X-ray crystallography brings pain pathways into view

X-ray crystallography brings pain pathways into view

February 16, 2015

February 16, 2015

Powerful opiate drugs are a mainstay in modern medicine, alleviating pain in both acute and chronic forms. These charms, however, bear a curse. Users quickly develop tolerance to their effects, requiring ever-increasing doses of the drug. Further, such opioid compounds lead to drug dependence, owing to their notoriously addictive qualities.

In a first of its kind study, Petra Fromme, Ph.D., a researcher at Arizona State University’s Biodesign Institute joins an international team using techniques of X-ray crystallography with high-speed lasers to home in on the detailed structure of opioid receptors and synthetic drugs that bind to these sites.

Their efforts pave the way for the development of powerful new analgesics, capable of blocking pain without generating tolerance or dependency. Their research findings appear in the current issue of the journal Nature Structural and Molecular Biology.

The international research group was led by Gustavo Fenalti (formerly of the Scripps Research Institute, now with Celgene Corporation, San Diego, California),  and includes researchers from the laboratory of Raymond C. Stevens, Ph.D., at the University of Southern California, as well as members of the SLAC National Accelerator Laboratory, Center for Free Electron Laser Science, Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany and others.

Researchers from the laboratory of Raymond C. Stevens, Ph.D., at the University of Southern California, as well as members of the SLAC National Accelerator Laboratory, Center for Free Electron Laser Science, Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany and others contributed to the international undertaking.

Fromme, director of Biodesign’s newly established Center for Applied Structural Discovery highlights the importance of the present study:

“Serial femtosecond crystallography permits detailed examination of vital biochemical details that have long eluded proper study. In this case, revealing the subtle interaction of a human opioid receptor with a binding peptide is a critical step for understanding the pharmacological profile of opioid drugs. The research opens the door to a new generation of improved treatments for pain.”

Ancient friend and foe

Opioids figure among the oldest known drugs, their therapeutic uses dating to prehistory. Such compounds are structurally similar to morphine and other natural alkaloid derivatives of the opium poppy. They work by binding to various opiate receptors, located primarily in the central and peripheral nervous systems and the gastrointestinal tract. While this much is known, many mysteries regarding their precise mode of action (and their troublesome side-effects) are still shrouded in mystery.

Opioid receptors belong to a large protein family known as G protein–linked receptors. These sensing molecules outside of cells trigger a cascade of cellular responses that affect the brain.

When an opioid binding agent or ligand binds with a receptor, the result is a dramatic attenuation of pain, often accompanied by a sense of intense euphoria, (a fact accounting for the popularity of opioid drugs, including opium and heroin, for recreational use and abuse).

Fromme’s group has a led a major initiative to better understand this protein family using a powerful new X-ray laser technology.

Three primary opioid receptors, labeled mu (μ), delta (δ), and kappa (κ) are known to bind with various naturally-occurring opioids produced by the body. These endogenous opioid ligands include endomorphins, enkephalins and dynorphins, (which bind with μ, δ and κ receptors, respectively). A more thorough understanding of endogenous opioids and their receptors is essential for drug discovery of new pain analgesics with more desirable properties.

Researchers hope to create novel synthetic opiate ligands, capitalizing on their powerful analgesic properties while reducing or eliminating side-effects. Although drugs like morphine are known to primarily engage the μ opioid receptors, intriguing evidence suggests that subtle interactions between the μ and δ receptors are responsible for opioid tolerance and dependence.

The current study examines a particular opioid ligand known as DIPP-NH2, which has already shown considerable promise as a tolerance-free, dependence-free analgesic. The effect appears to be due to DIPP-NH2’s ability to simultaneously act as a μ receptor agonist and a δ receptor antagonist. This means that the ligand binds with the μ receptor and blocks binding events at the δ receptor. For this reason, DIPP-NH2 is known as a bivalent ligand.

X-ray vision

A powerful method of exploring atomic and molecular scale structure involves the crystallization of a sample of interest and bombardment with a beam of incident X-rays. Crystalline atoms of the sample cause the incoming X-rays to diffract in many directions. Once the angles of diffraction are carefully measured, they may be assembled into a three-dimensional portrait of electron densities within the crystal and ultimately, detailed structural and functional properties are unlocked.

X-ray crystallography has been a vital tool for revealing the structure and function of a wide variety of biological molecules, including drugs, vitamins, proteins and nucleic acids such as DNA. The technique remains a primary method for characterizing the atomic structure of new materials and their properties—key ingredients in the eventual design and validation of new pharmaceutical drugs.

One shortcoming of traditional X-ray crystallography is that X-rays can damage or destroy the delicate crystal structures under investigation. In the current study, a pathbreaking method known as serial femtosecond crystallography was used. Here, a stream of nanocrystals are exposed to an extremely brief pulses from a device known as an X-ray Free Electron Laser (XFEL).

The use of XFEL permits high-dose irradiation of the sample, due to the brevity of the X-ray pulse, lasting just a millionth of a billionth of a second. The method allows much smaller crystals to be used, capturing critical structural information before the sample is destroyed. Each sample is exposed to a single femtosecond X-ray burst, producing a single diffraction pattern, which is read by a series of detectors.

In the current study, the team used serial femtosecond crystallography to solve the structure of the bifunctional opiate ligand DIPP-NH2 bound to a human opiate receptor (δ-OR). Observation of such peptide-receptor interactions is essential for developing a complete pharmacological profile of opioid peptides and development and refinement of improved analgesic drugs.

The study first required researchers to engineer and crystalize a receptor construct combining the human opioid receptor δ-OR with the ligand DIPP-NH2. Previous work had demonstrated that subtle chemical rearrangements of opioid ligands could affect their functionality, creating potent compounds with mixed μ agonist-δ antagonist properties.

The δ-OR construct was shown to bind with its ligand with similar affinity to naturally occurring δ-OR. The XFEL method provided unprecedented structural details revealing new molecular determinants of peptide interaction with the δ-OR receptor and identifying a key structure contributing to DIPP-NH2’s bifunctional activity.

Analysis of the δ-OR–DIPP-NH2 presents a platform for the examination of numerous peptides with pharmacological properties, including new ligands for the management of pain.

For this study, professor Fromme collaborated with colleagues at Scripps Research Institute, Arizona State University, Vrije Universiteit Brussel in Belgium, University of North Carolina Chapel Hill Medical School, SLAC National Accelerator Laboratory, Deutsches Elektronen-Synchrotron in Germany, the University of Hamburg, European XFEL GmbH in Germany, and the Clinical Research Institute of Montreal in Canada.

In addition to her appointment at the Biodesign Institute, Fromme is a professor at ASU’s Department of Chemistry and Biochemistry.

 

Written by: Richard Harth

Science Writer: Biodesign Institute

 

richard.harth@asu.edu