Synchrotron light proves effectiveness of several drugs in virus infections like SARS-CoV2

Microtubules are intracellular structures that function as true cellular highways for the transport of substances, vesicles, organelles and even viruses, in the case that a cell gets infected. In most viral infections, they are the transport routes to generate the viral factories, regions close to the nucleus where virus production is concentrated.

The idea is to design drugs that, by binding to microtubules, prevent viruses from using them during the infection process. In general, drugs that target microtubules are called MTAs (microtubule targeting agents). There are two types: stabilizers (MSA) and destabilizers (MDA). Both are widely available and most of these drugs are in the WHO Essential Medicines List, and hence, they are therapeutic alternatives that are affordable and available worldwide.

Researchers from CIB Margarita Salas selected 16 commercially available MTA (including 15 in clinical use) to analyse their capacity to inhibit the viral replication against 5 different virus: the human common cold coronavirus (HCoV), the pandemic SARS-CoV-2 coronavirus, the vesicular stomatitis virus, the poxvirus vaccinia and African swine fever virus.

Scientist confirmed that the MTA tested had an effect on virus replication and spreading and that this effect varies according to the virus dependency on the microtubular network. “The inhibitory effect obtained varied depending on the specific functions that viruses have developed throughout evolution to exploit cellular transport machinery”, explains Dra. Marian Oliva, researcher at CIB Margarita Salas-CSIC.

In particular, the most complex use of microtubules filaments might correspond to coronavirus (CoVs), such as the one responsible for the Covid-19 pandemic. Microtubules are necessary both for virus internalization and later at several levels of the formation of the viral replication site. In fact, S and M coronavirus proteins (located on the virus surface) interact with tubulin (protein that forms microtubules) during the infection, although their specific function is currently unknown. Various projects involving the use of the ALBA Synchrotron are under way to study deeper these aspects.

Read more on the ALBA website

Image: Image obtained at the XALOC beamline of ALBA. Drug mebendazole (MBZ) bounds to the protein that forms the microtubules: tubulin (T2RT and T1D).

Structural studies of SARS-CoV-2 nucleocapsid protein

Perspectives in relation to diagnosis and drug design

 A novel zoonotic coronavirus SARS-CoV-2 was originally explored in Wuhan, China in December 2019 and further regarded to the serious pandemic known as COVID-19. In early March 2022, the global COVID-19 pandemic has caused over 453 million confirmed cases and over 6 million deaths (John Hopkins Coronavirus Resource Center, https://coronavirus.jhu.edu).

 The COVID-19 virus and the emergence of new virus variants seriously threat to global public health. It is a strong requirement to develop the effective diagnostic tools which are able to quickly and reliably detect active SARS-CoV-2 infections.

 Structural proteins of the COVID-19 virus are very important to understand its pathogenic mechanism, thus leading to the development of antibodies, vaccines and drugs for targeting these proteins and viruses.

 SARS-CoV-2 comprised the four structural proteins; the spike (S), nucleocapsid (N), envelope (E) proteins and membrane glycoprotein (M). A complete virus particle (virion) is represented in Figure 1. Cryo-electron microscopy is one of the powerful tools to determine the overall structure of the S protein, thus presenting a unique crown or ‘corona’-like shape.

 Three viral proteins; the spike (S), envelope (E) and membrane (M) are embedded in the outer layer of the corona viral particle. The corona viruses protect themselves from the surrounding environment, then the ribonucleic acid (RNA) forms a stable packed in the lipid membrane. The nucleocapsid protein (nucleoprotein) is responsible for tightly wrap the RNA of viruses. However, the fatty membrane of SARS-CoV-2 is sensible to be destroyed by soap, detergent or surfactant.

 The nucleocapsid protein significantly involves in viral genomic RNA binding, thus protecting the coiled RNA as its genetic material inside the virus particle. Moreover, the N protein also plays an important role in the early stages of viral infection when the RNA genome is first released into the target host cell.

 X-ray crystal structures of the N-terminal (PDB entry 7CDZ) and C-terminal domains have been illustrated here (PDB entry 6WZO). Holo structure of N-terminal domain in complex with double strand RNA (PDB entry 7ACS) has been determined by Nuclear Magnetic Resonance Spectroscopy technique.

Read more on the Thai Synchrotron website

Image:  Three dimensional models of the SARS-CoV-2 virion and a schematic diagram of its four structural proteins. 

Credit: Figures were modified from coronavirusexplained     

Beaming in on Coronavirus details

User operation resumed at European XFEL end of March, and the first experiments to receive beamtime are those being carried out at the Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument. They will focus on getting deeper insights into the Coronavirus, and, if successful, can lead to a better understanding of the structure of key Coronavirus proteins. New information about the shapes of these proteins, which the virus needs to copy itself, will aid scientists in their quest to find ways to fight COVID.

“Three user collaborations have proposed experiments that will use two distinct approaches to study the Coronavirus. Two collaborations lead by scientists from DESY and Diamond Light Source will look at the structure and binding of ligands to the proteases of the Coronavirus,” says Adrian Mancuso, leading scientist at the SPB/SFX instrument. A ligand is a molecule that binds another specific molecule or atom. Some ligands deliver a signal during the binding process and can be thought of as signaling molecules, which interact with proteins in target cells called receptors. At the European XFEL, scientists can potentially observe the process of these ligands attaching to proteins at atomic resolution, however, first an ordered crystal of the relevant protein is required. “XFELs are uniquely positioned to watch how irreversible processes in proteins—such as binding of potential drug candidates—happen,” explains Mancuso.

Read more on the European XFEL website

Image: A shot from the control hutch showing one of the first COVID-related beamtimes at SPB/SFX

Credit: European XFEL

Promising candidates identified for COVID drugs

A team of researchers has identified several candidates for drugs against the coronavirus SARS-CoV-2 at DESY´s high-brilliance X-ray lightsource PETRA III. They bind to an important protein of the virus and could thus be the basis for a drug against Covid-19.

In a so-called X-ray screening, the researchers, under the leadership of DESY, tested almost 6000 known active substances that already exist for the treatment of other diseases in a short amount of time. After measuring about 7000 samples, the team was able to identify a total of 37 substances that bind to the main protease (Mpro) of the SARS-CoV-2 virus, as the scientists report online today in the journal Science. Seven of these substances inhibit the activity of the protein and thus slow down the multiplication of the virus. Two of them do this so promisingly that they are currently under further investigation in preclinical studies. This drug screening – probably the largest of its kind – also revealed a new binding site on the main protease of the virus to which drugs can couple.

Read more on the DESY website

Image: In the control hutch of the PETRA III beamline P11, DESY researcher Wiebke Ewert shows on a so-called electron density map where a drug candidate (green) binds to the main protease of the corona virus (blue).

Credit: DESY, Christian Schmid