Accelerating structure-based design of rapid uncharged reactivators - - PowerPoint PPT Presentation

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Accelerating structure-based design of rapid uncharged reactivators - - PowerPoint PPT Presentation

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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

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ORNL is managed by UT-Battelle, LLC for the US Department of Energy

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

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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

  • f ligand-free and paraoxon-

conjugated hAChE

 Design of uncharged bis-oximes

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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

Limitations of oxime therapy

Kovalevsky et al. 2016 Ann. N.Y. Acad. Sci. 1378, 41-49

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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 P31

Gerlits et al. 2019 Chem. Biol. Interact. 309, 108698

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The new crystal form of hAChE

face-to-face and 4-helix bundle dimers are formed

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Gerlits et al. 2019 J. Biol. Chem. 294, 10607-10618

RS-170B

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hAChE:RS-170B VX-hAChE:RS-170B

O P O CH3 O O CH3 CN O

2.8 Å resolution 2.8 Å resolution

2.8Å 2.6Å 3.4Å 2.9Å

W86 Y124 Y72 Y337 S203 H447 E334 W236 W286 Cl–

Room-temperature structures

G = +6 kcal/mol

Gerlits et al. 2019 J. Biol. Chem. 294, 10607-10618

RS-170B

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Room-temperature structures

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Low-temperature structures

hAChE:RS194B VX-hAChE:RS194B

Gorge entry Gorge entry

Gorecki et al. 2020 J. Biol. Chem. 295, 4079-4092

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His447 Trp86 Trp286 Trp338 Arg296 Phe297 Phe295 Ser203 Acyl loop: Residues 287-299 Glu334

POX conjugation alters acyl pocket loop conformation

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Neutrons are unique for probing protein H dynamics – both spatial and temporal resolution.

POX-hAChE hAChE Length Time

Protein vibrational dynamics on the picosecond timescale

CNCS instrument at SNS, ORNL

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some

Elastic Inelastic global global local local

Protein vibrational dynamics on the picosecond timescale

Pitch Amplitude

POX

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Design of uncharged bis-oximes

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Low-temperature structure of hAChE:LG-703 complex

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Gorge reshaping due to LG-703 binding

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Design of uncharged bis-oximes

Gorecki et al. 2020 J. Biol. Chem. 295, 4079-4092

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His0 His+ Lys0 Lys+ Arg+ Gln Tyr Water D3O+ D+

Scattering length

  • f D

Neutrons reveal atomic details

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Neutron diffraction image was collected on LADI instrument at the Institut Laue Langevin (Grenoble, France) for 16 hours.

Crystal growth of hAChE for neutron diffraction

hAChE:RS194B hAChE:RS194B hAChE:RS2-170B hAChE:4K-TMA

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  • A. Pre-flight

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.

PCG-6 SpX-11 mission In search for better diffracting crystals – towards micro gravity growth

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ORNL:

  • Dr. Oksana Gerlits (U of Tennessee,

Knoxville)

  • Dr. Kevin Weiss (CSMB)

IMAGINE:

  • Prof. Flora Meilleur
  • Dr. Dean Myles

Malcolm Cochran MaNDi:

  • Dr. Leighton Coates

CNCS:

  • Dr. Georg Ehlers

Acknowledgements

Funding:

NIH CounterAct

  • Dr. Lukáš Górecki

Department of Toxicology and Military Pharmacy Faculty of Military Health Sciences University of Defense Biomedical Research Center University Hospital 500 05 Hradec Kralove Czech Republic

  • Dr. Killian Oukoloff

Professor Carlo Ballatore SSPPS, UCSD

Bianca Pomar Stephanie Luedtke Yunshen Li Celine Bojo

UCSD:

  • Prof. Zoran Radic

U of Utah:

  • Prof. Donald Blumenthal

Ohio State U:

  • Prof. Xiaolin Cheng