Identification of Allosteric Ligands of N-methyl-D-aspartate receptor GluNR1: Potential Neuroprotective Treatment for Acute Ischaemic Stroke (AIS)

Acute ischaemic stroke (AIS) is a global burden. In the
UK, stroke is still one of the leading causes of death and
nearly 2/3 of stroke survivors leave hospital with a
disability. Effective neuroprotective drugs can meet the
significant need for AIS management by protecting
patients prior to a stroke insult, and aiding them in
recovery. Excitatory neurotransmission conducted by the
N-methyl-D-aspartate (NMDA) ionotropic glutamate
receptor is essential for the development and function of
the central nervous system (CNS). However, excessive
stimulation of these membrane receptors has been
implicated as one of the key contributors of neural injury
in AIS and several other neurodegenerative diseases.
Our aims are to use rational drug design and structure
based virtual screening methods to design small
molecule inhibitors which will target a short peptide
sequence of the ligand binding domain of the NMDA
receptor. This novel site was identified to bind to
antibodies and protect against ischaemic stroke in rodent
models. Using in silico methods we have identified
compounds that interact well with the peptide sequence.
Several compounds were tested for their efficacy and
safety profiles in in vitro assay that induces NMDAR
excitotoxicity such as LDH cytotoxicity assay using
primary cortical neurons from mice. In addition, we also
used the high content imaging analysis system
(Columbus) to assess nuclear morphology of the cells
when exposed to different concentrations of compounds
in presence of the toxin. We have completed the virtual
screening segment of the project, screening over ~25,000
compounds from commercial vendors and in-house
libraries. We have narrowed these down to 27
compounds that interact well with the novel site of
NMDAR, and tested them in our in vitro model. Two of
these compounds were shown to be neuroprotective with
reduction of cell death up to 10% at 10µM concentrations.
These properties are useful to characterise further in the
binding assay as well as in in vivo experiments to further
understand the mode of action of the compounds