Novel Pan-Coronavirus 3CL Protease Inhibitor MK-7845: Biological and Pharmacological Profiling.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).

Effect of TRPV4 Antagonist GSK2798745 on Chlorine Gas-Induced Acute Lung Injury in a Swine Model.

As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO2/FiO2), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO2/FiO2, but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

Adoptive transfer of CD49a+ Tissue resident memory cells reverses pulmonary fibrosis in mice.

Pulmonary fibrosis is a devastating disease with no effective treatments to cure, stop or reverse the unremitting, fatal fibrosis. A critical barrier to treating this disease is the lack of understanding of the pathways leading to fibrosis as well as those regulating the resolution of fibrosis. Fibrosis is the pathologic side of normal tissue repair that results when the normal wound healing programs go awry. Successful resolution of tissue injury requires several highly coordinated pathways, and this research focuses on the interplay between these overlapping pathways: immune effectors, inflammatory mediators and fibroproliferation in the resolution of fibrosis. Previously we have successfully prevented, mitigated, and even reversed established fibrosis using vaccinia vaccination immunotherapy in two models of murine lung fibrosis. The mechanism by which vaccinia reverses fibrosis is by vaccine induced lung specific Th1 skewed tissue resident memory (TRMs) in the lung. In this study, we isolated a population of vaccine induced TRMs - CD49a+ CD4+ T cells - that are both necessary and sufficient to reverse established pulmonary fibrosis. Using adoptive cellular therapy, we demonstrate that intratracheal administration of CD49a+ CD4+ TRMs into established fibrosis, reverses the fibrosis histologically, by promoting a decrease in collagen, and functionally, by improving lung function, without the need for vaccination. Furthermore, co-culture of in vitro derived CD49+ CD4+ human TRMs with human fibroblasts from individuals with idiopathic pulmonary fibrosis (IPF) results in the down regulation of IPF fibroblast collagen production. Lastly, we demonstrate in human IPF lung histologic samples that CD49a+ CD4+ TRMs, which can down regulate human IPF fibroblast function, fail to increase in the IPF lungs, thus potentially failing to promote resolution. Thus, we define a novel unappreciated role for tissue resident memory T cells in regulating established lung fibrosis to promote resolution of fibrosis and re-establish lung homeostasis. We demonstrate that immunotherapy, in the form of adoptive transfer of CD49a+ CD4+ TRMs into the lungs of mice with established fibrosis, not only stops progression of the fibrosis but more importantly reverses the fibrosis. These studies provide the insight and preclinical rationale for a novel paradigm shifting approach of using cellular immunotherapy to treat lung fibrosis.

Severe acute respiratory disease in American mink experimentally infected with SARS-CoV-2.

An animal model that fully recapitulates severe COVID-19 presentation in humans has been a top priority since the discovery of SARS-CoV-2 in 2019. Although multiple animal models are available for mild to moderate clinical disease, models that develop severe disease are still needed. Mink experimentally infected with SARS-CoV-2 developed severe acute respiratory disease, as evident by clinical respiratory disease, radiological, and histological changes. Virus was detected in nasal, oral, rectal, and fur swabs. Deep sequencing of SARS-CoV-2 from oral swabs and lung tissue samples showed repeated enrichment for a mutation in the gene encoding nonstructural protein 6 in open reading frame 1ab. Together, these data indicate that American mink develop clinical features characteristic of severe COVID-19 and, as such, are uniquely suited to test viral countermeasures.

Metabolic shifts modulate lung injury caused by infection with H1N1 influenza A virus.

Influenza A virus (IAV) infection alters lung epithelial cell metabolism in vitro by promoting a glycolytic shift. We hypothesized that this shift benefits the virus rather than the host and that inhibition of glycolysis would improve infection outcomes. A/WSN/33 IAV-inoculated C57BL/6 mice were treated daily from 1 day post-inoculation (d.p.i.) with 2-deoxy-d-glucose (2-DG) to inhibit glycolysis and with the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) to promote flux through the TCA cycle. To block OXPHOS, mice were treated every other day from 1 d.p.i. with the Complex I inhibitor rotenone (ROT). 2-DG significantly decreased nocturnal activity, reduced respiratory exchange ratios, worsened hypoxemia, exacerbated lung dysfunction, and increased humoral inflammation at 6 d.p.i. DCA and ROT treatment normalized oxygenation and airway resistance and attenuated IAV-induced pulmonary edema, histopathology, and nitrotyrosine formation. None of the treatments altered viral replication. These data suggest that a shift to glycolysis is host-protective in influenza.

Repeated Orotracheal Intubation in Mice.

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator's ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.