University of South Florida Health-University of Arizona team reports compounds simultaneously targeting M^pro and cathepsin L may improve treatment of COVID-19 infection

Doctoral student Michael Sacco (sitting) with Yu Chen, PhD, associate professor of molecular medicine, in Dr. Chen’s laboratory at the USF Health Morsani College of Medicine | Photo by Allison Long, USF Health Communications

TAMPA, Fla (Nov. 6, 2020) — SARS-CoV-2, the respiratory virus that causes COVID-19, attacks the body in multiple steps. Gaining entry into cells deep within the lungs and hijacking the human host cell’s machinery to churn out copies of itself are two of the earliest steps — both essential for viral infection.

A new study offers insight into designing antiviral drugs against COVID-19 by showing that some existing compounds can inhibit both the main protease (M^pro), a key viral protein required for SARS-CoV-2 replication inside human cells, and the lysosomal protease cathepsin L, a human protein important for viral entry into host cells. The study, led by researchers at the University of South Florida Health (USF Health) Morsani College of Medicine and the University of Arizona College of Pharmacy, was published Nov. 6 in Science Advances.

“If we can develop compounds to shut down or significantly reduce both processes – viral entry and viral replication – such dual inhibition may enhance the potency of these compounds in treating the coronavirus infection,” said study co-principal investigator Yu Chen, PhD, a USF Health associate professor of molecular medicine with expertise in structure-based drug design. “Metaphorically, it’s like killing two birds with one stone.”

The USF Health-University of Arizona (UA) collaborators built upon their previous work, which identified and analyzed several promising, existing antiviral drugs as candidates to treat COVID-19.  All the candidates chosen to pursue target M^pro to block the replication of SARS-CoV-2 within human cells grown in the laboratory.

Two of the compounds, calpain inhibitors II and XII, did not show as much activity against M^pro as another drug candidate called GC-376 in biochemical tests. However, the calpain inhibitors, especially XII, actually worked better than GC-376 at killing SARS-CoV-2 in cell cultures, said lead author Michael Sacco, a doctoral student in Dr. Chen’s laboratory.

“We figured if these calpain inhibitors were less effective at inhibiting the virus’s main protease, they must be doing something else to explain their antiviral activity,” Sacco said. They learned from research done by other groups, including collaborator and study co-principal investigator Jun Wang, PhD, of UA, that calpain inhibitors can block other proteases, including cathepsin L, a critical human host protease involved in mediating SARS-CoV-2 entry into cells.

X-ray crystal structures of the SARS-CoV-2 protein Mpro interacting with calpain inhibitors II (depicted above in orange) and XII (below in blue). Calpain inhibitor XII adopts an atypical inverted binding pose. | Images courtesy of Michael Sacco, USF Health.

In this latest study, the USF Health researchers used advanced techniques, particularly X-ray crystallography, to visualize how calpain inhibitors II and XII interacted with viral protein M^pro. They observed that the calpain II inhibitor fit as expected into the targeted binding sites on the surface of the SARS-CoV-2 main protease. Unexpectedly, they also discovered that the calpain XII inhibitor adopted a unique configuration – referred to as “an inverted binding pose” — to tightly fit into M^pro active binding sites. (A snug fit optimizes the inhibitor’s interaction with the targeted viral protein, decreasing the enzyme activity that helps SARS-CoV-2 proliferate.)

“Our findings provide useful structural information on how we can design better inhibitors to target this key viral protein in the future,” Dr. Chen said.

Besides the increased potency (desired drug effect at a lower dose) of targeting both viral protease M^pro and human protease cathepsin L, another benefit of dual inhibitors is their potential to suppress drug resistance, Dr. Chen said.

SARS-CoV-2 can mutate, or change, its targeted genetic sequence. These viral mutations trick the human cell into allowing the virus to attach to the cell’s surface membrane and insert its genetic material and can alter the shape of viral proteins and how they interact with other molecules (including inhibitors) inside the cell.

Dr. Chen, the study’s co-principal investigator, is applying his expertise in structure-based drug design to look for new ways to stop COVID-19 viral infection. He collaborates with researchers at the University of Arizona College of Pharmacy. | Allison Long, USF Health Communications

When the virus mutates so it can continue reproducing, it can become resistant to a particular inhibitor, reducing that compound’s effectiveness. In other words, if the genetic sequence of the viral target (lock) changes, the key (inhibitor) no longer fits that specific lock. But let’s say the same key can open two locks to help prevent COVID-19 infection; in this case the two locks are M^pro, the viral target protein, and cathepsin L, the human target protein.

“It’s harder for the virus to change both locks (two drug targets) at the same time,” Dr. Chen said. “So a dual inhibitor makes it more difficult for antiviral drug resistance to develop, because even if the viral protein changes, this type of compound remains effective against the human host protein that has not changed.”

The USF Health-University of Arizona research team continues to fine-tune existing antiviral drug candidates to improve their stability and performance, and hopes to apply what they’ve learned to help design new COVID-19 drugs. Their next steps will include solving how calpain inhibitors interact chemically and structurally with cathepsin L.

Jun Wang, PhD, UA associate professor of pharmacology and toxicology, was the corresponding author for the Science Advances paper, along with Dr. Chen as co-corresponding author. The collaborative work was supported in part by grants from the National Institutes of Health.

Michael Sacco, lead author of the Science Advances paper, is a doctoral student in the USF Health Department of Molecular Medicine. | Allison Long, USF Health Communications

A University of Arizona-University of South Florida team  identified and analyzed four promising antiviral drug candidates in the preclinical study

TAMPA, Fla. (July 6, 2020) — As the death toll from the COVID-19 pandemic mounts, scientists worldwide continue their push to develop effective treatments and a vaccine for the highly contagious respiratory virus.

University of South Florida Health (USF Health) Morsani College of Medicine scientists recently worked with colleagues at the University of Arizona College of Pharmacy to identify several existing compounds that block replication of the COVID-19 virus (SARS-CoV-2) within human cells grown in the laboratory. The inhibitors all demonstrated potent chemical and structural interactions with a viral protein critical to the virus’s ability to proliferate.

Yu Chen, PhD, an associate professor of molecular medicine with expertise in structure-based drug design, has turned toward looking for new or existing drugs to stop SARS-CoV-2.

The research team’s drug discovery study appeared June 15 in Cell Research, a high-impact Nature journal.

The most promising drug candidates – including the FDA-approved hepatitis C medication boceprevir and an investigational veterinary antiviral drug known as GC-376 – target the SARS-CoV-2 main protease (M^pro), an enzyme that cuts out proteins from a long strand that the virus produces when it invades a human cell. Without M^pro, the virus cannot replicate and infect new cells. This enzyme had already been validated as an antiviral drug target for the original SARS and MERS, both genetically similar to SARS-CoV-2.

“With a rapidly emerging infectious disease like COVID-19, we don’t have time to develop new antiviral drugs from scratch,” said Yu Chen, PhD, USF Health associate professor of molecular medicine and a coauthor of the Cell Research paper. “A lot of good drug candidates are already out there as a starting point. But, with new information from studies like ours and current technology, we can help design even better (repurposed) drugs much faster.”

Before the pandemic, Dr. Chen applied his expertise in structure-based drug design to help develop inhibitors (drug compounds) that target bacterial enzymes causing resistance to certain commonly prescribed antibiotics such as penicillin. Now his laboratory focuses its advanced techniques, including X-ray crystallography and molecular docking, on looking for ways to stop SARS-CoV-2.

Using 3D computer modeling, Michael Sacco (left), a doctoral student in the Department of Molecular Medicine, worked with Dr. Chen to determine the interactions between antiviral drug candidate GC-376 and COVID-19’s main protease.

M^pro represents an attractive target for drug development against COVID-19 because of the enzyme’s essential role in the life cycle of the coronavirus and the absence of a similar protease in humans, Dr. Chen said. Since people do not have the enzyme, drugs targeting this protein are less likely to cause side effects, he explained.

The four leading drug candidates identified by the University of Arizona-USF Health team as the best (most potent and specific) for fighting COVID-19 are described below. These inhibitors rose to the top after screening more than 50 existing protease compounds for potential repurposing:

• Boceprevir, a drug to treat Hepatitis C, is the only one of the four compounds already approved by the FDA. Its effective dose, safety profile, formulation and how the body processes the drug (pharmacokinetics) are already known, which would greatly speed up the steps needed to get boceprevir to clinical trials for COVID-19, Dr. Chen said.
• GC-376, an investigational veterinary drug for a deadly strain of coronavirus in cats, which causes feline infectious peritonitis. This agent was the most potent inhibitor of the M^pro enzyme in biochemical tests, Dr. Chen said, but before human trials could begin it would need to be tested in animal models of SARS-CoV-2. Dr. Chen and his doctoral student Michael Sacco determined the X-ray crystal structure of GC-376 bound by M^pro, and characterized molecular interactions between the compound and viral enzyme using 3D computer modeling. 

• Calpain inhibitors II and XII, cysteine inhibitors investigated in the past for cancer, neurodegenerative diseases and other conditions, also showed strong antiviral activity. Their ability to dually inhibit both M^pro and calpain/cathepsin protease suggests these compounds may include the added benefit of suppressing drug resistance, the researchers report.

All four compounds were superior to other M^pro inhibitors previously identified as suitable to clinically evaluate for treating SARS-CoV-2, Dr. Chen said.

Michael Sacco looks at COVID-19 viral protein crystals under a microscope.

A promising drug candidate – one that kills or impairs the virus without destroying healthy cells — fits snugly, into the unique shape of viral protein receptor’s “binding pocket.” GC-376 worked particularly well at conforming to (complementing) the shape of targeted M^pro enzyme binding sites, Dr. Chen said. Using a lock (binding pocket, or receptor) and key (drug) analogy, “GC-376 was by far the key with the best, or tightest, fit,” he added. “Our modeling shows how the inhibitor can mimic the original peptide substrate when it binds to the active site on the surface of the SARS-CoV-2 main protease.”

Instead of promoting the activity of viral enzyme, like the substrate normally does, the inhibitor significantly decreases the activity of the enzyme that helps SARS-CoV-2 make copies of itself.

Visualizing 3-D interactions between the antiviral compounds and the viral protein provides a clearer understanding of how the M^pro complex works and, in the long-term, can lead to the design of new COVID-19 drugs, Dr. Chen said. In the meantime, he added, researchers focus on getting targeted antiviral treatments to the frontlines more quickly by tweaking existing coronavirus drug candidates to improve their stability and performance.

Two viral protein images generated by Yu Chen, University of South Florida Health, using X-ray crystallography. Above: The protein dimer (one molecule is blue and the other orange) shows the overall structure of the COVID-19 virus’s main protease (M^pro), the researchers’ drug target. Below: Three configurations of active sites where inhibitor GC-376 binds with the M^pro viral enzyme, as depicted by 3D computer modeling.

Dr. Chen worked with lead investigator Jun Wang, PhD, UA assistant professor of pharmacology and toxicology, on the study. The work was supported in part by grants from the National Institutes of Health.

-Photos by Torie Doll, USF Health Communications and Marketing

Xingmin Sun, PhD

A team led by Xingmin Sun, PhD, assistant professor in the USF Health Department of Molecular Medicine, is working on vaccine options for Clostridium difficile infection, or CDI, a significant public health threat worldwide. Dr. Sun, a National Institutes of Health career development awardee,  is currently funded through a newly awarded $2 million NIH R01 grant and a NIH R21 grant to develop effective vaccines against CDI.

This bacterial infection can overwhelm a person’s intestine causing fever, vomiting, diarrhea, severe abdominal pain, and, in severe cases, life-threatening inflammation. The bug can be difficult to eradicate with antibiotics; in fact, most cases of CDI, occur after a course of antibiotics therapy, which wipes out the protective microbes normally populating the gut, allowing toxic spores to thrive.  The infection most commonly is acquired from health care facilities and providers; people over age 65 with compromised immune systems, and patients who have suffered one episode of CDI are at highest risk.

Dr. Sun was interviewed at the American Society for Microbiology (ASM) Microbe 2018 Conference, held June 7-11 in Atlanta, Ga.  The USF microbiologist shared his team’s progress on a vaccine for CDI infection and perspectives on what to consider when developing a vaccine to prevent or help treat this infection.  Watch the videos of his interviews below:

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//www.youtube.com/watch?v=hcBnjT9GlVQ

In addition to working on new therapeutics to treat or prevent Clostridium difficile, Dr. Sun’s laboratory is investigating antibiotic resistance in CDI and collaborating with other laboratories to develop more effective antibiotics against CDI.

Research by Laura Blair’s team seeking to untangle tau may lead to targeted treatments for Alzheimer’s, Parkinson’s and other neurodegenerative diseases

Both of USF Health neuroscientist Laura Blair’s grandmothers died from ALS, a debilitating neurodenerative disease that progressively weakens muscles and leads to paralysis.

Laura Blair, PhD, assistant professor in the Department of Molecular Medicine, at her laboratory in the USF Health Byrd Alzheimer’s Institute

“Seeing that firsthand really put in my heart to do everything I could to help people suffering from these devastating neurodegenerative diseases,” said Blair, PhD, an assistant professor in the Morsani College of Medicine’s Department of Molecular Medicine.

As an USF undergraduate student conducting research in chemist Bill Baker’s laboratory, she had the chance to work at the USF Health Byrd Alzheimer’s Institute with Chad Dickey, PhD, an accomplished and creative NIH-funded neuroscientist who was the first to find that proteins involved in learning and memory were selectively impaired in a mouse model of Alzheimer’s disease.

Blair jumped at the opportunity.  Dr. Dickey’s National Institutes of Health (NIH) work was focusing on defects in the removal of damaged proteins from cells, with promising studies of the key role “chaperone proteins” play in regulating brain cell function in Alzheimer’s disease and other neurodegenerative disorders.

“I was excited to be part of the translational work he was doing – seeking to understand how the cell’s natural defense, these chaperone proteins, might be harnessed to regulate (abnormal) misfolding proteins in order to help fix or prevent neurodegenerative disease,” Dr. Blair said.

That was nine years ago.

Dr. Blair pulls up microscopic images of stained neurons with doctoral student Lindsey Shelton, right. The stain is used to determine if treatments administered to mice —  for instance, overexpressing cochaperone protein Aha1 or human enzyme CyP40 — change how toxic tau becomes to nerve cells.  

 Dr. Laura Blair describes the focus of her laboratory’s research.

Carrying on a scientific legacy

Since then, Dr. Blair received her doctorate in medical sciences at USF in 2014 (she also earned bachelor’s and master’s degrees here) and continued to work in Dr. Dickey’s laboratory as a postdoctoral fellow.  When Dr. Dickey passed away last year at the age 40 following a courageous battle with cancer, Dr. Blair and colleague John Koren, PhD, assumed the leadership of Dickey’s team with heavy hearts. But they push forward with their mentor’s same dedication to seeking answers and sense of urgency to “publish, publish, publish.”

“Chad was always larger than life and one of the biggest influences in my life, so losing him has been surreal,” said Blair, who keeps Dr. Dickey’s nameplate on the office she now occupies. “It’s hard, but we will continue the work he started and carry on his scientific legacy.”

“He was a tremendous mentor who taught all of us how to think about a problem and ways to come up with solutions.” And she said, smiling at the memory of Dr. Dickey’s unrelenting drive to find solutions, “he taught us there’s no sense in waiting until tomorrow for an experiment that could have been started yesterday.”

Dr. Blair with Jeremy Baker, doctoral candidate in the Department of Molecular Medicine who was the lead author for a recent PLOS Biology paper reporting that a human enzyme can reduce neurotoxic amyloids in a mouse model of dementia.

Dr. Blair’s laboratory studies how chaperone proteins drive different states of the tau protein associated with Alzheimer’s disease and more than a dozen other tauopathies, including Parkinson’s disease and traumatic brain injury.  Just as their name suggests, “chaperone” proteins escort other proteins in the cell to the places they need to be, ensure these proteins do not interact with others that could be bad influences, and see to it that proteins degrade, “or are put to bed, so to speak,” when the time is right, Dr. Blair said. Under normal circumstances, chaperone proteins help ensure tau proteins are properly folded to maintain the healthy structure of nerve cells.

Pursuing how chaperones drive different states of tau

In particular, the USF researchers explore how various chaperone proteins interact, for better or worse, with various forms of tau – ranging from soluble tau protein that can spread from one brain cell to another to the aggregated, misfolded species of tau tangles inside brain cells.  (Sticky plaques of B-amyloid protein and toxic tangles of tau protein both accumulate in patients with Alzheimer’s disease, but recent studies suggest that tau deposits may be more closely linked to the actual death of neurons leading to memory loss and dementia.)

“If we can understand which states of tau are worse in terms of driving neurotoxicity,” Dr. Blair said, “maybe we can begin to shift tau into a better state so we can help delay disease progression, if not stop it in its tracks.”

 Dr. Blair comments on differences in Alzheimer’s disease.

Dr. Blair and colleague John Koren, PhD, assistant professor (far left), co-manage a research team focusing on how chaperone proteins drive different states of the tau protein associated with Alzheimer’s disease and more than a dozen other tauopathies.

.With a support of a new five-year, $1.5-million R01 grant from the National Institute on Aging, Dr. Blair, along with co-principal investigators Paula Bickford, PhD, of the USF Center of Excellence for Aging and Brain Repair, and Vladimir Urvesky of the Department of Molecular Medicine, is looking at a family of energy-independent, small heat shock proteins known to prevent harmful tau aggregation.  In an earlier paper published several years ago in the Journal of Neuroscience, Drs. Blair, Dickey and colleagues reported that high levels of one of these chaperone proteins, Hsp27, reduced tau accumulation in neurons and rescued learning and memory in a mouse model for Alzheimer’s disease.

Dr. Blair is also principal investigator of a second, five-year $1.36-million grant from the National Institute of Mental Health (the continuation of a grant originally awarded to Dr. Dickey) to develop a treatment blocking the effect of a stress-related protein genetically linked to depression, anxiety and other behaviors associated with post-traumatic stress disorder (PTSD). The grant builds upon earlier USF research, showing that as levels of this stress-related protein, known as FKBP51, increase in the brain with age it partners with chaperone protein Hsp90 to make tau more deadly to brain cells involved in memory formation. Using a new mouse model genetically engineered to overexpress FKBP51 and exhibit symptoms like those seen in humans with PTSD, Dr. Blair’s team will test various treatments on mice exposed to early-life environmental stresses.

Dr. Blair and Shelton, lead author of a USF-led paper published this September in Proceedings of the National Academy of Sciences demonstrating that Hsp90 cochaperone Aha1 boosted production of toxic tau aggregates in a mouse model of neurodegenerative disease. 

The ultimate goal of all this tau regulation research is to discover and commercialize targeted treatments that work, whether that’s drugs that inhibit or activate chaperone proteins, or gene therapies, or a combination. Currently no FDA-approved medications for Alzheimer’s disease specifically target beta amyloid or tau; they only help improve symptoms for some patients for a limited time.

A single-bullet therapeutic approach to a disease like Alzheimer’s that affects diffuse areas of the brain is unlikely, Dr. Blair said. “The multi-treatment option is probably going to be the most effective, but it’s difficult to address that until we have individual treatments moving forward.”

“Working toward treatments to help slow and prevent these devastating neurodegenerative diseases is what keeps our laboratory so motivated and determined.”

 On working in a building that bridges research and clinical care

Slides, containing stained nerve cells from mouse brain tissue, are used by the researchers to evaluate neuronal health following various treatments.

Identifying potential treatments for neurodegenerative diseases

Building on Dr. Dickey’s work with chaperone proteins, the USF researchers continue to make significant progress in identifying potential targets to help slow or prevent neurodegenerative disease progression.

The team recently identified cochaperone protein Aha1, which binds to and stimulates the activity of heat shock protein Hsp90, as one promising therapeutic target.  Hsp90 regulates the folding, degradation and accumulation of tau. In a study published this September in  Proceedings of the National Academy of Sciences, the researchers demonstrated that Hsp90 cochaperone Aha1 boosted production of toxic tau aggregates in a mouse model of neurodegenerative disease and led to neuron loss and memory impairment.

The researchers also found that inhibiting Aha1 prevented the dramatic accumulation of tau in cultured cells. “We think Aha1 inhibitors offer promise for effects similar to Hsp90 inhibitors with less side effects,” Dr. Blair said.

Dr. Blair and Shelton work at an automated stereotactic injector station. The equipment helps the researchers determine the effects of gene therapy in the brain.

This June, in the journal PLoS Biology, Dr. Blair (principal investigator), Jeremy Baker (lead author) and colleagues reported for the first time that a naturally-occurring human enzyme – called cyclophilin 40 or CyP40 – could break apart clumps of tau in a mouse model of dementia. The USF led study found that CyP40 reduced the amount of aggregated tau, converting it into a more soluble and less toxic form of amyloid. In a mouse model of an Alzheimer’s-like disease, experimental expression of CyP40 preserved brain neurons and rescued cognitive deficits. The same enzyme also disaggregated alpha-synuclein, an aggregate associated with Parkinson’s disease.

Exactly how CyP40 can untangle clumps of tau and alpha-synuclein is not yet clear, but, Blair said “our finding suggests that CyP40, or one of the more than 40 other proteins with similar activity, may have a role to play in treating neurodegenerative diseases.”

Dr. Blair points to the hippocampus on the map of a mouse brain. An area critical for learning and memory, the hippocampus is especially vulnerable to damage at early stages of Alzheimer’s disease.

 What Dr. Blair hopes she can impart to students as a mentor

Some things you might not know about Dr. Blair:

• Blair and her husband Tom belong to a local swing dancing community, where they enjoy dancing the Lindy Hop and Balboa to ‘40s jazz.
• Blair is a mother to 7-year-old Oliver, and a stepmother to 17-year-old Laney, who is a high school senior. They also have two dogs, a Shih Tzu named Oreo and a Shih Tzu-Yorkshire terrier mix named Pumpkin.
• Participating in the Great American Teach-in two years ago, Dr. Blair spoke to her son’s kindergarten class about her career as a neuroscientist, including working on brain teaser puzzles and discussing a video of a preclinical neurobehavioral test with the young students. But, she said, the kids were particularly impressed with the squishy plastic “stress-reliever brains” she brought along.

Research associate Leo Breydo loads protein into a gel.

Cultured cells are harvested for analysis.

-Photos by Sandra C. Roa and Eric Younghans, University Communications and Marketing

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two rare neurodegenerative diseases also considered as spectrum disorders, affect thousands of Americans every year.

ALS, a condition that effects nerves and muscles, and FTD, a disorder that causes changes in behavior and personality, language, motor skills and function, are associated with one another due to a common genetic mutation in multiple genes.

David Kang, PhD, professor of molecular medicine, in his laboratory at the USF Health Byrd Alzheimer’s Institute. He was the study’s lead author. 

However, researchers did not know exactly why that happened until a new research breakthrough at University of South Florida Health in Tampa, Florida.

A group of USF Health researchers found that a mutated ALS and FTD gene is pathologically linked to mitochondria dysfunction and TDP-43 pathology – causing problems for people affected by either disease.

The results of this study were published in Nature Communications Journal.

The study’s lead investigator David Kang, PhD, professor of molecular medicine and researcher at the USF Health Byrd Alzheimer’s Institute, said that this is a very important pathological link for ALS and FTD.

“Mutations in the ALS and FTD gene, called CHCHD10, are instigating dysfunction in the mitochondria, the cell powerhouse plants that produce the majority of the energy in the human body,” Dr. Kang said. “This gene allows TDP-43, a protein that’s part of the nuclear function, to exit the nucleus to get into the cytoplasm or cell body and cause TDP 43-pathology – which is relatively specific to ALS and FTD.”

While many genes cause ALS and FTD, this gene is the first one that’s been linked to mitochondria, Dr. Kang said. Dr. Kang and his team of researchers have worked on this study for two years, supported by grants from the Veterans Administration, National Institutes of Health and Florida Department of Health.

Dr. Kang (second from right) with some members of his research team. From left: JungA (Alexa) Woo, PhD, assistant professor; Courtney Trotter, graduate research assistant; and Tian Lu, PhD, postdoctoral research scholar, Department of Molecular Medicine, USF Health Morsani College of Medicine.

To come up with the results, the USF Health researchers studied worms, mammalian cell lines, primary neurons and mouse brains. The models allowed them to prove that the mutated gene is very important to the mitochondrial function, which the human body needs.

“We took the human gene, CHCHD10, and we put it into worms with a short life-span,” Dr. Kang said. “We used worms who lacked the CHCHD10 gene and had dysfunctional mitochondria, which, as a result, had motor problems and could not move properly. What happened was that the normal human gene helped the worms live longer – completely rescuing their abnormalities and restoring their mitochondria function and movement. However, when we put the single mutated gene that causes ALS and FTD into the worm, it did not rescue at all. They were still completely dysfunctional.”

The USF Health research team suggests that these results are critical to ALS and FTD research. This was the first study to show that the normal gene increases mitochondria function and the mutant gene increases mitochondria dysfunction. The normal gene is bound to TDP-43 protein and allows it to stay in the nucleus.

Alexa Woo, PhD

However, when TDP-43 pathology is outside the nucleus, it decreases mitochondria function and synaptic integrity, connecting points between neurons. Researchers said synapse loss occurs in all neurodegenerative diseases.

This is an important step into the right direction, but researchers at USF Health agree that there is more work to be done.

“I think ultimately, if we can understand how the neurodegenerative disease leads to aberrations at the molecular level, then we can potentially target specific molecules that induce pathology in ALS or FTD,” Dr. Kang said.

Photos by V.  Hysenlika

USF Health researcher Mack Wu, MD, studies what happens when the microvascular endothelial barrier controlling blood-tissue exchange is compromised during ischemia-reperfusion injury, a condition that can lead to irreversible tissue damage. He also investigates the molecular control of gut permeability, also known as “leaky gut,” in tissue injuries caused by trauma and severe burns.

His group’s work has broad implications for a variety of conditions including stroke, heart attack, thrombosis, sepsis, trauma or other inflammatory diseases associated with microvascular injury.

Mack Wu, MD, is a professor of surgery and molecular medicine at USF Health Morsani College of Medicine and a research physiologist at James A. Haley Veterans’ Hospital. On the monitor next to him are images of microvessels in the small intestine injected with fluorescent dye.

The closely connected endothelial cells lining the interior of blood vessel walls play a critical role in limiting the how much fluid, proteins and small molecules cross the wall of the tiny blood vessels, or microvessels. However when this protective endothelial barrier is damaged, excessive amounts of blood fluid, proteins and molecules leak outside the microvessels into nearby body tissue – a process known as microvascular hyperpermeability. If this breech of endothelial barrier is associated with a body-wide inflammatory response, it can trigger a chain of events leading to edema (swelling), shock from severe blood and fluid loss (hypovolemic shock), and ultimately multiple organ failure.

Pinpointing potential solutions for ischemia-reperfusion injury

Previous research by Dr. Wu’s laboratory and other groups discovered that ischemia-reperfusion injury can cause endothelial barrier damage leading to vascular hyperpermeability, or abnormally leaky blood vessels.

Ischemia-reperfusion injury is typically associated with conditions like organ transplantation, stroke, heart attack, or cardiopulmonary bypass where blood supply to a vital organ is temporarily cut off (ischemia), resulting in oxygen deprivation. For instance, a period of ischemia occurs while a donor organ is transported to a recipient in the operating room, or when a clot interrupts blood circulation to the brain. When blood supply is re-established with new blood returned to the previously oxygen-deprived area (reperfusion), tissue injury can worsen because the reperfusion itself causes inflammation and oxidative damage rather than restoring normal function. It its severest form, ischemia-reperfusion injury can result in multiple organ failure, or even death.

“I believe endothelial barrier injury is one of the key elements of ischemia-reperfusion injury, so my group is trying to find out which molecule is ultimately responsible for the endothelial barrier damage,” said Dr. Wu, a professor of surgery and molecular medicine at USF Health Morsani College of Medicine and a research physiologist at James A. Haley Veterans’ Hospital.

Dr. Wu with some members of his laboratory team. From left, Rebecca Eitnier, research assistant; Shimin Zhang, Department of Molecular Medicine graduate student; Ricci Haines, research associate; and Fang Wang, research assistant.

With the support of a $1.49-million, four-year R01 grant from the National Heart, Lung and Blood Institute, Dr. Wu’s team is zeroing in on a molecule known as focal adhesion kinase, or FAK, an enzyme that may play a role in weakening the microvascular endothelial barrier during ischemia-reperfusion injury.   Using cell models and a newly developed mouse model in which the endothelial-specific gene for FAK is knocked out, the USF researchers are testing whether selectively inhibiting FAK activity can rescue the endothelial barrier from such injury.

The work is critical because no FDA-approved treatment exists to prevent tissue damage following reperfusion. Identifying a new mechanism for the injury would provide potential targets for drug development, Dr. Wu said. So for instance, he said, after an initial stroke a new intravenously administered drug selectively targeting endothelial cells in the brain’s microvessels might stop further harmful swelling of the brain caused by stroke.

Defining molecular control of “leaky gut” in severe burn trauma

A second grant from the U.S. Department of Veterans Affairs funds Dr. Wu’s studies to define the underlying molecular mechanisms of leaky guts induced by traumatic injury associated with thermal (fire, scald or chemical) burns.  Massive burn trauma is a significant cause of injury and death in American soldiers. With a $960,000 VA Merit Award, Dr. Wu focuses on how intestinal epithelial barrier damage happens during severe burns, with the aim of developing targeted therapies to prevent posttraumatic complications.  In particular, he is working to determine the pathways by which the protein palmitoylation in gut epithelial cells are stimulated by burn injury.

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Epithelial cells line the interior of the small intestines, and after severe burn injury, this protective epithelial barrier commonly breaks down, causing bacteria and toxins to flow from the intestine into the circulating blood.  The result of this abnormal epithelial permeability, or “leaky gut,” can be deadly if sepsis ensues – a bacterial infection in the bloodstream sets up a body-wide inflammatory response leading to multiple organ failure.

While the role gut barrier failure plays in posttraumatic complications is well recognized, its cellular and molecular mechanisms remain poorly understood.  Currently, pushing IV fluids to help prevent hypovolemic shock and administering antibiotics and anti-inflammatories are the only therapies, mostly supportive, Dr. Wu said.

“More effective early therapeutic interventions to prevent leaky gut and systemic inflammatory response will be key to preventing sepsis,” he added, whether in soldiers with trauma or VA patients with inflammatory bowel diseases.

From industry to academia

Dr. Wu joined USF Health and the Haley VA Hospital in 2011.  He came from Sacramento, Calif, where he was an associate professor of surgery at the University of California at Davis School of Medicine and a research physiologist at Sacramento VA Medical Center.   Previously, Dr. Wu was a faculty member in the Department of Medical Physiology at Texas A&M University Health Science Center. He screened pharmaceutical compounds as a toxicologist in a biotechnology laboratory before joining Texas A&M, moving from industry to academia in 1995.

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Dr. Wu received his MD degree from Second Military Hospital in Shanghai, China, and conducted an internship at Shanghai Second Hospital.

One of his earliest and most highly cited studies, published in the American Journal of Physiology (1996), was first to report nitric oxide’s role in contributing to cardiovascular injury. The study showed an increase in nitric oxide induces vascular endothelial growth factor (VEGF) to promote leakage in tiny coronary veins.

Another more recent study in Shock (2012) provided direct evidence that thermal burn injury causes intestinal barrier disruption and inflammation characterized by intestinal mucosal permeability (leakage) and an infiltration of immune system cells known as neutrophils.

Something you may not know about Dr. Wu:

He loves deep-sea fishing. Dr. Wu has fished for sharks off the Golf coast of Texas, rockfish off the Pacific coast of California, and grouper off the west coast of Florida.

Dr. Wu is a member of the USF Health Heart Institute. His team’s work has broad implications for a variety of conditions including stroke, heart attack, thrombosis, sepsis, trauma or other inflammatory diseases associated with microvascular injury.

Photos by Eric Younghans, USF Health Communications and Marketing

The USF-led study suggests CyP40 or similar proteins may be potential therapeutics for Alzheimer’s and Parkinson’s diseases

TAMPA, Fla. (June 27, 2017) — A naturally occurring human enzyme –called cyclophilin 40 or CyP40– can unravel protein aggregates that contribute to both Alzheimer’s disease and Parkinson’s disease, reports a study led by researchers at the University of South Florida in Tampa and published today in the open access journal PLOS Biology. The finding may point toward a new therapeutic strategy for these diseases.

This is the first time that CyP40 has been shown to disaggregate, or dissolve, a toxic, soluble form of amyloid responsible for a neurodegenerative disease, according to Laura Blair, PhD, an assistant professor in the Department of Molecular Medicine at the USF Health Byrd Alzheimer’s Institute.  Blair and fellow USF researchers worked with colleagues from several institutions in Germany.

USF neuroscientist Laura Blair, PhD, principal investigator for the study, in her laboratory at the USF Health Byrd Alzheimer’s Institute.

The study found that CyP40 could reduce the amount of aggregated tau, converting it into a more soluble and less toxic form. In a mouse model of an Alzheimer’s-like disease, experimental expression of CyP40 preserved brain neurons and rescued cognitive deficits. The same enzyme also disaggregated alpha-synuclein, an aggregate associated with Parkinson’s disease.

In most neurodegenerative diseases, misfolded proteins accumulate abnormally to form an insoluble clump called amyloid. Many amyloid-forming proteins, including tau in Alzheimer’s disease and α-synuclein in Parkinson’s disease, contain the amino acid proline, which has a unique structure inducing a bend in the amino acid chain. Those bends contribute to stacking of adjacent regions of the protein promoting clumping. CyP40 may dissolve these insoluble clumps by interacting with prolines within the amyloid structure.

Exactly how CyP40 reduces aggregation is not yet clear, and the authors provide two possibilities. The enzyme may bind to aggregated protein and, by reversing the proline bend, help unstack and separate the amino acid chain. Support for this model comes from the observation that the enzyme was less effective at reducing aggregates when its action was inhibited. Alternatively, the enzyme may bind to the protein before it forms aggregates, sequestering it and thus preventing the potentially harmful clumping.

Dr. Blair with Jeremy Baker, a doctoral candidate in the Department of Molecular Medicine and a lead author on the PLOS Biology paper. Pictured on the monitor are tau oligomers.

Understanding more specifically how the enzyme works may help point toward a therapeutic strategy centered on proline’s role in amyloid formation.

“The finding that Cyp40 can untangle clumps of tau and alpha-synuclein suggests that it, or one of the more than 40 other human proteins with similar activity, may have a role to play in treating neurodegenerative disease,” Blair said.

The study was supported by grants from the National Institutes of Health, the Alzheimer’s Association and the Veterans Health Administration.

Citation:
Human cyclophilin 40 unravels neurotoxic amyloids; Jeremy D. Baker, Lindsey B. Shelton, Dali Zheng, Filippo Favretto, Bryce A. Nordhues, April Darling, Leia E. Sullivan, Zheying Sun1, Parth K. Solanki, Mackenzie D. Martin, Amirthaa Suntharalingam, Jonathan J. Sabbagh, Stefan Becker, Eckhard Mandelkow, Vladimir N. Uversky, Markus Zweckstetter, Chad A. Dickey, John Koren III, and Laura J. Blair; PLOS Biology; June 27, 2017; https://doi.org/10.1371/journal.pbio.2001336

– Photos by Eric Younghans, USF Health Communications and Marketing