Enteroviruses (EV) are a group of positive-strand RNA (+RNA) viruses that include many important human pathogens (e.g. poliovirus, coxsackievirus, echovirus, numbered enteroviruses and rhinoviruses). Fluoxetine was identified in drug repurposing screens as potent inhibitor of enterovirus B and enterovirus D replication. In this paper we are reporting the synthesis and the antiviral effect of a series of fluoxetine analogues. The results obtained offer a preliminary insight into the structure-activity relationship of its chemical scaffold and confirm the importance of the chiral configuration. We identified a racemic fluoxetine analogue, 2b, which showed a similar antiviral activity compared to (S)-fluoxetine. Investigating the stereochemistry of 2b revealed that the S-enantiomer exerts potent antiviral activity and increased the antiviral spectrum compared to the racemic mixture of 2b. In line with the observed antiviral effect, the S-enantiomer displayed a dose-dependent shift in the melting temperature in thermal shift assay, indicative for direct binding to the recombinant 2C protein.

There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine—which also targets 2C—but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

Estrogen deficiency induces bone loss by increasing bone resorption, in part through upregulation of receptor activator of nuclear factor-κB ligand (RANKL). RANKL is secreted by osteoblasts and osteocytes, but more recently bone marrow (pre)adipocytes have also been shown to express RANKL. Estrogen deficiency increases bone marrow adipose tissue (BMAT). The aim of this study was to determine the effect of ovariectomy (OVX) on RANKL protein expression by bone marrow adipocytes in C3H/HeJ mice. Fourteen-week-old female C3H/HeJ mice ( n = 20) were randomized to sham surgery (Sham) or OVX. After 4 wk animals were euthanized. BMAT volume fraction (BMAT volume/marrow volume) was quantified by polyoxometalate-based contrast-enhanced nano-computed tomography. The percentage of RANKL-positive bone marrow adipocytes (RANKL-positive bone marrow adipocytes/total adipocytes) and the percentage of RANKL-positive osteoblasts covering the bone surface (bone surface covered in RANKL-positive osteoblasts/total bone surface) were quantified in the distal metaphysis of immunohistochemically stained sections of the left femur. The effects of OVX were analyzed by Student’s t test or Mann–Whitney U test. RANKL was detected in osteoblasts, osteocytes, and bone marrow adipocytes. OVX significantly increased mean percentage of RANKL-positive bone marrow adipocytes [mean (SD): Sham 42 (18)%; OVX 64 (12)%; P = 0.029] as well as BMAT volume/marrow volume [median (interquartile range): Sham 1.4 (4.9)%; OVX 7.2 (7.3)%; P = 0.008] compared with Sham. We show that OVX increased both the percentage of RANKL-positive bone marrow adipocytes and the total BMAT volume fraction in C3H/HeJ mice. Therefore, RANKL produced by bone marrow adipocytes could be an important contributor to OVX-induced bone loss in C3H/HeJ mice.

Over half a century since the description of the first antiviral drug, “old” re‐emerging viruses and “new” emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co‐infections, make the war against viruses quite challenging. Herein we report a host‐targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS‐CoV‐2 at low micromolar and sub‐micromolar concentrations. However, while the anti‐hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS‐CoV‐2 entry/replication is debated.