Scientists at the National High Magnetic Field Laboratory (Maglab) (CSA CSM) have detailed the mechanism that activates influenza and permits it to reproduce in human cells. The research, published in Structure, shows for the first time how protons move through the virus’s M2 proton channel and portends the development of new drugs to treat the flu. “We were actually able to see the protons jumping from one site to another site within the molecular structure,” says Tim Cross, director of the Maglab’s nuclear magnetic resonance(NMR) facility. “And we had never anticipated being able to get to such detailed information about this channel.”
In 2009, the flu virus mutated after the swine flu epidemic, leaving ineffective numerous drugs used to combat it. “We actually have fewer drugs now than we did a decade ago to treat influenza,” says Cross. The mechanism within the M2 proton channel, however, remained unchanged, meaning new drugs developed to target it will remain effective against all strains of influenza. “We really don’t know when a deadly flu epidemic is going to hit next. We need backup to vaccines.”
Cross’s team used the MagLab’s 900 MHz nuclear magnetic resonance ultrawide bore magnet to get a clear image of the channel. The 900 was originally conceived shortly after the lab’s founding in 1990. At the time, a 900 MHz magnet had not been demonstrated and 750 MHz was state of the art. “In the beginning, I took out my pencil and did some numbers to see not only what a 900 would look like in a standard bore configuration but how large a magnet might eventually be required,” says Denis Markiewicz, senior magnet designer at the Maglab. His team ultimately decided to develop a 20 T (tesla) large outer magnet capable of reaching 25 or even 30 T when paired with HTS inner coils. It was an ambitious objective, according to Markiewicz. “I’m not sure that I can say that everyone appreciated the implications of what was being suggested,” he says. “Perhaps the enthusiasm extended beyond the rational constraints that are normally applied.”
The magnet’s bore diameter is 105 mm, a size that provides space for not only additional coils, gradients and probes but also samples weighing up to 350 g, the size of an adult rat. It operates at 1.7K, kept cool by a cryostat containing 2,400 liters of liquid helium. “This has been an incredible test bed for developing NMR probe technology,” says Cross. “Here at the lab we’ve now produced something like 57 different probes that represent the highest sensitivity, the highest resolution, the highest performance probes in the world.”
Probe development is just one example of how Maglab engineers keep the 900 in state-of-the-art condition. Next up for the magnet is a plan to enhance imaging capabilities by replacing its gradient coils with stronger versions. Researchers also hope new HTS materials will increase the 900’s functionality and continue to attract new users to the Maglab.
Since coming online in 2004, researchers using the 900 have published nearly 70 papers according to Cross, including several NMR structures of disease. Vladimir Ladizhansky from the University of Guelph, for example, used the 900 to examine sensory rhodopsins, while Tatyana Polenova from the University of Delaware used it to structure the viral capsid of HIV. Both scientists used the 900 from a distance as part of a remote access program at the Maglab. “This is something that we are very excited about because it will allow a large number of users from around the country to utilize the magnet without having to pay for travel costs,” says Cross.
Last year the Maglab hosted 249 users from 75 institutions. About one-third of those used the 900, according to Cross. “I have to get in line to get access to these magnets,” he says. “To get an NMR spectrum of a large protein requires several days of signal averaging. So time on the instruments is in great demand.”
It took about six months of spectrometer time for Cross’s team to image the M2 proton channel. Initial imaging was completed on lower field and less expensive magnets at the lab, but the 900 was used exclusively for the final published spectra. “We go to the 900 for sensitivity,” Cross says. “We also go there because as you go up in field strength the different proton signals [and] the different carbon signals get spread out a little bit more in the spectral space, the frequency space. And so you get a little bit better spectral resolution.”
Cross’s influenza research is now close to completion. He says there are some plans to investigate structural changes caused by the 2009 mutation, but that research into mycobacterium tuberculosis is now his focus. “What’s really novel about tuberculosis is that when you challenge the bug with the current drugs that are available it goes into a hibernating state.”
Current treatments require at least six months to kill the bacteria. Cross hopes to dramatically reduce treatment time by identifying the mechanism that facilitates the hibernation. “We have our first structure out in this project,” he says, “but we don’t know how it functions yet.”