High throughput screening for SARS-CoV-2 helicase inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z' = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC50 <10 µM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral.

F4/80hi Resident Macrophages Contribute to Cisplatin-Induced Renal Fibrosis.

Background: Cisplatin-induced kidney injury remains a major obstacle in utilizing cisplatin as a chemotherapeutic for solid-organ cancers. Thirty percent of patients treated with cisplatin develop acute kidney injury (AKI), and even patients who do not develop AKI are at risk for long-term declines in kidney function and development of chronic kidney disease (CKD). Modeling cisplatin-induced kidney injury in mice has revealed that repeated low doses of cisplatin lead to development of kidney fibrosis. This model can be used to examine AKI-to-CKD transition processes. Macrophages play a role in some of these processes, including immune response, wound healing, and tissue remodeling. Depleting macrophage populations in the kidney reduced fibrosis development in other models of renal fibrosis. Methods: We used either C57BL/6 mice with a Ccr2 genetic knockout or liposome encapsulated clodronate (Clodrosome) to deplete macrophage populations during repeated 9 mg/kg cisplatin treatments. We assessed how immune cell populations were altered in the blood and kidney of these mice and how these alterations affected development of renal fibrosis and kidney injury. Results: We found that Clodrosome treatment decreased collagen deposition, myofibroblast accumulation, and inflammatory cytokine production, whereas Ccr2 genetic knockout had no effect on these markers after cisplatin treatment. Additionally, Ccr2-/- mice had decreased levels of F4/80lo infiltrating macrophages in the kidney after cisplatin treatments, but Clodrosome treatment depleted F4/80hi resident and CD206+ M2 macrophages. Conclusions: These data suggest that Clodrosome depletion of F4/80hi and M2 macrophages in the kidney attenuates development of renal fibrosis after repeated low doses of cisplatin.

Pharmacological inhibitors of autophagy have opposite effects in acute and chronic cisplatin-induced kidney injury.

The nephrotoxicity of cisplatin remains a major hurdle in the field of oncology. Thirty percent of patients treated with cisplatin develop acute kidney injury, and all patients are at risk for long-term impacts on kidney function. There are currently no Federal Drug Administration-approved agents to prevent or treat cisplatin-induced kidney injury. The dosing regimen used in preclinical models of nephrotoxicity may impact the success of therapeutic candidates in clinical trials. Here, we demonstrated that pharmacological inhibitors of autophagy have opposite effects when used as interventions in two different models of cisplatin-induced kidney injury. Eight-week-old male C57BL/6 mice were treated with either one dose of 20 mg/kg cisplatin or weekly doses of 9 mg/kg cisplatin for 4 wk or until body weight loss exceeded 30%. Concurrently, mice were administered multiple doses of 60 mg/kg chloroquine or 15 mg/kg 3-methyladenine attempting to globally inhibit autophagy. Mice that received a single high dose of cisplatin had worsened kidney function, inflammation, and cell death with the addition of chloroquine. 3-Methlyadenine did not impact the development of acute kidney injury in this model. In contrast, mice that received repeated low doses of cisplatin showed improved kidney function, reduced inflammation, and reduced fibrosis when treated with either chloroquine or 3-methyladenine. This study highlights how therapeutic candidates can have drastically different effects on the development of cisplatin-induced kidney injury depending on the dosing model used. This emphasizes the importance of choosing the appropriate model of injury for preclinical studies.NEW & NOTEWORTHY This study examined how inhibition of autophagy has opposite effects on the development of acute and chronic kidney injury. Autophagy inhibition exacerbated the development of acute kidney injury following a single high dose of cisplatin but prevented the development of injury and fibrosis following repeated low doses of cisplatin.

Deletion of arylamine N-acetyltransferase 1 in MDA-MB-231 human breast cancer cells reduces primary and secondary tumor growth in vivo with no significant effects on metastasis.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. Previous studies showed that inhibition or depletion of NAT1 in breast cancer cells diminishes anchorage-independent growth in culture, suggesting that NAT1 contributes to breast cancer growth and metastasis. To further investigate the contribution of NAT1 to growth and cell invasive/migratory behavior, we subjected parental and NAT1 knockout (KO) breast cancer cell lines (MDA-MB-231, MCF-7, and ZR-75-1) to multiple assays. The rate of cell growth in suspension was not consistently decreased in NAT1 KO cells across the cell lines tested. Similarly, cell migration and invasion assays failed to produce reproducible differences between the parental and NAT1 KO cells. To overcome the limitations of in vitro assays, we tested parental and NAT1 KO cells in vivo in a xenograft model by injecting cells into the flank of immunocompromised mice. NAT1 KO MDA-MB-231 cells produced primary tumors smaller than those formed by parental cells, which was contributed by an increased rate of apoptosis in KO cells. The frequency of lung metastasis, however, was not altered in NAT1 KO cells. When the primary tumors of the parental and NAT1 KO cells were allowed to grow to a pre-determined size or delivered directly via tail vein, the number and size of metastatic foci in the lung did not differ between the parental and NAT1 KO cells. In conclusion, NAT1 contributes to primary and secondary tumor growth in vivo in MDA-MB-231 breast cancer cells but does not appear to affect its metastatic potential.

Will Protective Innovations Like the "Airway Box" Become Routine Practice After the Pandemic?: An Opinion Survey.

Background Tracheal intubation carries an elevated risk of exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to the generation of aerosols containing high concentrations of the virus. An airway box was designed to mitigate the exposure of healthcare professionals performing intubations. Aim We evaluated usability and sustainability in the routine practice of the "airway box" as a protective device during high-risk airway procedures.  Materials and methods After institutional review board approval, clinicians were educated on using the device through simulation, intranet learning modules, and emailed resources. The airway box was made available in the emergency department, critical care units, perioperative area, and operating rooms. QR codes affixed to the box, emailed, and displayed in common areas provided easy access to complete a REDcap survey (Vanderbilt University Nashville, USA) eliciting providers' experience. Data was collected and analyzed between April 1 and July 31, 2020, on REDcap, and the results were analyzed. Results 687 emergent intubations took place. 232 were performed by anesthesiologists, 315 by emergency department providers, and 140 by critical care specialists. 39 surveys were completed, 29 from intubations in the operating room, three from the critical care units, five from interventional radiology suites, and two perioperatively. Providers found the device to be readily available, with a score of 4.51/5, and the majority of providers, 60%, found the device easy to use, rating it either a 4 or 5 out of 5. Providers acquired a mean Mallampati score of 1.75 and 1.40 mean laryngoscopic grade view. Conclusion Intubation boxes may effectively mitigate high-risk viral exposure during airway procedures. Survey responses show that devices were easy to use and did not significantly affect visualization of the airway. Similar to mask use, enclosure devices in clinical practice could become a vital part of medical protective equipment even after the SARS-CoV-2 pandemic if they are effectively implemented.

C57BL/6 mice require a higher dose of cisplatin to induce renal fibrosis and CCL2 correlates with cisplatin-induced kidney injury.

C57BL/6 mice are one of the most commonly used mouse strains in research, especially in kidney injury studies. However, C57BL/6 mice are resistant to chronic kidney disease-associated pathologies, particularly the development of glomerulosclerosis and interstitial fibrosis. Our laboratory and others developed a more clinically relevant dosing regimen of cisplatin (7 mg/kg cisplatin once a week for 4 wk and mice euthanized at day 24) that leads to the development of progressive kidney fibrosis in FVB/n mice. However, we found that treating C57BL/6 mice with this same dosing regimen does not result in kidney fibrosis. In this study, we demonstrated that increasing the dose of cisplatin to 9 mg/kg once a week for 4 wk is sufficient to consistently induce fibrosis in C57BL/6 mice while maintaining animal survival. In addition, we present that cohorts of C57BL/6 mice purchased from Jackson 1 yr apart and mice bred in-house display variability in renal outcomes following repeated low-dose cisplatin treatment. Indepth analyses of this intra-animal variability revealed C-C motif chemokine ligand 2 as a marker of cisplatin-induced kidney injury through correlation studies. In addition, significant immune cell infiltration was observed in the kidney after four doses of 9 mg/kg cisplatin, contrary to what has been previously reported. These results indicate that multiple strains of mice can be used with our repeated low-dose cisplatin model with dose optimization. Results also indicate that littermate control mice should be used with this model to account for population variability.

Rapamycin attenuates liver injury caused by vinyl chloride metabolite chloroethanol and lipopolysaccharide in mice.

Vinyl chloride (VC) is a prevalent environmental toxicant that is rapidly metabolized within the liver. Its metabolites have been shown to directly cause hepatic injury at high exposure levels. We have previously reported that VC metabolite, chloroethanol (CE), potentiates liver injury caused by lipopolysaccharide (LPS). Importantly, that study showed that CE alone, while not causing damage per se, was sufficient to alter hepatic metabolism and increase mTOR phosphorylation in mice, suggesting a possible role for the mTOR pathway. Here, we explored the effect of an mTOR inhibitor, rapamycin, in this model. C57BL/6 J mice were administered CE, followed by rapamycin 1 h and LPS 24 h later. As observed previously, the combination of CE and LPS significantly enhanced liver injury, inflammation, oxidative stress, and metabolic dysregulation. Rapamycin attenuated not only inflammation, but also restored the metabolic phenotype and protected against CE + LPS-induced oxidative stress. Importantly, rapamycin protected against mitochondrial damage and subsequent production of reactive oxygen species (ROS). The protective effect on mitochondrial function by rapamycin was mediated, by restoring the integrity of the electron transport chain at least in part, by blunting the deactivation of mitochondrial c-src, which is involved mitochondrial ROS production by electron transport chain leakage. Taken together, these results further demonstrate a significant role of mTOR-mediated pathways in VC-metabolite induced liver injury and provide further insight into VC-associated hepatic damage. As mTOR mediated pathways are very complex and rapamycin is a more global inhibitor, more specific mTOR (i.e. mTORC1) inhibitors should be considered in future studies.