Enzyme provides an inroad

Scientists at Scripps discover enzyme for treating brain inflammation in patients with PHARC, Parkinson’s, Alzheimer’s, ALS and MS

Lori Lesko
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LA JOLLA, Calif.—A team led by scientists at The Scripps Research Institute (TSRI) has identified an enzyme that produces a high level of inflammatory lipid molecules in the brain, which seems to trigger a rare, mysterious inherited neurodegenerative disorder known as PHARC, named for its unique symptoms (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract).
For the past several years, Scripps researchers have worked with enzymes not just to treat PHARC, but the more common and tragic neurodegenerative diseases such as Alzheimer’s, Parkinson’s, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease).
The TSRI team’s breakthrough, published Jan. 12 in Nature Chemical Biologys advance online publication, indicates the PHARC disorder may be treatable if researchers can develop suitable drug candidates that inhibit the enzyme known as ABHD16A.
“This finding is a good example of what can be gained from studying enzymes linked to rare human genetic disorders,” according to Dr. Benjamin F. Cravatt III, chair of TSRI’s department of chemical physiology and member of TSRI’s Skaggs Institute for Chemical Biology. “We began the project actually setting out to look for the enzyme that produces lysophosphatidylserine based on previous work showing this lipid is potentially relevant for PHARC.”
It’s too early to start clinical studies on human subjects, however, “since much better ABHD16A inhibitors will need to be developed and tested in preclinical models to confirm or disconfirm whether ABHD16A is a good drug target for PHARC,” Cravatt tells DDNews.
“First, we would like to generate and study ABHD12-ABHD16A double knockout mice and generated centrally active ABHD16A inhibitors to directly test whether disruption of ABHD16A blocks or reverses the neuroinflammatory and sensorimotor defects associated with PHARC,” he says. “Our long-term goal of this study is to develop a safe and effective treatment for PHARC and other neuroinflammatory/neurodegenerative disorders.”
First described by Norwegian researchers in 2009, PHARC usually manifests in early adolescence and progressively worsens with age.
In September 2010, lead author Torunn Fiskerstrand described PHARC in the American Journal of Genetics as a “neurodegenerative disease marked by early-onset cataract and hearing loss, retinitis pigmentosa and involvement of both the central and peripheral nervous systems, including demyelinating sensorimotor polyneuropathy and cerebellar ataxia.”
In 2010, PHARC was linked to gene mutations that inactivate the enzyme ABHD12. The finding prompted Cravatt and his laboratory to develop a mouse model of the disease in which the ABHD12 gene is similarly inactivated.
Studies of those “PHARC mice” revealed that the ABHD12 enzyme normally breaks down immune-signaling lipid molecules called lysophosphatidylserines (lyso-PSs) in the brain. ABHD12’s absence in the PHARC mice leads to an abnormal buildup of lyso-PSs and subsequent neuroinflammation.
Having identified an enzyme that normally breaks down lyso-PS molecules, Cravatt and his team set out to find an enzyme that makes lyso-PSs. In principle, such an enzyme could be targeted with compounds that inhibit its activity to dial down lyso-PS levels in the nervous system—and thus treat PHARC.
To find a lyso-PS-making enzyme, Cravatt lab Research Associate Siddhesh S. Kamat led the drive to develop a sophisticated test for detecting the enzyme-mediated conversion of precursor phosphatidylserine molecules to lyso-PSs.
“Using this test we discovered a cryptic, yet distinct, lyso-PS-making enzyme activity in the mouse brain,” Kamat says. “The tests revealed especially high levels of this activity in the cerebellum, a brain region strongly impacted in PHARC.”
The Cravatt lab has pioneered the chemoproteomic technique: activity based protein-profiling, and used it in conjunction with metabolomics approaches to successfully validate and annotate function to several serine hydrolase enzymes, particularly those implicated in human diseases, according to TSRI.
The team also found that this lyso-PS-making activity could be powerfully blocked in the lab dish by the weight-loss drug tetrahydrolipstatin (THL, also known as Orlistat and Xenical). THL is a known inhibitor of multiple enzymes, and the team was able to link the lyso-PS-making activity in mouse brain specifically to one of these enzymes: a previously uncharacterized enzyme called ABHD16A.
“The identification of ABHD16A as the major lyso-PS-making enzyme, and that inhibition of ABHD16A in ABHD12-null systems (PHARC models) restores normalcy to the elevated lyso-PS levels, was the ‘ah-ha’ moment for me,” Kamat tells DDNews.
“Humans and mice both have ABHD16A (lyso-PS-making enzyme) expression in the brain, and given the high sequence similarity (approximately 96 percent) between the human and mouse enzyme, we think that ABHD16A might perform the same function in the human brain as well,” Kamat says.
In further work, Cravatt’s team collaborated with the laboratory of chemist Amy R. Howell at the University of Connecticut to find a more potent and selective inhibitor of ABHD16A, initially as a tool for studying the enzyme.
Dr. Howell “very generously allowed us to screen her library of THL-related compounds for ABHD16A inhibitors,” Cravatt says, where the team eventually isolated a small-molecule compound, KC01, that disrupts ABHD16A activity in mammalian cells more selectively than THL does.
“Using this inhibitor with advanced chemical proteomic and metabolomics techniques, we were able to confirm that ABHD16A is a major producer of lyso-PS molecules in several different mammalian cells,” Kamat says.
The team found that blocking ABHD16A activity with KC01 markedly reduced secreted lyso-PS levels in culture and brought elevated lyso-PS levels back down almost to normal in cells derived from PHARC patients. The treatment also greatly reduced the secretion of inflammatory compounds by PHARC-mouse immune cells (macrophages) following exposure to a bacterial toxin.
Finally, the team confirmed the role of ABHD16A by breeding a line of mice whose ABHD16A gene was inactivated. The ABHD16A-knockout animals grew up with lower than usual brain levels of lyso-PSs, and their macrophages showed a correspondingly muted response to immune stimulation.
“Currently we are validating whether ABHD16A is a therapeutic target for PHARC and other neurological disorders,” Kamat says.
Cravatt hopes to conduct future research in collaboration with the Howell laboratory to develop a better ABHD16A inhibitor that, unlike THL and KC01, can reach the brain from the bloodstream and thus can be tested in live mice—and perhaps in patients someday.
“We also think there is a potential for applying the lyso-PS-lowering strategy more broadly against neurological and immunological disorders,” Cravatt notes.
Other co-authors of the peer-reviewed paper, “Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay” were Kaddy Camara of the Howell Laboratory at the University of Connecticut; Dong-Hui Chen and Thomas D. Bird of the University of Washington in Seattle who supplied cell samples from PHARC patients; and William H. Parsons and Melissa M. Dix of the Cravatt laboratory at TSRI.

Lori Lesko

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