Diamond Annual Review 2021/22

8 9 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Coronaviruses have relatively large single-stranded RNA genomes and to maintain the fidelity of these genomes they rely on a complex of two non- structural proteins (nsp14 and nsp10). The study allowed a biochemical characterisation of the nsp14- nsp10 complex and developed assay systems that could screen for inhibitors. This work highlighted domains that could be possible targets for antiviral drugs and identified several drugs and molecules that could be developed or re-purposed to inhibit the nuclease activity of the nsp14-nsp10 complex. These included ebselen which has been shown to inhibit the SARS-CoV-2 major protease (M pro ) and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment. Efficacy of the vaccine The rapid development of vaccines and mAbs against COVID-19 through intensive international collaboration has been one of the positive highlights of the pandemic. Although current vaccines elicit neutralising antibody responses to the virus spike derived from early isolates, new strains have emerged with multiple mutations. Many studies are ongoing to assess the impact of these therapies against the new variants of the virus and the ability of these different variants to evade antibody responses . Recent studies include data collected automatically at Diamond on macromolecular crystallography beamline I03 (see MX intro section showing how previously infected or vaccinated individuals respond to the new variants and antibodies). A large international group studied the ability of mAbs and convalescent and vaccine sera to neutralise two Delta variant viruses (B.1.617.1 and B.1.617.2) that were first reported in India but spread globally 4 . The group used beamline I03 at Diamond to determine the crystal structures of the variants. Although neutralisation of both viruses was reduced compared with earlier strains there was no evidence of widespread antibody escape, suggesting that the current generation of vaccines will provided protection against B.1.617, although reduced titres may lead to some breakthrough infections. However, there was concern that some unvaccinated individuals infected with earlier variants may be more at risk of reinfection with B.1.617.2. These findings provide important new insights to influence immunisation policy. The Beta variant has been particularly difficult to neutralise using serum induced by the early pandemic. To understand this, an international collaboration performed a study to understand the antigenic landscape of the Beta variant 5 . The study included an in-depth structure-function analysis of potent mAbs from volunteers infected with the Beta variant and generated 674 mAbs from beta infected individuals. The authors also performed a detailed structure-function analysis of the 27 most potent mAbs: one binding the spike N-terminal domain (NTD), the rest the receptor-binding domain (RBD). Structural analyses took place using data gathered on beamline I03 and this will add to the increased understanding of the structure and behaviour of the different variants, which will have an important impact on the design and use of available vaccines. This ongoing research will also assist the development of future vaccination strategies. This group performed analysis on different variants including P.1 from Brazil and Beta from South Africa (B.1.351) and Alpha from the UK (B.1.1.7) 6 . Structural analysis at Diamond showed howmAbs neutralise all three variants, and that P.1 is easier to neutralise than other similar receptor-binding domain (RBD) mutations. The study showed that the South African variant B.1.351 was of greatest concern. In another recent paper in Cell an international research group studied the neutralisation of Omicron by a large panel of sera collected from early infected individuals and those vaccinated with three doses of the Oxford/Astra Zeneca or Pfizer vaccines 7 . The rapid emergence of the Omicron variant in the background of high Beta variant immunity implies that the virus may have evolved to escape neutralisation by Beta-specific serum. The study mapped the Omicron RBD mutations and revealed Omicron to be structurally similar but the most antigenically distant variant and that the variant escaped neutralisation by most mAbs. The researchers commented that although the large number of mutational changes seen in Omicron leads to a fall in vaccine effectiveness, it is likely that protection from severe infection will be maintained. The study demonstrated that a third vaccine dose is effective in boosting neutralisation titres against Omicron and that booster vaccination programmes should add considerable protection against Omicron infection. Drug development (both repurposing and new drugs) To defeat the current pandemic and manage the impact of SARS-CoV-2 in the future, a combination of new drugs, repurposed drugs and other therapies will be required. Diamond is playing a key role supporting research groups around the world to develop effective therapies. Major screening studies are taking place to identify new drug candidates, repurposed antivirals are being investigated and there is ongoing development of vaccines that can tackle new variants. The urgency of the current global pandemic demands rapid action. Throughout the pandemic a research collaboration between Diamond, Exscientia (Oxford), Scripps Research Institute in California and Leuven University has aimed to accelerate the path to clinical trials for potential antiviral treatments, both now and in the future. Two potential drug targets are themain protease (M pro ) and the papain-like protease (PL pro ) which are essential for the coronavirus replication cycle. The international research group conducted extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents 8 . The library was developed at Calibr, the drug development division of the Scripps Research Institute with funding from the Bill and Melinda Gates foundation. The screens identified two potential drug candidates, the caspase-1 inhibitor SDZ 224015, which was found to be a potent irreversible inhibitor of M pro , and tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor was an inhibitor of PL pro . This is the first reported PL pro inhibitor with drug-like properties. Diffraction data were collected from crystals cryo-cooled at Diamond with X-ray diffraction data collected at beamlines I04-1 and I24. As both compounds have already undergone safety evaluation in humans, they are trial candidates for rapid clinical evaluation against COVID-19. A collaboration between Diamond and Israeli research groups designed a computational pipeline to help identify irreversible inhibitors based on structures of targets with non-covalent binders 9 . The so-called covalentiser was developed to identify covalent analogues of non-covalent binders. Covalent irreversible inhibitors have become increasingly popular over the last decade as chemical probes and drugs. These offer several advantages over non-covalent inhibitors such as prolonged residence time, improved selectivity, and an ability to compete with high affinity natural substrates, but designing new covalent inhibitors is challenging. The group used a pre-compiled database of covalentiser results to look for possible candidate inhibitors for SARS-CoV-2 proteins. Using the protocol allowed discovery of several covalent kinase inhibitors and optimised a potent covalent COVID-19 protease inhibitor, with a low-cost, modular, and fast synthesis. The protocol was prospectively validated against various kinases, as well as in a rapid drug discovery campaign against the SARS-CoV-2 main protease. Co- crystal structural data were collected at Diamond on beamline I04-1 to confirm the computational model. These results point to many other potential targets that may be targeted by a covalent analogue of a known ligand, potentially shortening the time and effort in finding future covalent inhibitors. Other therapies An international collaboration continued their work on neutralising single domain antibodies (nanobodies) which have significant potential as an effective treatment against COVID-19 10 . They appear to offer considerable advantages over conventional antibodies as their small size and stability mean they can be formulated for respiratory administration which results in improved bioavailability, easier administration, and improved therapeutic compliance. They can also be manufactured using readily available microbial systems. The research group isolated four nanobodies that bind different epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. These neutralise the virus in vitro with picomolar potency. X-ray crystallography diffraction data were collected and processed at beamlines I03, I04 and I24 at Diamond. Cryo-EM results were obtained at eBIC. In a Syrian hamster model of COVID-19, the group showed that treatment either systemically (intraperitoneal route) or via the respiratory tract (intranasal route) with a single dose of the most potent nanobody prevented disease progression. The researchers suggested that combinations of nanobodies that target different epitopes may improve resilience in combating new variants of the virus. References 1. Mendonça, L. et al . Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress. Nat Commun. 12, 4629 (2021). DOI: 10.1038/s41467-021-24887-y 2. Walker, A. et al . The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme. Nucleic Acids Research 49, 13019–13030 (2021) DOI: 10.1093/ nar/gkab1160 3. Baddock, H. et al . Characterisation of the SARS-CoV-2 ExoN (nsp14ExoN– nsp10) complex: implications for its role in viral genome stability and inhibitor identification. Nucleic Acids Research 50, 1484–1500 (2022). DOI: 10.1093/nar/gkab1303 4. Liu, C. et al . Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum. Cell 184, 16 P4220-4236 (2021). DOI: 10.1016/j.cell.2021.06.02 5. Liu, C. et al . The antibody response to SARS-CoV-2 Beta underscores the antigenic distance to other variants. Cell Host & Microbe 30, P53-68 (2021). DOI: 10.1016/j.chom.2021.11.013 6. Dejnirattisai, W. et al . Antibody evasion by the P.1 strain of SARS-CoV-2. Cell 184, P2939-2954(2021). DOI: 10.1016/j.cell.2021.03.055 7. Dejnirattisai, W. et al. SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses. Cell 185, P467-484.E15 (2022). DOI: 10.1016/j.cell.2021.12.046 8. Redhead, M. et al . Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19. Scientific Reports 11, 13208 (2021). DOI: 10.1038/s41598-021-92416-4 9. Zaidman, D. et al . An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor. Cell Chemical Biology 28, P1795-1806 (2021). DOI: 10.1016/j.chembiol.2021.05.018 10. Huo, J. et al . A potent SARS-CoV-2 neutralizing nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19. Nature Communications 12, 5469 (2021). DOI: 10.1038/s41467-021- 25480-z Crystal structure of SARS-CoV-2 M pro with a pre-clinical candidate from the COVID Moonshot bound (shown in yellow). The COVID Moonshot, of which Diamond is a founding member, has received funding of £8 million fromWellcome, on behalf of the COVID-19 Therapeutics Accelerator to develop a globally accessible antiviral.