Loading...

Daily news and top headlines for life science research professionals

FREE Email Newsletter View Sample


New Inhibitors of Elusive Enzymes Promise to Be Valuable Scientific Tools

Featured In: Academia News

Wednesday, October 31, 2024

See today's top life science stories and headlines - Sign up now!

newsvine diigo google
slashdot
Share
Loading...

Scientists at The Scripps Research Institute (TSRI) have discovered the first selective inhibitors of an important set of enzymes. The new inhibitors, and chemical probes based on them, now can be used to study the functions of enzymes known as diacylglycerol lipases (DAGL), their products, and the pathways they regulate. Early tests in mouse macrophages suggest that DAGL-inhibiting compounds might also have therapeutic uses, for they suppress the production of a pro-inflammatory molecule that has been implicated in rheumatoid arthritis and related conditions.

“We’ve developed the first set of chemical probes that effectively allows one to study these DAGL enzymes in living cell and animal models,” said Benjamin F. Cravatt, chairman of the Department of Chemical Physiology, professor in the Dorris Neuroscience Center and member of the Skaggs Institute for Chemical Biology at TSRI. Cravatt and his laboratory conducted the new study, published in the current issue of the journal Nature Chemical Biology.

Important But Poorly Understood
DAGL enzymes have been of interest mainly because of their role in making 2-AG (2-Arachidonoylglycerol), an important cannabinoid that is naturally produced in humans and other mammals. Cannabinoids are named for Cannabis (marijuana) plants, because they stimulate the same cellular receptors that are hit by marijuana’s active ingredients. Drugs that can enhance 2-AG’s signaling in the nervous system are being developed as treatments for pain, depression and anxiety.

But 2-AG exists in various tissues throughout the body, and on the whole, its functions are not well understood. Until now researchers have lacked enzyme inhibitors that can usefully probe those functions by selectively shutting off 2-AG’s production. “Existing DAGL inhibitors block many other enzymes, are not very potent, and do a poor job of getting into cells,” Cravatt said. “There has been a need for better chemical tools in this area.”

Cravatt’s laboratory had previously developed a set of compounds that act as potent inhibitors of serine hydrolases—the broad enzyme family to which DAGL enzymes belong. In the new study, Cravatt’s team, including first author Ken Hsu, a Hewitt Foundation postdoctoral researcher in the Cravatt laboratory, screened a library of these compounds for specific activity as DAGL inhibitors.

A Big Improvement
After finding a promising lead compound, Hsu and his colleagues chemically optimized it to obtain KT109 and KT172. The former selectively inhibits DAGLβ, the main enzymatic producer of 2-AG outside the nervous system. KT172 inhibits both DAGLβ and DAGLα, which is principally responsible for making 2-AG within the nervous system.

In a big improvement over previously described DAGL inhibitors, KT109 and KT172 are highly selective (i.e., they do not block many other, non-DAGL enzymes) and active in cells and animals. By analyzing the structures of their initial DAGL inhibitors, the team was also able to devise a new DAGL-tailored activity-based probe that binds to the active site of DAGLs and fluorescently labels these low-abundance and difficult-to-detect enzymes in cell or tissue samples. “Without the DAGL-specific probe, we would have found it very difficult to develop, optimize and confirm target engagement for our DAGL inhibitors,” Hsu said.

In neuron-like mouse cells, human prostate cancer cells, and mouse liver cells and macrophages (a type of immune cell that is frequently involved in inflammatory conditions), the DAGL inhibitors were able to inactivate DAGLβ activity. “At the optimal doses used, we were able to achieve selective and near-complete inhibition of the enzyme,” said Hsu. In these cell and animal studies, the inhibitors also reduced levels of 2-AG as well as arachidonic acid, another bioactive lipid that DAGL enzymes can regulate.

New Questions
2-AG is known to have an anti-inflammatory effect when it activates cannabinoid receptors on macrophages. Thus, one might expect that knocking down 2-AG production with a DAGL inhibitor would have a pro-inflammatory effect. Instead, Hsu, Cravatt and their colleagues found that blocking DAGL in mouse macrophages that had been stimulated with pro-inflammatory agents markedly lowered their secretion of TNFα, a major inflammatory signaling molecule.

Blocking DAGL has potential effects on multiple lipid signaling pathways in cells, and the researchers aren’t yet certain which of these effects explains the surprising suppression of TNFα. “The effect is dependent on DAGLβ, though, because we see the same result in DAGLβ knockout mice,” said Hsu. Cravatt added that their observations of the unexpected DAGL-inhibition effects in mouse macrophages could be due to the suppression of pro-inflammatory eicosanoids that derive from downstream metabolites regulated by DAGLβ.

TNFα is a key instigator of the inflammation seen in rheumatoid arthritis, and antibodies directed against TNFα are now front-line therapies for the condition. “What we’ve done so far is just early-stage cell biology, but conceivably the further optimization of our DAGL inhibitors could result in a new type of anti-inflammatory drug that also works against arthritis and related conditions,” Cravatt said.

Cravatt and his team are now studying the pathways through which the new inhibitors have this anti-inflammatory effect. They also plan to develop new inhibitors that will selectively block DAGLα and 2-AG production in the central nervous system.

Source: The Scripps Research Institute

Join the Discussion
Rate Article: Average 0 out of 5
register or log in to comment on this article!

0 Comments

Add Comment

Text Only 2000 character limit

Page 1 of 1

Research Exchange

Live Cell Analysis of Autophagy

Sep 4

Analysis of autophagosome formation using fluorescent cellular imaging and lentiviral biosensors.

Deconstructing 3D Cell Culture

Aug 31

3D culture models offer the potential to significantly reduce drug failure.

Safety Without Sacrifice

Safety Without Sacrifice

Jun 26

Liquid handling ergonomics and performance considerations

ELNs: The Beating Heart of a Scientist’s World

ELNs: The Beating Heart of a Scientist’s World

May 22

As ELNs deliver more benefits to researchers, the cost of changing working practice is outweighed by access to high quality, high context collaboration.

Evaluation of a New Nano-Type UV-Vis Spectrophotometer

Mar 3 2011

Analysis of one- to four-microliter size samples for nucleic acids has become routine in many life science laboratories. However, until now, available instruments require considerable manipulation of the instrument and sample; some require manually recording the data. The user must typically lower and raise the arm manually, then wipe the sample manually from the target after each analysis. And fiberoptics used in some of these instruments are subject to deterioration.

Production of Recombinant Proteins and Monoclonal Antibodies in Hollow Fiber Bioreactors

Jan 25 2011

While well-understood, robust and convenient, classical batch-style 2-D culture on non-porous supports or 3-D suspension culture in other devices are really not very biologically relevant models. Cell culture conditions can affect the quality of the antibody or protein produced.

Selecting Robots for Use in Drug Discovery and Testing

Dec 6 2010

Drug discovery and testing, with their need for speed, repeatability and verification, are ideally suited to benefit from robot automation. It is therefore not surprising that robots have been at the forefront of automation developments in both these areas.

HP Scalable Network Storage Systems for Life Sciences

Sep 13 2010

Life sciences research today is advancing exponentially, each step bringing us closer to the realization of truly personalized medicine–preventive care and treatments designed specifically for each individual. In the near future, PCPGM healthcare researchers expect to be able to use predictive genetic testing to create custom treatment plans for individuals and deliver dramatic improvements over today’s one-size-fits-all approach. But research capabilities are only part of the equation; current storage and operating capacities must also evolve to accommodate ever-expanding amounts of data before the goal of personalized medicine can be realized.

Using the Tecan Genesis Workstation to Automate a Cytometric Bead Array (CBA) Immunoassay

Mar 11 2010

The poster describe the process involved in automating a Cytometric Bead Array (CBA) immunoassay developed to measure relative concentrations of serum antibodies against Tetanus (TT), Sperm Whale Myoglobin (SWM) and Keyhole Limpet Hemocyanin (KLH) in KLH-immunized volunteers.

Ensuring Quality in Assays Performed with Automated Liquid Handlers

Feb 2 2010

The focus of this presentation is to highlight the need of ensuring quality in important assays performed with automated liquid handlers. Nearly all assays performed within a laboratory are volume-dependent. In turn, all concentrations of biological and chemical components in these assays, as well as the associated dilution protocols, are volume-dependent. Because analyte concentration is volume-dependent, an assay’s results might be falsely interpreted if liquid handler variability and inaccuracies are unknown or if the system(s) go unchecked for a long period.

Inkjet System for Protein Crystallography

Feb 1 2010

X-ray crystallography is used routinely by scientists to obtain the three dimensional structure of a biological molecule of interest.Such information can be used to determine how a pharmaceutical interacts with a protein target and what changes might improve functionality. However, the crystallization of macromolecules still remains a serious hindrance in structural determination despite impressive advances in screening methods and technologies.

Attention Deficit & Hyperactivity in a Drosophila Memory Mutant

Attention Deficit & Hyperactivity in a Drosophila Memory Mutant

Nov 9 2009

Action selection is modulated by external stimuli either directly or via memory retrieval. In a constantly changing environment, animals have evolved attention-like processes to effectively filter the incoming sensory stream. These attention-like processes, in turn, are modulated by memory. The neurobiological nature of how attention, action selection and memory are inter-connected is unknown. We describe here new phenotypes of the memory mutant radish in the fruit fly Drosophila.

Red Meat Consumption and Mortality: Results From 2 Prospective Cohort Studies

Mar 16

BACKGROUND: Red meat consumption has been associated with an increased risk of chronic diseases. However, its relationship with mortality remains uncertain. METHODS: We prospectively observed 37 698 men from the Health Professionals Follow-up Study (1986-2008)...

Structural analysis of eyespots: dynamics of morphogenic signals that govern elemental positions in butterfly wings.

Mar 15

ABSTRACT: BACKGROUND: To explain eyespot colour-pattern determination in butterfly wings, the induction model has been discussed based on colour-pattern analyses of various butterfly eyespots. However, a detailed structural analysis of eyespots that can serve as...

Prokariotic Cell Collection in Denmark

Nov 6 2009

I would like to know about a prokariotic cell collection in Denmark. Is there a cell bank in this country? I need a Lactobacillus strain for a fermentation assay and this information about the bank is very helpful for me.

Request for Entries

Oct 16 2009

Ask the Experts is your chance to get the answers to questions on applications, materials, methods, processes, and technologies. Email you question to bst_web@advantagemedia.com, and the editors of Bioscience Technology will find an appropriate expert to answer it. Watch this space in the future to see the questions your colleagues are posting.

STAY INFORMED: SUBSCRIBE TO

Magazine and E-mail Newsletters

Loading...
Loading...

Free Life Science Industry
Subscriptions

Magazine

wireless week

Newsletters

newsletters

Sign up now



MULTIMEDIA

Video:

Epigenetics

Aug 30

Hank & his clone Circus Hank explain the power of epigenetics, which studies the factors that determine how much or whether some genes are expressed in your body.

Podcasts:

Futures In Biotech 93: Snyder's Omics

May 17

Host: Marc Pelletier Co-Host: Andre Nantel, Ph.D. The technologies that our guest has developed are playing an important role in changing the world, not like the car, the microwave and the cell phone, but as in Drs. Flox, McCoy, and Crusher. Guest: Dr. Michael Snyder We invite...

Top Stories and Headlines
EVERY DAY!

FREE Email Newsletter

Information: