In March 2020, when the COVID-19 pandemic started to take hold, long-term collaborators Professor Francesco Spinozzi from Marche Polytechnic University and Professor Alessandro Paciaroni from the University of Perugia were working on an experiment at the ISIS neutron and muon source, a stone's throw from Diamond. They decided that they wanted to use their skills to help fight the virus outbreak, but as Europe went into Lockdown, it became impossible for them to travel outside Italy.
Under normal circumstances, Profs Spinozzi and Paciaroni investigate the structure of proteins using Small Angle X-ray Scattering (SAXS). They had no experience of working with coronaviruses, but looked for a way they could put their skills to good use.
Meanwhile, the beamline staff at Diamond were working hard to keep the facility open and available to scientists involved in COVID research. However, the UK Lockdown meant users couldn't come to Diamond in person.
SAXS allows us to investigate the structure of matter, and it's a technique that lends itself to examining everything from aeroplane components to proteins. Experiments on B21's sister beamline, I22, can be tailored to meet the needs of individual users, but this is a time-consuming process. On B21, Principal Beamline Scientist Nathan Cowieson and his team specialise in SAXS on liquid samples. This allows them to have a much higher throughput, collecting data very quickly. B21 is particularly suited to structural biology studies of proteins in solution.
B21 already offered an option for users to mail their samples to Diamond so that their experiments could be run by beamline staff. A small number of users were using the mail-in route. As you can imagine, it's hard for researchers to hand over their precious samples to someone else to run their experiment! Profs Spinozzi and Paciaroni are regular users on the B21 beamline. As it happens, they had prior experience of using the mail-in route. So they had complete confidence that it would work when they came up with their experiment proposal. Their investigation into the structure of the COVID-19 protease Mpro, recently published in Scientific reports, gives us vital information that will inform the development of new drugs and therapies to fight COVID-19 and future coronavirus outbreaks.
When viruses infect a host organism, they use its cells to replicate themselves. Coronaviruses use the host cells to produce copies of long polyproteins - all the separate proteins the virus needs, joined together. For the virus to become active, the polyprotein needs to be broken up into its component proteins. One enzyme, the Mproprotease, controls many of these 'cleaving' events. If we could stop Mprofrom performing its vital function, we could stop the virus from spreading. Mprois, therefore, a good target for anti-viral drugs, particularly because this enzyme is well-conserved across the whole family of coronaviruses.
Mproexists in two forms - as a single protein (monomer) and a pair of identical proteins (a dimer). The Mprodimer is the active form, but we don't fully understand the process by which it forms from the monomer. If we did, we may be able to develop compounds that interfere with this dimerisation, which would potentially be broad-spectrum anti-virals.
Prof Spinozzi and Prof Paciaroni devised a series of experiments to investigate dimerisation in Mpro and the effect of several inhibitor compounds, which were chosen by researchers at the University of Palermo.
Prof Spinozzi explains:
With SAXS you don't need to have your crystal as a protein, so it removes that restriction from experiments. It also allows you to get much closer to physiological conditions, for example in terms of temperature. You can add different molecules to the solution, and change the pH and the salt concentrations. It offers a wide range of possibilities.
Prof Paciaroni added:
SAXS offers great flexibility, compared to other techniques such as diffraction. It's the only technique capable of studying the dimerisation process, which takes place in solution. Critically, it can distinguish between Mpromonomers and dimers, which is the most neglected feature of Mpro. SAXS also allows us to see precisely what effect the inhibitors are having, in terms of whether they are interacting with the monomers or the dimers. The devil is in the details!
By June 2020, B21 was running a full user programme, despite restrictions meaning that only one staff member could be at the beamline at any time. All experiments had to use the mail-in route, producing a heavy workload.
Nathan Cowieson, Principal Beamline Scientist on B21, said:
We get emails from the users detailing exactly how they want us to handle their samples. We're developing new logistics and database capabilities.
We have around half a dozen groups using the beamline for COVID research, and they've typically each done a couple of virtual visits. Some are doing antibody work, so they're looking at potential vaccines and immunity. Others are focusing on nanoparticles and liposomes, that's drug delivery vectors. This particular work concentrated on inhibitors, which is something Diamond is working on internally, as well. We've had excellent results from using the mail-in route, and in fact, the beamline had its highest ever number of publications last year.
Profs Spinozzi and Paciaroni are continuing their work on understanding and inhibiting Mpro dimerisation. They're currently planning a new series of experiments on B21, using another batch of promising inhibitors - including an anti-viral drug that targets HIV and one used to treat a feline coronavirus. Repurposing existing drugs that also have an effect on COVID-19 is much quicker than developing and testing new anti-viral compounds, although both approaches are needed as we move towards a future where COVID is endemic.
To find out more about the B21 beamline or discuss potential applications, please contact Principal Beamline Scientist Nathan Cowieson: nathan.cowieson@diamond.ac.uk.
Silvestrini L et al. The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors. Scientific reports 11.1 (2021): 1-16. DOI:10.1038/s41598-021-88630-9.
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