Kamlendra Singh

Competing with COVID: Researcher suggests varying from vaccines to fight virus

March 16, 2022 By Penquite, Cara (MU-Student) in Research Tags: #MizzouResearch, COVID-19, virology

CC_COVID — COVID-19 virus particles have spike proteins, represented in red, that attach to receptors on host cells. Antivirals block the receptors on host cells so the virus cannot infect more cells. | Creative Commons Photo

By Cara Penquite | Bond LSC

Vaccines were the light at the end of the tunnel throughout the COVID-19 pandemic, but virus mutations threaten to extinguish hope of a quick end to the pandemic. Kamlendra Singh turns towards antivirals as the next step.

“There will be a time we will find an antiviral which will be very difficult for the virus to mutate [and avoid],” Singh said, “That’s what we are after.”

The Singh lab studied COVID-19 antiviral compounds that prevent binding between the virus and host cells with help from Siddappa Byrareddy, professor and vice-chair of research in the Department of Pharmacology Experimental Neuroscience at the University of Nebraska Medical Center (UNMC). The first COVID-19 antiviral compound Singh’s team discovered during their in a preliminary study conducted in mouse models, has been filed for a patent while they continue to search for the antivirals that target different proteins of the virus. These compounds would prevent the virus from entering cells even after exposure to the virus.

The antiviral compound disrupts the interaction between the ACE2 receptor on the surface of host cells and the spike protein on the virus so the virus cannot infect cells. ACE2 acts as a doorway into the cell where COVID-19 binds, enters and takes over the cell. Once inside, the virus hijacks the cell and uses it to create more virus. Additionally, the virus releases its genome into the host cells, activating cell defense mechanisms which can be more dangerous than the infection itself.

While vaccines prepare the immune system to fight off COVID-19, Singh’s antivirals simply block this doorway.

“The very first thing was to find the compound that can inhibit viral entry because it is the first step of the infection,” Singh said. “If you can block the very first step then you can block everything else.”

Vaccines act as a practice round for the body where the immune response learns which antibodies are effective against the virus. But when the virus mutates, antibodies built up from vaccination or prior infection may be less effective against the mutated virus.

The antiviral compounds discovered by Singh and his team do not have this problem since they bind to the host cell’s receptors where few mutations occur. Additionally, the compounds can change their shape in response to those mutations that may occur.

“We call it ‘wiggling and jiggling,’” Singh said. “The compound has the capability to change it’s shape conformation.”

This ability to change shape is not unique to COVID-19 antivirals. Drawing on previous experience working on HIV antivirals, Singh explains that shape changing properties are common in molecules with single bonds between their component atoms.

“If you have a single bond somewhere, then they can change or they can reorient and bind someplace,” Singh said.

Singh believes antivirals may be the key to fighting COVID-19 even with emerging mutants. While vaccines mitigated COVID-19 rates and hospitalizations, they also might have played a role in the creation of new mutants.

“It’s probably too early to say, but it looks like these variants are probably evolving under the pressure of antibodies,” Singh said. “Those antibodies may have been induced by either direct infusions giving the antibodies [to patients], or they may have been induced by other vaccines or by previous infection.”

While viruses also mutate under the pressure of antivirals, Singh hopes to find an antiviral that is difficult for the virus to avoid through mutation.

“We have been working on developing a better compound using the compounds we discovered, and we have found one more compound that has at least 10 times better efficacy against [SARS-Cov-2],” Singh said.

Kamlendra Singh is a research assistant professor of molecular microbiology and immunology and assistant director of the Molecular Interactions Core at Bond LSC. Singh wished to express his sincere gratitude to Prof. Byrareddy (University of Nebraska Medical School). Without their collaboration, the discovery of the antiviral compounds would not be possible. Singh is also thankful to two students – Saathvik Kannan (a Hickman High School student) and Austin (a Mizzou undergrad). Without the help of these two talented young scientists, the research would not have been so successful. Finally, Singh mentioned that the support from the Bond Life Sciences Center has been extremely valuable in his research.

Outlining Omicron: researchers determine key mutations in the latest COVID-19 variant

February 2, 2022 By Penquite, Cara (MU-Student) in Research Tags: computer science, COVID-19, genetics

Bond LSC and UNMC scientists explain mutations unique to the Omicron variant

Austin Spratt Protein Models — Austin Spratt, undergraduate mathematics student in the Kamlendra Singh lab, shows protein models of the Omicron spike protein and the receptor it attaches to when infecting cells. “The genetic codes are used to identify the mutations, and then we use the structure to see how it would change over time. It’s going to give us more information about new mutations that occur,” Spratt said. | Photo by Cara Penquite, Bond LSC

By Cara Penquite | Bond LSC

It took eight days for Kamlendra Singh and his team to crack the code behind the Omicron COVID-19 variant. Although usually characterized by months of waiting, the peer-review process matched their pace, publishing their research a week later.

“That’s how quick it went because the editors and the general [scientific community] recognize that it’s very timely and important research,” said the Bond Life Sciences Center principal investigator.

Discovering 46 genetic mutations in the Omicron variant, their research explains Omicron’s ability to efficiently infect cells and evade antibodies. Their explanation predicted Omicron’s possibility for increased transmissibility a couple weeks before the beginning of the late December surge of COVID-19 cases in the U.S. The work published online December 10, 2021, in the Journal of Autoimmunity.

“You will see a lot of infections now because the vaccine-induced immune responses are not able to control the [Omicron] infection,” said Siddappa Byrareddy, a collaborator on the project and Professor and Vice-Chair of Research in the Department of Pharmacology Experimental Neuroscience at the University of Nebraska Medical Center (UNMC).

Genetic mutations to the spike protein on the surface of the virus increase Omicron’s ability to infect cells and evade antibodies. In Omicron, the mutations allow for a stronger bond between the spike protein and the healthy cell. The spike protein acts as a key enable the virus to get inside the cell by attaching to receptors on the surface of healthy cells. Once inside, the virus hijacks the cell and uses it to create more copies of the virus.

“What happens is, these mutations are in the receptor-binding domain. Which means, when these two things come together, when the spike protein tries to attach to the ACE2 receptor, these mutations cause it to bind more. When that happens, we have increased transmissibility or increased infectivity,” said Saathvik Kannan, a Hickman High School sophomore, and lead author from the Singh lab involved in identifying the mutations.

The spike protein is also the primary point of contact for antibodies designed to neutralize the virus. Since antibodies are not universal, the immune system must be exposed to a virus, either through infection or vaccination, before creating antibodies to neutralize that virus. Omicron’s mutations in the spike protein make it different from other variants of COVID-19, so antibodies that can neutralize other variants seem to be less effective in preventing Omicron infection.

“Antibodies are very specific. Any one mutation can completely abolish the binding,” Singh said.

While antibodies from previous infection or vaccination may not prevent Omicron transmission, Byrareddy emphasized that they are still decreasing hospitalization.

“Even though the vaccines are not effective [against Omicron] transmission, they still help the immune response to fight against the virus. That’s why we don’t [see] a lot of hospitalization,” Byrareddy said.

One potential solution to mitigate Omicron’s effect would be to create an Omicron-specific vaccine. While a new vaccine could decrease Omicron transmission, Singh warns that it would not necessarily prevent the emergence of further variants. This is a particular concern with Omicron because of its many versions.

“We’re seeing different Omicron versions where you have the Omicron, but with a couple changes. Those couple changes are making a big impact.” said Austin Spratt, another Singh lab researcher and MU undergraduate studying mathematics involved in the project.

Spratt’s role in the project was to create computer simulations of the spike proteins to see models of the mutations.

“[Omicron] has bigger mutations and different flavor mutations [than previous variants], and there are starting to be subcategories of that,” Spratt said.

Singh and Byrareddy’s labs are taking their research in the direction of antivirals with the hope of creating a drug that could reduce COVID-19 symptoms. The antiviral would block receptors on the surface of healthy cells so that the virus’ spike protein will not fit anymore. Without a key to get into the cells, the virus would not take over the cells.

While antivirals would not cut out all variants, Spratt believes they could help the problem as they did with other pandemics such as the Spanish Flu.

“I think it’s possible that they create antivirals that will slowly but surely cut out variants over time,” Spratt said.

This research published in the Journal of Autoimmunity December 10, 2021, under the title “Omicron SARS-CoV-2 variant: Unique features and their impact on pre-existing antibodies.”

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.

#IAmScience Gilberto Perez

August 31, 2018 By Erica Overfelt in #IAmScience Tags: virology

By Erica Overfelt | Bond LSC

Research is like a puzzle, and figuring out how the pieces fit together is Gilberto Perez’s favorite part of research.

“In research you have to think about the steps,” Perez said. “Most likely, I’m not going to complete the puzzle in my lifetime, but it will help someone else put a piece in it and hopefully, down the road, it will be finished.”

Before coming to Mizzou, Perez had mindless jobs in high school and that’s why he likes the idea of a puzzle. He came here unsure of what he wanted to be involved in until he was introduced to a major in biochemistry. He took classes with Stefan Sarafianos then soon got involved with his lab at Bond LSC. When Sarafianos left Perez was under the direction Kamlendra Singh in the lab.

“Once I started to figure out how important research was, it showed me an important aspect of finding cures and vaccinations,” Perez said. “I like mysteries, and it’s a big giant mystery on what works and what doesn’t.”

When Perez first joined the lab his sophomore year he made solutions and buffers, now he uses those same buffers to make reaction mixes to see the efficacy in drug resistance in HIV. The lab tests which drugs react best to HIV viruses.

“I really like the work I do with virology,” Perez said. “It interests me how viruses work.”

Two years later, Perez sees how research extends outside of the lab.

“I always figured research was about taking steps to figure out what to do next, such as in life,” Perez said. “You take little chances here and there and figure out the outcomes, but sometimes you have to take a different approach. You see failures and it is not your fault but it is just life. It is about the persistence of life.”

Along with a new way of seeing life, the lab has also introduced Perez to different perspectives on various cultures.

“Our lab consists of three people and our cultures are all very different,” Perez said. “There are diverse minds, so it really helps out with communicating. They are more patient to be able to hear what I am saying, there are times I don’t make sense. Because of their patience I am able to articulate slowly.”

However, a new perspective isn’t the only thing the lab has given him. Perez was president of Association of Latina America Students last year and credits the lab for the confidence to run.

“I have become more social and have taken a lot of leadership roles,” Perez said. “I am becoming more independent, before I was very dependent on others. I wouldn’t take the initiative. I was really scared of failure and that is something research teaches you. I just have to accept all the mess ups I make.”