Kinesin Inhibitors Research Articles

Abstract 2684: Drug sensitivity profiling of BT-474 breast cancer cell line for identification of novel therapies targeting HER2-positive breast cancer

Abstract Developing targeted therapy for breast cancer expressing hormone receptors such as estrogen receptor (ER) and progesterone receptor (PR) as well as human epidermal growth factor receptor 2 (HER2) is essential. However, breast cancer has high susceptibility to spread despite the targeted therapies and metastases often turn resistant to the used treatment. Therefore, new therapies are needed especially at the stage when cancer becomes resistant to hormonal therapy. The aim of the study was to investigate drug sensitivity of BT-474 breast cancer cell line and to recognize possible vulnerabilities especially in HER2+ breast cancer. BT-474 cell line (ER+, PR+, HER2+) is originally derived from human solid, invasive ductal carcinoma of the breast. Drug sensitivity of the cell line was assessed by screening of a large compound library consisting of many clinically available and emerging anti-cancer drugs including e.g. conventional chemotherapy, metabolic modifiers, kinesin inhibitors, apoptotic modulators, immunomodulators and kinase inhibitors such as HER2, pan-HER2 and EGFR-HER2 inhibitors. A panel of 525 compounds was tested in five concentrations covering a 10.000-fold drug-relevant concentration range in 384-well format. Cells were seeded to pre-drugged plates, followed by cell viability measurements (CellTiter-Glo) after 72 hours. Maximal and minimal responses to drugs were analyzed, the EC50 values were calculated and Drug Sensitivity Score (DSS) was calculated for each drug as a measure of reduced viability. A selective DSS (sDSS) was calculated to identify the selective drug response pattern. DSS analysis of BT-474 cell line showed sensitivity to conventional chemotherapy including paclitaxel and docetaxel and to kinase inhibitors such as EGFR and HER2 inhibitors neratinib, afatinib, mubritinib, poziotinib, dacomitinib and lapatinib with nanomolar EC50 values. Moreover, BT-474 cells showed sensitivity to PI3K and mTOR inhibitors, such as copanlisib and omipalisib, and to HSP90 and HDAC inhibitors, such as tanespimycin and abexinostat. Screening of large compound libraries combined with DSS and sDSS analysis enables drug sensitivity profiling of BT-474 breast cancer cells for discovery of novel therapies against HER+ breast cancer. Furthermore, drug sensitivity profiling enables repurposing of existing drugs to new indications and identification of vulnerabilities in different types of cancer cells. Citation Format: Jenni H. Mäki-Jouppila, Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli, Jani Saarela, Katja Fagerlund. Drug sensitivity profiling of BT-474 breast cancer cell line for identification of novel therapies targeting HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2684.

KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that (i) KIF15 motility differs significantly from Eg5; (ii) KIF15-IN-1 is a potent inhibitor of KIF15 motility; (iii) MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and (iv) pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.

Tension-Induced Error Correction and Not Kinetochore Attachment Status Activates the SAC in an Aurora-B/C-Dependent Manner in Oocytes

Cell division with partitioning of the genetic material should take place only when paired chromosomes named bivalents (meiosis I) or sister chromatids (mitosis and meiosis II) are correctly attached to the bipolar spindle in a tension-generating manner. For this to happen, the spindle assembly checkpoint (SAC) checks whether unattached kinetochores are present, in which case anaphase onset is delayed to permit further establishment of attachments. Additionally, microtubules are stabilized when they are attached and under tension. In mitosis, attachments not under tension activate the so-named error correction pathway depending on Aurora B kinase substrate phosphorylation. This leads to microtubule detachments, which in turn activates the SAC [1-3]. Meiotic divisions in mammalian oocytes are highly error prone, with severe consequences for fertility and health of the offspring [4, 5]. Correct attachment of chromosomes in meiosis I leads to the generation of stretched bivalents, but-unlike mitosis-not to tension between sister kinetochores, which co-orient. Here, we set out to address whether reduction of tension applied by the spindle on bioriented bivalents activates error correction and, as a consequence, the SAC. Treatment of oocytes in late prometaphase I with Eg5 kinesin inhibitor affects spindle tension, but not attachments, as we show here using an optimized protocol for confocal imaging. After Eg5 inhibition, bivalents are correctly aligned but less stretched, and as a result, Aurora-B/C-dependent error correction with microtubule detachment takes place. This loss of attachments leads to SAC activation. Crucially, SAC activation itself does not require Aurora B/C kinase activity in oocytes.

Structural basis of small molecule ATPase inhibition of a human mitotic kinesin motor protein

Kinesin microtubule motor proteins play essential roles in division, including attaching chromosomes to spindles and crosslinking microtubules for spindle assembly. Human kinesin-14 KIFC1 is unique in that cancer cells with amplified centrosomes are dependent on the motor for viable division because of its ability to cluster centrosomes and form bipolar spindles, but it is not required for division in almost all normal cells. Screens for small molecule inhibitors of KIFC1 have yielded several candidates for further development, but obtaining structural data to determine their sites of binding has been difficult. Here we compare a previously unreported KIFC1 crystal structure with new structures of two closely related kinesin-14 proteins, Ncd and KIFC3, to determine the potential binding site of a known KIFC1 ATPase inhibitor, AZ82. We analyze the previously identified kinesin inhibitor binding sites and identify features of AZ82 that favor binding to one of the sites, the α4/α6 site. This selectivity can be explained by unique structural features of the KIFC1 α4/α6 binding site. These features may help improve the drug-like properties of AZ82 and other specific KIFC1 inhibitors.

Is inflammatory micronucleation the key to a successful anti-mitotic cancer drug?

Paclitaxel is a successful anti-cancer drug that kills cancer cells in two-dimensional culture through perturbation of mitosis, but whether it causes tumour regression by anti-mitotic actions is controversial. Drug candidates that specifically target mitosis, including inhibitors of kinesin-5, AurkA, AurkB and Plk1, disappointed in the clinic. Current explanations for this discrepancy include pharmacokinetic differences and hypothetical interphase actions of paclitaxel. Here, we discuss post-mitotic micronucleation as a special activity of taxanes that might explain their higher activity in solid tumours. We review data showing that cells which exit mitosis in paclitaxel are highly micronucleated and suffer post-mitotic DNA damage, and that these effects are much stronger for paclitaxel than kinesin-5 inhibitors. We propose that post-mitotic micronucleation promotes inflammatory signalling via cGAS–STING and other pathways. In tumours, this signalling may recruit cytotoxic leucocytes, damage blood vessels and prime T-cell responses, leading to whole-tumour regression. We discuss experiments that are needed to test the micronucleation hypothesis, and its implications for novel anti-mitotic targets and enhancement of taxane-based therapies.

Abstract 4207: Selective drug sensitivity score (DSS) for indolent and aggressive prostate cancer cell lines

Abstract Prostate cancer (PC) is the most common malignancy in men and the second leading cause of cancer-related deaths. The majority of the PCs are classified as adenocarcinomas characterized by the expression of androgen receptor (AR) and prostate-specific antigen (PSA). Two of the most commonly used cell lines are LNCaP and PC-3 cells, derived from lymph node and bone metastases, respectively. Also VCaP cells, derived from vertebral metastases, are widely used in prostate cancer research. It has been well established that LNCaP and VCaP cells represent the conventional indolent form of PC expressing AR and PSA and are androgen-dependent. PC-3 cells, on the other hand, do not express AR and PSA, are androgen-independent, and represent the highly aggressive form. The drug sensitivity of the cell lines was assessed by applying a large panel of drugs covering cancer chemotherapeutics and clinically available and emerging drugs including conventional chemotherapy, kinase inhibitors, metabolic modifiers, rapalogs, differentiating/epigenetic modifiers, kinesin inhibitors, apoptotic modulators, NSAIDs, hormone therapy, immunomodulators and HSP inhibitors. A panel of 460 compounds was tested in five concentrations covering a 10.000-fold drug-relevant concentration range in 384-well format. Cells were seeded to pre-drugged plates, followed by cell viability measurements (CellTiter-Glo) after 72 hours. Maximal and minimal responses to drugs were analyzed, the EC50 values were calculated and Drug Sensitivity Score (DSS) was calculated for each drug as a measure of reduced viability. A selective Drug Sensitivity Score (sDSS) was calculated to identify the selective drug response pattern of each three cancer cell lines. As expected, the results indicate that LNCaP and VCaP cells in general were more sensitive to drugs of different categories than PC-3 cells. According to DSS analysis, all three cell lines showed sensitivity to conventional chemotherapy and kinase inhibitors. However, PC-3 cells were more sensitive to kinase inhibitors than conventional chemotherapy. Determining sDSS revealed specific sensitivities of each cell line. LNCaP cells were sensitive to kinase inhibitors, such as mTOR and AKT inhibitors. Also VCaP cells showed selective sensitivity to kinase inhibitors, especially Aurora kinase and IGF1R inhibitors. In addition to kinase inhibitors, VCaP cells were selectively sensitive to HDAC inhibitors. Furthermore, PC-3 cells were sensitive to e.g. CDK inhibitors. We conclude that the cell-based compound screening combined with DSS and sDSS analysis provides a possibility to profile cellular responses to an extensive collection of anti-cancer compounds enabling repurposing of existing drugs to new indications, identification of vulnerabilities in different types of cancer cells and functional investigation of cellular pathways behind drug sensitivity or resistance. Citation Format: Jenni Mäki-Jouppila, Jenni Bernoulli, Johanna Tuomela, Mari I. Suominen, Jussi M. Halleen, Sanna Timonen, Elina Huovari, Katja Suomi, Swapnil Potdar, Päivi Östling, Jani Saarela, Katja M. Fagerlund. Selective drug sensitivity score (DSS) for indolent and aggressive prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4207. doi:10.1158/1538-7445.AM2017-4207

Abstract 3838: Drug sensitivity profile of 5TGM1 murine multiple myeloma cell line emphasizes the translational potential of the syngeneic in vivo model

Abstract Multiple myeloma (MM) is the second most common hematologic malignancy that originates from B-cells (plasma cells) and causes 2% of cancer-related deaths. Symptoms of MM include bone pain caused by multiple osteolytic lesions, pathologic fractures, and hypercalcemia. Typically, MM has a low growth fraction and it is highly dependent on the microenvironment. These properties have made it hard to target by conventional chemotherapy, but could now be exploited by novel stroma-targeting drugs and immunotherapy. These new approaches underline the need for well characterized models with functional immune system and appropriate tumor microenvironment. To gain additional information supporting the use of the syngeneic 5TGM1 murine multiple myeloma model in drug development, we tested drug sensitivity of 5TGM1 cells by screening an extensive panel of drugs. The compound library consisting of 460 compounds included conventional chemotherapy, kinase inhibitors, metabolic modifiers, rapalogs, differentiating/epigenetic modifiers, kinesin inhibitors, apoptotic modulators, NSAIDs, hormone therapy, immunomodulators and HSP inhibitors. The compounds were tested in five concentrations covering a 10.000-fold drug-relevant concentration range in 384-well format. Cells were seeded to plates with a compound library, followed by cell viability measurements (CellTiter-Glo) after 72 hours. Maximal and minimal responses to drugs were analyzed, and the EC50 values were calculated. Drug Sensitivity Score (DSS) was calculated for each drug as a measure of reduced viability. According to DSS analysis, 5TGM1 cells showed sensitivity to conventional chemotherapy, such as antimitotic drugs, and kinase inhibitors, such as MEK1/2 inhibitors. In addition, the cells showed particular sensitivity to several HSP90 inhibitors currently in phase I/II clinical development for MM. Lenalidomide and pomalidomide, efficient in treating multiple myeloma in humans, both gave low DSS value indicating that 5TGM1 cells are not sensitive to these drugs, which is expected because they do not bind to murine form of the target cereblon. In contrast, 5TGM1 cells were highly sensitive to the proteasome inhibitor bortezomib (DSS 32.2), which is currently in clinical use. In conclusion, the murine 5TGM1 cells show sensitivity to various MM drugs used in the clinic and under development. Evaluating the effects of the microenvironment on the growth and drug sensitivity of 5TGM1 cells in vitro and in vivo will be essential. Furthermore, the cell-based compound screening combined with DSS analysis provides a possibility to profile cellular responses to an extensive collection of anti-cancer compounds enabling identification of vulnerabilities in cancer cells and functional investigation of cellular pathways behind drug sensitivity or resistance. Citation Format: Jenni Mäki-Jouppila, Jenni Bernoulli, Mari I. Suominen, Tiina Kähkönen, Jussi M. Halleen, Sanna Timonen, Elina Huovari, Katja Suomi, Swapnil Potdar, Maria Nurmi, Päivi Östling, Jani Saarela, Katja M. Fagerlund. Drug sensitivity profile of 5TGM1 murine multiple myeloma cell line emphasizes the translational potential of the syngeneic in vivo model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3838. doi:10.1158/1538-7445.AM2017-3838

Kinesin-5 Blocker Monastrol Protects Against Bortezomib-Induced Peripheral Neurotoxicity.

Neurotoxicity is a relevant side effect of bortezomib treatment. Previous reports have shown that the development of peripheral neuropathy caused by anti-neoplastic agents may be a result of reduced axonal transport. Based on evidence from prior studies that the kinesin-5 inhibitor monastrol enhances axonal transport and improves neuronal regeneration, we focused on the neuroprotective role of monastrol during the chemotherapeutic treatment with bortezomib. Prolonged treatment of C57BL/6 mice with bortezomib induced a length-dependent small-fiber neuropathy with axonal atrophy and loss of sensory nerve fibers. The administration of monastrol substantially alleviated morphological features of axonal injury and functional measures of sensory neuropathy. Cytotoxicity studies in leukemia and multiple myeloma cell lines showed no interference of monastrol with the cytostatic effects of bortezomib. Our data indicate that the novel approach of targeting microtubule turnover by monastrol provides protection against bortezomib-induced neurotoxicity. The favorable cytotoxic profile of monastrol makes it an interesting candidate as neuroprotective agent in combined chemotherapy regimens that warrants further consideration.

Novel Allosteric Pathway of Eg5 Regulation Identified through Multivariate Statistical Analysis of Hydrogen-Exchange Mass Spectrometry (HX-MS) Ligand Screening Data

The mitotic kinesin Eg5 is an important target in cancer chemotherapy. A structurally diverse collection of canonical loop L5 inhibitors engage an allosteric pathway that includes elements of its microtubule binding region. However, recent evidence suggests that Eg5 may permit alternative allosteric mechanisms. Terpendole E, a natural-product Eg5 inhibitor, is active against mutants resistant to canonical loop L5 inhibitors and appears to offer a unique mode of inhibition. To investigate the variety of inhibitor responses, the structure-function properties of eighteen kinesin inhibitors were quantified with hydrogen-exchange mass spectrometry (HX-MS), functional analysis and molecular modeling. A unique strategy for high-density data analysis was implemented, based on a scalable multivariate statistical method, as current HX-MS routines have a limited capacity to guide a characterization of ligands when additional functional data is available. Inhibitor evaluation was achieved using orthogonal partial least squares projection to latent structures discriminant analysis (OPLS-DA). The strategy generated a model that identified functionally-significant conformational elements involved in kinesin inhibition, confirming the canonical allosteric pathway and identifying a novel response pathway. Terpendole E is demonstrated to be an atypical L5 site inhibitor, where binding induces an allosteric effect mediated by a destabilization in the β-sheet core of the molecular motor, an element involved in mechanochemical coupling for structurally-related kinesins. The analysis suggests that a different approach to inhibitor development may be fruitful.

Eg5 Inhibitors have Contrasting Effects on Microtubule Stability and Spindle Integrity Depending on their Modes of Action

The goal of this work was to investigate the mechanisms underlying different Eg5 (kinesin-5) inhibitors and to assess their effects on Eg5-mediated stabilization of dynamic microtubules. Antimitotic Eg5 inhibitors were grouped into those that bind Loop-5 and produce an ADP-like state (monastrol, STLC, ispinesib and filanesib) and those that produce a rigor-like state (BRD9876). To understand their contrasting effects on Eg5 mechanics, inhibitors were analyzed in mixed-motor gliding assays consisting of different ratios of kinesin-1 and Eg5 motors, in which Eg5 “braking” dominates motility. Loop-5 inhibitors increased gliding speeds, consistent with the compounds inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with Eg5 being locked in a rigor state. Quantitative critical detachment force analysis (Arpag et al. Biophys J. 2014) indicates that L5 inhibitors enhance Eg5 detachment whereas the rigor inhibitor BRD9876 generates an Eg5 species with decreased susceptibility of detachment. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with a KI of 4 nM. Consistent with its recently reported microtubule polymerase activity (Chen and Hancock, Nature Comm. 2015:8160) and its dominant two-head-bound strong-binding population (Chen et al., JBC 2016:M116), Eg5 was shown to stabilize microtubules against depolymerization in vitro following taxol washout. This stabilization was eliminated in monastrol but was enhanced by BRD9876, consistent with strong binding of Eg5 to microtubules enhancing microtubule stability. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors inhibit spindle formation through contrasting mechanisms and rigor inhibitors are predicted to paradoxically stabilize microtubule arrays in cells.

Abstract 2722: Reviving kinesin-5 inhibitors: Evidence for a powerful combinatorial strategy

Abstract Mitosis serves as a powerful opportunity for therapeutic intervention in the treatment of neoplastic diseases like cancer. Anti-mitotic pharmacological agents tame the proliferative capacity of tumor cells by interfering with proper function of the mitotic spindle. Several next generation anti-mitotics target kinesins involved in assembling the mitotic spindle. Of these mitotic kinesins, the kinesin-5 Eg5 (also known as KSP) is perhaps the most compelling because of its primacy during spindle assembly in animal cells. Indeed, Eg5-inhibitors (K5Is) induce a potent and lethal mitotic arrest in cell culture and murine xenografts; however, K5Is failed to induce tumor regression during Phase II trials. This devastating clinical performance has halted further development of K5I monotherapies and motivated investigation into the underlying reason(s) for K5I inefficacy. We previously showed that tumor cells in culture are capable of acquiring K5I resistance by employing parallel spindle assembly pathways. By studying a limited number of K5I-resistant cell lines, we showed that resistance involved the kinesin-12 Kif15; however, the generality of this finding was unclear. In this study we capitalize on the latest methodologies in targeted genome editing to study mechanisms of K5I resistance on a broader cell population level. We find that cells require Kif15 in all cases of K5I resistance, and that rescue is sensitive to the timing and extent of Kif15 expression. We propose that K5Is steer cells into an evolutionary bottleneck that requires Kif15, and that adaptive changes occur during passage through the bottleneck that enable populations to recover and diversify. These findings prompt a search for pharmacological inhibitors of Kif15 to test in combination with K5Is as an anti-mitotic chemotherapeutic regimen. Citation Format: Emma G. Sturgill, Ryoma Ohi. Reviving kinesin-5 inhibitors: Evidence for a powerful combinatorial strategy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2722.

Kinesin-5 inhibitor resistance is driven by kinesin-12.

The microtubule (MT) cytoskeleton bipolarizes at the onset of mitosis to form the spindle. In animal cells, the kinesin-5 Eg5 primarily drives this reorganization by actively sliding MTs apart. Its primacy during spindle assembly renders Eg5 essential for mitotic progression, demonstrated by the lethal effects of kinesin-5/Eg5 inhibitors (K5Is) administered in cell culture. However, cultured cells can acquire resistance to K5Is, indicative of alternative spindle assembly mechanisms and/or pharmacological failure. Through characterization of novel K5I-resistant cell lines, we unveil an Eg5 motility-independent spindle assembly pathway that involves both an Eg5 rigor mutant and the kinesin-12 Kif15. This pathway centers on spindle MT bundling instead of Kif15 overexpression, distinguishing it from those previously described. We further show that large populations (∼10(7) cells) of HeLa cells require Kif15 to survive K5I treatment. Overall, this study provides insight into the functional plasticity of mitotic kinesins during spindle assembly and has important implications for the development of antimitotic regimens that target this process.

Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity

Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human neurological disorders. Homozygous nonsense mutations in kinesin-binding protein (KBP)/KIAA1279 cause the neurological disorder Goldberg-Shprintzen syndrome (GOSHS), which is characterized by intellectual disability, microcephaly, and axonal neuropathy. Here, we show that KBP regulates kinesin activity by interacting with the motor domains of a specific subset of kinesins to prevent their association with the MT cytoskeleton. The KBP-interacting kinesins include cargo-transporting motors such as kinesin-3/KIF1A and MT-depolymerizing motor kinesin-8/KIF18A. We found that KBP blocks KIF1A/UNC-104-mediated synaptic vesicle transport in cultured hippocampal neurons and in C. elegans PVD sensory neurons. In contrast, depletion of KBP results in the accumulation of KIF1A motors and synaptic vesicles in the axonal growth cone. We also show that KBP regulates neuronal MT dynamics by controlling KIF18A activity. Our data suggest that KBP functions as a kinesin inhibitor that modulates MT-based cargo motility and depolymerizing activity of a subset of kinesin motors. We propose that misregulation of KBP-controlled kinesin motors may represent the underlying molecular mechanism that contributes to the neuropathological defects observed in GOSHS patients.

Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy.

The kinesin class of microtubule-associated motor proteins present attractive anti-cancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms, and explore the implications for future anticancer drug development against these targets.

The natural diterpene tonantzitlolone A and its synthetic enantiomer inhibit cell proliferation and kinesin-5 function

Tonantzitlolone A, a diterpene isolated from the Mexican plant Stillingia sanguinolenta, shows cytostatic activity. Both the natural product tonantzitlolone A and its synthetic enantiomer induce monoastral spindle formation in cell experiments which indicates inhibitory activity on kinesin-5 mitotic motor molecules. These inhibitory effects on kinesin-5 could be verified in in vitro single-molecule motility assays, where both tonantzitlolones interfered with kinesin-5 binding to its cellular interaction partner microtubules in a concentration-dependent manner, yet with a larger effect of the synthetic enantiomer. In contrast to kinesin-5 inhibition, both tonantzitlolone A enantiomers did not affect conventional kinesin-1 function; hence tonantzitlolones are not unspecific kinesin inhibitors. The observed stronger inhibitory effect of the synthetic enantiomer demonstrates the possibility to enhance the overall moderate anti-proliferative effect of the lead compound tonantzitlolon A by chemical modification.

Trapping the Transition State of Kinesin-5 Produces a Different Multimotor Force Outcome than Inhibiting Product Release

Kinesin-5 (Eg5) is an essential mitotic motor that works in concert with multiple motors to form the bipolar spindle. Acute interest in the human kinesin-5 stems from the potential to target it for clinical therapy; many small-molecule inhibitors bind to an allosteric loop-5 (L5) pocket, inhibit ADP release, and reduce Eg5 motion to a diffusive mode that does not require ATP hydrolysis. Herein, we show that not all L5-directed drugs have the same catalytic effect nor do they have equivalent impacts upon the motor-microtubule complex. Linear free energy relationships, derived from steady state kinetics measurements of wildtype and eight L5 mutant proteins, show that monastrol is a transition state inhibitor, uniquely through an allosteric mechanism, whereas STC and ispinesib are not. Unexpectedly, in vitro “tug of war” assays between human Eg5 and human Kinesin-1 (KHC) revealed that these two categories of small chemical inhibitors also elicit dramatically different effects on Eg5 motility. Inhibitors of product release allow diffusive microtubule sliding. In contrast, trapping the transition state reveals bimodal behaviour that ultimately causes Eg5 to function as a brake that resists microtubule sliding by KHC. We conclude that ADP state inhibition of Eg5 allows the other kinesin forces on the microtubule to be unchecked while inhibition at the transition state of Eg5 hinders the entire multi-motor ensemble. It is possible that these disparate force outcomes are the result of variation in how drug inhibition at different catalytic intermediates can affect inter-head communication both within Eg5 and between motors in the ensemble. Our work highlights that not all L5 directed drugs are equal, and suggests kinesin inhibitors trapping the transition state should be further explored for their therapeutic potential.

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5.

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a "Humanized" <em>Drosophila</em> S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Mitotic slippage and expression of survivin are linked to differential sensitivity of human cancer cell-lines to the Kinesin-5 inhibitor monastrol.

The mitotic Kinesin-5 motor proteins crosslink and slide apart antiparallel spindle microtubules, thus performing essential functions in mitotic spindle dynamics. Specific inhibition of their function by monastrol-like small molecules has been examined in clinical trials as anticancer treatment, with only partial success. Thus, strategies that improve the efficiency of monastrol-like anticancer drugs are required. In the current study, we examined the link between sensitivity to monastrol and occurrence of mitotic slippage in several human cell-lines. We found that the rank of sensitivity to monastrol, from most sensitive to least sensitive, is: AGS>HepG2>Lovo>Du145≥HT29. We show correlation between the sensitivity of a particular cell-line to monastrol and the tendency of the same cell-line to undergo mitotic slippage. We also found that in the monastrol resistant HT29 cells, prolonged monastrol treatments increase mRNA and protein levels of the chromosomal passenger protein survivin. In contrast, survivin levels are not increased by this treatment in the monastrol-sensitive AGS cells. We further show that over-expression of survivin in the monastrol-sensitive AGS cells reduces mitotic slippage and increases resistance to monastrol. Finally, we show that during short exposure to monastrol, Si RNA silencing of survivin expression reduces cell viability in both AGS and HT29 cells. Our data suggest that the efficiency of anti-cancer treatment with specific kinesin-5 inhibitors may be improved by modulation of expression levels of survivin.

Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors

Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.