Microtubule Dynamics Research Articles

Microtubule‐targeting agents for cancer treatment: Seven binding sites and three strategies

AbstractMicrotubules are pivotal in diverse cellular functions encompassing cell signaling, morphology, intracellular trafficking, and cell mitosis/division. They are validated targets for disease treatment, notably hematological cancers and solid tumors. Microtubule‐targeting agents (MTAs) exert their effects by modulating microtubule dynamics, impeding cell proliferation, and promoting cell death. Recent advances in structural biology have unveiled novel perspectives for investigating multiple binding sites and mechanisms of action used by MTAs. In this review, we first provide an overview of the intricate structure and dynamics of microtubules. Then we explore the seven binding sites and the three primary strategies (stabilization, destabilization, and degradation) harnessed by MTAs. Furthermore, we introduce the emerging domain of microtubule‐targeting degraders, exemplified by PROteolysis TArgeting Chimeras and small‐molecule degraders, which enable precise degradation of specific microtubule‐associated proteins implicated in cancer pathogenesis. Additionally, we discuss the promising realm of precision‐targeted approaches, including antibody–drug conjugates and the utilization of photopharmacology in MTAs. Lastly, we provide a comprehensive overview of the clinical applications of microtubule‐targeting therapies, assessing their efficacy and current challenges. We aim to provide a global picture of MTAs development as well as insights into the microtubule‐targeting drug discovery for cancer treatment.

The genesis of cardiovascular risk in inflammatory arthritis: insights into glycocalyx shedding, endothelial dysfunction, and atherosclerosis initiation.

This narrative review provides a comprehensive examination of the complex interplay between inflammatory arthritis (IA) and cardiovascular pathology. It particularly illuminates the roles of atherosclerosis initiation, endothelial dysfunction, and glycocalyx shedding. IA not only provokes tissue-specific inflammatory responses, but also engenders a considerable degree of non-specific systemic inflammation. This review underscores the accelerating influence of the chronic inflammatory milieu of IA on cardiovascular disease (CVD) progression. A focal point of our exploration is the critical function of the endothelial glycocalyx (EG) in this acceleration process, which possibly characterizes the earliest phases of atherosclerosis. We delve into the influence of inflammatory mediators on microtubule dynamics, EG modulation, immune cell migration and activation, and lipid dysregulation. We also illuminate the impact of microparticles and microRNA on endothelial function. Further, we elucidate the role of systemic inflammation and sheddases in EG degradation, the repercussions of complement activation, and the essential role of syndecans in preserving EG integrity. Our review provides insight into the complex and dynamic interface between systemic circulation and the endothelium.

Incidence and Resolution of Eribulin-Induced Peripheral Neuropathy (IRENE) in Locally Advanced or Metastatic Breast Cancer: Prospective Cohort Study.

Eribulin, a halichondrin-class microtubule dynamics inhibitor, is a preferred treatment option for patients with advanced breast cancer who have been pretreated with an anthracycline and a taxane. Peripheral neuropathy (PN) is a common side effect of chemotherapies for breast cancer and other tumors. The Incidence and Resolution of Eribulin-Induced Peripheral Neuropathy (IRENE) noninterventional postauthorization safety study assessed the incidence and severity of PN in patients with breast cancer treated with eribulin. IRENE is an ongoing observational, single-arm, prospective, multicenter, cohort study. Adult patients (≥18 years of age) with locally advanced or metastatic breast cancer and disease progression after 1-2 prior chemotherapeutic regimen(s) for advanced disease were treated with eribulin. Patients with eribulin-induced PN (new-onset PN or worsening of preexisting PN) were monitored until death or resolution of PN. Primary endpoints included the incidence, severity, and time to resolution of eribulin-induced PN. Secondary endpoints included time to disease progression and safety. In this interim analysis (data cutoff date: July 1, 2019), 67 (32.4%) patients experienced any grade eribulin-induced PN, and 12 (5.8%) patients experienced grade ≥3 eribulin-induced PN. Median time to resolution of eribulin-induced PN was not reached. Median time to disease progression was 4.6 months (95% CI, 4.0-6.5). Treatment-emergent adverse events (TEAEs) occurred in 195 (93.8%) patients and serious TEAEs occurred in 107 (51.4%) patients. The rates of any grade and grade ≥3 eribulin-induced PN observed in this real-world study were consistent with those observed in phase III randomized clinical trials. No new safety findings were observed.

TUBB3 and KIF21A in neurodevelopment and disease.

Neuronal migration and axon growth and guidance require precise control of microtubule dynamics and microtubule-based cargo transport. TUBB3 encodes the neuronal-specific β-tubulin isotype III, TUBB3, a component of neuronal microtubules expressed throughout the life of central and peripheral neurons. Human pathogenic TUBB3 missense variants result in altered TUBB3 function and cause errors either in the growth and guidance of cranial and, to a lesser extent, central axons, or in cortical neuronal migration and organization, and rarely in both. Moreover, human pathogenic missense variants in KIF21A, which encodes an anterograde kinesin motor protein that interacts directly with microtubules, alter KIF21A function and cause errors in cranial axon growth and guidance that can phenocopy TUBB3 variants. Here, we review reported TUBB3 and KIF21A variants, resulting phenotypes, and corresponding functional studies of both wildtype and mutant proteins. We summarize the evidence that, in vitro and in mouse models, loss-of-function and missense variants can alter microtubule dynamics and microtubule-kinesin interactions. Lastly, we highlight additional studies that might contribute to our understanding of the relationship between specific tubulin isotypes and specific kinesin motor proteins in health and disease.

Abstract P2068: β Iv -spectrin/stat3 Are Responsible For Concentric Versus Eccentric Cardiac Hypertrophy

Hypertrophy is critical to development of heart failure following cardiac stress. In pressure overload, hypertrophy is divided by early concentric and chronic eccentric orientations, however, the mechanisms regulating this transition and the impact on heart function are largely unknown. Previous work identified the structural protein β IV -spectrin and its interaction with the transcription factor, STAT3, in coordinating hypertrophic profiles. Specifically, mice expressing truncated β IV -spectrin lacking interaction with the kinase CaMKII ( qv 3J mice) resisted β IV -spectrin degradation and maintained concentric hypertrophy and systolic function compared to WT following 6 weeks transaortic constriction (TAC). Alternatively, mutant β IV -spectrin mice lacking the β IV -spectrin/STAT3 binding domain ( qv 4J ) expressed reduced EF and LV dilation, consistent with eccentric remodeling, suggesting β IV -spectrin/STAT3 is a regulator of cardiac profiles. This relationship was examined by adenoviral expression of the hypertrophic driver MEK1/2 on isolated myocytes, where MEK increased the width of WT cells but length in qv 4J . Notably, application of STAT3 inhibitor S3I-201 on qv 4J myocytes restored growth in width. Additionally, in vivo delivery of S3I-201 to WT mice after 6 weeks TAC reduced LV dilation and recovered systolic function. To enable hypertrophy, recent work identified the need for microtubules to traffic and locally translate sarcomeric transcripts. Therefore, we tested the hypothesis that β IV -spectrin/STAT3 direct orientation by altering microtubule stability and sarcomeric transcript trafficking. Isolated myocytes from WT and qv 4J mice were treated with the microtubule depolymerizing drug Nocodazole and then washed to evaluate microtubule repolymerization. Microtubules restored faster in qv 4J myocytes but could be reduced to WT levels by treating with S3I-201. Furthermore, localization of the sarcomeric gene, actc1 (α-actinin) by RNAScope showed more peripheral localization in qv 4J myocytes, compared to WT, and was normalized by S3I-201. Together, these data identify β IV -spectrin/STAT3 as modulators of microtubule dynamics, sarcomeric transcript distribution, hypertrophic orientation, and systolic function.

Abstract P2016: Extracellular Stiffness Induces Glucose Dysregulation Via Microtubule-dependent Mechanism

Previous studies have reported profound changes in metabolic fuel utilization pathways during the development of heart failure, including changes in glucose uptake, glucose transporter mislocalization and/or Insulin-resistance. insulin stimulates glucose transport in muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Multiple reports have shown that the microtubules in adipocytes and muscle cells play an important role in the insulin-stimulated glucose transport process. We recently demonstrated, using a tunable stiffness substrate mimicking disease myocardium, that we could induces early, cell-autonomous remodeling of adult cardiomyocytes that is dependent on detyrosination of α-tubulin. We therefore employed this culture substrate to define how adult isolated rat cardiomyocytes glucose uptake response is to Insulin when cultured on stiff vs. soft. At the same time, we studied the localization and expression of Glut4 transporter. We found that 48h on stiff substrate was sufficient to induce a loss of insulin sensitivity in our isolated cardiomyocytes. Immunofluorescence reveals a decrease of Glut4 at the Intercalated disc (ID) but show increase of aggregates of the Glut4 transporter at the cell surface. Western Blot analyses shows no change in the total expression of the transporter. Finally, knowing the importance of microtubules network in the trafficking of Glut4, we overexpressed of tubulin tyrosine ligase (TTL) to prevent the detyrosination of microtubules. Surprisingly, TTL not only restored Glut4 at the ID and decreased the number of aggregates, but it also significantly increased glucose uptake as well as the total amount of Glut4 by more than 2-fold compared to soft control. In conclusion, we demonstrated the importance of microtubule dynamics in cardiomyocytes for the Insulin-dependent trafficking of Glut4 to the membrane.

Effect of Colchicine Binding Site Inhibitors on the Tubulin Intersubunit Interaction.

Microtubules are dynamic, non-covalent polymers consisting of α- and β-tubulin subunits that are involved in a wide range of intracellular processes. The polymerization and dynamics of microtubules are regulated by many factors, including small molecules that interact with different sites on the tubulin dimer. Colchicine binding site inhibitors (CBSIs) destabilize microtubules and inhibit tubulin polymerization, leading to cell cycle arrest. Because of their therapeutic potential, the molecular mechanism of CBSI function is an area of active research. Nevertheless, important details of this mechanism have yet to be resolved. In this study, we use atomistic molecular dynamics simulations to show that the binding of CBSIs to the tubulin heterodimer leads to the weakening of tubulin intersubunit interaction. Using atomistic molecular dynamics simulations and binding free energy calculations, we show that CBSIs act as protein-protein interaction inhibitors and destabilize interlinkage between α and β subunits, which is crucial for longitudinal contacts in the microtubule lattice. Our results offer new insight into the mechanisms of microtubule polymerization inhibition by colchicine and its analogs.

Plasmodium microtubule-binding protein EB1 is critical for partitioning of nuclei in male gametogenesis.

Sexual reproduction of the malaria parasites is critical for their transmission to a mosquito vector. Several signaling molecules, such as kinases and phosphatases, are known to regulate this process. We previously demonstrated that Plasmodium falciparum (Pf) Ca2+-dependent protein kinase 4 (CDPK4) and serine/arginine-rich protein kinase 1 (SRPK1) are critical for axoneme formation during male gametogenesis, with genetic deletion of either gene causing a complete block in parasite transmission to the mosquito. A comparative phospho-proteome analysis of Pfcdpk4- and RNA-seq analysis of Pfsrpk1- gametocytes showed that these kinases regulate similar biological processes linked to both microtubule (MT) dynamics and cell motility. One of these proteins was a nuclear MT-associated End Binding protein 1 (EB1), which was hypophosphorylated in Pfcdpk4- gametocytes. To study the functional relevance of EB1, we created gene deletion parasites for EB1. We further demonstrate that Pfeb1- parasites like WT NF54 parasites proliferate normally as asexuals and undergo gametocytogenesis and gametogenesis. Strikingly, these parasites suffer a severe defect in nuclear segregation and partitioning of nuclei into emerging microgametes. Further genetic crosses utilizing male- and female-sterile parasites revealed that Pfeb1- parasites only suffer a male fertility defect. Overall, our study reveals an essential function for PfEB1 in male gamete nuclear segregation and suggests a potential therapeutic avenue in the design of transmission-blocking drugs to prevent malaria transmission from humans to mosquito. IMPORTANCE Gametogenesis and subsequent gamete fusion are central to successful transmission of the malaria parasites to a female Anopheles mosquito vector and completion of the sexual phase of the parasite life cycle. Male gametogenesis involves the formation of axonemes inside male gametes from male gametocytes via active cytoskeleton remodeling. The tubulin and tubulin-binding proteins are, thus, attractive anti-malarial drug targets. In the present study, we demonstrate that a microtubule-binding protein PfEB1 is essential for male gamete fertility, specifically for the inheritance of nuclei from activated male gametocytes. Targeting PfEB1 function may provide new avenues into designing interventions to prevent malaria transmission and disease spread.

In vivo impact of tubulin polymerization promoting protein (Tppp) knockout to the airway inflammatory response

Microtubule dysfunction has been implicated as a mediator of inflammation in multiple diseases such as disorders of the cardiovascular and neurologic systems. Tubulin polymerization promoting protein (Tppp) facilitates microtubule elongation and regulates tubulin acetylation through inhibition of cytosolic deacetylase enzymes. Pathologic alterations in microtubule structure and dynamics have been described in cystic fibrosis (CF) and associated with inflammation, however the causality and mechanism remain unclear. Likewise, Tppp has been identified as a potential modifier of CF airway disease severity. Here we directly assess the impact of microtubule dysfunction on infection and inflammation by interrogating wild type and a Tppp knockout mouse model (Tppp − / −). Mice are challenged with a clinical isolate of Pseudomonas aeruginosa-laden agarose beads and assessed for bacterial clearance and inflammatory markers. Tppp − / − mouse model demonstrate impaired bacterial clearance and an elevated inflammatory response compared to control mice. These data are consistent with the hypothesis microtubule dysregulation is sufficient to lead to CF-like airway responses in mice.

HDAC-6 inhibition ameliorates the early neuropathology in a mouse model of Krabbe disease.

In Krabbe disease (KD), mutations in β-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, leads to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport. We tested whether inhibiting the α-tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice. Our data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria. Overall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction.

Cell division visualized: barley reporter lines for chromosome and microtubule dynamics.

Cell division visualized: barley reporter lines for chromosome and microtubule dynamics.

Tau, microtubule dynamics, and axonal transport: New paradigms for neurodegenerative disease.

The etiology of Tauopathies, a diverse class of neurodegenerative diseases associated with the Microtubule Associated Protein (MAP) Tau, is usually described by a common mechanism in which Tau dysfunction results in the loss of axonal microtubule stability. Here, we reexamine and build upon the canonical disease model to encompass other Tau functions. In addition to regulating microtubule dynamics, Tau acts as a modulator of motor proteins, a signaling hub, and a scaffolding protein. This diverse array of functions is related to the dynamic nature of Tau isoform expression, post-translational modification (PTM), and conformational flexibility. Thus, there is no single mechanism that can describe Tau dysfunction. The effects of specific pathogenic mutations or aberrant PTMs need to be examined on all of the various functions of Tau in order to understand the unique etiology of each disease state.

Nutritional stress-induced regulation of microtubule organization and mRNP transport by HDAC1 controlled α-tubulin acetylation

In response to nutritional stress, microtubules in cells of the Drosophila female germline are depleted from the cytoplasm and accumulate cortically. This triggers aggregation of mRNPs into large processing bodies (P-bodies) and oogenesis arrest. Here, we show that hyperacetylation of α-tubulin at lysine 40 (K40) alters microtubule dynamics and P-body formation. We found that depletion of histone deacetylase 1 (HDAC1) by RNAi phenocopies the nutritional stress response, causing α-tubulin hyperacetylation and accumulation of maternally deposited mRNPs in P-bodies. Through in vitro and in vivo studies, we identify HDAC1 as a direct regulator of α-tubulin K40 acetylation status. In well-fed flies, HDAC1 maintains low levels of α-tubulin acetylation, enabling the microtubule dynamics required for mRNP transport. Using quantitative phosphoproteomics we identify nutritional stress-induced changes in protein phosphorylation that act upstream of α-tubulin acetylation, including phosphorylation of HDAC1 at S391, which reduces its ability to deacetylate α-tubulin. These results reveal that Drosophila HDAC1 senses and relays the nutritional status, which regulates germline development through modulation of cytoskeleton dynamics.

Microtubule Dynamics Deregulation Induces Apoptosis in Human Urothelial Bladder Cancer Cells via a p53-Independent Pathway.

Bladder cancer (BLCA) is the sixth most common type of cancer and has a dismal prognosis if diagnosed late. To identify treatment options for BLCA, we systematically evaluated data from the Broad Institute DepMap project. We found that urothelial BLCA cell lines are among the most sensitive to microtubule assembly inhibition by paclitaxel treatment. Strikingly, we revealed that the top dependencies in BLCA cell lines include genes encoding proteins involved in microtubule assembly. This highlights the importance of microtubule network dynamics as a major vulnerability in human BLCA. In cancers such as ovarian and breast, where paclitaxel is the gold standard of care, resistance to paclitaxel treatment has been linked to p53-inactivating mutations. To study the response of BLCA to microtubule assembly inhibition and its mechanistic link with the mutational status of the p53 protein, we treated a collection of BLCA cell lines with a dose range of paclitaxel and performed a detailed characterization of the response. We discovered that BLCA cell lines are significantly sensitive to low concentrations of paclitaxel, independently of their p53 status. Paclitaxel induced a G2/M cell cycle arrest and growth inhibition, followed by robust activation of apoptosis. Most importantly, we revealed that paclitaxel triggered a robust DNA-damage response and apoptosis program without activating the p53 pathway. Integration of transcriptomics, epigenetic, and dependency data demonstrated that the response of BLCA to paclitaxel is independent of p53 mutational signatures but strongly depends on the expression of DNA repair genes. Our work highlights urothelial BLCA as an exceptional candidate for paclitaxel treatment. It paves the way for the rational use of a combination of paclitaxel and DNA repair inhibitors as an effective, novel therapeutic strategy.

Cystathionine β-synthase affects organization of cytoskeleton and modulates carcinogenesis in colorectal carcinoma cells.

Cystathionine β-synthase (CBS), one of three enzymes that endogenously produce hydrogen sulfide, is extensively studied for its relevance in the cells of various tumors. In our previous work, we observed that the immunofluorescence pattern of CBS is very similar to that of tubulin and actin. Therefore, we focused on the potential interaction of CBS with cytoskeletal proteins β-actin and β-tubulin and the functional relevance of the potential interaction of these proteins in colorectal carcinoma cell lines. To study the potential interaction of CBS with cytoskeletal proteins and its functional consequences, a CBS-knockout DLD1 (DLDx) cell line was established by using the CRISPR/Cas9 gene editing method. The interaction of the selected cytoskeletal protein with CBS was studied by immunoprecipitation, Western blot analysis, immunofluorescence, and proximity ligation assay. The functional consequences were studied by proliferation and migration assays and by generation of xenografts in SCID/bg mice. We have found that CBS, an enzyme that endogenously produces H2S, binds to cytoskeletal β-tubulin and, to a lesser extent, also to β-actin in colorectal carcinoma-derived cells. When CBS was knocked out by the CRISPR/Cas9 technique (DLDx), we observed a de-arranged cytoskeleton compared to the unmodified DLD1 cell line. Treatment of these cells with a slow sulfide donor GYY4137 resulted in normal organization of the cytoskeleton, thus pointing to the role of CBS in microtubule dynamics. To evaluate the physiological importance of this observation, both DLD1 and DLDx cells were injected into SCID/bg mice, and the size and mass of the developed xenografts were evaluated. Significantly larger tumors developed from DLDx compared to the DLD1 cells, which correlated with the increased proliferation of these cells. Taken together, in colorectal cancer DLD1 cells, CBS binds to the cytoskeleton, modulates microtubule dynamics, and thus affects the proliferation and migration in the colorectal carcinoma stable cell line.

Subcellular positioning during cell division and cell plate formation in maize.

During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.

Maintaining asymmetry in cell division.

Promoting asymmetric division through microtubule dynamics establishes cell fate.

Spatial organization and dynamics of chromosomes and microtubules during barley mitosis.

Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time-lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre-prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.

The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton

Completion of neuronal migration is critical for brain development. Kif21b is a plus-end-directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex. Invivo analysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of newborn neurons independently of its motility on microtubules. We show that Kif21b directly binds and regulates the actin cytoskeleton both invitro and invivo in migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.