Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules

Kinetochore-microtubule attachments represent a fundamental requirement for the accurate segregation of genetic material during the complex process of eukaryotic cell division. Prior research has shown that the conserved Ndc80 protein complex serves as the primary physical link between the mitotic spindle and the chromosomal kinetochore structure. The dynamic nature of these attachments necessitates a highly regulated system where microtubule binding can be reversibly inhibited to facilitate physiological error correction. Maintaining genomic stability depends heavily on the ability of the cell to resolve improper connections before the final stages of chromosome separation occur. Small molecule inhibitors capable of modulating these specific protein-protein interactions offer significant potential for both fundamental mechanistic research and the development of novel therapeutic interventions. Despite the biological importance of this interaction, the availability of chemical tools that can precisely target the Calponin-homology (CH) domain of the protein remains insufficient. This absence of evidence motivated the current investigation into supramolecular strategies for disrupting these essential molecular interfaces.

This investigation develops a novel class of rationally designed inhibitors for the Ndc80 Calponin-homology (CH) domain by leveraging advanced principles of Supramolecular Chemistry. The researchers sought to construct lysine-specific molecular tweezers that could effectively compete with microtubule filaments for binding sites on the surface of the protein. The experimental design focused on a multiple-click approach to assemble these supramolecular units into various multivalent configurations ranging from covalently fused dimers to pentamers. Each synthesized construct possessed a unique overall size, degree of preorganization, and structural stiffness to determine the optimal parameters for protein surface recognition. The primary objective involved identifying specific multivalent architectures that could disrupt the interaction between the protein and microtubules without interfering with other cellular processes. The team also aimed to elucidate how the spatial arrangement of these tweezers influences their ability to target multiple lysine residues simultaneously across the protein. By characterizing these interactions, the study intended to establish a framework for the design of high-affinity binders for challenging protein-protein interaction interfaces.

The research team utilized a multiple-click chemical approach to synthesize a library of multivalent molecular tweezers consisting of covalently fused dimers, trimers, tetramers, and pentamers. These supramolecular assemblies were engineered with varying degrees of structural preorganization to evaluate how conformational rigidity impacts the efficiency of protein binding. Nuclear Magnetic Resonance (NMR) spectroscopy provided a high-resolution method for identifying the specific lysine residues on the protein surface that interact with the tweezers. Enhanced sampling Molecular Dynamics (MD) simulations were conducted to provide a detailed mechanistic rationale for the binding modes observed during the experimental assays. These computational models allowed the scientists to analyze the role of secondary interactions and spatial preorganization in targeting multiple sites on the protein surface. The investigators performed biochemical assays to measure the disruption of the protein-microtubule interaction at low micromolar concentrations while monitoring the stability of the filaments. Statistical frameworks were applied to the data to compare the binding affinities of the different multivalent constructs and identify the most effective inhibitor designs.

Two specific dimers and one trimer were identified as the most efficient binders of the Ndc80 Calponin-homology (CH) domain among the synthesized supramolecular library. These multivalent molecular tweezers successfully disrupted the interaction between the protein and microtubule filaments when applied at low micromolar concentrations in the experimental setup. The results demonstrated that the inhibition of the protein-microtubule complex occurred without causing any measurable changes to the underlying dynamics of the microtubule filaments themselves. Nuclear Magnetic Resonance (NMR) spectroscopy data pinpointed lysine (Lys) residues 160 and 204 as the preferred sites for the interaction between the tweezers and the protein. Molecular dynamics simulations revealed that the degree of pre-organization in the multivalent constructs significantly influences their ability to bind multiple lysine residues effectively. The analysis showed that secondary interactions between the tweezers and the protein surface contribute to the overall stability and specificity of the binding event.

The successful disruption of the Ndc80-microtubule interaction by multivalent molecular tweezers establishes a new paradigm for the rational design of protein-protein interaction inhibitors. These findings suggest that supramolecular chemistry can provide highly specific tools for investigating the complex mechanisms of chromosome segregation during eukaryotic cell division. The identification of lysine residues 160 and 204 as key interaction sites highlights the potential for targeting specific surface-exposed amino acids in drug development. The researchers conclude that the principles of multivalent binding and structural preorganization are essential for creating effective inhibitors of large protein-protein interfaces. Future applications of this technology may include the development of therapeutic agents designed to correct errors in the distribution of genetic material during mitosis. The study provides a foundation for exploring how similar supramolecular constructs could be adapted to target other proteins containing calponin-homology domains or similar motifs.