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

Aida Attar,Frank-Gerrit Klärner,Gal Bitan,Thomas Schrader,Wai-Ting Coco Chan

doi:10.1186/2050-6511-15-23

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.

Highlights

The “molecular tweezer” CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues
We reported that the molecular tweezer, CLR01, is a novel, broad-spectrum inhibitor of abnormal protein self-assembly, which acts by a “process-specific” mechanism and inhibits the aggregation and toxicity of multiple amyloidogenic proteins [3,4,5]
To further examine the putative process-specific mechanism and toxicity profile of CLR01, here we evaluated the effect of the compound in vitro on a physiologic protein self-assembly process—tubulin polymerization—and in vivo using wild-type (WT) mice to which CLR01 was administered at high doses either as a one-time bolus or daily for 1 month

Summary

Introduction

The “molecular tweezer” CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. Inhibition of Recently, we reported that the molecular tweezer, CLR01, is a novel, broad-spectrum inhibitor of abnormal protein self-assembly, which acts by a “process-specific” mechanism and inhibits the aggregation and toxicity of multiple amyloidogenic proteins [3,4,5]. Lys is the only proteinaceous amino acid side-chain that effectively forms both types of interactions – hydrophobic interactions involving the butylene chain, and Coulombic attraction/repulsion of its ε-NH3+ group. Both types of interactions are important in aberrant protein self-assembly. CLR01 competes for the same interactions that are key to nucleation and aggregation by most amyloidogenic proteins [11,12]

Methods

Results

Discussion

Conclusion