Safety and pharmacological characterization of the molecular tweezer CLR01 – a broad-spectrum inhibitor of amyloid proteins’ toxicity

Abstract

BackgroundThe “molecular tweezer” CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimer’s disease and other amyloidoses, here we studied its safety and pharmacokinetics.MethodsToxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimer’s disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5 - 72 h post administration.ResultsExamination of CLR01’s effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compound’s putative “process-specific” mechanism of action.A single-injection of 100 mg/kg CLR01 in mice – 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin.The brain penetration of CLR01 was found to be 1 - 3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h.ConclusionEstimation of brain levels compared to amyloid β-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01.The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses.

Molecular tweezers for lysine and arginine – powerful inhibitors of pathologic protein aggregation

“…125 In the acute setting, administration of 100 mg/kg CLR01 (2,500-fold above the dose used in the 3×Tg mice, 83-fold above the dose in the TTR mice) initially caused obvious signs of distress to the mice (mainly freezing and hunching), which began to resolve after 30 min and resolved completely within 2 h. No mouse died until the time point of euthanasia at 24 h. Histological and serological analyses showed liver injury (Figure 9), which would be expected for such a high dose, but no damage to other tissues, including heart, lungs, kidney, or brain. 125 In the 10-mg/kg group there were no signs of distress and no significant serological findings. In one out of the 8 mice in this group there were mild signs of liver degeneration.…”

“…125 In addition to the size and negative charge of CLR01, which likely restrict its oral absorption, CLR01 may be metabolized in the gastrointestinal tract. To begin to explore this possibility, we hypothesized that the most likely metabolism of CLR01 would be dephosphorylation.…”

Molecular Tweezers Inhibit Islet Amyloid Polypeptide Assembly and Toxicity by a New Mechanism

“…36,57,60 The mechanism of action of CLR01 involves micromolar-affinity binding to K residues and interfering with hydrophobic and electrostatic interactions that are important for the initial self- assembly of amyloidogenic proteins and/or their interaction with cellular targets. 30,58,61,62 Initially, we found that CLR01 inhibited formation of β-sheet-rich IAPP fibrils and IAPP-induced toxicity. However, although we observed complete inhibition of aggregation at IAPP/CLR01 concentration ratio 10:1, inhibition of toxicity required a ratio of ∼1:100, suggesting that blocking the formation of β-sheet-rich aggregates was not sufficient for inhibition of IAPP toxicity.…”

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

“…Inhibition of aggregation by MTs is selective for amyloidogenic proteins but is not specific to a particular protein (15). For example, the MT, CLR01 (Fig.…”

“…For example, the MT, CLR01 (Fig. 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).…”