Animal studies suggest that palmitoylation inhibitor reduces SARS-CoV-2 virulence

Since the outbreak of coronavirus disease in the world in 2019 (COVID-19), more than 76 million people have been infected, with more than 1.68 million deaths. The virus that causes this pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), one of the seven pathogenic coronaviruses that have hit the world so far. Even while the first vaccines are being phased out, the search for specific and safe treatments is still ongoing, as new variants emerge, forcing new locks in several regions.

New study overprint of bioRxiv * the server demonstrates the ability of fatty acid synthase inhibitors to improve the survival of mice infected with murine hepatitis virus. In this way, it is added to the sum of knowledge about the connection of SARS-CoV-2 with the metabolism of cholesterol in the host cell.

Objectives of the study

Coronaviruses target multiple tissues in the human host, including the lungs and upper respiratory tract cells, intestinal tissues, kidneys, and liver. They bind to host cells via angiotensin 2-converting enzyme receptor (ACE2) and possibly other receptors such as neuropylin-1 (NRP1). Once the virus binds to the ACE2 receptor, proteolytic cleavage occurs using host cell proteases. This generates a S2 fragment that stimulates the fusion of the cell membrane of the viral host, allowing the virus to enter the cytosol.

Most potential therapies target this binding step. Currently, some promising lead compounds include protease inhibitors and soluble ACE2 molecules that act as bait, preventing the virus from invading host cell receptors. The high mutation rate of positive RNA viruses gives them an advantage allowing them to avoid the specific inhibition caused by these molecules.

The current study aimed to understand how to develop a broad-spectrum antivirus program that targets basic aspects of the host-virus interaction. This could help fight future coronaviruses. The present study examined the possibility of using palmitoylation inhibitors to treat SARS-CoV-2 infection.

Palmitoylation of the spike necessary for new virions

Earlier reports showed that a protein called palmitoyltransferase ZDHHC5, together with its binding partner Golga7, interacted with the helical SARS-CoV-2 protein. Their research, which used affinity purification and mass spectrometry, suggests that the tail protein of the spikes, which is located in the cytosolic domain, undergoes palmitoylation after formation in the host cell. This is similar to the process that occurs in murine hepatitis virus (MHV), whose spike protein must have C-terminal palmitoylation in order for the virus to replicate successfully.

If palmitoylation is prevented, the protein class cannot be assembled into new virions. The mutated spike protein was also unable to stimulate membrane fusion to form syncytial cell fusion, unlike wild-type virus.

Multiple sites of palmitoylation

In many coronaviruses that infect both human and mouse cells, the C-terminal domains have shown enrichment of cysteine ​​residues in the last 20 residues, near the membrane. With SARS-CoV-2, 10 of the first 20 residues are cysteine, allowing plenty of room for palmitoylation.

The researchers used HEK293T cells, allowing them to express the SARS-CoV-2 spike protein labeled with the C9 epitope at the C-terminal end. They found that these cells contain numerous types of protein classes, some full-length, some of which contain the S2 subunit, and some multimers of class proteins.

These cells were then grown in the presence of an analogous palmitate donor compound, termed 15-hexadecinoic acid (15-HDYA). The researchers wanted to find out if this palmitite analog could covalently bind to a protein class, whether it is a full-length, S2 or multimeric species.

They then used acyl-polyethylene glycol (PEG) (APE) exchange, a method that involves switching each class of palmitoyl group to a 5-kDa PEG fat plate. This would help measure how much protein-based palmitoylation would occur. They found that protein spike can be modified on a number of sites. Looking at individual proteins, there were at least four palmitoyl groups.

Spike palmitoylation is required for cell fusion.  (A) Western blot SARS-CoV-2 Spike-C9 of ten mutants Cys to Ser.  (B) Immunofluorescence of SARS-CoV-2 Spike and SpikeC-> S-C9 mutant.  Scale bars, 10 μm (C) Co-cultures of HEK cells expressing Spike-C9 and ACE2 form syncytium.  HEK293T cells cotransfected with mCherry-C1 (magenta) and SARS-CoV-2 Spike-C9 (left and center) or SARS-CoV-2 SpikeC-> S-C9 (right) co-cultured with EGFP-C1 transfected cells left, cyan) or and myc-ACE2 (middle, right).  Measuring rods, 20 µm.  The tips of the arrows indicate fused cells.” height =”782″ src =”https://d2jx2rerrg6sh3.cloudfront.net/image-handler/picture/2020/12/2020.12.20.423603v1_—_1200_width.jpg” srcset =”https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20201224124450/ri/1200/picture/2020/12/2020.12.20.423603v1_—_1200_width.jpg 1200w, https://d2jx2rerrg6sh3.cloudfront.net/ image-handler / ts / 20201224124450 / ri / 1150 / slika / 2020/12 / 2020.12.20.423603v1 _ — _ 1200_width.jpg 1150w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20201224124450/ ri / 950 / slika / 2020/12 / 2020.12.20.423603v1 _ — _ 1200_width.jpg 950w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20201224124450/ri/750/picture/2020/ 12 / 2020.12.  20.423603v1 _ — _ 1200_width.jpg 750w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20201224124450/ri/550/picture/2020/12/2020.12.20.423603v1_—_1200_width.jpg 550w, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20201224124450/ri/450/picture/2020/12/2020.12.20.423603v1_—_1200_width.jpg 450w” sizes =”(min-width: 1200px) 673px, (min-width: 1090px) 667px, (min-width: 992px) calc (66.6vw – 60px), (min-width: 480px) calc (100vw – 40px), calc 100vw) – 30px)” title =”Spike palmitoylation is required for cell fusion.  (A) Western blot SARS-CoV-2 Spike-C9 of ten mutants Cys to Ser.  (B) Immunofluorescence of SARS-CoV-2 Spike and SpikeC-> S-C9 mutant.  Scale bars, 10 μm (C) Co-cultures of HEK cells expressing Spike-C9 and ACE2 form syncytium.  HEK293T cells cotransfected with mCherry-C1 (magenta) and SARS-CoV-2 Spike-C9 (left and center) or SARS-CoV-2 SpikeC-> S-C9 (right) co-cultured with EGFP-C1 transfected cells left, cyan) or and myc-ACE2 (middle, right).  Measuring rods, 20 µm.  The tips of the arrows indicate fused cells.” width =”1200″/><meta itemprop=

Spike palmitoylation is required for cell fusion. (A) Western blot SARS-CoV-2 Spike-C9 of ten mutants Cys to Ser. (B) Immunofluorescence of SARS-CoV-2 Spike and SpikeC-> S-C9 mutant. Scale bars, 10 μm (C) Co-cultures of HEK cells expressing Spike-C9 and ACE2 form syncytium. HEK293T cells cotransfected with mCherry-C1 (magenta) and SARS-CoV-2 Spike-C9 (left and center) or SARS-CoV-2 SpikeC-> S-C9 (right) co-cultured with EGFP-C1 transfected cells left, cyan) or and myc-ACE2 (middle, right). Measuring rods, 20 µm. The tips of the arrows indicate fused cells.

Palmitoylation required to form syncytia

Further, they used a site-directed mutagenesis induction technique to convert all ten cysteine ​​residues to serine. They used fluorescence microscopy to determine whether spike-C9 cells and cysteine> serine spike proteins were equally capable of catalyzing the fusion of cell cells with ACE2-expressing cells. As expected, they found that a number of large syncinations occurred with spike-C9, but not with the cysteine ​​serine variant of spike. “The results show that Cys residues are required for the Spike protein to facilitate syncytial formation.”

Silencing ZDHHC5 reduces the formation of viral deposits

There are 23 ZDHHCs (palmitoyltransferases containing the Asp-His-His-Cys zinc domain of the Asp-His-His-Cys finger zinc domain in the human genome, of which only ZDHHC5 palmitoylates the spike protein SARS-CoV-2). genes were effective in limiting the spread of the virus in culture, they used a less lethal virus, human CoV 229E, which was suitable to work with biosafety level 2. It has a cytosolic tail that resembles SARS-CoV-2 and can be palmitoylated at multiple sites Like the SARS-CoV-2 spike, the 229E spike is capable of ZDHHC5 palmitoylation.

Using a cell test for 229E with muted ZDHHC5, they found that fewer plaques formed compared to controls. To determine whether this was due to attenuated infection or subsequent spread of infection through a single layer of cells, cells were incubated with the virus in liquid culture. This showed that ZDHHC5 was needed, not for the initial infection, but for the collection and release of infectious viral particles.

Inhibition of FASN prevented palmitoylation of helical SARS-CoV2

There is currently no specific ZDHHC5 inhibitor, but the fatty acid synthase inhibitor (FASN) cerulenin blocks the addition of palmitoyl NOD1 and NOD2, which are substrates of ZDHHC5. Being a toxic compound, they tested another molecule of a FASN inhibitor, called TVB-3166, and bromopalmitate, a nonspecific inhibitor of the ZDHHC enzyme. They found that the palmitoylation of the spikes was reduced for both viruses, while in unique plaque formation tests the number of plaques formed from 229E was 86% lower.

The FASN inhibitor increases the survival of mice infected with MHV

Compound TVB-3166 can be administered orally, and researchers tested it on mice infected with 229E virus. They found that while control mice showed signs of disease within two days of infection, which continued to progress, treated mice survived longer periods, and many survived. “The results show that a reduction in palmitoyl-CoA levels is useful during CoV infection because this reduction promotes survival in mice.”

Implications and future guidance

The study therefore suggests that palmitoylation of the S2 subunit is necessary for viral membrane fusion in SARS-CoV-2, MHV, and SARS viruses. In MHV, palmitoylation of the spike is really needed to allow it to interact with a membrane protein that allows it to assemble the fitting into new viral particles. The addition of TVB-3166, either in cell tests or in infected mice, reduced the production of new infectious virus particles and increased mouse survival.

The mechanism of action of this molecule indicates the role of altered lipid metabolism in coronavirus infections. Some of the signs of this are changes in serum apolipoproteins and lipids, as well as increased levels of free fatty acids. These lipid changes may promote virus replication. In the ectodomain SARS-CoV-2, there is also a pocket that binds linoleic acid, as well as other pathogenic coronaviruses, which indicates the need to investigate the connection between the metabolism of fatty acids and lipids with coronavirus infection.

Further studies to investigate the possible therapeutic role of TVB-2640, a related FASN inhibitor already in clinical trials for cancer and non-alcoholic liver disease, are available orally and are well tolerated in patients. Because the FASN enzyme is the host, viral escape mutations are unlikely to occur. “Identification and approval of orally available pan-CoV treatment or prophylaxis would be useful in the short term and in the future against other zoonotic CoVs.”

* Important notice

bioRxiv publishes preliminary scientific reports that have not been reviewed and should therefore not be considered final, guide clinical practice / health-related behavior, or treat it as established information.

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