Sirius’s protein crystallography beamline analyzed more than 200 protein crystals from the novel coronavirus as they were exposed to tiny fragments of widely used drugs. If a compound fits perfectly into a target protein, its action can be blocked in the virus (André Godoy and Aline Nakamura position SARS-CoV-2 protein crystal for analysis at Sirius; photo: CNPEM)
Published on 03/16/2021
By Maria Fernanda Ziegler | Agência FAPESP – A powerful beam of synchrotron light has enabled scientists to determine the structure of more than 200 protein crystals from the novel coronavirus SARS-CoV-2.
The investigation conducted in Brazil by researchers at the University of São Paulo’s São Carlos Institute of Physics (IFSC-USP) is important to the development of a possible drug to treat COVID-19. It is also the first of its kind.
Conducted by Aline Nakamura and André Godoy, the experiment inaugurated the first research station at fourth-generation particle accelerator Sirius, Brazil’s most complex science facility, hosted by the Brazilian Center for Research in Energy and Materials (CNPEM) in Campinas, in the state of São Paulo.
“We had the good fortune to be the first to try out the protein crystallography beamline, called Manacá. This opportunity has significantly expedited our study,” said Glaucius Oliva, principal investigator at the Center for Innovation in Biodiversity and Drug Discovery (CIBFar) and lead researcher for antiviral drug discovery relating to COVID-19. “Since the pandemic began, Sirius and all other synchrotron light sources worldwide have focused entirely on experiments relating to COVID-19. Sirius is still in the commissioning stage, but we were given the chance to use it because our experiment was very much associated with research on the novel coronavirus.”
Manacá is Tupi for flower and an acronym for “MAcromolecular Micro and NAno CrystAllography”. CIBFar is hosted by IFSC-USP and is one of the Research, Innovation and Dissemination Centers (RIDCs) supported by FAPESP.
Besides the partnership with CNPEM, the project supported by FAPESP in search of drugs to treat COVID-19 also involves researchers affiliated with the University of São Paulo’s Biomedical Sciences Institute (ICB-USP), São Carlos Institute of Chemistry (IQSC-USP) and Ribeirão Preto School of Pharmaceutical Sciences (FCFRP-USP), as well as researchers at São Paulo State University (UNESP) and the University of Campinas (UNICAMP).
“We can now do in minutes what took hours on CNPEM’s old electron accelerator,” rejoiced Godoy, a researcher with ten years of experience in analysis using other synchrotron light sources around the world.
The inaugural experiment lasted three days, but analysis using Sirius is set to become more efficient in future. “Commissioning is still underway, so the machine isn’t operating at full power, but we had X-rays exiting the beamline window and falling on the crystals frozen at cryogenic temperatures, while the detector measured diffraction of the synchrotron light by the crystals to obtain the structure of the proteins of which they’re made,” Oliva said.
However, robotic arms to position the crystals had not yet been fitted, for example. “So we had to swap out the crystals ready for the next analysis by hand,” he explained. “You can’t use all the nominal power while the synchrotron ring is being commissioned, but even so it was very efficient and the tests were most valuable.” Oliva was the first researcher to test UVX, the second-generation synchrotron light source designed and built by Brazilians in the 1990s and now replaced by Sirius.
The fourth-generation accelerator’s beamlines reveal the atomic structure of organic and inorganic materials, he said. Its advanced magnets, vacuum, and control systems ensure that the subatomic particles are accelerated to near-light speed, emitting synchrotron light as their trajectories are deflected.
The significance of determining the structure of tiny protein crystals in the search for drugs to treat COVID-19 can be understood by imagining a jigsaw puzzle: the proteins are the picture to be assembled (the targets inside the virus), and the pieces to be slotted into each position are chemical compounds. If a chemical compound fits perfectly into a target protein, this means the protein’s action can be blocked to prevent the virus from invading human cells or replicating inside them.
The researchers used the Manacá beamline to pinpoint the positions of the thousands of atoms in two viral proteins so as to determine their structure. One of the proteins is the virus’s main protease (Mpro), known as non-structural protein 5 (nsp5), which cleaves the long chain of proteins synthesized in the invaded cell using the information contained in the viral genome, making them active and functional for replication of the virus. The other protein the researchers studied is nsp15, the enzyme that cleaves the viral RNA. Its behavior in human cells is still being investigated.
“Proteins have a convoluted surface due to the way their atoms are organized,” Oliva said. “Their grooves are where their catalytic function is performed, in the virus and in infected cells. Our goal in this study is to determine the structure of these two proteins and to find molecules, compounds, or substances that can be candidate drugs because they fit perfectly into the catalytic sites and can therefore block the virus’s action.”
The team of researchers decided to go much farther than looking randomly for candidate drugs or testing substances in cultured cells. They opted for a strategy known as fragment screening, in which hundreds of tiny fragments of widely used drugs are placed in contact with crystals of the target proteins to find out whether they bind and if so where. “The information gleaned from these experiments can be used to synthesize larger and more complex molecules that connect the previously identified fragments,” Oliva said. “True candidates for antiviral drugs can be developed in this manner.”
If candidates are identified by protein crystallography and fragment screening, the next step will be to test them in cultured cells, in an animal model, and in humans, following all the phases of trials required for drug discovery and development. Successful compounds will be used to treat patients infected by SARS-CoV-2.
Besides Macaná, Sirius will have 13 beamlines when commissioning is complete, with up to 38 research stations optimized for a wide array of experiments performed simultaneously in such fields as healthcare, energy, new materials, and environmental management, among many others.
“A great deal of work remains to be done, but each advance of Sirius proves ever more emphatically that we have the capabilities to take science and technology in Brazil to a higher level. The Brazilian scientific community is extremely competent, and it’s our mission to support our scientists by offering them conditions to do groundbreaking research. The machine we’re finalizing is globally competitive, designed by Brazilians, and built in partnership with locally owned engineering firms,” said Antonio José Roque da Silva, Director-General of CNPEM and head of Project Sirius.
* With information from CNPEM.