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Researchers have produced a high-resolution crystal structure of an enzyme essential to the survival of SARS-CoV-2, the virus that causes COVID-19. The discovery could lead to the design of critically needed new antivirals to combat current and future coronaviruses. A team of Mount Sinai researchers has produced a high-resolution crystal structure of an enzyme essential to the survival of SARS-CoV-2, the virus that causes COVID-19. The discovery could lead to the design of critically needed new antivirals to combat current and future coronaviruses. The enzyme, known as nsp14, has a crucially important region known as the RNA methyltransferase domain, which has eluded previous attempts by the scientific community to characterize its three-dimensional crystal structure. A paper describing the innovative process was published inthe September 8 online edition of Nature Structural & Molecular Biology. "Being able to visualize the shape of the methyltransferase domain of nsp14 at high resolution gives us insights into how to design small molecules that fit into its active site, and thus inhibit its essential chemistry," says senior author Aneel Aggarwal, PhD, Professor of Pharmacological Sciences at the Icahn School of Medicine at Mount Sinai. "With this structural information, and in collaboration with medicinal chemists and virologists, we can now design small molecule inhibitors to add to the family of antivirals that go hand-in-hand with vaccines to combat SARS-CoV-2." Prescription antivirals that target key enzymes of SARS-CoV-2 include nirmatrelvir for the main protease (MPro) enzyme, and molnupiravir and remdesivir for the RNA polymerase (nsp12) enzyme. Research to develop new antivirals targeting different enzymatic activities has been accelerating in laboratories around the world, and Mount Sinai's discovery has added significantly to that effort. "Part of what drives our work," says Dr. Aggarwal, "is the knowledge gained from treating HIV -- that you typically need a cocktail of inhibitors for maximum impact against the virus." The Mount Sinai team actually developed three crystal structures of nsp14, each with different cofactors, from which they identified the best scaffold for the design of antivirals for inhibiting the RNA methyltransferase activity that the enzyme enables and the virus needs to survive. According to their scheme, the antiviral would take the place of the natural cofactor S-adenosylmethionine, thus preventing the methyltransferase chemistry from occurring. The crystal structures that the team has elucidated have been made available to the public and will now serve as guides for biochemists and virologists globally to engineer these compounds. Making the discovery possible was the ability of researchers to clear a hurdle that had prevented others in the past from creating three-dimensional crystals of the nsp14 methytransferase domain. "We employed an approach known as fusion-assisted crystallization," explains lead author Jithesh Kottur, PhD, a postdoctoral fellow at Icahn Mount Sinai, and a crystallographer and biochemist. "It involves fusing the enzyme with another small protein that helps it to crystalize." Dr. Aggarwal, an internationally recognized structural biologist, underscores the importance of ongoing investigative work by researchers in his field against a virus that has led to millions of deaths globally. "The virus evolves so quickly that it can develop resistance to the antivirals now available, which is why we need to continue developing new ones," he observes. "Because of the high sequence conservation of nsp14 across coronaviruses and their variants (meaning it does not mutate much), our study will aid in the design of broad-spectrum antivirals for both present and future coronavirus outbreaks."

As coronavirus variants continue to fuel the ongoing pandemic, Gritstone bio is pressing ahead with its second-generation Covid-19 vaccine, now with financing from the Coalition for Epidemic Preparedness Innovations (CEPI) to support additional clinical trials. Gritstone has reached an agreement with CEPI for up to $20.6 million in funding to bring its vaccine into a Phase 1 test, initially in South Africa. Combined with the biotech’s own Phase 1 tests, the company said it has four candidates being evaluated for vaccination of the young, the elderly, those previously vaccinated, and patients who are immunocompromised. If the Gritstone vaccine works in the South African study, it would be made available globally through the World Health Organization’s COVAX initiative. Emeryville, California-based Gritstone calls its second-generation Covid vaccine program CORAL. Like the first generation of Covid vaccines, Gritstone’s candidates are delivering the spike protein to trigger an immune response. But the biotech’s vaccines are also delivering other parts of an antigen that are “conserved,” meaning that they don’t change much as the virus mutates. A vaccine that addresses conserved regions of a virus makes it more likely to retain efficacy even as the virus changes. The CORAL vaccines are delivered to cells by a chimpanzee adenovirus, self-amplifying messenger RNA, or both. Self-amplifying mRNA (SAM) is comprised of RNA that encodes the target antigens, plus RNA polymerase, which is an enzyme that copies a DNA sequence into an RNA sequence during the transcription process. The Gritstone vaccine is given as an intramuscular injection. The company says in its regulatory filings that after the vaccine is taken up by a patient’s cells, the RNA is translated into protein and the RNA polymerase starts to replicate the RNA from the injection, which amplifies the copies of RNA in the cells. Consequently, large amounts of target antigens are produced. Gritstone says that unlike mRNA vaccines, its SAM vaccines produce large quantities of antigen that in turn drive strong T cell responses. “We believe Gritstone’s CORAL vaccine candidate has the potential to improve both T cell and antibody responses to Spike and other viral proteins,” the company said in its 2020 annual report. “Additionally, by targeting several viral antigens, some of which are highly conserved between viral strains (such as SARS and SARS-CoV-2), we believe our vaccine candidate may have pan-SARS/coronavirus potential to protect against future coronavirus pandemics.” The planned Phase 1 study in South Africa will evaluate two SAM vaccines, each targeting both the spike protein and other parts of the virus. Both vaccines are designed to elicit robust responses from B cells and T cells. Gritstone expects to start this study by the end of this year. According to terms of the agreement announced Tuesday, CEPI will fund the multi-arm study, which will evaluating the vaccines in those who have not been infected, convalescent subjects who are currently healthy but previously had SARS-CoV-2 infection based on antibody tests, and HIV positive patients. In March, a separate Phase 1 study supported by the National Institute of Allergy and Infectious Diseases began dosing patients. Gritstone expects this study will report data in the fourth quarter of this year. In addition, Gritstone is preparing company-sponsored clinical trials evaluating CORAL as a booster to the Covid vaccines that are currently authorized for emergency use, as well as for those with weakened immune systems. Image: Maksim Tkachenko, Getty Images