Viral ‘molecular scissor’ is next COVID-19 drug target

American and Polish scientists, reporting Oct. 16 in the journal Science Advances, laid out a novel rationale for COVID-19 drug design—blocking a molecular “scissor” that the virus uses for virus production and to disable human proteins crucial to the immune response.

The researchers are from The University of Texas Health Science Center at San Antonio (UT Health San Antonio) and the Wroclaw University of Science and Technology. Information gleaned by the American team helped Polish chemists to develop two molecules that inhibit the cutter, an enzyme called SARS-CoV-2-PLpro.

SARS-CoV-2-PLpro promotes infection by sensing and processing both viral and human proteins, said senior author Shaun K. Olsen, Ph.D., associate professor of biochemistry and structural biology in the Joe R. and Teresa Lozano Long School of Medicine at UT Health San Antonio.

“This enzyme executes a double-whammy,” Dr. Olsen said. “It stimulates the release of proteins that are essential for the virus to replicate, and it also inhibits molecules called cytokines and chemokines that signal the immune system to attack the infection,” Dr. Olsen said.

SARS-CoV-2-PLpro cuts human proteins ubiquitin and ISG15, which help maintain protein integrity. “The enzyme acts like a molecular scissor,” Dr. Olsen said. “It cleaves ubiquitin and ISG15 away from other proteins, which reverses their normal effects.”

Dr. Olsen’s team, which recently moved to the Long School of Medicine at UT Health San Antonio from the Medical University of South Carolina, solved the three-dimensional structures of SARS-CoV-2-PLpro and the two inhibitor molecules, which are called VIR250 and VIR251. X-ray crystallography was performed at the Argonne National Laboratory near Chicago.

“Our collaborator, Dr. Marcin Drag, and his team developed the inhibitors, which are very efficient at blocking the activity of SARS-CoV-2-PLpro, yet do not recognize other similar enzymes in human cells,” Dr. Olsen said. “This is a critical point: The inhibitor is specific for this one viral enzyme and doesn’t cross-react with human enzymes with a similar function.”

Specificity will be a key determinant of therapeutic value down the road, he said.

The American team also compared SARS-CoV-2-PLpro against similar enzymes from coronaviruses of recent decades, SARS-CoV-1 and MERS. They learned that SARS-CoV-2-PLpro processes ubiquitin and ISG15 much differently than its SARS-1 counterpart.

“One of the key questions is whether that accounts for some of the differences we see in how those viruses affect humans, if at all,” Dr. Olsen said.

Source: Read Full Article

Potential target identified for migraine therapy

Migraines affect millions of people worldwide, often lasting days and severely disrupting lives. More than simply super-intense headaches, some migraines actually result from pathological excitation of neurons in the brain. A new study in mice led by Kohichi Tanaka at Tokyo Medical and Dental University (TMDU) shows that susceptibility to migraines could be related to a molecular transporter that normally works to prevent excessive excitation of neurons.

Neurons in the brain communicate with each other by passing along molecules called neurotransmitters. After a neurotransmitter takes care of business, it is transported away from the synapse—the space between two neurons—so that it cannot be used over and over again. This process is called reuptake, and is one of many ways in which over-excitation of neurons in the brain is prevented. Migraines are related to a condition called cortical depression, in which a large wave of hyperactivity spreads across the brain, followed by a wave of inhibition, or depressed brain activity. Tanaka and his team hypothesized that susceptibility to cortical spreading depression is related to disrupted transport of glutamate, the most common excitatory neurotransmitter.

In turns out that mammals have four molecules that transport glutamate, and three of them are in the cerebral cortex. To determine which of these, if any, is related to cortical spreading depression, the researchers created three strains of knockout mice, each of which lacked one of the three cortical glutamate-transporter genes. They found that when mice lacked the GLT-1 transporter, cortical spreading depression occurred more frequently and spread more quickly than in control mice or in the other knockout mice.

“We know that 90% of glutamate is transported by GLT-1 back into astrocytes, not neurons,” says Tanaka. “Our findings thus highlight another important function of glial cells in the brain as they support neuronal function.”

To confirm their findings, the team then measured the amount of glutamate outside of cells using a platinum-iridium electrode coated with glutamate oxidase. When glutamate oxidase interacts with glutamate, it creates a negative current that can be detected by the electrode very quickly, allowing almost real-time measurements of glutamate concentration in the region.

“A fast biosensor is critical,” explains Tanaka, “because cortical spreading depression only lasts about 5 minutes, and the changes in glutamate concentration could never be found using conventional methods that take minutes to hours of sampling.” When testing the three knockout mice, only the GLT-1 knockout mice produced current that differed from that of the control mice. This means that the greater and faster accumulation of glutamate outside of neurons resulted from impaired uptake by astrocytes.

Source: Read Full Article

Two existing drugs point to a potential new target against COVID-19

New lab-based studies show that two existing drugs, including one developed by a researcher at Harvard Medical School and Boston Children’s Hospital, inhibit SARS-CoV-2—the virus that causes COVID-19—from infecting human cells in a dish.

Both drugs, vacuolin-1 and apilimod, originally developed years ago, target a large enzyme called PIKfyve kinase.

Before this study, little was known about this enzyme’s role in COVID-19 infection. The work, which will need to be replicated in human trials, suggests a potential new target for COVID-19 therapies.

Findings were published Aug. 6 in PNAS.

“Our findings show that targeting this kinase through a small-molecule antiviral against SARS-CoV-2 may be an effective strategy to lessen the progression or seriousness of COVID-19,” said study co-senior author Tomas Kirchhausen, professor of cell biology in the Blavatnik Institute at HMS and professor of pediatrics at Boston Children’s.

Kirchhausen discovered vacuolin-1 16 years ago. Apilimod was developed by a company called LAM Therapeutics.

Ebola and beyond

When Kirchhausen first found vacuolin-1, he published a paper describing what it does in a variety of cell types.

Several years later, he began a long collaboration with HMS colleagues in the Center for Excellence in Translational Research focused on small molecules against emerging viruses.

They showed that vacuolin-1 and apilimod, which have a similar chemistry, were both effective inhibitors against the Ebola virus. They did not publish their results at the time.

When COVID-19 began to hit the U.S. hard in early March, Kirchhausen’s lab ramped down like many others in the country. Before turning out the lights, however, he remembered that the kinetics of cell entry of Ebola virus were similar to those of coronaviruses like SARS-CoV-2.

Kirchhausen reached out to co-senior author Sean Whelan, who had been part of the Center for Excellence team at HMS but had since moved to Washington University. The duo performed cell biology studies with SARS-CoV-2 virus in Whelan’s lab.

“Within a week, we knew apilimod worked extremely well in preventing SARS-CoV-2 infection in human cells in the lab,” says Kirchhausen, who initially published this discovery on the bioRxiv pre-print website in April 2020.

That pre-print also included a review of apilimod’s effectiveness against Ebola and SARS-CoV-2.

“We found that like apilimod, vacuolin-1 is a very strong inhibitor for viral infection in the lab,” said Kirchhausen.

In an unexpected coincidence, an unrelated group posted a paper showing that, in a screen of 12,000 clinical-stage or FDA-approved small molecules, apilimod was one of the best drugs for inhibiting SARS-CoV-2 virus replication. That paper has since been published in Nature.

Now in clinical trials

Apilimod’s parallel development ultimately landed with AI Therapeutics after it failed to show any benefit in phase I and II clinical trials for treatment of autoimmune conditions, its original purpose.

Although those trials were not successful, apilimod’s clinical testing in 700 healthy volunteers and patients showed it did not produce significant side effects even when given to patients for more than a year at high doses.

This spring, using some of the data from Kirchhausen’s bioRxiv paper as well as information from drug screens by others, AI Therapeutics received FDA approval to see whether apilimod reduces the seriousness of COVID-19.

In late July, AI Therapeutics announced a new randomized, double-blind, placebo-controlled study with apilimod, known as LAM-002 in the study. It will test apilimod’s safety, tolerability and efficacy in reducing the amount of virus in about 140 patients with confirmed early-onset COVID-19.

Looking forward, Kirchhausen hopes to identify other drugs to be given in addition to a PIKfyve kinase inhibitor.

Source: Read Full Article

Scientists discover novel drug target for pancreatic cancer

Scientists at Sanford Burnham Prebys Medical Discovery Institute have uncovered a novel drug target, a protein called PPP1R1B, that stops the deadly spread of pancreatic cancer, called metastasis, when inhibited in mice. Published in Gastroenterology, the findings are a first step toward a potential treatment for one of the deadliest cancers known today.

“Our study uncovers a protein, called PPP1R1B, that is completely new to pancreatic cancer researchers and that drives tumor metastasis, the major reason the cancer is so lethal,” says Anindya Bagchi, Ph.D., associate professor in the Tumor Initiation and Maintenance Program at Sanford Burnham Prebys and senior author of the study. “With this proof-of-concept data, we can start drug screens that identify an inhibitor of PPP1R1B, which, if successful, may help more people survive pancreatic cancer.”

Pancreatic cancer is one of the deadliest cancers: Fewer than 10% of people with this type of cancer remain alive five years later. The tumor is difficult to detect because symptoms often don’t appear until the disease has already metastasized. However, if the tumor is contained in the pancreas, the five-year survival rate increases to nearly 40%, according to the American Cancer Society. For unknown reasons, pancreatic cancer is on the rise and predicted to become the second-leading cause of cancer-related deaths in the U.S. by 2030.

A surprising finding

In the study, the scientists set out to understand how pancreatic cancer responds to oxygen deprivation (hypoxia). Cancer researchers have long wondered how pancreatic cancers are able to thrive in such a harsh environment—and speculated that increased production of hypoxia inducible factor 1 alpha (HIF1A), a gene triggered by hypoxia, can stimulate tumor growth. Drugs that inhibit HIF1A are being explored for many hypoxic cancers, but until now the protein’s role in pancreatic cancer was unclear—presenting a hurdle to clinical trials evaluating these potentially promising drugs.

As a first step, the scientists created mice with pancreatic tumors that do not produce HIF1A. They expected that removing this protein would be beneficial and allow the mice to become cancer free. However, to their surprise, these mice had more aggressive tumors—with more invasion into nearby organs, greater metastasis and shorter survival times.

“Our original hypothesis was that if we remove HIF1A, a supposed driver of tumor survival, growth should be delayed or we should be curing the cancer,” says Bagchi. “Instead, we got the exact opposite results. When we saw this, we knew that we may have hit something really interesting, and needed to nail down exactly why we are seeing this effect.”

New drug target revealed

Digging deeper, the scientists discovered that these mice had increased levels of a protein called PPP1R1B. When they removed the gene that codes for this protein, the mice had fewer metastases—suggesting that a drug that inhibits the protein would stop pancreatic cancer from spreading.

“Our data also showed that tumor samples from people with metastatic pancreatic cancer had increased levels of PPP1R1B, adding further evidence that the protein has therapeutic potential,” says Ashutosh Tiwari, Ph.D., postdoctoral associate in the Bagchi lab at Sanford Burnham Prebys and first and co-lead author of the study. “Elevated levels of PPP1R1B have also been found in colon, lung and prostate cancers, and might also be seen in other hypoxic tumors, so an inhibitor may have benefits beyond pancreatic cancer.”

Next, the scientists plan to start drug screens that seek to identify compounds that inhibit PPP1R1B. These activities will take place at the Institute’s Conrad Prebys Center for Chemical Genomics, one of the most advanced drug discovery centers in the nonprofit world.

“The path to a successful treatment for pancreatic cancer begins with a strong scientific understanding of what is driving the tumor’s growth and aggressiveness,” says Lynn Matrisian, Ph.D., chief science officer at the Pancreatic Cancer Action Network (PanCAN), who wasn’t involved in the study. “This study has uncovered a promising drug target that, following additional research, may one day result in a treatment that helps more people fight the world’s toughest cancer.”

A team effort

Source: Read Full Article