Niklas Arnberg

Infections by influenza A virus (IAV) are a global health concern, aggravated by emerging drug-resistant strains. We report the identification of a series of dihydrothiazolo-ring-fused 2-pyridones bearing an extended carboxylic acid motif as promising antiviral scaffolds. Initial phenotypic screening yielded compound 9 (IC50 = 28 µM), which guided structure-activity optimization at C‐6, C‐7, and C‐8. This yielded compound 35d, which showed moderate potency (IC50 = 21 µM), but with minimal cytotoxicity and a broad Selectivity Index (>19). A time‐of‐addition study indicates that 35d disrupts an early post‐attachment stage of the influenza virus life cycle, possibly preventing its entry into the host cell. Notably, 35d also demonstrated broad‐spectrum antiviral activity across multiple viral families. A hydroxamic acid bioisostere (74) retained moderate potency, supporting the scaffold’s versatility. In silico human ADME/PK predictions for 10 representative analogs indicated favorable properties, including moderate oral bioavailability (≈40–60%), short–moderate half‐life (≈2–12 h), CYP2C9‐driven metabolism, low plasma unbound fraction (≈0.5–7%), modest tissue distribution, and minor renal/biliary clearance. These findings establish the extended carboxylic acid 2-pyridone scaffold as a strong foundation for advancing both targeted anti-influenza A agents and broader-spectrum antiviral therapeutics.

Human norovirus (HuNoV) is a leading cause of acute viral gastroenteritis, but despite high impact on public health and healthcare, the mechanisms of viral attachment to and entry into target cells are not yet fully understood. Recent reports indicate that saliva and bile contribute to the transmission of HuNoV. For example, human bile acids increase cell surface ceramide levels in human enteroids, which improves norovirus entry into cells, resulting in enhanced replication. Bile acids can also interact directly with the norovirus capsid, but it is not known whether bile or other gastrointestinal body fluids directly affect HuNoV attachment to host cells. In this study, we investigated the effects of patient-derived gastric juice, pancreatic juice, and bile on HuNoV GII.4 virus-like particle (VLP) attachment to and entry into a human duodenal cell line, HuTu-80. We show that while gastric juice and pancreatic juice do not affect viral attachment or entry, bile—in particular, hydrophobic bile acids—significantly enhance cellular attachment and subsequent entry of GII.4 VLPs into cells. In addition, we show that hydrophobic bile acids induce the accumulation of viral particles in the vicinity of cells. These results suggest the presence of a new en masse infection mechanism, where bile acids aggregate virions and allow direct and more efficient attachment to and entry into target cells. IMPORTANCE: Viruses transmitted by the fecal-oral route encounter secreted host factors in gastrointestinal fluids. Some host factors can be exploited by the virus to facilitate infection. Human bile acids indirectly promote norovirus entry into and infection of human enteroids, but the direct effect of bile acids on attachment and uptake, along with the impact of other gastrointestinal fluids, remains unknown. Here, we investigated the direct effects of human body fluids on cellular attachment of norovirus VLPs. We show that human bile and hydrophobic bile acids induce an accumulation of norovirus VLPs, which is associated with significantly enhanced attachment and entry into human duodenal cell lines. These results highlight the differential effects of gastrointestinal body fluids on viral attachment and entry while providing useful information on the complex HuNoV-host interactions that facilitate infection.

Top 30 markers of clusters in CAF subset of single cell data

Lists of genes used to calculate signature scores for cytolytic activity, T cell activation and M1 vs M2 macrophage polarization in single cell data analysis

Orthotopic transplantation of gal 4–KO cell lines identifies differences in CD4+ and CD8+ cell infiltration and function. A, Schematic of the orthotopic transplantation experiments using gal 4–KO and R26 control cell lines transplanted into immune competent C57BL6/J mice. B, Representative IHC images of gal 4 in R26 control and gal 4–KO tumors (top scale bar, 50 μm; bottom, scale bar, 20 μm). A total of 10 tumors were stained, 5 of each condition. C–E, Representative IHC images (left) and quantification (right) of CD4+ cells (P = 0.0032; C), CD8+ cells (P = <0.0001; D), and FOXP3+ cells (ns., no significant difference; E), in orthotopically transplanted tumors with gal 4 (R26 controls) and no gal 4 expression (Gal 4–KO); scale bar, 100 μm. Significance calculated using the Mann–Whitney test. Error bars show mean ± SEM. F, Representative contour plots illustrating gating strategies of intratumoral CD8+ T cells (left). Scatter plot with bar graph showing the fraction of CD8+ cells out of the total amount of CD45+ CD3+ cells in R26 control and gal 4–KO tumors (right; P = 0.0001). Significance calculated using the Mann–Whitney test. Error bars show mean±SEM. G Representative contour plots of intratumoral CD45+, CD3+, CD8+ cells (CD8+ T cells gated as in Fig. 3 F) expressing the exhaustion markers PD-1 (x-axis) and Tim-3 (y-axis) in R26 control and gal 4–KO tumors (left). Scatter plot with bar graph s (right) showing quantification of CD8+ T cells exhaustion (PD1+ and Tim-3 cells in Q2) in R26 control and gal 4–KO tumors (P = 0.046). Significance calculated using the Mann–Whitney test. Error bars show mean with SEM. **, P < 0.01; ***, P < 0.001.

Top 30 markers of clusters in Immune subset of single cell data

Galectin 4 is a cancer cell–derived marker of disease progression and prognosis in pancreatic cancer, with an association to reduced immune activity. A, Representative ISH images of cytokeratin 18 (Krt18, blue, top image) and fibroblast activation protein alpha (Fap, blue, the bottom image), together with gal 4 (Lgals4, in red), in mouse model of PDAC tumor tissue. 4 tumors analyzed in total; scale bar, 10 μm. B, Violin plot of single-cell RNA-seq data from Peng et al. (40) showing LGALS4 expression in cell types from human PDAC tumors. C, Western blot and quantification (semiquantitative) of gal 4 protein in normal murine ductal organoid (N, two different cell lines), PanIN organoid (P, two different cell lines), and tumor organoid (T, three different cell lines) cell lines. Vinculin was used as loading control. D, Kaplan–Meier survival curve adapted from the TCGA data set (26), with patients grouped into high (n = 26) and low (n = 49) gal 4 mRNA expression (groups defined in Materials and Methods) and relationship to survival in patients with PDAC with moderately differentiated tumors. *, Significance (P = 0.0254) calculated using the Mantel–Cox test. E, Z-score comparison of high (n = 26) and low (n = 49) gal 4-expressing tumors within the group of moderately differentiated PDAC tumors in the TCGA data set. Genes associated with cytolytic effect of immune cells analyzed in the left graph (P = 0.036), and CD8 activation scores analyzed in the right graph (P = 0.0253). Genes used for CD8 activation and cytolytic effect are listed in Supplementary Table S2. *, Indicates significance calculated using an unpaired t test. Error bar indicates mean ± SEM.

Raw read count, normalized expression values (TPM) and sample metadata for bulk RNA sequencing data