viruses

Treating COVID-19: Bond LSC lab explores effectiveness of remdesivir and other potential drugs

April 30, 2020 By Jerome Duggan in Research Tags: COVID-19, Kamal Singh, viruses

By Jerry Duggan | Bond LSC

As countries hang their hopes on the drug remdesivir for battling COVID-19, recent modeling and computer-aided drug evaluation at the University of Missouri caution to keep an open mind to other drug treatments.

Kamlendra Singh at MU’s Bond Life Sciences Center assessed remdesivir and several other drugs for long-term success in treating coronavirus causing the pandemic across the world. His results were published April 26 in the journal Pathogens.

“Remdesivir is working against COVID-19, but these other drugs are in no way inferior to it,” said Singh, the assistant director of the MU Molecular Interactions Core, Bond LSC investigator and MU associate research professor of Molecular Microbiology and Immunology in the School of Medicine. “They are all FDA approved and we believe, based on our research, that they are worth looking into as a form of treatment.

Working with colleagues at the Karolinska Institutet in Stockholm, Sweden, where he has an appointment, Singh and his team started their COVID-19 specific research in earnest on February 20. Through a combination of bioinformatics, molecular modeling and computer-aided drug design, they evaluated antiviral medicines remdesivir, favipiravir, 5-Fluorouracil and ribavirin for their effectiveness against COVID-19, and possible resistance under pressure of these compounds.

While Singh agrees that remdesivir works against COVID-19, he sees potential weaknesses of the drug and believes alternatives could supplement its use.

The team first assessed at the Gilead pharmaceutical remdesivir to inhibit SARS-CoV-2 viral polymerase, an antiviral drug originally used to treat Ebola. Remdesivir is one of the most highly touted drugs at this point in the fight against COVID-19. But Singh’s findings indicate SARS-CoV-2, — the causative agent of COVID-19 — is capable of mutating — that is, developing alterations in its genome — that could help it develop resistance to the drug.

Armed with this conclusion, Singh’s team looked at three additional compounds to treat the virus. With T-705, also known as favipiravir, they concluded COVID-19 could also develop resistance, but its past effectiveness as a broad-spectrum inhibitor of RNA viruses made it worth a look as a potential option to treat COVID-19. Favipiravir, which has been approved in Japan as an anti-influenza drug, was recently approved in Italy and China for treatment of COVID-19. Favipiravir is currently undergoing testing regarding its viability against COVID-19 and results are pending.

Additionally, Singh’s lab conducted research on two other broad-spectrum RNA polymerase inhibitors: 5-Fluorouracil and ribavirin. Singh examined 5-Fluorouracil because RNA polymerase (the enzyme that copies the virus genome) from SARS-CoV-2 is structurally similar to the RNA polymerases from human Rhinovirus and Foot-and-Mouth disease viruses and 5-FU shows effectiveness against these viruses. Due to this structural similarity, it stood to reason that 5-Fluorouracil may also have some effectiveness in treating COVID-19.

Ribavirin — one of the most widely used, broad spectrum inhibitors of RNA-viruses — shows unique properties that make it a prime candidate to bind to the active site of COVID-19. By binding to the active site, ribavirin theoretically could stop the replication of the viral genome and slow the spread of COVID-19 within an individual’s body. Still, doubt remains regarding its effectiveness since the virus can likely develop resistance to ribavirin through mutations or other mechanisms.

Singh has been working on coronaviruses — a broader category of viruses of which COVID-19 is just one specific manifestation — since his arrival at MU in 2009, so he is no stranger to this area of research. His broad goal is to ultimately have his research discoveries “make a difference in the lives of patients.”

He started his work on COVID-19 with the same goal in mind.

“I did not start my research on COVID-19 in order to write this paper and have it published,” he said. “I just wanted to do my part and spend some time researching compounds that could potentially be effective against COVID-19.”

As detailed as Singh’s findings are, he is quick to acknowledge that he is part of a global scientific community and had a team of professionals within his lab, especially Dr. Kyle Hill (a postdoctoral associate) that helped him to complete this work.

“This progress is not made by individuals, but by teams,” he said. “I have a talented group of individuals working with me and collaborating with me. I am well aware of their expertise and I know how to best utilize their skills.”

Ujjwal Neogi, who works at Karolinska Institutet, spearheaded the bioinformatics portion of the research. Ujjwal also helped in conceptualizing the idea of exploring already existing drugs to treat COVID-19. This work involved analyzing genetic code of the viruses, binding of the drugs with the target, and analyzing sequences of DNA, RNA, or protein to identify regions of similarity to find the most unchanging, widespread target for using a drug. This is important work since the smallest of differences in nucleotide sequence can result in mutations that would render these proposed COVID-19 treatments ineffective. Neogi was assisted with these tasks by Kyle Hill at the Bond LSC.

Anoop Ambikan (at Karolinska Institute) used R, a statistical computing and graphics program, to help put together the models and graphics behind this research. Xiao Heng, a member of the faculty at MU, used her expertise in biochemistry to help Singh conceptualize the research trajectory. Thomas Quinn, director of MU’s Molecular Interactions Core in the Bond LSC, helped with editing and advising throughout the process. Siddappa Byrareddy (University of Nebraska Medical Center, Omaha, Nebraska) brought previous experience with the topic of treating SARS, a form of coronavirus. Anders Sönnerborg, Singh’s longtime collaborator at Karolinska, provided funding for this work and is the one who takes the drugs to the clinic if they demonstrate potential. His involvement was important from the start of the process. Stefan Sarafianos, a former Bond LSC investigator and current collaborator had engaged with Singh in similar viral research in the past, and he imparted a lot of knowledge to Singh that enabled this research.

Taking a deeper look at potential drugs in an analytical way like this contributes to the University of Missouri System’s NextGen Precision Health Initiative. The NextGen Initiative aims to improve large-scale interdisciplinary collaboration in pursuit of life-changing precision health advancements and research.

While Singh concedes that none of his team’s work constitutes a guaranteed effective form of treatment against the virus, he feels that researchers have an obligation to continue working on a cure, and should not accept the status quo of hundreds of thousands of deaths globally. He also makes clear that for now this is all dry lab work. But plans are already in place to test these drugs in the wet lab for the validation once the appropriate protocols are established. Funding from the Karolinska Institute and the submission of two recent COVID related VA grant applications with Drs. Deutscher and Whaley-Connell will support compound validation and additional novel compound discovery and characterization.

“These treatments, if they turn out to be effective, all have limitations,” he admitted. “But, in the midst of a global pandemic, they are worth taking a deeper look at, because we have reason to believe, based on our research, that all of these drugs could potentially be effective in treating COVID-19.”

Kamlendra Singh is Assistant Director of the MU Molecular Interactions Core and an MU Associate Research Professor of Veterinary Pathology in the Collage of Veterinary Medicine (as of May 1, 2020).

Read more details about this research in “Feasibility of Known RNA Polymerase Inhibitors as Anti-SARS-CoV-2 Drugs,” published April 26 in the journal Pathogens.

In a viral game, the fight isn’t fair

October 30, 2017 By Samantha Kummerer in Research Tags: viruses

Lab explores how parvo wins in tug of war with cells

Kinjal Majumder and David Pintel examine the protein levels in mouse cells during MVM infection. Each black band represents the amount of viral protein in infected cells over time. | Photo by Samantha Kummerer, Bond LSC

By Samantha Kummerer | Bond LSC

At the start of any tug of war game, the battle is even. But it doesn’t stay that way for long. After a back and forth, the inevitable happens — the stronger team gives the rope one last tug and send the losers toppling over, claiming their dominance.

This is a game cells and viruses know well. In their version of tug of war, the virus eventually overtakes the cell and not only topples it but causes a consequence far worse than a few scraped knees.

This is how post-doctoral fellow Kinjal Majumder thinks of the interaction between parvoviruses and the dividing cells it conquers.

Majumder and others in the lab of David Pintel at Bond Life Sciences Center recently gained insight into how the virus achieves victory over the cell. These findings could improve human therapies and even play a role in treating cancer.

Meet the Champion

Parvoviruses are some of the smallest, simplest viruses. With only two genes, it has fewer DNA base pairs than most other viruses, up to ten times smaller in some cases. The virus’ size and simplicity, however, do not make it any easier to understand.

Pintel’s lab works at the “nitty gritty” level of the virus to study its basic molecular mechanism.

The group understands a little about how the virus operates but is working on the why.

Like a sly culprit, the virus uses its tiny nature to sneak inside the cell. The cell recognizes the presence of the virus as a foreign piece of DNA and responds to try to remove it. Once the cell responds, the virus begins replicating inside until it overtakes the cell. But, the tug of war game ends up more one-sided, so perhaps viewing the virus as a ruthless conqueror would be more accurate.

Latest Developments

Majumder’s experiments specifically focus on DNA damage response, an intrinsic function of cells. The DNA damage response constantly works to protect us from cancer by continuously repairing broken DNA to prevent harmful mutations in cells . This response uses a network of cellular pathways to monitor and provide checkpoints in the cell cycle to prevent damage from being passed on to the next generation of cells. But parvoviruses also tricks cells to begin a DNA damage response, which they use to eventually take over the host cell.

In July, the lab published the latest finding from a series of papers exploring a type of these parvoviruses called minute virus of mice (MVM). The discovery began when the team found that the virus stops cells from dividing within infected cells.

To do this, MVM uses the DNA damage response to stop cells from dividing, but still allows virus replication to continue. The group developed a system to examine how the virus took over the cell. Further experiments revealed the virus transcriptionally regulates cell cycle genes.

The team used CRISPR to target a cellular gene that the virus must inactivate for it to replicate. Expression of this gene is required for the cell to divide. They discovered the virus actually blocks the transcription of this gene so it cannot make its protein. Blocking this function also prevents the cell from dividing. Without cell division, the virus is free to rapidly replicate inside the cell.

Post-doctoral fellow, Kinjal Majumder, points to the results of a recent experiment. When MVM is mutated at particular sites, the protein levels produced by the virus decrease, but the virus continues to replicate efficiently. | Photo by Samantha Kummerer. Bond LSC

Majumder said parvo’s manipulation of infected cell cycles is different from other viruses because it can only replicate in cells that are actively synthesizing DNA. It eventually halts the process of cell division in infected cells by dysregulating transcription factors that regulate cell cycle gene expression. That’s what makes this discovery unique.

Endless Possibilities

This discovery only scratches the surface.

Majumder said the lab constantly thinks of new experiments to explore parvovirus biology. The simplicity of its DNA expedites the process of culturing and growing the virus, leaving more time for what Majumder calls the “fun experiments.”

“We try to be thorough and confident of our findings, so we attack experiments from many different angles,” Majumder said.

Imagine viewing an object from multiple angles under varying light conditions. The change in perspective reveals something different about it with each new look. This approach expands their understanding of parvoviruses.

Majumder explained the lab makes use of everything from high-resolution imaging and CRISPR technology to proteomics and deep sequencing to study the tug-of-war between MVM and the DNA damage response.

“The thing about being a postdoc is you kind of have to be a jack of all trades,” Majumder said about his ability to conduct the range of experiments.

Wider Implications

While parvoviruses are not a deadly threat to humans, understanding it has major implications for humans.

Parvoviruses are used to develop gene therapy tools to treat disorders like muscular dystrophy and spinal muscular atrophy. The Pintel lab collaborates with labs such as the Lorson and Sarafianos lab in Bond LSC, to explore its therapeutic potential.

Majumder explained the lab is also interested in understanding how the virus could improve cancer treatment. Parvo’s tendency to replicate in dividing cells links it to how cells divide uncontrollably in cancer. Majumder explained those scientists are trying to use that function of the virus to target cancer cells.

This makes work in the Pintel lab that much more important. But, before the virus can fully improve humans’ conditions, researchers better grasp its capabilities. And that means more of the daily experiments within the Pintel lab.

“What we learn from studying a simple virus can be expanded to more complicated viruses because there are some viruses that can make a dozen different proteins, so that’s a more complicated system,” Majumder said. “Before you can get to the more complicated systems, you need to be able to understand the one that makes just a few proteins.”

For now, scientists will play their own tug of war as they go back and forth in their findings and experiments to uncover the mystery surrounding this small, unique virus.

Holding on: Bond LSC scientist discovers protein prevents release of HIV and other viruses from infected cells

August 26, 2014 By Roger Meissen in Research Tags: Bond Life Sciences Center, Bond LSC, HIV, host cell, Minghua Li, MU School of Medicine, proteins, Shan-Lu Liu, viruses

Shan-Lu Liu and Minghua Li, HIV Research at the Bond Life Sciences Center — Shan-Lu Liu, Bond LSC scientist and associate professor in the MU School of Medicine’s Department of Molecular Microbiology and Immunology. Courtesy Justin Kelley, University of Missouri Health System.

Shan-Lu Liu initially thought it was a mistake when a simple experiment kept failing.

But that serendipitous accident led the Bond Life Sciences Center researcher to discover how a protein prevents mature HIV from leaving a cell.

Proceedings of the National Academy of Sciences published this research online Aug. 18.

“It’s a striking phenomena caused by this particular protein,” Liu said. “The HIV is already assembled inside the cell, ready for release, but this protein surprisingly tethers this virus from being released.”

The TIM – T-cell/transmembrane immunoglobulin and mucin – family of proteins hasn’t received much attention from HIV researchers, but recent research shows the protein family plays a critical role in viral infections. From Ebola and Dengue to Hepatitis A and HIV, these proteins aid in the entry of viruses into host cells.

But its ability to stop the virus from leaving cells remained unknown until now. Liu’s lab stumbled onto this finding in November 2011 when trying to create stable cells for a different experiment. After two months of troubleshooting the HIV lentiviral vector – where genes responsible for creating TIM-1 proteins were inserted into a cell to create a stable cell line that expresses the protein – Liu was confident the vector’s failure was not only interesting but also important.

The lab spent the next two years trying to figure out what was happening. Minghua Li, an MU Area of Pathobiology graduate student, carried out experiments that confirmed the protein’s power to inhibit HIV-1 release from cells, reducing normal viral infection. His experiments showed TIM proteins prevent normal deployment of HIV, created by an infected cell, into the body to propagate.

TIM proteins stand erect like topiary on the outside and inside surfaces of T-cells, epithelial cells and other cells. When a virus initially approaches a cell, the top of each TIM protein binds with fats – called phosphatidylserine (PS) – covering the virus surface. This allows a virus, such as Ebola virus and Dengue virus, to enter the cell, infect and replicate, building up a population inside.

But as the virus creates new copies of itself, the host cell’s machinery also incorporates TIM proteins into new viruses. That causes problems for HIV as it tries to leave the cell. Now these proteins cause the viruses to bind to each other, clumping together and attaching to the cell surface.

“We see this striking phenotype where the virus just accumulates on the cell surface,” said Liu, who is also an associate professor in the MU School of Medicine’s Department of Molecular Microbiology and Immunology. “We consider this an intrinsic property of cellular response to viral infection that holds the virus from release.”

This model shows the interaction between TIMs and PS among the round HIV virions, as well as that between viral producer cells. This collectively leads to accumulation of HIV virions on the plasma membrane on the outside of the cell. Courtesy Mingua Li. — This model shows the interaction between TIMs and PS among the round HIV virions, as well as that between viral producer cells. This collectively leads to accumulation of HIV virions on the plasma membrane on the outside of the cell. Courtesy Minghua Li.

Further research is needed to determine overall benefit or detriment of this curious characteristic, but this discovery provides insight into the cell-virus interaction.

“This study shows that TIM proteins keep viral particles from being released by the infected cell and instead keep them tethered to the cell surface,” said Gordon Freeman, Ph.D., an associate professor of medicine with Harvard Medical School’s Dana-Farber Cancer Institute, who was not affiliated with the study. “This is true for several important enveloped viruses including HIV and Ebola. We may be able to use this insight to slow the production of these viruses.”

The National Institutes of Health and the University of Missouri partially supported this research. Additional collaborators include Eric Freed, PhD, senior investigator with the National Cancer Institute (NCI) HIV Drug Resistance Program; Sherimay Ablan, biologist with the NCI HIV Drug Resistance Program; Marc Johnson, PhD, Bond LSC researcher and associate professor in the MU Department of Molecular Microbiology and Immunology; Chunhui Miao and Matthew Fuller, graduate students in the MU Department of Molecular Microbiology and Immunology; Yi-Min Zheng, MD, MS, senior research specialist with the Christopher S. Bond Life Sciences Center at MU; Paul Rennert, PhD, founder and principal of SugarCone Biotech LLC in Holliston, Massachusetts; and Wendy Maury, PhD, professor of microbiology at the University of Iowa.

Read the full study on the PNAS website and browse the supplementary data for this work. See more news on this research from the MU School of Medicine.

Viruses as Vehicles: Finding what drives

August 4, 2014 By Paige Blankenbuehler in Research Tags: AIDS, Bond Life Sciences Center, decoding science, gene therapy, HIV, Johnson Lab MU, Marc Johnson, research, science, University of Missouri, viruses

Graduate students Yuleam Song and Dan Salamango inoculate a bacteria culture in Johnson's lab. The inoculation takes a small portion of a virus and multiplies the sample, allowing researchers to custom-make viruses. — Graduate students Yuleam Song and Dan Salamango inoculate a bacteria culture in Johnson’s lab. The inoculation takes a small portion of a virus and multiplies the sample, allowing researchers to custom-make viruses.

By Madison Knapp | Bond Life Sciences Center summer intern

Modern science has found a way to turn viruses —tiny, dangerous weapons responsible for runny noses, crippling stomach pains and worldwide epidemics such as AIDS— into a tool.

Gene therapy centers on the idea that scientists can hijack viruses and use them as vehicles to deliver DNA to organs in the body that are missing important genes, but the understanding of virus behavior is far from exhaustive.

Marc Johnson, researcher at the Christopher S. Bond Life Sciences Center and associate professor of molecular microbiology and immunology in the MU School of Medicine, has been building an understanding of viral navigation mechanisms which allow a virus to recognize the kind of cell it can infect.

Johnson’s research specifically explores the intricacies of the viral navigation system and could improve future direction of gene therapy, he said.

Marc Johnson (left) with a post doctoral student that works in his lab. The lab does important research on the basic function and mechanisms of viral navigation and transport. — Marc Johnson (left) with Dan Salamango, a graduate student that works in his lab. The lab does important research on the basic function and mechanisms of viral navigation and transport.

Turning a virus into a tool

Conceptualized in the 1970s, gene therapy was developed to treat patients for a variety of diseases, including Parkinson’s, leukemia and hemophilia (a genetic condition that stops blood from clotting).

To treat disease using gene therapy, a customized virus is prepared. A virus can be thought of as a missile with a navigation system and two other basic subunits: A capsule that holds the ammunition and the ammunition itself.

The viral genetic material can be thought of as the missile’s ammunition. When a cell is infected, this genetic material is deployed and incorporated into the cell’s DNA. The host cell then becomes a factory producing parts of the virus. Those parts assemble inside the cell to make a new virus, which then leaves the cell to infect another.

The capsule is made of structural protein that contains the genetic material, and the navigation system is a protein that allows the virus to recognize the kind of cell it can infect.

Viral navigation

Gene therapy uses viruses to solve many problems by utilizing a virus’ ability to integrate itself into a host cell’s DNA; to do this successfully, researchers need to provide a compatible navigation component.

In the body, viruses speed around as if on a busy highway. Each virus has a navigation system telling it which cells to infect. But sometimes if a virus picks up the wrong type of navigation system, it doesn’t know where to go at all.

“What you can do is find a virus that infects the liver already, steal its navigation protein and use that to assemble the virus you want to deliver the gene the liver needs,” Johnson said. “You can basically take the guidance system off of one and stick it onto another to custom design your virus.”

But this doesn’t always work because of incompatibility among certain viruses, he said.

Johnson and his lab are working to understand what makes switching out navigation proteins possible and why some viruses’ navigation systems are incompatible with other viruses.

“I’m trying to understand what makes it compatible so that hopefully down the road we can intelligently make others compatible,” Johnson said.

The right map, the right destination

Johnson creates custom viruses by introducing the three viral components—structural protein, genetic material, and navigation protein—to a cell culture. The structural protein and genetic material match, but the navigation component is the wild card. It could either take to the other parts to produce an infectious virus, or it could be incompatible.

Johnson uses a special fluorescent microscope to identify which viruses assembled correctly and which didn’t.

A successful pairing is like making a match. If a navigation protein is programmed to target liver cells, it’s considered a successful pairing when the virus arrives at the liver cell target location.

The scope of gene therapy continues to widen. Improved mechanisms for gene therapy, and greater knowledge of how a navigation protein drives a virus could help more people benefit from the vehicles viruses can become.

Johnson uses several high-profile model retroviruses, including human immunodeficiency virus (HIV), which affects an estimated 35 million people worldwide each year, according to the World Health Organization.

Understanding nuances of HIV in comparison to other viruses allows Johnson to pick out which behaviors might be common to all retroviruses and others behaviors that might be specific to each virus.

Johnson said his more general approach makes it easier to understand more complex viral features.

“If there are multiple mechanisms at work, it gets a little trickier,” Johnson said. “My angle is more generic, which makes it easier to tease them apart.”

Supervising editor is Paige Blankenbuehler