X-rays shed light on how anti-asthmatic drugs work

A new study uncovers how a critical protein binds to drugs used to treat asthma and other inflammatory diseases.

By studying the crystal structure of an important protein when it was bound to two drugs widely prescribed to treat asthma, an international team of scientists has discovered unique binding and signaling mechanisms that could lead to the development of more effective treatments for asthma and other inflammatory diseases.

The protein, called cysteinyl leukotriene receptor type 1 (CysLT1R), controls the dilation and inflammation of bronchial tubes in the lungs. It is therefore one of the primary targets for anti-asthma drugs, including the two drugs studied: zafirlukast, which acts on inflammatory cells in the lungs, and pranlukast, which reduces bronchospasms due to allergic reactions.

Using the Linac Coherent Light Source (LCLS) X-ray free-electron laser at the Department of Energy’s SLAC National Accelerator Laboratory, the team bombarded tiny crystals of CysLT1R-zafirlukast with X-ray pulses and measured its structure. They also used X-rays from the European Synchrotron Radiation Facility in Grenoble, France to collect data about CysLT1R-pran crystals. They published their findings in October in Science Advances.

The researchers gained a new understanding of how CysLT1R interacts with these anti-asthma drugs, observing surprising structural features and a new activation mechanism. For example, the study revealed major differences between how the two drugs attached to the binding site of the protein. In comparison to pranlukast, the zafirlukast molecule jammed open the entrance gate of CysLT1R’s binding site into a much wider configuration. This improved understanding of the protein suggests a new rationale for designing more effective anti-asthma drugs.

The study was performed by a collaboration of researchers at SLAC; Moscow Institute of Physics and Technology, Russia; University de Sherbrooke, Canada; University of Southern California; Research Center Juelich, Germany; Universite Grenoble Alpes-CEA-CNRS, France; Czech Academy of Sciences, Czech Republic; and Arizona State University.

Citation: Aleksandra Luginina et al., Science Advances, 09 October 2019 (10.1126/sciadv.aax2518).

For questions or comments, contact the SLAC Office of Communications at communications@slac.stanford.edu.

Image caption: Using X-rays, researchers uncovered details about two drugs widely prescribed to treat asthma: pranlukast (shown up top) and zafirlukast (shown beneath). Their results revealed major differences between how the two drugs attached to the binding site of the receptor protein. In comparison to pranlukast, the zafirlukast molecule jammed open the entrance gate of protein’s binding site into a much wider configuration. (10.1126/sciadv.aax2518)

This is a reposting of my SLAC news story, courtesy of SLAC Linear Accelerator Laboratory.

To battle mosquito-borne disease, SLAC x-ray laser provides new view of insecticides

Illustration, of LCLS x-ray pulses blasting BinAB nanocrystals composed of protein BinA (yellow) and BinB (blue), courtesy of SLAC National Accelerator Laboratory.
Illustration of LCLS x-ray pulses blasting BinAB nanocrystals composed of protein BinA (yellow) and BinB (blue), courtesy of SLAC National Accelerator Laboratory.

Mosquitoes continue to spread devastating diseases such as malaria, West Nile virus, dengue fever and Zika virus throughout the world. Sadly, there are no medications or vaccines for many of these deadly diseases, so it’s critical to prevent mosquito bites.

A cost effective way to eliminate these disease-bearing insects is the use of specialized insecticides that target against the larval stage of a mosquito. These larvicides, like BinAB, kill some mosquito species, but they are currently ineffective against Aedes mosquitoes that transmit Zika and dengue fever. Now, an international team of researchers is working to develop a new toxin that will kill a broader range of mosquito species, including Aedes.

The existing larvicide BinAB is composed of two proteins, BinA and BinB, which pair together to form nanocrystals inside Lysinibacillus sphaericus soil bacteria. When these bacteria are distributed on the surface of stagnant water locations where mosquitoes breed, the mosquito larvae eat the bacteria — dissolving the nanocrystals that bind to their gut, activating the deadly BinAB toxin and killing the larvae.

The proteins are toxic to the mosquitoes, but harmless to humans and other animals. Unfortunately, previous research has shown that BinAB is also harmless to an Aedes mosquito, because the protein never binds to the insect’s gut so the toxin isn’t activated.

“Part of the appeal is that the larvicide’s safe because it’s so specific, but that’s also part of its limitation,” said Michael Sawaya, PhD, a scientist at the UCLA-DOE Molecular Biology Institute, in a recent news release.

Now, the researchers are adapting the BinAB toxin to attack mosquito species that are insecticide resistant. In order to do this, they needed to understand the 3-D structure of the BinAB proteins and how they work. This was a challenge, because the nanocrystals were so tiny and their structural details were a mystery.

The research team increased the size of the nanocrystals using genetic engineering, and then blasted them with an intense beam of bright, fast pulses of light using the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. This allowed the team to collect detailed structural data from the tiny crystals and create 3-D maps of the electron density of the BinAB protein, as reported in a recent paper in Nature.

The LCLS experiments helped the researchers fully understand how the BinAB protein forms and functions. They are now engineering a modified version of the protein that will kill a broader range of mosquito species.

“The most immediate need is to now expand the spectrum of action of the BinAB toxin to counter the progression of Zika, in particular,” said Jacques-Philippe Colletier, PhD, a scientist at the Institut de Biologie Structurale in France, in the news release. “BinAB is already effective against Culex [carrier of West Nile encephalitis] and Anopheles [carrier of malaria] tos. With the results of the study, we now feel more confident that we can design the protein to target Aedes mosquitoes.”

This is a reposting of my Scope blog story, courtesy of Stanford School of Medicine.

Researchers Study How Metal Contamination Makes Gasoline Production Inefficient

SSRL X-rays are focused to illuminate a small sample of catalysts inside a movable cylindrical holder. A lens magnifies the resulting sample image onto a screen, a CCD camera captures the 2-D image, and software is used to reconstruct a 3-D image of the single catalyst particle from a series of these 2-D images. (Florian Meirer/Utrecht University)
SSRL X-rays are focused to illuminate a small sample of catalysts inside a movable cylindrical holder. A lens magnifies the resulting sample image onto a screen, a CCD camera captures the 2-D image, and software is used to reconstruct a 3-D image of the single catalyst particle from a series of these 2-D images. (Florian Meirer/Utrecht University)

Scientists at SLAC and Utrecht University have identified key problems in the crude oil refining process in an effort to increase the production yield of gasoline.

Their recent experiments at SLAC National Accelerator Laboratory studied catalysts that crack apart the long-chain hydrocarbons in crude oil into smaller, more valuable hydrocarbons like gasoline. The efficiency of this refinement process decreases as the catalysts age.

The researchers used X-ray beams at SLAC’s Stanford Synchrotron Radiation Lightsource to image whole catalyst particles and their internal structure with high resolution – like taking a landscape photograph where you can see a panoramic view and zoom in to see the ants.

To learn more about this research, check out my communications article for SLAC National Accelerator Laboratory.

Researchers at SLAC Study Promising Alternative to Morphine

Morphine is a common narcotic pain reliever with significant side effects (sfxeric/Flickr).
Morphine is a common narcotic pain reliever with significant side effects (sfxeric/Flickr).

Morphine is a powerful narcotic, commonly used to treat moderate to severe pain from surgery, injury and chronic health conditions like cancer or osteoarthritis. However, morphine has many negative side effects. It can cause drowsiness, nausea, vomiting, dizziness and constipation. More troubling, people can become dependent on it.

According to the Centers for Disease Control and Prevention, narcotic dependence and overdose deaths are a growing health problem in the United States. Narcotic sales quadrupled from 1999 to 2010 and narcotic-related deaths more than tripled from 1999 to 2012. More than 2 million people in the U.S. currently abuse narcotics.

In order to address this problem, researchers have long searched for alternative drugs that effectively relieve pain without inducing dependence as morphine does. A team of researchers now believe they’ve found a good candidate for such a drug, although it will be a long time before this drug, if it proves to be effective, is available in stores.

These researchers are studying their new kind of pain reliever using an ultrabright, ultrafast X-ray source called the Linac Coherent Light Source (LCLS), which is located at SLAC National Accelerator Laboratory.

The LCLS creates the brightest X-ray source on the planet, which researchers need to accurately study the structure of tiny, nanoscale drug compounds.

How Narcotics Work

Narcotics are also called opioid pain relievers, because they block the sensation of pain by binding to opioid receptors within the pain pathway of the brain. A drug can provide pain relief by binding to one of three types of opioid receptors in the brain: delta, mu and kappa. Narcotics like morphine target the opioid mu receptors.

The problem is that long-term use of morphine reduces the number of available opioid mu receptors. As a result, tolerance develops so a higher dose of the drug is needed to achieve the same level of pain relief. Ultimately, prolonged use leads to physical dependence — which is when the neurons adapt to the presence of the drug and function normally only when it’s in the body.

Previous research has shown that administering morphine with another drug that simultaneously blocks the opioid delta receptors prevents this morphine-induced tolerance and dependence. Researchers just need to find the right drug combination with minimal side effects.

Recently, a team of researchers, led by University of Southern California chemistry professor Vadim Cherezov, developed a drug that activates opioid mu receptors while blocking opioid delta receptors. Their drug is derived from a specialized peptide – a naturally occurring chain of amino acids. Their research results were reported in the February issue of Nature Structural and Molecular Biology.

Studying Tiny Crystals

Unfortunately, it is very difficult to study new opioid compounds like the one Cherezov recently developed.

In order to understand the structure of new compounds, scientists usually grow crystals of the compound and then hit them with X-rays. By measuring the angles and intensity of how the X-rays bounce off the crystals, they can produce a three-dimensional picture of the crystal structure.

However, it can be difficult to grow crystals large enough to use this standard method, called X-ray crystallography. Plus, you typically need to freeze the crystal to make it more rigid, rather than study it in natural conditions and temperatures. On top of that, a conventional X-ray beam might blast and destroy the small crystal before you can collect enough data.

Instead, Cherezov’s team used the Linac Coherent Light Source to perform their experiment on tiny crystals at room temperature. LCLS X-ray pulses last just a quadrillionth of a second, or 100 times faster than it takes light to travel the width of a human hair. Yet they are a billion times brighter than a conventional X-ray source.

Using this unique X-ray source with higher-intensity, very short pulses, Cherezov was able to use smaller crystals and still collect terabytes of structural data before the crystals vaporized.

Each crystal the research team prepared was a millionth of a meter and contained many copies of their new opioid compound bound to an opioid receptor. The team placed the crystals in a toothpaste-like gel to simulate the receptors’ natural environment, and then injected a thin stream of this gel into the path of the LCLS X-ray beam.

The resulting structural model was precise enough to show how the new drug molecules bind with the receptor. This atomic-scale map should help scientists develop pain-relieving drugs with fewer negative side effects. It’s likely to to take years, though, before it can be tested in humans.

“This work will provide a solid basis for the design of a new generation of pain relievers with reduced dependency,” Cherezov said in a press release.

Cherezov’s experiment is just one of many performed at the Linac Coherent Light Source.

The LCLS is a Department of Energy User Facility where approximately 600 scientists conduct groundbreaking experiments each year, across many fields, including chemistry, biology, material science, technology and energy science.

This is a repost of my KQED Science blog.

Ultrafast Laser Synchronization Breakthrough

AMO experiment schematic
Set-up of a nitrogen pulse-pump experiment that uses pulse arrival time information from a cross-correlator mounted downstream from the experiment. Figure courtesy of SLAC National Accelerator Laboratory.

A journal article, just published in Applied Physics Letters, details a major breakthrough for experiments at SLAC’s Linac Coherent Light Source (LCLS).

LCLS delivers intense ultrashort x-ray pulses that can be used to study the motion of atoms as they respond to external triggers, such as an optical laser. In these “pump-probe” experiments, the optical laser “pump” pulse starts a reaction in the material, while the x-ray “probe” pulse investigates the state of the material after a defined time delay. A sequence of x-ray pulses, with different time delays between the laser and x-ray pulses, is used to “film” the reaction in the material.

LCLS ultrafast x-ray pulses basically act like high-speed flashes of a camera strobe, allowing scientists to capture images with a “shutter speed” of less than 100 femtoseconds – the time it takes light to travel the width of a human hair.

In order to be able to “film” optically-induced ultrafast processes, however, scientists need more than just ultrashort x-ray and laser pulses. They also need to synchronize the x-ray pulses to the optical laser pulses with almost femtosecond accuracy, in order to have snapshots with good time resolution (“sharp focus”). This is a major challenge, since the main laser system for the x-ray free electron laser is a kilometer away from the optical laser and experiment.

State-of-the-art synchronization is performed at LCLS by accurately measuring the arrival times of the electron bunches (and corresponding x-ray pulses) relative to the radiofrequency that drives the accelerator, since the optical laser is locked to this reference radiofrequency. The best time resolution so far achieved with this approach is 280 fs (full width at half maximum, FWHM).

Recently, scientists at the LCLS Atomic, Molecular and Optical Science Instrument (AMO) dramatically improved the time resolution for their pump-probe experiments. Their new synchronization strategy is to directly measure the relative arrival time of both the x-ray and optical laser pulses at the experiment on a shot-by-shot basis. They do this by introducing into the x-ray beam what they call a cross-correlator, which is mounted downstream of the main experiment.

AMO scientists split their laser beam, sending it to both a pump-probe experiment and the cross-correlator (with a time delay). In the cross-correlator, the laser beam is reflected off a Si3N4 thin film. The spot of the laser pulse is then imaged with a long-distance microscope on a CCD camera. X-ray pulses also hit the same surface of the Si3N4 film, quasi-instantaneously changing the surface reflectivity.

The x-ray pulse very briefly changes the surface reflectivity. By imaging and measuring the position of this reflectivity change with the reflected laser, AMO scientists can directly measure the relative arrival time of the x-ray and optical laser pulses at their experiment. The scientists then use this pulse arrival time information from the cross-correlator to correct their corresponding experimental data on a shot-by-shot basis.

The AMO team demonstrated their improved time resolution with a nitrogen pump-probe experiment. With the time information from the cross-correlator, they were able to decrease the time resolution of their nitrogen experiment down to only 50 fs (FWHM). That’s almost down to the theoretical limit, allowing scientists to investigate all sorts of new ultrafast science.