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cryo-electron microscopy

Pursuing Safe and Effective Anti-Viral Drugs for COVID-19

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Senior hospital patient on a ventilator
Stock photo/SoumenNath

Right now, the world is utterly focused on the coronavirus outbreak known as COVID-19. That’s certainly true for those of us at NIH. Though I am working from home to adhere rigorously to physical distancing, I can’t remember ever working harder, trying to do everything I can to assist in the development of safe and effective treatments and vaccines.

Over the past several weeks, a mind-boggling array of possible therapies have been considered. None have yet been proven to be effective in rigorously controlled trials, but for one of them, it’s been a busy week. So let’s focus on an experimental anti-viral drug, called remdesivir, that was originally developed for the deadly Ebola virus. Though remdesivir failed to help people with Ebola virus disease, encouraging results from studies of coronavirus-infected animals have prompted the launch of human clinical trials to see if this drug might fight SARS-CoV-2, the novel coronavirus that causes COVID-19.

You may wonder how a drug could possibly work for Ebola and SARS-CoV-2, since they are very different viruses that produce dramatically different symptoms in humans. The commonality is that both viruses have genomes made of ribonucleic acid (RNA), which must be copied by an enzyme called RNA-dependent RNA polymerase for the virus to replicate.

Remdesivir has an affinity for attaching to this kind of polymerase because its structure is very similar to one of the RNA letters that make up the viral genome [1]. Due to this similarity, when an RNA virus attempts to replicate, its polymerase is tricked into incorporating remdesivir into its genome as a foreign nucleotide, or anomalous letter. That undecipherable, extra letter brings the replication process to a crashing halt—and, without the ability to replicate, viruses can’t infect human cells.

Would this work on a SARS-CoV-2 infection in a living organism? An important step was just posted as a preprint yesterday—a small study showed infusion of remdesivir was effective in limiting the severity of lung disease in rhesus macaques [2]. That’s encouraging news. But the only sure way to find out if remdesivir will actually help humans who are infected with SARS-CoV-2 is to conduct a randomized, controlled clinical trial.

In late February, NIH’s National Institute of Allergy and Infectious Diseases (NIAID) did just that, when it launched a randomized, controlled clinical trial to test remdesivir in people with COVID-19. The study, led by NIAID’s Division of Microbiology and Infectious Diseases, has already enrolled 805 patients at 67 testing sites. Most sites are in the United States, but there are also some in Singapore, Japan, South Korea, Mexico, Spain, the United Kingdom, Denmark, Greece, and Germany.

All trial participants must have laboratory-confirmed COVID-19 infections and evidence of lung involvement, such as abnormal chest X-rays, rattling sounds when breathing (rales) with a need for supplemental oxygen, or a need for mechanical ventilation. They are randomly assigned to receive either a round of treatment with remdesivir or a harmless placebo with no therapeutic effect. To avoid bias from creeping into patient care, the study is double-blind, meaning neither the medical staff nor the participants know who is receiving remdesivir.

There is also an early hint from another publication that remdesivir may benefit some people with COVID-19. Since the end of January 2020, Gilead Sciences, Foster City, CA, which makes remdesivir, has provided daily, intravenous infusions of the drug on a compassionate basis to more than 1,800 people hospitalized with advanced COVID-19 around the world. In a study of a subgroup of 53 compassionate-use patients with advanced complications of COVID-19, nearly two-thirds improved when given remdesivir for up to 10 days [3]. Most of the participants were men over age 60 with preexisting conditions that included hypertension, diabetes, high cholesterol, and asthma.

This may sound exciting, but these preliminary results, published in the New England Journal of Medicine, come with major caveats. There were no controls, participants were not randomized, and the study lacked other key features of the more rigorously designed NIH clinical trial. We can all look forward to the results from the NIH trial, which are are expected within a matter of weeks. Hopefully these will provide much-needed scientific evidence on remdesivir’s safety and efficacy in people with COVID-19.

In the meantime, basic researchers continue to learn more about remdesivir and its interaction with the novel coronavirus that causes COVID-19. In a recent study in the journal Science, a research team, led by Quan Wang, Shanghai Tech University, China, mapped the 3D atomic structure of the novel coronavirus’s polymerase while it was complexed with two other vital parts of the viral replication machinery [4]. This was accomplished using a high-resolution imaging approach called cryo-electron microscopy (cryo-EM), which involves flash-freezing molecules in liquid nitrogen and bombarding them with electrons to capture their images with a special camera.

With these atomic structures in hand, the researchers then modeled exactly how remdesivir binds to the polymerase of the novel coronavirus. The model will help inform future efforts to tweak the structure of the drug and optimize its ability to disrupt viral replication. Such detailed biochemical information will be vital in the weeks ahead, especially if data generated by the NIH clinical trial indicate that remdesivir is a worthwhile lead to pursue in our ongoing search for anti-viral drugs to combat the global COVID-19 pandemic.

References:

[1] Nucleoside analogues for the treatment of coronavirus infections. Pruijssers AJ, Denison MR. Curr Opin Virol. 2019 Apr;35:57-62.

[2] Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2. Williamson B, Feldmann F, Schwarz B, Scott D, Munster V, de Wit E et. al. BioRxiv. Preprint posted 15 April 2020.

[3] Compassionate use of remdesivir for patients with severe Covid-19. Grein J, Ohmagari N, Shin D, Brainard DM, Childs R, Flanigan T. et. al. N Engl J Med. 2020 Apr 10. [Epub ahead of publication]

[4] Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Gao Y, Yan L, Liu F, Wang Q, Lou Z, Rao A, et al. Science. 10 April 2020. [Epub ahead of publication]

Links:

Coronavirus (COVID-19) (NIH)

Accelerating COVID-19 Therapeutic Interventions and Vaccines (NIH)

NIH Clinical Trial of Remdesivir to Treat COVID-19 Begins (National Institute of Allergy and Infectious Diseases/NIH)

Developing Therapeutics and Vaccines for Coronaviruses (NIAID)

COVID-19, MERS & SARS (NIAID)

NIH Support: National Institute of Allergy and Infectious Diseases


Electricity-Conducting Bacteria May Inspire Next-Gen Medical Devices

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Nanowires
Credit: Edward H. Egelman

Technological advances with potential for improving human health sometimes come from the most unexpected places. An intriguing example is an electricity-conducting biological nanowire that holds promise for powering miniaturized pacemakers and other implantable electronic devices.

The nanowires come from a bacterium called Geobacter sulfurreducens, shown in the electron micrograph above. This rod-shaped microbe (white) was discovered two decades ago in soil collected from an unlikely place: a ditch outside of Norman, Oklahoma. The bug can conduct electricity along its arm-like appendages, and, in the hydrocarbon-contaminated, oxygen-depleted soil in which it lives, such electrical inputs and outputs are essentially the equivalent of breathing.

Scientists fascinated with G. sulfurreducens thought that its electricity had to be flowing through well-studied microbial appendages called pili. But, as the atomic structure of these nanowires (multi-colors, foreground) now reveals, these nanowires aren’t pili at all! Instead, the bacteria have manufactured unique submicroscopic arm-like structures. These arms consist of long, repetitive chains of a unique protein, each surrounding a core of iron-containing molecules.

The surprising discovery, published in the journal Cell, was made by an NIH-funded team involving Edward Egelman, University of Virginia Health System, Charlottesville. Egelman’s lab has had a long interest in what’s called a type 4 pili. These strong, adhering appendages help certain infectious bacteria enter tissues and make people sick. In fact, they enable bugs like Neisseria meningitidis to cross the blood-brain barrier and cause potentially deadly bacterial meningitis. While other researchers had proposed that those same type 4 pili allowed G. sulfurreducens to conduct electricity, Egelman wasn’t so sure.

So, he took advantage of recent advances in cryo-electron microscopy, which involves flash-freezing molecules at extremely low temperatures before bombarding them with electrons to capture their images with a special camera. The cryo-EM images allowed his team to nail down the atomic structure of the nanowires, now called OmcS filaments.

Using those images and sophisticated bioinformatics, Egelman and team determined that OmcS proteins uniquely fit into the nanowires’ long repetitive chains, spacing their iron-bearing cores at regular intervals to transfer electrons and convey electricity. In fact, bacteria unable to produce OmcS proteins make filaments that conduct electricity 100 times less efficiently.

With these cryo-EM structures in hand, Egelman says his team will continue to explore their conductive properties. Such knowledge might someday be used to build biologically-inspired nanowires, measuring 1/100,000th the width of a human hair, to connect miniature electronic devices directly to living tissues. This is one more example of how nature’s ability to invent is pretty breathtaking—surely one wouldn’t have predicted the discovery of nanowires in a bacterium that lives in contaminated ditches.

Reference:

[1] Structure of Microbial Nanowires Reveals Stacked Hemes that Transport Electrons over Micrometers. Wang F, Gu Y, O’Brien JP, Yi SM, Yalcin SE, Srikanth V, Shen C, Vu D, Ing NL, Hochbaum AI, Egelman EH, Malvankar NS. Cell. 2019 Apr 4;177(2):361-369.

Links:

Electroactive microorganisms in bioelectrochemical systems. Logan BE, Rossi R, Ragab A, Saikaly PE. Nat Rev Microbiol. 2019 May;17(5):307-319.

High Resolution Electron Microscopy (National Cancer Institute/NIH)

Egelman Lab (University of Virginia, Charlottesville)

NIH Support: National Institute of General Medical Sciences; National Institute of Allergy and Infectious Diseases; Common Fund


MicroED: From Powder to Structure in a Half-Hour

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MicroED determines structure in 30 min

Credit: Adapted from Jones et al. ChemRxiv.org

Over the past few years, there’s been a great deal of excitement about the power of cryo-electron microscopy (cryo-EM) for mapping the structures of large biological molecules like proteins and nucleic acids. Now comes word of another absolutely incredible use of cryo-EM: determining with great ease and exquisite precision the structure of the smaller organic chemical compounds, or “small molecules,” that play such key roles in biological exploration and drug development.

The new advance involves a cryo-EM technique called microcrystal-electron diffraction (MicroED). As detailed in a preprint on ChemRxiv.org [1] and the journal Angewandte Chemie [2], MicroED has enabled researchers to take the powdered form of commercially available small molecules and generate high-resolution data on their chemical structures in less than a half-hour—dramatically faster than with traditional methods!


A Lean, Mean DNA Packaging Machine

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Three views of bacteriophage T4

Credit: Victor Padilla-Sanchez, The Catholic University of America, Washington, D.C.

All plants and animals are susceptible to viral infections. But did you know that’s also true for bacteria? They get nailed by viruses called bacteriophages, and there are thousands of them in nature including this one that resembles a lunar lander: bacteriophage T4 (left panel). It’s a popular model organism that researchers have studied for nearly a century, helping them over the years to learn more about biochemistry, genetics, and molecular biology [1].

The bacteriophage T4 infects the bacterium Escherichia coli, which normally inhabits the gastrointestinal tract of humans. T4’s invasion starts by touching down on the bacterial cell wall and injecting viral DNA through its tube-like tail (purple) into the cell. A DNA “packaging machine” (middle and right panels) between the bacteriophage’s “head” and “tail” (green, yellow, blue spikes) keeps the double-stranded DNA (middle panel, red) at the ready. All the vivid colors you see in the images help to distinguish between the various proteins or protein subunits that make up the intricate structure of the bacteriophage and its DNA packaging machine.


What a Year It Was! A Look Back at Research Progress in 2017

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I want to wish everyone a Happy New Year! Hope your 2018 is off to a great start.

Over the holidays, the journal Science published its annual, end-of-the-year list of research breakthroughs, from anthropology to zoology. I always look forward to seeing the list and reflecting on some of the stunning advances reported in the past 12 months. Last year was no exception. Science’s 2017 Breakthrough of the Year, as chosen by its editors, was in the field of astrophysics. Scientists were able to witness the effects of the collision of two neutron stars—large stars with collapsed inner cores—smacking into each other 130 million light years away. How cool is that!

Numbered prominently among the nine other breakthroughs were five from biomedicine: gene therapy, gene editing, cancer immunotherapy, cryo-EM, and biology preprints. All involved varying degrees of NIH support, and all drew great interest from readers. In fact, three of the top four vote-getters in the “People’s Choice” category came from biomedicine. That includes the People’s 2017 Breakthrough of the Year: gene therapy success. And so, in what has become a Director’s Blog tradition, I’ll kick off our new year of posts by taking a closer look at these biomedical breakthroughs—starting with the little girl in the collage above, and moving clockwise around the images:


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