in mice receiving free vancomycin. Together, these findings support a therapeutic strategy that repurposes nystatin and taurocholate, in combination with antibiotics and nanoparticle-based delivery, to target intracellular spore persistence and reduce the incidence of C. difficile recurrence..