Pre-clinical validating of RAR-M NVG-645 for disease-modification of ALS

Paul Chazot¹, Andrew Whiting¹, Peter McCaffery^2
1Durham University, ²Aberdeen University

Introduction/Background & aims The initial suggestion that RAR (retinoid receptors) ligands may be therapeutic for ALS arose from the increased evidence for disrupted retinoid signalling and deficiency in the disease pathology. Our novel approach involves retinoic acid receptor-modulators (RAR-M), with both genomic activity (known effects of retinoids), together with non-genomic activity through cytoplasmic RARs that, for instance, activate ERK1/2 kinase and control translation. Our innovation involves design and synthesis of RAR-M, with optimal potency against both activities, which have potential as ALS therapeutics. We recognised that dual-acting RAR-M are necessary, activating gene transcription, but also controlling multiple, rapid, non-genomic pathways. Most existing RAR-modulators are lipophilic, allowing penetration of the CNS; however, excessively lipophilic drugs are often non-selective and poorly soluble. Molecular design allowed us to retain sufficient amphiphilic properties for optimal permeability and solubility, as to ensure good CNS exposure. Starting with ATRA, we designed lipophilic, stable RAR-M prototype EC23; then a library of 70 new chemical entities, identifying NVG645, which has ideal properties and is our lead candidate.

Method/Summary of work Maximal RAR-M neurotrophic activity requires both genomic and non-genomic activity. Supporting growth of neurons is a key property for ALS therapeutics, hence, we screened our library using neurite outgrowth in human neuronal SH-SY5Y cells to determine neurotrophic activity. We have then tested the ability of NVG645 at 10nM to  to support neurite outgrowth in human motor neurons (derived from iPSCs) and mouse clonal motor neurons, protect versus glutamate-mediated neurotoxicity, modulate expression of AMPAR subunit proteins, and neuroprotective and inflammatory genes, and finally the numbers and sizes of stress granules in vitro, using standard immunobiochemical and qPCR methods. ADMET studies were performed for lead drug NVG645 versus ATRA properties, including potency, metabolic stability, solubility, selectivity, permeability/efflux and minimal toxicity.

Results/Discussion Compared to control, NVG-645 significantly induced neurite outgrowth in human clonal SH-SY5Y cells , and clearly demonstrating the ability to induce growth of human and mouse motor neurons at low concentrations. NVG-645 (10 nM) up-regulated AMPA receptor subunits (GluA1 and GluA2), and ameliorated glutamate-induced excitotoxicity of primary cortical neurons, a proposed mechanism of neuronal death in ALS. Furthermore, we find our novel RAR-M NVG645: (i) has potent neurotrophic activity in human neural cell lines and iPSC derived motor neurons; (ii) supported neuromuscular function in an ex vivo rat assay; and (iii) induced neuroprotective and repress inflammatory genes and proteins. Furthermore,  NVG0645 reduces the number of stress-induced stress granules (SGs) in neuronal cells: NVG0645 at 10 nM reduces in number oxidative stress induced SGs; particularly importantly, this includes large harmful (>100 mM size) SGs. We have shown this for the first time for any RAR ligand – indeed very few drugs of any type have this activity. Crucially, NVG645 displays selectivity for RARß and RARγ receptors, little affinity for RXRs, and has excellent drug pharmacodynamics and pharmacokinetic properties, superior to ATRA. In terms of target engagement, NVG645 has very high blood-brain barrier penetration into the spinal cord and frontal cortex, no efflux liability, and concentrates in the spinal cord and motor cortex, major sites of motor neuron death in ALS. 

Conclusion(s) Herein, we report an increasing selection  of evidence using a range of neuronal cell culture and in vivo rodent models, that NVG645 displays excellent pharmacodynamic and pharmacokinetic properties, superior to ATRA, and crucially, the three key “N” properties required by a disease-modifying drug, for a wide range of complex neurodegenerative diseases, including ALS, AD and PD, namely Neuroprotection, Neuroplasticity and Neurorepair.

Reference(s)

1. Retinoic acid receptor-targeted drugs in neurodegenerative disease. Clark JN, Whiting A, McCaffery P. Opin Drug Metab Toxicol. 2020 Nov;16(11):1097-1108

2. Design of synthetic retinoids. Chisholm DR, Whiting A. Methods Enzymol. 2020;637:453-491.

3. Genomic and non-genomic pathways are both crucial for peak induction of neurite outgrowth by retinoids. Khatib T, Marini P, Nunna S, Chisholm DR, Whiting A, Redfern C, Greig IR, McCaffery P. Cell Commun Signal. 2019;17:40.