Molnupiravir combined with different repurposed drugs further inhibits SARS-CoV-2 infection in human nasal epithelium in vitro

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in late 2019, has caused a worldwide pandemic with unprecedented economic and societal impact. Currently, several vaccines are available, and multitudes of antiviral treatments have been proposed and tested. Although many of the vaccines show high clinical efficacy, they are not equally accessible worldwide. Additionally, due to the continuous emergence of new virus variants, and generally short duration of immunity, the development of safe and effective antiviral treatments remains of the utmost importance. Since the emergence of SARS-CoV-2, substantial efforts have been undertaken to repurpose existing and approved drugs for accelerated clinical testing and potential emergency use authorizations. However, drug-repurposing using high throughput screenings in cellular assays, often identify hits that later prove ineffective in clinical studies. Our approach was to evaluate the activity of compounds that have either been tested clinically or already undergone extensive preclinical profiling, using a standardized in vitro model of human nasal epithelium. Secondly, we evaluated drug combinations using sub-maximal doses of each active single compound. Here, we report the antiviral effects of 95 single compounds and 30 combinations. The data show that selected drug combinations including 10 μM of molnupiravir, a viral RNA-dependent RNA polymerase (RdRp) inhibitor, effectively inhibit SARS-CoV-2 replication. This indicates that such combinations are worthy of further evaluation as potential treatment strategies against coronavirus disease 2019 (COVID-19).

Phosphoantigens are Molecular Glues that Promote Butyrophilin 3A1/2A1 Association Leading to Vγ9Vδ2 T Cell Activation

Tumor cells and pathogen-infected cells are presented to human γδ T cells based on "inside-out" signaling in which metabolites called phosphoantigens (pAgs) inside target cells are recognized by the intracellular domain of a butyrophilin protein (BTN3A1), leading to an extracellular conformational change. Here, we report that pAgs function as molecular "glues" that initiate a heteromeric association between the intracellular domains of BTN3A1 and the structurally similar BTN2A1. Working with both exogenous and endogenous pAgs, we used x-ray crystallography, mutational studies, cellular assays, synthetic probe as well as molecular dynamics investigations to determine how pAgs glue intracellular BTN3A1 and BTN2A1 together for the "inside-out" signaling that triggers γδ T cell activation. This γδ T cell-specific mode of antigen sensing creates opportunities for the development of alternative immunotherapies against cancer and infectious diseases that do not involve αβ T cells.

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbVQ40V/T66K/V70F-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.

Targeted Disruption of E6/p53 Binding Exerts Broad Activity and Synergism with Paclitaxel and Topotecan against HPV-Transformed Cancer Cells

High-risk human papillomaviruses (HR-HPV) are the etiological agents of almost all cervical cancer cases and a high percentage of head-and-neck malignancies. Although HPV vaccination can reduce cancer incidence, its coverage significantly differs among countries, and, therefore, in the next decades HPV-related tumors will not likely be eradicated worldwide. Thus, the need of specific treatments persists, since no anti-HPV drug is yet available. We recently discovered a small molecule (Cpd12) able to inhibit the E6-mediated degradation of p53 through the disruption of E6/p53 binding in HPV16- and HPV18-positive cervical cancer cells. By employing several biochemical and cellular assays, here we show that Cpd12 is also active against cervical cancer cells transformed by other HR-HPV strains, such as HPV68 and HPV45, and against a HPV16-transformed head-and-neck cancer cell line, suggesting the possibility to employ Cpd12 as a targeted drug against a broad range of HPV-induced cancers. In these cancer cell lines, the antitumoral mechanism of action of Cpd12 involves p53-dependent cell cycle arrest, a senescent response, and inhibition of cancer cell migration. Finally, we show that Cpd12 can strongly synergize with taxanes and topoisomerase inhibitors, encouraging the evaluation of Cpd12 in preclinical studies for the targeted treatment of HPV-related carcinomas.

Synthesis of Tyrosol and Hydroxytyrosol Glycofuranosides and Their Biochemical and Biological Activities in Cell-Free and Cellular Assays

Tyrosol (T) and hydroxytyrosol (HOT) and their glycosides are promising candidates for applications in functional food products or in complementary therapy. A series of phenylethanoid glycofuranosides (PEGFs) were synthesized to compare some of their biochemical and biological activities with T and HOT. The optimization of glycosylation promoted by environmentally benign basic zinc carbonate was performed to prepare HOT α-L-arabino-, β-D-apio-, and β-D-ribofuranosides. T and HOT β-D-fructofuranosides, prepared by enzymatic transfructosylation of T and HOT, were also included in the comparative study. The antioxidant capacity and DNA-protective potential of T, HOT, and PEGFs on plasmid DNA were determined using cell-free assays. The DNA-damaging potential of the studied compounds for human hepatoma HepG2 cells and their DNA-protective potential on HepG2 cells against hydrogen peroxide were evaluated using the comet assay. Experiments revealed a spectrum of different activities of the studied compounds. HOT and HOT β-D-fructofuranoside appear to be the best-performing scavengers and protectants of plasmid DNA and HepG2 cells. T and T β-D-fructofuranoside display almost zero or low scavenging/antioxidant activity and protective effects on plasmid DNA or HepG2 cells. The results imply that especially HOT β-D-fructofuranoside and β-D-apiofuranoside could be considered as prospective molecules for the subsequent design of supplements with potential in food and health protection.

The luminal AAA+ ATPase torsinA mediates distinct mechanisms of nuclear-cytoplasmic communication by adopting different functional assembly states.

Chemical and mechanical nuclear-cytoplasmic communication across the nuclear envelope (NE) is largely mediated by the nuclear pore complex (NPC) and the linker of nucleoskeleton and cytoskeleton (LINC) complex, respectively. While NPC and LINC complex assembly are functionally related, the mechanisms responsible for this relationship remain poorly understood. Here, we investigated how the luminal ATPases associated with various cellular activities (AAA+) protein torsinA promotes NPC and LINC complex assembly using fluorescence fluctuation spectroscopy (FFS), quantitative photobleaching analyses, and functional cellular assays. We report that torsinA controls LINC complex-dependent nuclear-cytoskeletal coupling as a soluble hexameric AAA+ protein and interphase NPC biogenesis as a membrane-associated helical polymer. These findings help resolve the conflicting models of torsinA function that were recently proposed based on in vitro structural studies. Our results will enable future studies of the role of defective nuclear-cytoplasmic communication in DYT1 dystonia and other diseases caused by mutations in torsinA.

The neutrophil protein CD177 is a novel PDPN receptor that regulates human cancer-associated fibroblast physiology

The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.

A semi-automated instrument for cellular oxidative potential evaluation (SCOPE) of water-soluble extracts of ambient particulate matter

Several automated instruments exist to measure the acellular oxidative potential (OP) of ambient particulate matter (PM). However, cellular OP of the ambient PM is still measured manually, which severely limits the comparison between two types of assays. Cellular assays could provide a more comprehensive assessment of the PM-induced oxidative stress, as they incorporate more biological processes involved in the PM-catalyzed reactive oxygen species (ROS) generation. Considering this need, we developed a semi-automated instrument, the first of its kind, for measuring the cellular OP based on a macrophage ROS assay using rat alveolar macrophages. The instrument named SCOPE – semi-automated instrument for cellular oxidative potential evaluation – uses dichlorofluorescein diacetate (DCFH-DA) as a probe to detect the OP of PM samples extracted in water. SCOPE is capable of analyzing a batch of six samples (including one negative and one positive control) in 5 h and is equipped to operate continuously for 24 h with minimal manual intervention after every batch of analysis, i.e., after every 5 h. SCOPE has a high analytical precision as assessed from both positive controls and ambient PM samples (coefficient of variation (CoV)<17 %). The results obtained from the instrument were in good agreement with manual measurements using tert-butyl hydroperoxide (t-BOOH) as the positive control (slope =0.83 for automated vs. manual, R2=0.99) and ambient samples (slope =0.83, R2=0.71). We further demonstrated the ability of SCOPE to analyze a large number of both ambient and laboratory samples and developed a dataset on the intrinsic cellular OP of several compounds, such as metals, quinones, polycyclic aromatic hydrocarbons (PAHs) and inorganic salts, commonly known to be present in ambient PM. This dataset is potentially useful in future studies to apportion the contribution of key chemical species in the overall cellular OP of ambient PM.

Differential recognition of opioid analgesics by µ opioid receptors: Predicted interaction patterns correlate with ligand-specific voltage sensitivity

The µ opioid receptor (MOR) is the key target for analgesia, but the application of opioids is accompanied by several issues. There is a wide range of opioid analgesics, differing in their chemical structure and their properties in receptor activation and subsequent effects. A better understanding of ligand-receptor interactions and resulting effects is important. Here, we calculated the respective binding modes for several opioids and analyzed fingerprints of ligand-receptor interactions. We further corroborated the binding modes experimentally by cellular assays. As ligand-induced modulation of activity due to changes in membrane potential was displayed by MOR, we further analyzed the effects of voltage sensitivity of this receptor. With a combined in silico and in vitro approach, we defined discriminating interaction patterns for the ligand-specific voltage sensitivity. With this, we present new insights for interactions likely in ligand recognition and their specific effects on activation of the MOR.

Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.