Abstract

NMDA receptors (NMDAR) are the main
calcium-permeant ligand-gated channels in the brain. They are activated by glutamate, the
major excitatory transmitter in the CNS. NMDA receptor-regulated neuronal
nitric oxide synthase (nNOS) is
implicated in multiple neurological conditions. The scaffold protein, PSD95,
brings nNOS close to activated NMDARs, in turn activating nNOS and
downstream signalling. Protein-protein interactions in this pathway
are attractive drug targets because NMDAR and nNOS have not been found to be tractable therapeutic targets. A peptide that uncouples PSD95 from NMDARs
was the first successful neuroprotectant in a phase II clinical trial for
treatment of stroke (1).

We previously revealed that NOS1AP
(aka CAPON) is a druggable mediator of disease-associated signalling downstream of the NMDAR/PSD95/nNOS
complex. We designed the first inhibitor of nNOS-NOS1AP interaction. This peptide disrupts the interaction and reduces brain damage in a neonatal hypoxia-ischemia
animal model (2) and suppresses neuropathic pain in mice (3). A putative prodrug inhibitor of NOS1AP-nNOS interaction is also efficaceous in models of neuropathic pain (4) and anxiety (5).

Recently we developed an ultra-high sensitivity
assay (uHS) to screen small molecule libraries for modulation of NOS1AP-nNOS interaction. Compared with other PPI
assays - fluorescence polarization (FP) and microscale thermophoresis (MST) - our uHS assay is much more sensitive, eliminating laborious recombinant protein needs, reducing costs and time. It is more reliable (z' ~0.8), reducing false positives and false negatives. The assay was validated in accordance with the plate uniformity and signal variability standards of the National Center for Advancing Translational Sciences (NCATS)
(6). This assay was first applied to i) a cherry-picked
list of hits from a 140000 compound in silico library and ii) a library of >2000
drugs and related molecules. It will now be applied to a larger library of blood brain barrier permeable inhibitors. The assay can also be used in intact living cells to
demonstrate target engagement. It is generalizable, and we have extended it
to other protein-protein interaction sites.

(1)
Hill MD, et al. Lancet Neurol. 2012 Nov;11(11):942-50.

(2) Li LL, et al. J Neurosci. 2013 May 8;33(19):8185-201.

(3) Lee WH, et al. Pain. 2018 May;159(5):849-863.

(4) Lee WH, et al. Mol Pain. 2018 Jan-Dec;14:1744806918801224.

(5) Zhu LJ, et al. Nat Med. 2014 Sep;20(9):1050-4.

(6) Iversen PW, et al. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-