Molecular tweezers prevent tau deposition in mouse brains.

The “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.

Protein regions that are involved in protein–protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein–protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular “Janus” ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein–protein interface, “freezes” one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.

Self-assembly of the microtubule-associated protein tau into neurotoxic oligomers, fibrils, and paired helical filaments, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer’s disease (AD) and other tauopathies. Modulating the self-assembly process and inhibiting formation and spreading of such toxic species are promising strategies for therapy development. A challenge in investigating tau self-assembly in vitro is that unlike most amyloidogenic proteins, tau does not aggregate in the absence of posttranslational modifications (PTM), aggregation inducers, or pre-formed seeds. The most common induction method is addition of polyanions, such as heparin, yet this artificial system may not represent adequately tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in-vitro data that do not reflect the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, methods for in-vitro phos-phorylation of tau to produce aggregation-competent forms recently have been introduced (Despres et al., Identifica-tion of the Tau phosphorylation pattern that drives its aggregation, Proc. Natl. Acad. Sci. USA, 2017). However, the oli-gomerization, seeding, and interaction with assembly modulators of the different forms of tau have not been studied to date. To address these knowledge gaps, we compared here side-by-side the self-assembly and seeding activity of heparin-induced tau with two forms of in-vitro phosphorylated tau and tested how the molecular tweezer CLR01, a negatively charged compound, affected these processes. Tau was phosphorylated by incubation either with activated extracellular signal-regulated kinase 2 or with a whole rat-brain extract. Seeding activity was measured using a FRET-based biosensor-cell method. We also used solution-state NMR to investigate the binding sites of CLR01 on tau and how they were impacted by phosphorylation. Our systematic structure-activity relationship study demonstrates that heparin-induced tau behaves differently from in-vitro phosphorylated tau. The aggregation rates of the different forms are distinct as is the intracellular localization of the induced aggregates, which resembles brain-derived tau strains suggesting that heparin-induced tau and in-vitro phosphorylated tau have different conformations, proper-ties, and activities. CLR01 inhibits aggregation and seeding of both heparin-induced and in-vitro-phosphorylated tau dose-dependently, although heparin induction interferes with the interaction between CLR01 and tau.