Abstract
Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm. Despite demonstration of their efficacy in multiple cellular and animal models, important questions regarding their mechanism of action, including cell penetrance and intracellular distribution, have not been answered to date. The main impediment to answering these questions has been the low intrinsic fluorescence of the main compound tested to date, called CLR01. Here, we address these questions using new fluorescently labeled molecular tweezers derivatives. We show that these compounds are internalized in neurons and astrocytes, at least partially through dynamin-dependent endocytosis. In addition, we demonstrate that the molecular tweezers concentrate rapidly in acidic compartments, primarily lysosomes. Accumulation of molecular tweezers in lysosomes may occur both through the endosomal-lysosomal pathway and via the autophagy-lysosome pathway. Moreover, by visualizing colocalization of molecular tweezers, lysosomes, and tau aggregates we show that lysosomes likely are the main site for the intracellular anti-amyloid activity of molecular tweezers. These findings have important implications for the mechanism of action of molecular tweezers in vivo, explaining how administration of low doses of the compounds achieves high effective concentrations where they are needed, and supporting the development of these compounds as drugs for currently cureless proteinopathies.
Highlights
Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm
The concentration dependence of the fluorescence of the two compounds was assessed by a simple titration, which showed a linear increase of the fluorescence with concentration, as expected, both for the fluorescent appendage, TAMRA at 535 nm or FAM at 518 nm (Supplementary Fig. 1a, b, respectively), and for the horseshowlike structure of the molecular tweezers (MTs) (335 nm)
The plateau CLR16 fluorescence was reduced by nearly 30% in cytoplasm, and 40% in the lysosomes in the presence of 40-fold excess CLR01. These results indicated that only part of the reduction in fluorescence could be attributed to the quenching observed in cell-culture medium, suggesting that CLR01 competed with CLR16 for entry into the cells and colocalization with lysosomes
Summary
Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm Despite demonstration of their efficacy in multiple cellular and animal models, important questions regarding their mechanism of action, including cell penetrance and intracellular distribution, have not been answered to date. A key feature contributing to the low toxicity of CLR01 is its highly labile binding to Lys (and Arg) residues[11] and the fact that these residues tend to be more exposed in unstructured and misfolded polypeptides than in natively folded proteins[3] These characteristics allow CLR01 to disrupt effectively the relatively weak hydrophobic and electrostatic interactions mediating abnormal protein self-assembly, without affecting normal physiological processes.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
