Accelerating structure-based design of rapid uncharged reactivators of organophosphate- inhibited human acetylcholinesterase by joint X- ray/neutron mechanistic studies Andrey Kovalevsky Neutron Scattering Division Oak Ridge National Laboratory Oak Ridge, TN ORNL is managed by UT-Battelle, LLC for the US Department of Energy 1 Open slide master to edit 1
Talk outline New crystal form of hAChE amenable for growing neutron- diffraction quality crystals Low- and room-temperature X-ray crystallography of RS-170B complexes Low-temperature structures of RS194B complexes Neutron vibrational spectroscopy of ligand-free and paraoxon- conjugated hAChE Design of uncharged bis-oximes 2 Open slide master to edit 2
Limitations of oxime therapy Many oximes have low penetration levels across the blood-brain barrier Aged conjugates are resistant to current oxime reactivators Half-lives of aging for different conjugates: Slow reactivation rates (a few reactions per minute) For soman-hAChE ~ 2 min For sarin-hAChE several hours Reactivation efficiency depends on structures of OPs and reactivators For VX-hAChE over 30 hours HI-6 is an efficient reactivator of AChE conjugated to sarin but not to tabun RS2-170B is more effective than HI-6 for soman conjugate Kovalevsky et al. 2016 Ann. N.Y. Acad. Sci. 1378 , 41-49 3 Open slide master to edit 3
The new crystal form of hAChE lack of glycosylation on Asn350 allows a different type of crystal packing a = 126 Å b = 126 Å c = 131 Å P3 1 Gerlits et al. 2019 Chem. Biol. Interact. 309 , 108698 4 Open slide master to edit 4
The new crystal form of hAChE face-to-face and 4-helix bundle dimers are formed 5 Open slide master to edit 5
RS-170B Gerlits et al. 2019 J. Biol. Chem. 294 , 10607-10618 6 Open slide master to edit 6
Room-temperature structures hAChE:RS-170B VX-hAChE:RS-170B E334 O P O CH 3 W236 O CH 3 2.8Å 2.9Å O Y337 CN H447 O 2.6Å S203 Cl – 3.4Å W286 G = +6 kcal/mol Y124 W86 Y72 2.8 Å resolution 2.8 Å resolution RS-170B Gerlits et al. 2019 J. Biol. Chem. 294 , 10607-10618 7 Open slide master to edit 7
Room-temperature structures 8 Open slide master to edit 8
Low-temperature structures hAChE:RS194B VX-hAChE:RS194B Gorge entry Gorge entry Gorecki et al. 2020 J. Biol. Chem. 295 , 4079-4092 9 Open slide master to edit 9
POX conjugation alters acyl pocket loop conformation Glu334 Trp338 His447 Phe295 Ser203 Arg296 Trp86 Acyl loop: Residues 287-299 Phe297 Trp286 10 Open slide master to edit 10
Protein vibrational dynamics on the picosecond timescale POX-hAChE hAChE Time Length Neutrons are unique for probing protein H dynamics – CNCS instrument at SNS, ORNL both spatial and temporal resolution . 11 Open slide master to edit 11
Protein vibrational dynamics on the picosecond timescale Amplitude Inelastic Pitch Elastic POX global global local local some 12 Open slide master to edit 12
Design of uncharged bis-oximes 13 Open slide master to edit 13
Low-temperature structure of hAChE:LG-703 complex 14 Open slide master to edit 14
Gorge reshaping due to LG-703 binding 15 Open slide master to edit 15
Design of uncharged bis-oximes Gorecki et al. 2020 J. Biol. Chem. 295 , 4079-4092 16 Open slide master to edit 16
Neutrons reveal atomic details His 0 His + Tyr Arg + Scattering length Lys 0 of D D + Lys + Gln D 3 O + Water 17 Open slide master to edit 17
Crystal growth of hAChE for neutron diffraction hAChE:RS194B hAChE:RS194B hAChE:RS2-170B Neutron diffraction image was collected on LADI instrument at the Institut Laue Langevin (Grenoble, France) for 16 hours. hAChE:4K-TMA 18 Open slide master to edit 18
In search for better diffracting crystals – towards micro gravity growth A. Pre-flight PCG-6 SpX-11 mission All hardware transferred to a +12°C refrigerator following hardware integration. The flight hardware turned over to Cold Stowage on May 30, 2017 and loaded into the DCB on the same day. 19 Open slide master to edit 19
Acknowledgements ORNL: Dr. Lukáš Górecki Department of Toxicology and Military Pharmacy Faculty of Military Health Sciences Dr. Oksana Gerlits (U of Tennessee, University of Defense Knoxville) Dr. Kevin Weiss (CSMB) Biomedical Research Center IMAGINE: University Hospital Prof. Flora Meilleur UCSD: 500 05 Hradec Kralove Dr. Dean Myles Czech Republic Malcolm Cochran Prof. Zoran Radic Dr. Killian Oukoloff MaNDi: Professor Carlo Ballatore Dr. Leighton Coates SSPPS, UCSD CNCS: Dr. Georg Ehlers Bianca Stephanie Yunshen Celine Pomar Luedtke Bojo Li Funding: NIH CounterAct Ohio State U: U of Utah: Prof. Donald Blumenthal Prof. Xiaolin Cheng 20 Open slide master to edit 20
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