Covid19: a pandemics. In December 2019 a number of cases of atypical pneumonia were reported in the city of Wuhan, China. Few weeks later it turned into an epidemic of international concern and in march 2020 the world health organization (WHO) declared it a pandemics, spread everywhere in the world. Scientists identified the agent causing Covid-19 disease as a virus of the coronavirus type and named it SARS-CoV2.
Other respiratory outbreaks in the recent past. Other recent viral respiratory outbreaks were generated by two viruses of the coronavirus type: the SARS-CoV, causing SARS (sever acute respiratory syndrome) in 2002, and MERS-CoV (middle east respiratory syndrome) causing MERS in 2012. In both cases, viruses originated from bats and then infected humans (maybe through intermediate hosts such as civet and camel, respectively). SARS-CoV and SARS-CoV2 viruses show a high degree of similarity.
Coronavirus: the virus type responsible for Covid-19 pandemic. Coronaviruses are viruses known to infect birds and mammals. There are four types of coronaviruses: alpha and beta which are known to infect mammals, while gamma and delta usually infect birds. Coronaviruses are also known agents causing the common cold.
Coronaviruses have a molecule of single strand RNA, which represents their genome, and a number of structural proteins and accessory proteins. The four structural proteins are: spike proteins (S), located on the surface, membrane proteins (M), envelope proteins (E), and the nucleocapsid protein (N). These proteins are essential for SARS-CoV2 infection. In particular, spike proteins are key for virus binding to host cells: a specific region of the spike protein, the so called receptor binding domain, binds to a receptor expressed in human cells of the lung, the ACE2 receptor. After binding ACE2, the virus enters epithelial cells of the lung. Upon entry, the virus starts replicating quickly and triggers a strong response of the immune system, accompanied by lung tissue damage.
Currently, routine test to detect Covid-19 is real time RT-PCR on samples collected by nasopharyngeal or oropharyngeal swabs.
The joint effort of scientists worldwide against covid19. The world emergency and the lack of effective treatments against the disease have led to a huge joint effort of clinicians and researchers worldwide. However, the social distancing rules, applied nearly everywhere in the world to limit contagion, have imposed changes in scientists’ habits, who found new ways to share results with the scientific community before publishing in peer review journals. Each new piece of information, indeed, could be extremely useful for other researchers, to avoid duplicating effort and thus contributing to quickly gain insight into virus biology and pathology. For instance, Columbia University recently shared online a virtual Covid-19 meeting, where scientists involved in Covid-19 research could present and discuss their latest results.
Insights into SARS-CoV2 emergence and functioning.
Coronaviruses can evolve either by recombination with other viruses, quickly acquiring new functions making them more adapted to the new host -human beings-, or by acquiring and accumulating mutations over time. About 10-15 years ago a recombination event between the ancestor of SARS-CoV2 and a bat virus led to the acquisition of the receptor binding domain (the region within the spike protein responsible for binding ACE2 receptor), making the bat virus able to infect humans. Additional later mutations enabled to refine the interaction with human receptor. Studies show that SARS-CoV2 acquires about one new mutation every two weeks and the genome of the viruses isolated in different parts of the world are slightly different, demonstrating that the virus is evolving differently.
Virus can modulate interaction with the host also by “mimicry”, namely by means of viral proteins whose structure is very similar to that of some host proteins. Computational approaches showed that coronaviruses exploit this strategy more than any other infecting virus. They have about 30 proteins only, mimicking many human proteins. For instance, human proteins PARP9 and PARP13 (PARP proteins are involved in a variety of different cell processes, such as cell death and DNA repair) are mimicked by the viral protein NSP3. In vitro drug screens strongly suggest an effect of PARP inhibitors against coronavirus infections. Importantly, vaccines should not be designed against viral protein mimicking human proteins.
Potentially effective new approaches against Covid-19 disease.
Lipopeptides. Exciting results come from the use of lipopeptides, namely short protein fragments linked to lipid molecules, that can work as SARS-CoV2 inhibitors. The lipopeptide approach aims at inhibiting virus fusion with host cell membrane to block virus entry. Preliminary tests have shown that both airway and subcutaneous administration significantly increase animal survival upon infection with different virus types in hamster. For instance, lipopeptides against MERS virus significantly inhibit SARS-CoV2. Lipid conjugation is critical for peptide efficiency, improving efficacy of the inhibitory protein. Importantly, specific anti-SARS-CoV2 lipopeptides have shown to significantly inhibit virus entry in vitro. Notably, the effect is even higher when ACE2 receptor is not expressed in host cells, further demonstrating the known key role of ACE2 in viral entry. The ultimate goal would be to translate these findings into an approach in which lipopeptides are administered daily by an intranasal spray.
SARS-CoV2 antibodies. Another promising approach is the exploitation of anti SARS-CoV2 antibodies isolated from convalescent patients, to neutralize the virus. Studies are ongoing to isolate antibodies specifically binding the virus spike proteins.
Targeting master regulators of viral infection. Another approach aims at identifying drugs specifically targeting and regulating the activity of the so called master regulators, namely proteins mostly responsible for the biological processes of viral infection. Guided by a computational approach -already successfully used in cancer research- scientists were able to identify master regulators in virus infection. Identifying drugs targeting the master regulators, interfering with key processes of viral infection, would thus enable to revert viral infection. Preliminary experiments in SARS-CoV infected lung cells show time dependent changes in master regulator protein activity upon viral infection, meaning that some pathways are activated a 12 hours and gradually replaced by other programs at 24 and 48 hours. Interestingly, the same master regulators involved in virus induced changes are implicated in lung cancer malignant transformation. Once identified the master regulators involved in SARS-CoV infection, scientists focused on the identification of drugs able to interfere with their activity in order to revert infection. In vitro analysis of 450 drugs -FDA approved and late stage investigational- identified Selinexor as one of the best in shutting down activated master regulators and inverting viral infection. The target of Selinexor is XPO1 protein and the XPO1 interacting proteins are differentially active in response to SARS-CoV infection.
Targeting TMPRSS2 protein. As previously mentioned, virus entry requires the binding of the virus spike protein to the ACE2 receptor expressed in lung cells. Therefore, ACE2 might seem an ideal target to stop virus spread in the body. However, in mice, ACE2 is critical for survival, suggesting that while ACE2 inhibition may protect from virus infection, it may potentially be dangerous for the host itself. On the other hand, TMPRSS2 host protein has been found critical for viral infection without being crucial for mouse survival, suggesting that interfering with expression of this protein may represent a more successful approach than targeting ACE2. Several drugs downregulating TMPRSS2 are available. Many of these also involve oestrogen and androgen (hormones regulating male and female sexual development) pathway. Drugs interfering with androgen and oestrogen pathways down regulate TMPRSS2, suggesting it may indeed represent an effective approach.
Using nucleotide analogues to halt virus replication. Once entered human lung cells, SARS-CoV2 virus quickly replicates to spread infection in the human body. Virus replication requires duplication of the RNA molecule that represents the viral genome. RNA duplication occurs thanks to the activity of a protein called RNA-dependent-RNA-polymerase. Nucleotides are the “bricks” that the polymerase uses to synthesizes other RNA molecules. However, if the polymerase would use the “wrong bricks”, namely nucleotide analogues (which are similar enough to the normal nucleotides to be incorporated into growing RNA, yet different enough to stop RNA synthesis) virus replication could be arrested. Five nucleotide analogues have been identified to be potentially used as therapeutics for Covid-19. 1) Sovaldi, which is FDA approved for Hepatitis C treatment; 2) Vemlidy, which is FDA approved for the treatment of HIV and HBV; 3) Descovy and Truvada, FDA approved for HIV and HBV, and as chemoprevention for HIV; 4) Azt, FDA approved for HIV; 5) Alovudine, drug currently under experimentation for HIV. Experiments showed that four of the tested drugs (Sovaldi, Alovudine, Azt, Vemlidy) are able to terminate SARS-CoV2 polymerase reaction and Descovy and Truvada could potentially be a chemoprevention agent for Covid-19.
Targeting ER stress. Recent studies have highlighted that depletion of viral proteins NSP4 and ORF8 reduces replication of SARS virus. The same RNA region coding for these two proteins is also present in SARS-CoV2. NSP4 is involved in the formation of vesicles on which RNA synthesis occurs, and ORF8 is located in the Endoplasmic Reticulum (ER, a cellular organelle involved in maturation and sorting of synthesized proteins). Both proteins interact with similar proteins in the ER. Analysis of protein interacting with ORF8 and NSP4 showed that they are all linked with viral replication. During viral replication, cells undergo ER stress as the virus exploits these proteins for its replication, proposing them as potential alternative drug targets.
Lab models to study Covid-19.
Organoids. Studying mechanism of action of SARS-CoV2 infection requires proper models. Studies have shown that lung organoids (cell aggregates that in vitro are able to self-organize in structures recapitulating a specific tissue) derived from human multipotent stem cells firstly grown in suspension can be either developed in vivo, by injection in mice, where they result into human lung development, or in vitro, in a substance called matrigel where they develop a lung structure reminiscent of the lung of human foetus. Organoids can be infected with SARS-Cov2 and used as reliable model system.
Organoids can be useful not only for identifying effective treatments against SARS-CoV2 infection, but also for treating complication of the lung such as pneumonia and respiratory acute distress. Indeed, after SARS epidemics in 2003, many survivors developed pulmonary dysfunction and a consequent decline in quality of life after recovery. This is likely also for Covid-19. Thus mutations associated with lung injury similar to the damage caused by the virus may be introduced in organoids to be modelled and investigated. Cells of the immune system, such as macrophages, may be introduced to better mimic adult physiological situation.
New assays for drug discovery. Identifying an effective anti Covid-19 drug is now the priority. However, it should be considered that hundreds of different coronaviruses are circulating in animals; so it would be extremely useful to identify one drug broadly acting against many coronaviruses. Therefore, a more general approach exploiting yeast has been developed to simultaneously test drugs against different coronaviruses. Viral proteins do not grow in yeast, so upon expression of a viral protein, yeast cultures do not grow. However, if yeast cultures are treated with effective drugs against the viral protein, yeast grow well. Viral proteins are expressed in yeast with a “tag” to identify which yeast cells are infected with a certain viral protein (eg viral protein of MERS) and which are infected with the viral protein of another virus (eg SARS). Yeast cells can be grown together and treated with the same drug. Surviving yeast cells will be identified by the tag, enabling to draw conclusions on the efficacy of a certain drug tested against that specific viral protein. This approach enabled to confidently identify drugs against HIV.
Clinical aspects of covid-19.
Covid-19 causes severe pneumonia, in turn leading to severe respiratory distress. SARS-CoV2 associated pneumonia has a rapid onset (within 5-10 days) and 30-50% mortality. Receptors involved in virus entry are located in the airway. Upon virus entry, the disease involves specific areas of the lung, the alveoli (sites of oxygen/carbon dioxide exchange between inhaled air and blood). In alveoli there are i. Alveolar type 2 cells, which secrete a substance (surfactant) preventing fluid accumulation in body tissue (oedema). Failure of Alveolar type-2 cells increases the risk of oedema; and ii. alveolar macrophages, that regulate resolution of the inflammation elicited by viral infection.
Preliminary results on the analysis of data collected in international database (OHDSI) showed a major incidence of Covid-19 disease, in new York, in 60-80 year old patients, with no major difference between men and women. People with hypertension and heart disease have shown the worst outcome. However, so far -in the New York cohort- no major detriment has been observed by being on therapy with ACE2 (crucial for virus entry into the cell) inhibitors, common therapeutic approach for hypertension. Finally, no major side effects have been observed so far by administration of Hydroxyxhloroquine (anti-malaria drug currently being tested as a potential anti Covid-19 treatment) in Covid-19 patients.
References:
https://www.sciencedirect.com/science/article/pii/S0924857920301011?via%3Dihub
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Columbia University Virtual symposium, April 1st 2020: https://www.youtube.com/playlist?list=PL_H_pQ44HPJ8vkJMGXfhSLdmzEsZ7OSro