Background: Coronavirus disease (COVID-19) is an infectious disease discovered in 2019 and currently in outbreak across the world. Lung injury with severe respiratory failure is the leading cause of death in COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there still lacks efficient treatment for COVID-19 induced lung injury and acute respiratory failure.
Methods: Inhibition of angiotensin-converting enzyme 2 (ACE2) caused by the spike protein of SARS-CoV-2 is the most plausible mechanism of lung injury in COVID-19. We performed drug repositioning analysis to identify drug candidates that reverse gene expression pattern in L1000 lung cell line HCC515 treated with ACE2 inhibitor. We confirmed these drug candidates by similar bioinformatics analysis using lung tissues from patients deceased from COVID-19. We further investigated deregulated genes and pathways related to lung injury, as well as the gene-pathway-drug candidate relationships.
Results: We propose two candidate drugs, COL-3 (a chemically modified tetracycline) and CGP-60474 (a cyclin-dependent kinase inhibitor), for treating lung injuries in COVID-19. Further bioinformatics analysis shows that 12 significantly enriched pathways (P-value <0.05) overlap between HCC515 cells treated with ACE2 inhibitor and human COVID-19 patient lung tissues. These include signaling pathways known to be associated with lung injury such as TNF signaling, MAPK signaling and chemokine signaling pathways. All 12 pathways are targeted in COL-3 treated HCC515 cells, in which genes such as RHOA, RAC2, FAS, CDC42 have reduced expression. CGP-60474 shares 11 of 12 pathways with COL-3 and common target genes such as RHOA. It also uniquely targets other genes related to lung injury, such as CALR and MMP14.
Conclusions: This study shows that ACE2 inhibition is likely part of the mechanisms leading to lung injury in COVID-19, and that compounds such as COL-3 and CGP-60474 have potential as repurposed drugs for its treatment.
The inhibition of ACE2 promotes lung injury via the renin–angiotensin system (RAS)33. In pulmonary RAS, ACE2 converts angiotensin II (Ang II), an octapeptide hormone, to Ang-(1-7), an heptapeptide hormone. Ang II triggers pulmonary inflammation and activates the TNF signaling pathway and MAPK signaling pathway to promote lung injury. On the other hand, Ang-(1–7) inhibits inflammation and protects lungs from injury36 by inhibiting the MAPK signaling pathway37, lowering cytokine release38 and downregulating the RHOA/ROCK pathway. Thus, inhibition of ACE2 will increase Ang II levels, decrease Ang-(1–7), and deregulate various downstream pathways, such as TNF and MAPK signaling pathways to promote lung injury. Our pathway analysis on the HCC515 lung cell line confirmed that inhibition of ACE2 by moexipril can deregulate TNF signaling, MAPK signaling and cytokine signaling pathways. We further showed that these pathways are also deregulated in human lung tissues of deceased COVID-19 patients (Table 2). Moreover, inhibition of ACE2 induced similar expression patterns of lung injury markers to that in human lung tissues of deceased COVID-19 patients. This evidence suggests that inhibition of ACE2 may indeed be part of the molecular mechanisms of lung injury in COVID-19. Moreover, other pathways related to cancers (e.g. ‘viral carcinogenesis’ and ‘proteoglycans in cancer’), or cardiovascular diseases (e.g. viral myocarditis) also show up significantly enriched in the results. These results may help to explain the increased risks of fatality among COVID-19 patients with underlying conditions (cancers, heart diseases). Additionally, myocarditis has been clinically observed in a patient with COVID-1942, showing a direct link between the two conditions.
Our drug repositioning analysis suggested five possible drugs based on RNA-Seq data from patients deceased from COVID-19. Among them, clinical trial has started for treating patients with COVID-19 pneumonia with sirolimus (NCT04341675). Two other drugs (or compounds), COL-3 and CGP-60474, also have additional evidence of effectiveness from the L1000 data of the lung HCC515 cell line treated with ACE2 inhibitor moexipril. Moreover, both COL-3 and CGP-60474 could reverse the expression patterns of lung injury markers in HCC515 cells with ACE2 inhibitor inhibition and human COVID-19 patient lung tissues . This phenotypic evidence suggests that COL-3 and CGP-60474 may be effective in treating lung injury in COVID-19. Therefore, we further analyzed the target genes and pathways of these two drugs in treating lung injury in COVID-19.
COL-3, also known as incyclinide or CMT-3, is a chemically modified tetracycline. It reversed the expression patterns of many lung injury related genes and pathways, such as RHOA, RAC2 and FAS in the chemokine signaling pathway, TNF signaling pathway and MAPK signaling pathway. RHOA, also known as ras homolog family member A, is a member of the Rho family of small GTPases. The activation of RHOA is crucial for lung injury43. Inhibition of RHOA is a promising approach to acute lung injury treatment44. RAC2, also known as Ras-related C3 botulinum toxin substrate 2, is a member of the Ras superfamily of small guanosine triphosphate (GTP)-metabolizing proteins. Rac2 plays an important role in inflammation-mediated lung injury. FAS, also known as Fas cell surface death receptor, is a member of the TNF-receptor superfamily. FAS activation is essential in inducing acute lung injury. Small interfering RNA targeting Fas reduced lung injury in mice48. Previous results from many pre-clinical animal models have supported the role of COL-3 in reducing lung injury and improves survival of experimented animals. For example, COL-3 prevented lung injury and acute respiratory distress syndrome (ARDS) in a clinically applicable porcine model. It also improved acute respiratory distress syndrome (ARDS) survival in an ovine model56. Given all the evidence, COL-3 may be an attractive candidate for a clinical trial treating severe viral pneumonia related lung injury with respiratory failure in COVID-19.
CGP-60474, on the other hand, is an inhibitor of cyclin-dependent kinase. CGP-60474 not only shared target genes with COL-3, such as RHOA, WAS, HSPA1A, SNX2, RAB8A, IL2RG, MMP3, BCL2L1, JUN, HIST1H2BK, GNG11, IQGAP1 and MYL12B, but also has unique target genes that related to lung injury, like CALR and MMP14. Blocking CALR activity attenuated murine acute lung injury by inducing polarization of M2 subtype macrophages, which are anti-inflammatory. MMP14 was shown to trigger the anti-inflammatory proteolytic cascade to prevent lung injury in mice. Interestingly, so far only a few studies have reported some biological functions of CGP-60474. One drug reposition study using L1000 data also pointed to CGP-60474 as the most potent drug based on anti-inflammatory effects61. The authors then experimentally showed that CGP-60474 alleviated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in activated macrophages, downregulated the NF-κB activity, and reduced the mortality rate in lipopolysaccharide induced endotoxemia mice. Another in silico drug prediction study suggested that CGP-60474 could target multiple cancers, though no experiments were conducted. Although cyclin-dependent kinase inhibition by another drug, seliciclib, reduced lung damage in a mouse model of ventilator-induced lung injury, further in vivo investigation of CGP-60474 is needed to test its role in treating lung injury.
In summary, we propose two candidate drugs, COL-3 and CGP-60474, which can reverse the gene expression patterns in COVID-19 lung injury and a lung cell line with ACE2 being inhibited. We further analyzed potential molecular and biological mechanisms of lung injury in COVID-19. The work will hopefully gain the interest of the biomedical and clinical community for further validations in vivo for both candidate drugs, and even possibly clinical trials on COL-3 to save lives from severe respiratory failure in COVID-19.
Reference & Source information: https://f1000research.com/
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