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Mimicking synuclein toxicity in plant cells to identify novel neuroprotective leads

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 2R44NS108804-02
Agency Tracking Number: R44NS108804
Amount: $1,859,037.00
Phase: Phase II
Program: SBIR
Solicitation Topic Code: 999
Solicitation Number: PA19-272
Timeline
Solicitation Year: 2019
Award Year: 2020
Award Start Date (Proposal Award Date): 2020-09-30
Award End Date (Contract End Date): 2023-08-31
Small Business Information
KTRDC-UK 1401 UNVERSITY DR
Lexington, KY 40546-0001
United States
DUNS: 196165877
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 JOHN LITTLETON
 (859) 257-1127
 john.littleton@uky.edu
Business Contact
 DAVID GIBBS
Phone: (502) 583-7454
Email: dgibbs@blacksheepllc.com
Research Institution
N/A
Abstract

Abstract
A general mechanism for neurodegeneration, including Parkinson's disease and Alzheimer's dementia, is the
breakdown and subsequent aggregation of misfolded neuronal proteins. In the “synucleinopathies” for example
neurotoxicity is associated with the cleavage of mis-folded alpha-synuclein (ASYN), probably mainly by
asparagine endopeptidase (AEP) [Zhang et al 2017]. This generates neurotoxic peptides that then aggregate
with ASYN in lysosomes, forming the Lewy inclusion bodies associated with neurodegeneration. A very similar
mechanism exists in plant cells in which plant AEP breaks down misfolded proteins to produce vacuolar
aggregates associated with programmed cell death (PCD) [Hatsugai et al, 2015]. However, plants do not
contain ASYN, so, in order to mimic ASYN toxicity, plant cells were transformed to express the misfolding-
prone A53T variant of human ASYN (phase I). Thymoquinone was then used to trigger plant PCD [Hassanien
et al 2013] and the plant cells expressing ASYN-A53T were shown to be significantly more susceptible to this
toxicity than controls. In phase II a mutant population of these transgenic (ASYN) plant cells will be selected for
survival under this procedure. This is an example of “target-directed evolution” in which mutants that survive
selection should “evolve” toward increased biosynthesis of metabolites that inhibit AEP and/or ASYN toxicity.
Individual mutant plant cell clones with ASYN-protective activity will be identified by screening extracts of
resistant cultures, and micro-analytical methods [Kelley et al, 2019] will then be used to identify active
metabolites as leads. Lobelia cardinalis cell cultures were used in phase I because we had previously
transformed these with the human dopamine transporter gene to mimic MPP+-induced dopaminergic
neurotoxicity [Brown et al 2016]. In phase II we will also use the medicinal plant, Polygonum multiflorum, which
contains a stilbene that inhibits ASYN toxicity [Zhang et al 2018]. Phase II aims to identify novel leads that
engage the AEP and ASYN targets, and to test the most promising of these in cellular and animal models of
synucleinopathy. Leads will be developed with the University of Kentucky Parkinson's Disease Research
Center and a pharmaceutical partner. Identification of leads that engage these targets will also support target-
directed evolution in mutant plant cells as a commercial platform for drug discovery.

* Information listed above is at the time of submission. *

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