ABSTRACT
Background: TGF-beta signaling plays an essential role in tissue fibrosis and mediates profibrotic programs after SARS-CoV-2 infection in the kidney and lung. SARS-CoV-2 also induces humoral immune responses controlled by cytokines, including TGF-beta. Studies have found that the incidence of SARS-CoV-2 infection and the severity of Covid-19 in cystic fibrosis (CF) patients is lower than the general population. We studied how SARS-CoV-2 regulates TGF-beta-mediated gene expression in the CF airway. Method(s): Small RNAseq was performed in human bronchial epithelial cells CFBE41o-from a patient homozygous for the F508del mutation in the CFTR gene on Illumina NextSeq 500's. Pathway analysis was done by Ingenuity Pathway Analysis (IPA) software (QIAGEN) and miRNet browser. IPA was used for analyzing coronavirus associated pathways affected by differentially regulated miRNAs. miRNAs predicted to target the coronavirus associated genes were collected from TargetScan Human release 7.2, miRmap, Diana-TarBase v.8, and miRBase bioinformatics tools. Anti-miRNA oligonucleotide miRCURY LNATM Power Inhibitors or control (Exiqon) were used. Cells expressing F508del or wild type CFTR were used to compare the results in CF and non-CF models. Findings were validated in primary human bronchial epithelial (HBE) cells. Result(s): Compared to vehicle control, TGF-beta1 dysregulated 48 miRNAs;38 and 19 pathways were uniquely affected by the upregulated and downregulated miRNAs, respectively. We found 43 miRNAs targeting 119 different mRNA of the proteins associated with coronavirus pathogenesis pathway and 21 miRNAs targeting 21 different mRNA of the proteins associated with coronavirus replication pathway. Two miRNAs upregulated by TGF-beta1 target the host receptor for SARS-CoV-2 invasion, angiotensin converting enzyme 2 (ACE2). We confirmed the results by qRT-PCR that TGF-beta1 increased expression of specific miRNAs targeting ACE2 mRNA. Upregulation of the miRNAs was followed by inhibition of ACE2 mRNA and protein levels and the effect was blocked by specific anti-miRNA oligonucleotides. The above results differed between the CF and non-CF cells. Conclusion(s): miRNAs may be important effectors of TGF-beta modulating SARSCoV-2 pathogenicity and replication in the CF airway. Ongoing studies focus on elucidating the mechanisms of SARS-CoV-2 invasion of kidney cells.
ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused high mortality and enormous economic loss. Thus, in addition to effective vaccines, there is an urgent need of effective drug molecules to fight against COVID-19. This necessity motivated us to explore potential compounds targeting main protease (Mpro) enzyme of SARS-CoV-2, which is essential for processing the polyproteins that are translated from the viral RNA. In this work, we have developed a highly permeable to drugs and extremely stable, inducible Mpro expression system causing a strong change in yeast phenotype. The yeast was successfully engineered with the CRISPR / Cas9 method both to introduce the gene encoding Mpro and to remove the genes responsible for multi-drug resistance. Our system has been screened for Mpro inhibitors using nearly 2000 thousand drugs, and 800 plant and fungal extracts. Our screening results showed only one drug called ebselen (also called PZ 51, DR3305, or SPI-1005) that was able to restore the growth of yeast cells expressing Mpro. The selection of only one drug from among several proposed Mpro inhibitors may indicate a very rigorous nature of our yeast-based screening assay, allowing for the selection of the drug with the strongest Mpro inhibitory effect, as well as the lowest cytotoxic effect.