A USF Health-led team tests 40 million compounds and finds lead candidate that selectively relaxes airway smooth muscle cells with no detectable drug desensitization

TAMPA, Fla. (Dec. 2, 2021) – Beta-agonists (β-agonists) are the only drugs that directly open narrowed airways and make it easier to breathe for millions of people with asthma, a chronic respiratory disease. These inhaled medications activate the β₂-adrenergic receptors (β₂AR ) on airway smooth muscle cells and relax them, dilating airways and increasing air flow.

However, for a significant proportion of asthmatics, the effectiveness of existing β-agonists is insufficient to open tightly constricted airways and the clinical benefits realized appear to wane over time, leaving them constantly struggling with the disease.

“A lack of more effective therapies to treat or prevent shortness of breath is a major issue for patients with severe-to-moderate asthma,” said Stephen Liggett, MD, vice dean for research and a professor of medicine, molecular pharmacology and physiology, and biomedical engineering at the University of South Florida Health (USF Health) Morsani College of Medicine. “As regular use of β-agonists increases, the body becomes less sensitive to these bronchodilators.”

This process, known as tachyphylaxis or drug desensitization, contributes to insufficient asthma control, which leads to increased emergency department visits and hospitalizations — impacting the quality of life and extracting an economic toll in increased medical costs and missed days of work and school. Dr. Liggett’s laboratory works with collaborators across the country to understand the mechanisms of tachyphylaxis, with the aim of improving β-agonists.

Over the last three years, a multi-institutional research team led by USF Health studied 40 million compounds to identify those that activated β₂AR (β-agonists) without causing tachyphylaxis. The investigators found one such agonist, which was structurally distinct from all known traditional β-agonists. Their preclinical research suggests that a different class of β-agonists, known as biased agonists, offers promise for selectively treating asthma and other obstructive lung diseases. Such biased agonists offer a therapeutic option without causing the rapid turndown of these receptors (β₂AR) when the drug is used on an as-needed basis, or the even greater loss of effectiveness observed with chronic use.

The drug discovery study, published online recently in the Proceedings of the National Academy of Sciences of the United States (PNAS), was conducted by scientists with expertise in biochemistry, physiology, and computational biology. The team used molecular modeling driven by high speed, high-capacity supercomputers to define how this atypical agonist, named C1-S, works at the molecular level.

“This is the first β-agonist ever known to relax airway smooth muscle and treat asthma without any detectable tachyphylaxis and represents a significant breakthrough in asthma therapy,” said principal investigator Dr. Liggett, the PNAS paper’s senior author.

USF Health’s Stephen Liggett, MD, led a multi-institutional research team that studied 40 million compounds to identify those that activated β₂AR (beta-agonists) without causing tachyphylaxis (drug desensitization). — Photo by Allison Long, USF Health Communications and Marketing

β2-adrenergic receptors are G protein-coupled receptors (GPCR), present in airway smooth muscle cells to mediate various functions. The existing β-agonists used to treat asthma are all unbiased. That means the drug equally favors activating a G-protein signaling pathway that promotes airway smooth muscle cell relaxation (thus easier breathing) as well as engaging a beta arrestin (β-arrestin) signaling pathway that leads to the unwanted outcome of tachyphylaxis.

“Beta-arrestin is a protein that upon interaction with the G protein-coupled receptor begins to uncouple (inhibit) the receptor from stimulating the clinically important signaling pathway we want to preserve,” Dr. Liggett explained. “With unbiased beta agonists you have these dueling signaling processes essentially competing with each other.”

Research is underway to design biased agonists to help alleviate pain without addiction and to better treat certain cardiovascular conditions with minimal side effects; however, no GPCR-biased agonists are yet being developed for asthma.

The researchers approached this massive study with “no preconceived notions” about what compounds might work best, Dr. Liggett said. Among their key findings:

• Of the 40 million compounds screened, 12 agonists activated the target receptor (β₂AR), stimulating cyclic AMP production that causes airway smooth muscle relaxation. But only one of these 12 (C1-S) appeared to be strongly biased away from the b-arrestin signaling that limits airway smooth muscle response (and thus drug effectiveness) due to receptor desensitization.
• Through a series of biochemical experiments, the researchers verified for the first time that it was possible for an agonist to “split the signal” mediated by a G coupled-protein receptor (β₂AR). This split preferentially activates, or switches on, a signaling pathway beneficial for treating obstructive lung disease rather than a pathway believed to be physiologically harmful, Dr. Liggett said.
• In addition to measuring signaling at the cellular level, the researchers employed the magnetic twisting cytometry, a method pioneered by co-author Steven An, PhD, at Rutgers University that measures changes in human airway smooth muscle cell relaxation and contraction. All the biochemistry results correlated with the physiological response the researchers expected — relaxation of airway smooth muscle without desensitization.
• Computer modeling and docking was performed by investigators at Caltech (William Goddard III, PhD, and now graduate student Alina Tokmakova). These studies helped identify molecular contact points between the receptor and biased agonist C1-S; some of these binding sites were not seen with any other agonist before and thus point to the basis of the properties of this unique drug. The collection of 40 million compounds was assembled and maintained by Marc Giulianotti, PhD, of Florida International University.

As regular use of β-agonists increases, the body becomes less sensitive to these inhaled bronchodilators, a process known as as tachyphylaxis (drug desensitization) that contributes to insufficient asthma control.

The researchers plan to evaluate the safety and efficacy of their lead drug candidate C1-S for potential use in humans, Dr. Liggett said.

“Every day we see breakthrough asthma symptoms in patients using albuterol, a beta-2 receptor agonist that is the cornerstone of treatment. When exacerbated, these symptoms sometimes require hospitalization, use of a ventilator, and occasionally even result in death,” said Kathryn S. Robinett, MD, assistant professor of medicine at the University of Maryland School of Medicine’s Division of Pulmonary and Critical Care Medicine, who was not involved in the research. “A new class of beta-agonists that do not cause tachyphylaxis, like the one characterized in this study, could provide rapid relief and add a powerful tool to our belt in the treatment of asthma.”

The study’s co-lead authors were Donghwa Kim, PhD, of the USF Health Morsani College of Medicine, and Alina Tokmakova, currently a graduate student at University of California San Francisco.

The work was supported by grants from the National Heart, Lung, and Blood Institute, part of the National Institutes of Health.

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USF Health’s mission is to envision and implement the future of health. It is the partnership of the USF Health Morsani College of Medicine, the College of Nursing, the College of Public Health, the Taneja College of Pharmacy, the School of Physical Therapy and Rehabilitation Sciences, the Biomedical Sciences Graduate and Postdoctoral Programs, and USF Health’s multispecialty physicians group. The University of South Florida is a high-impact global research university dedicated to student success. Over the past 10 years, no other public university in the country has risen faster in U.S. News and World Report’s national university rankings than USF. For more information, visit health.usf.edu

A University of South Florida Health preclinical study indicates that FKBP51-progesterone receptor binding plays a critical role in stress-induced preterm birth

Babies born before 37 weeks, particularly those born before 34 weeks, have more health problems and may face long-term complications like heart and lung diseases and neurodevelopmental delays.

Tampa, FL (March 8, 2021) — Preterm birth is a leading cause of infant deaths and illness in the U.S. — yet its underlying molecular causes remain largely unclear. About 40 to 50% of preterm births, defined as births before 37 weeks of pregnancy, are estimated to be “idiopathic,” meaning they arise from unexplained or spontaneous labor. And, maternal stress linked to depression and post-traumatic stress disorders as well as fetal stress have been strongly implicated in preterm births with no known cause.

Now, for the first time, a University of South Florida Health (USF Health) preclinical study has uncovered a mechanism to help explain how psychological and/or physiological stress in pregnant women triggers idiopathic preterm birth. A research team at the USF Health Morsani College of Medicine Department of Obstetrics and Gynecology shows how cortisol — the “fight-or-flight” hormone critical for regulating the body’s response to stress — acts through stress-responsive protein FKBP51 binding to progesterone receptors to inhibit receptor function in the uterus. This reduced progesterone receptor activity stimulates labor.

The findings were published online first March 8 in Proceedings of the National Academy of Sciences (PNAS).

“This new study fills in some longstanding mechanistic gaps in our understanding of how normal labor begins and how stress causes preterm birth,” said the paper’s senior author Charles J. Lockwood, MD, senior vice president of USF Health, dean of the USF Health Morsani College of Medicine, and a professor of obstetrics and gynecology specializing in maternal-fetal medicine.

Dr. Lockwood was a co-principal investigator for the study along with the paper’s lead author Ozlem Guzeloglu-Kayisli, PhD, a USF Health associate professor of obstetrics and gynecology. Nihan Semerci, MSc, a senior biological scientist, shares the lead authorship with Dr. Guzeloglu-Kayisli.

The preclinical study providing molecular insights into maternal stress and preterm birth of unknown cause was led by USF Health’s Charles J. Lockwood, MD, (above) and Ozlem Guzeloglu-Kayisli, PhD. | Photos by Freddie Coleman and Allison Long, USF Health Communications

Progesterone reduces contractions of the uterus and sustained levels are essential to prevent a baby from being born too early. Reduced uterine progesterone receptor expression and signaling stimulates labor. In the brain, elevated FKBP51 expression has been strongly associated with increased risk for stress-related disorders.

Previous work by the USF Health team showed that normal human labor starting at term (between 37 and 42 weeks of pregnancy) was associated with reduced expression of progesterone receptors and increased expression of FKBP51, specifically in maternal decidual cells (specialized cells lining the uterus).

For the current study focused on maternal stress-induced idiopathic preterm birth, the researchers combined experiments in human maternal decidual cells and a mouse model in which FKBP5, the gene that makes FKBP51, had been removed, or “knocked out.” Altogether, their results revealed a novel functional progesterone withdrawal mechanism, mediated by maternal stress-induced uterine FKBP51 overexpression and enhanced FKPB51-progesterone receptor binding, that decreased progestational effects and triggered preterm birth. The researchers found that Fkbp5 knockout mice (with depletion of the gene encoding for FKBP51) exhibit prolonged gestation and are completely resistant to maternal stress-induced preterm birth.

Among the USF Health team’s key findings:

– FKPB51 levels were greater and FKPB51 binding to progesterone receptors was significantly increased in the decidual cells of women with idiopathic preterm birth, compared to decidual cells of gestational age-matched controls.

– The study reports for the first time that Fkbp5-deficient (knockout) mice are completely resistant to maternal stress-induced preterm birth and exhibit prolonged pregnancies accompanied by slower decline in systemic progesterone levels. This indicates that FKBP51 plays a crucial role in the length of pregnancy and initiation of labor and delivery.

– In contrast, mice with the FKPB5 gene intact and normal levels of FKPB51 protein (wild type mice) delivered earlier when exposed to maternal stress than either non-stressed wild type mice or FKPB5 knockout mice under nonstressed or stressed conditions.

“Collectively, these results suggest that FKBP51 plays a pivotal role both in term labor and stress-associated preterm parturition (birth) and that inhibition of FKBP51 may prove to be a novel therapy to prevent idiopathic preterm birth,” the study authors conclude.

Dr. Guzeloglu-Kayisli (center) in the Ob-Gyn research team’s laboratory, USF Health Morsani College of Medicine, with MCOM maternal-fetal medicine fellow Anthony Kendle, MD (left) and biological scientist Xiaofang Guo (right), who are isolating tissue samples | Photo by Allison Long, USF Health Communications.

Currently, injectable progesterone is the only drug approved to help prevent preterm birth in high-risk women who have had a previous preterm birth. However, its effectiveness was not confirmed by a recent large clinical trial, sparking debate in the health care community.  The authors finding that progesterone receptor activity was reduced in idiopathic preterm birth may explain the apparent lack of effectiveness of supplemental progesterone.

Babies born before 37 weeks, particularly those born before 34 weeks, have more health problems and may face long-term health complications, including childhood lung or heart disease and neurodevelopmental delays, Dr. Guzeloglu-Kayisli said. The likelihood of poor outcomes decreases as gestational age (length of the pregnancy) increases.

“Prevention of idiopathic preterm birth by extending gestation even two or three weeks can benefit the newborn, because it provides critical time needed for the fetus’s lungs and brain to mature,” Dr. Guzeloglu-Kayisli said. “Our research indicates the importance of investigating the potential use of FKBP51 inhibitors as a targeted therapy to reduce the risk of stress-related preterm birth.”

The USF Health study was supported in part by The March of Dimes Prematurity Research Center Ohio Collaborative grant.

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A USF Health-Johns Hopkins Medicine team shows that nanoparticles targeting the blood-clotting protein reduces necrotizing enterocolitis-like injury in neonatal mice

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TAMPA, Fla. (May 4, 2020) — Necrotizing enterocolitis (NEC), a rare inflammatory bowel disease, primarily affects premature infants and is a leading cause of death in the smallest and sickest of these patients. The exact cause remains unclear, and there is no effective treatment. Since no test can definitively diagnose the devastating condition early, infants with suspected NEC are carefully monitored and administered supportive care, such as IV fluids and nutrition, and antibiotics to fight infection caused by bacteria invading the gut wall. Surgery must be done to excise damaged intestinal tissue if the condition worsens.

A new preclinical study by researchers at the University of South Florida Health (USF Health) Morsani College of Medicine and Johns Hopkins University School of Medicine offers promise of a specific treatment for NEC, one of the most challenging diseases confronting neonatologists and pediatric surgeons.  The team found that inhibiting the inflammatory and blood-clotting molecule thrombin with targeted nanotherapy can protect against NEC-like injury in newborn mice.

Their findings were reported May 4 in the Proceedings of the National Academy of Sciences.

“Our data identified the inflammatory molecule thrombin, which plays a critical role in platelet-activated blood clotting, as a potential new therapeutic target for NEC,” said coauthor Samuel Wickline, MD, professor of cardiovascular sciences at Morsani College of Medicine and director of the USF Health Heart Institute. “We showed that anti-thrombin nanoparticles can find, capture and inactivate all the active thrombin in the gut, thereby preventing or reducing the small blood vessel damage and clotting that accelerates NEC.”

The PNAS paper’s senior author is Akhil Maheshwari, MD, professor of pediatrics and director of neonatology at the Johns Hopkins University School of Medicine. Before joining Johns Hopkins Medicine (Baltimore) in 2018, Dr. Maheshwari’s group at USF Health was the first to demonstrate that platelet activation is an early, critical event in causing NEC, and therapeutic measures to block these platelets might be a new way to prevent or reduce intestinal injury in NEC.

The nanotherapy platform created by Dr. Wickline and USF Health biomedical engineer Hua Pan, PhD, delivers high drug concentrations that specifically inhibit thrombin from forming blood clots on the intestinal blood vessel wall without suppressing the (clotting) activity needed to prevent bleeding elsewhere in the body. This localized treatment is particularly important for premature infants, Dr. Wickline said, because the underdeveloped blood vessels in their brains and other vital organs are still fragile and susceptible to rupture and bleeding.

Samuel Wickline, MD, and Hua Pan, PhD, of the USF Health Heart Institute, created the treatment platform used to deliver anti-thrombin nanoparticles to the site of gut damage.

For this study the researchers used a model they created — infant mice, or pups, induced to develop digestive tract damage resembling human NEC, including the thrombocytopenia commonly experienced by premature infants with NEC. Thrombocytopenia is characterized by low counts of blood cell fragments known as platelets, or thrombocytes, which normally stop bleeding from a cut or wound by clumping together to plug breaks in injured blood vessels.

The molecule thrombin plays a key role in the bowel inflammation driven by overactive platelets. While investigating role of platelet depletion in NEC-related thrombocytopenia, the USF-Johns Hopkins researchers were surprised to find that thrombin mediates platelet-activated blood clotting early in the pathology of NEC-like injury – before bacteria leaks from inside the gut to circulating blood or other organs. This clotting clogs small blood vessels and restricts blood flow to the inflamed bowel. Eventually, the lining of the damaged intestinal wall can begin to die off.

The investigative therapy essentially works “like a thrombin sponge” that is exponentially more potent than current agents used to inhibit clotting, Dr. Wickline explained. “It literally puts trillions of nanoparticles at that damaged (intestinal wall) site to sponge up all the overactive thrombin, which tones down the clotting and inflammation processes promoted by thrombin.”

“We are so excited about finding this new way to attenuate intestinal injury in NEC,” Dr. Maheshwari said.

The same approach has also been shown in preclinical studies to inhibit the growth of atherosclerotic plaques and certain kidney injuries without causing systemic bleeding problems, Dr. Wickline added. “The nanoparticles can be tailored to other inflammatory diseases highly dependent on thrombin for their progression.”

The study authors conclude that their experimental targeted treatment for NEC merits further evaluation in clinical trials. Grants from the National Institutes of Health supported the project.

New demands on universities go beyond research and teaching to basic research and technology transfer, NAI paper says

Tampa, FL  (April 28, 2014) —  Should universities change their faculty tenure and promotion calculus based on research and publication to one that recognizes and includes faculty research activities that translate into patents, licensing and commercialization of products?

“Yes,” say a group of university officials from across the nation whose paper “Changing the academic culture: Valuing patents and commercialization toward tenure and career advancement” is published in the current issue of The Proceedings of the National Academy of Sciences.

USF’s Paul R. Sanberg is lead author of the NAI paper

“Over the past several decades, university missions have expanded from teaching and research to include economic development and translating university-based research into real products that benefit society,” says paper lead author Paul R. Sanberg, Distinguished University Professor and senior vice president for research and innovation at the University of South Florida (USF), and president of the National Academy of Inventors (NAI).

Dr. Sanberg is also executive director of the Center of Excellence for Aging and Brain Repair at the USF Health Morsani College of Medicine.

The authors — from USF, the California Institute of Technology, the University of Delaware, the University of Minnesota, The University of Alabama at Birmingham, Purdue University, and the University of Missouri St. Louis — say that, while research activities that translate to product commercialization should not replace traditional scholarly pursuits such as teaching, mentoring students and publishing research, patent and commercialization activities should be considered equally in decisions related to faculty tenure and academic advancement.

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