Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) - 08.23.17

Overview | Description | Applications | Operations | Results | Publications | Imagery

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Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) produces crystals of acetylcholinesterase, a medically important neurotransmitter enzyme. Crystals grown in microgravity are larger and higher-quality and can be used for the technique called macromolecular neutron crystallography (MNC) to locate hydrogen atoms in the crystal structure. These hydrogen atoms play critical roles in the enzyme function and knowing their location clarifies that function. This advances development of better antidotes to fatal Organophosphate nerve agents, which act by inhibiting acetylcholinesterase in the nervous system.
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The following content was provided by Andrey Kovalevsky, and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom:

Principal Investigator(s)
Andrey Kovalevsky, Oak Ridge National Laboratory, Oak Ridge, TN, United States

Co-Investigator(s)/Collaborator(s)
Zoran Radic, University of California San Diego, CA, United States

Developer(s)
Center for the Advancement of Science in Space (CASIS), Rockledge, FL, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Earth Benefits

ISS Expedition Duration
April 2017 - September 2017; September 2017 - February 2018

Expeditions Assigned
51/52,53/54

Previous Missions
SpX-11 is the first flight of this investigation. The Handheld HDPCG hardware has previously flown on SpX-3, SpX-4, SpX-6, SpX-8 and will fly on SpX-10.

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Experiment Description

Research Overview

  • The experiment goal of Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) is to obtain optimized crystals of a human neurotransmitter enzyme called acetylcholinesterase to determine its structure at atomic level.
  • Hydrogen atoms play critical roles in the function of acetylcholinesterase but are rarely observed in structures obtained using X-ray crystallography. Hydrogen makes up approximately 50% of the atoms in this enzyme; obtaining information about the location of the hydrogen atoms using macromolecular neutron crystallography (MNC) is critical to understanding the protein’s structure and function.
  • Organophosphates (OPs) are commonly used as pesticides, but also as chemical weapons. Human exposure to OPs results in acute poisoning manifested in salivation, tremors, respiratory paralysis, and in the case of exposure to chemical weapons usually death, unless treatments with antidotes are immediately available.
  • Through studies conducted for more than half a century on chemical compounds known as oximes, antidotes have been developed. However, their efficiency is limited and novel oximes need to be designed that react with the OP-inhibitied human acetylcholinesterase much faster. This experiment seeks to obtain a neutron crystal structure of human acetylcholinesterase in the hopes that structure leads to a new class of oximes.

Description

The specific aim of the Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) experiment is to use the microgravity environment on the International Space Station (ISS) to grow large crystals of enzyme Human Acetylcholinesterase (hAChE) in the ligand-free form amenable to neutron diffraction experiments. Macromolecular neutron crystallography (MNC) combined with X-ray diffraction in a joint X-ray/neutron approach resolves the exact coordinates of all protons in hAChE and its complexes with organophosphates and oximes that are made by soaking experiments on Earth. The results allow for the understanding of the inhibition and reactivation reaction systems and use this data to derive mechanistic criteria essential for efficient reactivation of organophosphate-conjugated hAChE. The optimal geometry required for reactivation is revealed, including position of the oxime moiety relative to the proton abstracting enzyme groups and orientation of the anchoring portion of the reactivator molecule.
 
MNC has the unique ability to locate hydrogen atoms in a macromolecular structure even at medium resolutions of 2.0-2.5Å, thus providing essential insights into enzyme mechanisms and ligand binding through direct determination of the protonation states of active site residues and direct visualization of critical hydrogen bonds. However, even with recent advances in instrumentation, MNC still requires crystals that are orders of magnitude larger than those necessary for X-ray diffraction at synchrotrons.
 
Human acetylcholinesterase is the enzyme responsible for degrading the neurotransmitter acetylcholine in synapses. Organophosphate nerve agents such as sarin, soman and VX act by inhibiting the enzyme in the peripheral and central nervous system by covalently modifying the enzyme’s active site. . The chemical modification renders acetylcholinesterase inactive, leading to a life-threatening increase of acetylcholine concentration at the synapses of the nervous system. Exposure to organophosphate nerve agents is fatal if not treated with antidotes. Antidotes work by reactivating hAChE but there are several therapeutic challenges involved, especially when used in a mass casualty scenario. These include the need for antidotes that are orally active and that can cross the blood-brain barrier. Past research yielded small molecule antidotes that reactivate hAChE, but the rates of reactivation are far slower than the catalytic rate of acetylcholine hydrolysis. By investigating the limitations for the reactivation, it would be possible to design accelerated reactivators. The rational and expeditious development of antidotes with the desired properties, particularly the ability to reactivate aged acetylcholinesterase, requires information regarding the fine detail of catalytic mechanisms of organophosphate hydrolysis on the enzyme that is currently not available.

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Applications

Space Applications
Crystals obtained on the ground have not been of sufficient size or quality for neutron crystallographic experiments aimed at locating hydrogen atoms in order to understand the structure of the enzyme. This investigation depends on the microgravity environment aboard the space station and also builds on previous research and use of the station as a long-term scientific platform.

Earth Applications
Organophosphate nerve agents act by inhibiting the enzyme acetylcholinesterase in the peripheral and central nervous system. Exposure to these agents is fatal without antidote treatment. This experiment provides insight into the molecular structures of the enzyme and the underlying limitations in reactivating it after such exposure. This could lead to antidotes that would work much faster than those currently available.

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Operations

Operational Requirements and Protocols
Handheld High Density Protein Crystal Growth (HDPCG) hardware units launch at +12°C and transfer to ISS. A crew member uses an activation tool to rotate the protein reservoir above the precipitant reservoir, allowing vapor diffusion to begin. Following experiment activation, the HDPCGs are transferred to a +10°C cold stowage asset on ISS for the experiment duration. Just prior to return, the crew member again uses the activation tool, this time to rotate the protein reservoir away from the precipitant reservoir and deactivate the experiment. The HDPCGs return at +4°C.

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Decadal Survey Recommendations

Information Pending

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Results/More Information

Information Pending

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Related Websites

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Imagery

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NASA Image: ISS052E000508 - Astronaut Jack Fischer (FE-2) works with the Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) investigation in the Japanese module of the ISS.

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NASA Image: ISS052E000503 - Astronaut Jack Fischer (FE-2) works with the Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) investigation in the Japanese module of the ISS.

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NASA Image: ISS052E000504 - Astronaut Jack Fischer (FE-2) works with the Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) investigation in the Japanese module of the ISS.

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NASA Image: ISS052E000515 - Astronaut Jack Fischer (FE-2) works with the Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) investigation in the Japanese module of the ISS.

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image
NASA Image: ISS052E000516 - Astronaut Jack Fischer (FE-2) works with the Neutron Crystallographic Studies of Human Acetylcholinesterase for the Design of Accelerated Reactivators (CASIS PCG 6) investigation in the Japanese module of the ISS.

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