COOPERATIVE RNA MODULATION TO IMPROVE IRON HOMEOSTASIS SPECIFIC TO NEURONS WHILE PROVIDING ANTI-AMYLOID EFFICACY AS A POTENTIAL ALZHEIMER’S DISEASE THERAPY

Xudong Huang, Catherine M. Cahill, Kevin J. Hodgetts, Jin Gao, Yadong Wang, Yanyan Liu, Kazuhide Hayakawa, Nigel H. Greig, Fudi Wang, Debomoy K. Lahiri and Jack T. Rogers

Department of Psychiatry-Neuroscience and Department of Radiology, Massachusetts General Hospital, Charlestown, MA, USA and Department of Neurology, Brigham and Women’s Hospital, Cambridge, MA, USA

Abstract

Our low molecular weight neurotrophin is the newly identified lead agent ‘BL-1’, a benzimidazole, that was first identified after an HTS screen for 5’UTR inhibitors of Prion Protein (PrP) mRNA conducted at the Broad Institute (Cambridge, MA). BL-1 was in the pipeline to be medicinally advanced as a selective translation blocker of the amyloid co-seeding PrP as associated with transmissible spongioform encephalopathies. However, its therapeutic profile soon became diversified when we discovered, by secondary Western blot assays, that BL-1 activated translation of the ferritin light and heavy chains to safely store iron and prevent toxic buildup of iron-catalyzed oxidative radicals. BL-1’s capacity for a protective activity in condions of iron overload was found to be specific to neurons. This agent exhibited little or no off target induction of ferritin in monocytic (THP-1) or in cervical cancer (Hela) cell lines. BL-1 did not induce ferritin expression in oligodendroglia, which exhibited constitutively highl expression of this iron storage protein. These results serve as as further proof of BL-1’s iron mediated selectivity to neuronal lineages when promoteing safe storage of intracellular Fe likely sto prevent ferroptosis and slow down cellular aging. In neurons, BL-1 was highly selective to IRE-like targets since this benzimidazole did not change the expression of a battery of metabolic proteins (i.e., GAPDH, LDH and cis-aconitase) and β-actin and β-tubulin levels were unchanged.

Early developmental Lead Pb exposure is an added epigenic risk for later stage Alzheimer’s disease progression. To address this area of therapy, we have designed fifteen novel analogs of BL-1 (K.H.), four of which were tested for their potential neuroprotective action to shield neurons from Lead (Pb) and manganese (Mn) toxicity. We routinely observed these two metals caused 30-70% reduction in cell viability by MTS assays (250 μM for 48 h). Our BL-1 analogs were then detemined to protect SH-SY5Y cells from both Mn and Pb dependent loss of viability in a dose-dependent manner (e.g., 4-methoxy 494, the 4-H 495, and pyridyl analogs 498 and 499. In particular the 494 analog of BL-1 consistently offset neurotoxicity from Pb by 75% generating increased viability compared to untreated cells at 0.05 mM concentrations of Pb during exposure. This 494 analog of BL-1 is more metabolically stable than the 4-thiomethyl group of BL-1 (e.g., no oxidation of the thiomethyl group).

The drug-like properties particularly of BL-1, and its potentially better-tolerated 494 analog, will be evaluated in vivo. BL-1 limited tau phosphorylation without influencing tau and alpha-synuclein expression in neuronal cells lines and it improved cognitive performance of mice that underwent control cortical impact injury (CCI) (CCI is known to activate an AD-like pathology of increased amyloidosis, tau phosphorylation and increase brain iron burden). In collaboration with the Laboratory for Drug Discovery and Neurodegeneration (BWH, Harvard) we will medicinally diversify BL-1 as our lead small molecule activator of ferritin translation into a drug that will combat conditions of iron overload in the brain, improve neuronal health and boost cognition in established mouse models for AD.

Supported by grants from Aria and QR Pharmaceuticals, Alzheimer’s association (Zenith), and NINDS/NIH.