Protection of primary neurons and mouse brain from Alzheimer's pathology by molecular tweezers

Abstract

Alzheimer's disease is a devastating cureless neurodegenerative disorder affecting >35 million people worldwide. The disease is caused by toxic oligomers and aggregates of amyloid β protein and the microtubule-associated protein tau. Recently, the Lys-specific molecular tweezer CLR01 has been shown to inhibit aggregation and toxicity of multiple amyloidogenic proteins, including amyloid β protein and tau, by disrupting key interactions involved in the assembly process. Following up on these encouraging findings, here, we asked whether CLR01 could protect primary neurons from Alzheimer's disease-associated synaptotoxicity and reduce Alzheimer's disease-like pathology in vivo. Using cell culture and brain slices, we found that CLR01 effectively inhibited synaptotoxicity induced by the 42-residue isoform of amyloid β protein, including ∼80% inhibition of changes in dendritic spines density and long-term potentiation and complete inhibition of changes in basal synaptic activity. Using a radiolabelled version of the compound, we found that CLR01 crossed the mouse blood-brain barrier at ∼2% of blood levels. Treatment of 15-month-old triple-transgenic mice for 1 month with CLR01 resulted in a decrease in brain amyloid β protein aggregates, hyperphosphorylated tau and microglia load as observed by immunohistochemistry. Importantly, no signs of toxicity were observed in the treated mice, and CLR01 treatment did not affect the amyloidogenic processing of amyloid β protein precursor. Examining induction or inhibition of the cytochrome P450 metabolism system by CLR01 revealed minimal interaction. Together, these data suggest that CLR01 is safe for use at concentrations well above those showing efficacy in mice. The efficacy and toxicity results support a process-specific mechanism of action of molecular tweezers and suggest that these are promising compounds for developing disease-modifying therapy for Alzheimer's disease and related disorders.

Modulators of amyloid protein aggregation and toxicity: EGCG and CLR01

“…However, this was found not Lys residues with micromolar affinity, which is relatively weak but has been found to be sufficient for modulating the assembly and inhibit the toxicity of multiple amyloidogenic proteins without causing toxicity in cell culture unless substantially higher concentrations were used [69]. These data are consistent with in vitro enzyme inhibition studies, which required MT concentrations at least an order of magnitude higher than those needed for inhibition of amyloidogenic proteins [68,70] and with data showing beneficial effects with no associated toxicity in animal models [71,72].…”

“…In vitro studies of CLR01 inhibition of five major CYP enzymes showed IC 50 values of 1.5 μM for 2C19, 1.7 μM for 3A4, 2.2 μM for 2C9, 3.6 μM for 2D6, and >20 μM for 1A2 [71]. Inhibitory potencies are thought to have an increased risk for drug-drug interaction complications if the IC 50 is <1 μM [206], which was not found for CLR01 with any of the 5 isoforms studied.…”

“…In vitro studies of CLR01 activation of the CYP system found minimal activation up to 50 μM [71]. [207][208][209].…”

“…On the other hand, other studies have reported a decrease in the antioxidant activity of methylated catechins [217]. one hour showed 100% stability [71]. The initial plasma concentration measured following an intravenous injection of 1 mg/kg CLR01 to mice was 10-11 μM and the half-life was ~2.5 h in mice [205].…”

The Lys-Specific Molecular Tweezer, CLR01, Modulates Aggregation of the Mutant p53 DNA Binding Domain and Inhibits Its Toxicity

“…2a), disrupted in vitro the aggregation and toxicity of multiple disease-related proteins, such as amyloid β-protein (Aβ), tau and α-synuclein at equimolar or sub-equimolar concentration ratios (14), whereas disruption of tubulin polymerization required ~55-fold excess of CLR01 (15) and inhibition of enzymatic activity, e.g., of alcohol dehydrogenase, required ~850-fold excess CLR01 (16). In agreement with these observations, in vivo CLR01 suppressed α-synuclein aggregation in neurons and rescued the phenotype and survival of zebrafish embryos (17), cleared existing Aβ and tau aggregates in the brain of Alzheimer's disease transgenic mice, and decreased transthyretin aggregation in a mouse model of familial amyloidotic polyneuropathy, without any side effects (17)(18)(19). Moreover, CLR01 was found to have a high safety margin in mice (15), supporting its selective action as an inhibitor of abnormal protein aggregation.…”