Biology subjects

Prescott, R. A.

Publications and source records attributed to Prescott, R. A..

3 recordsLinked to original sources

bioRxiv2023

A comparative study of in vitro air-liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution

The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial cultures at air-liquid interface (HAE) are a physiologically relevant in vitro model of this heterogeneous tissue, enabling numerous studies of airway disease1-7. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining capacity for differentiation to HAE5. However, gene expression and innate immune function in HAE derived from BCi-NS1.1 versus primary cells have not been fully characterized. Here, combining single cell RNA-Seq (scRNA-Seq), immunohistochemistry, and functional experimentation, we confirm at high resolution that BCi-NS1.1 and primary HAE cultures are largely similar in morphology, cell type composition, and overall transcriptional patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1 HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

cell biology↗

bioRxiv2022

In silico docking screen identifies airway host protease targets for human SERPINs

Serine Protease Inhibitors (SERPINs) regulate protease activity in various physiological processes such as inflammation, cancer metastasis, angiogenesis, and neurodegenerative diseases. However, their potential in combating viral infections, where proteases are also crucial, remains underexplored. This is due to our limited understanding of SERPIN expression during viral-induced inflammation and of the SERPINs full spectrum of target proteases. Here, we demonstrate widespread expression of human SERPINs in response to respiratory virus infections, both in vitro and in vivo, alongside classical antiviral effectors. Through comprehensive in-silico docking with full-length SERPIN and protease 3D structures, we confirm known inhibitors of specific proteases; more importantly, the results predict novel SERPIN-protease interactions. Experimentally, we validate the direct inhibition of key proteases essential for viral life cycles, including the SERPIN PAI-1s capability to inhibit select cysteine proteases such as cathepsin L, and the serine protease TMPRSS2. Consequently, PAI-1 suppresses spike maturation and multi-cycle SARS-CoV-2 replication. Our findings challenge conventional notions of SERPIN selectivity, underscore the power of in-silico docking for SERPIN target discovery, and offer potential therapeutic interventions targeting host proteolytic pathways to combat viruses with urgent unmet therapeutic needs. SIGNIFICANCESerine protease inhibitors (SERPINs) play crucial roles in various physiological processes, including viral infections. However, our comprehension of the full array of proteases targeted by the SERPIN family has traditionally been limited, hindering a comprehensive understanding of their regulatory potential. We developed an in-silico docking platform to identify new SERPIN target proteases expressed in the respiratory tract, a critical viral entry portal. The platform confirmed known and predicted new targets for every SERPIN examined, shedding light on previously unrecognized patterns in SERPIN selectivity. Notably, both key proteases for SARS-CoV-2 maturation were among the newly predicted targets, which we validated experimentally. This underscores the platforms potential in uncovering targets with significance in viral infections, paving the way to define the full potential of the SERPIN family in infectious disease and beyond.

microbiology↗

bioRxiv2020

Comparative study of a 3CLpro inhibitor and remdesivir against both major SARS-CoV-2 clades in human airway models

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models. ImportanceThe arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

microbiology↗