Eg5 Inhibitor Research Articles

Phase I Study to Assess the Safety, Tolerability and Pharmacokinetics of AZD4877 in Japanese Patients with Solid Tumors.

IntroductionAZD4877 is a potent Eg5 inhibitor that has been shown to have an acceptable tolerability profile in a Phase I study of Western patients with solid tumors. This study was conducted to evaluate the safety, pharmacokinetic (PK) profile, maximum tolerated dose (MTD) and efficacy of AZD4877 in a Japanese population with solid tumors.MethodsIn this Phase I, open-label, dose-escalation study, AZD4877 (10, 15, 20 or 25 mg) was administered as a 1-hour intravenous infusion on days 1, 8 and 15 of repeated 28-day cycles to Japanese patients with advanced solid tumors. Adverse events (AEs) were evaluated according to Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. PK variables were assessed pre- and post dosing. The MTD of AZD4877 was determined by evaluating dose-limiting toxicities (DLTs). Efficacy was evaluated by assessing best response according to Response Evaluation Criteria In Solid Tumors version 1.0.ResultsOf the 21 patients enrolled, 18 received at least one dose of AZD4877 (N = 3 in both the 10 and 15 mg cohorts, N = 6 in both the 20 and 25 mg cohorts). The most commonly reported AEs were fatigue and nausea (39% of patients each). One patient in each of the 20 and 25 mg cohorts experienced a DLT (neutropenia and febrile neutropenia). Dose escalation was halted at 25 mg and the MTD was not defined in this population. CTCAE grade ≥3 abnormal laboratory findings/vital signs were reported in 12 patients, with neutropenia (56%) and leukopenia (44%) being the most commonly reported. Exposure to AZD4877 was not fully dose proportional and AZD4877 clearance and elimination half-life appeared independent of dose. The best response to AZD4877 was stable disease in five of 16 evaluable patients.ConclusionAZD4877 up to doses of 25 mg was well tolerated in Japanese patients. There was little evidence of clinical efficacy.

Structure−Activity Relationship and Multidrug Resistance Study of New S-trityl-l-Cysteine Derivatives As Inhibitors of Eg5

The mitotic spindle is a validated target for cancer chemotherapy. Drugs such as taxanes and vinca alkaloids specifically target microtubules and cause the mitotic spindle to collapse. However, toxicity and resistance are problems associated with these drugs. Thus, alternative approaches to inhibiting the mitotic spindle are being pursued. These include targeting Eg5, a human kinesin involved in the formation of the bipolar spindle. We previously identified S-trityl-L-cysteine (STLC) as a potent allosteric inhibitor of Eg5. Here, we report the synthesis of a new series of STLC-like compounds with in vitro inhibition in the low nanomolar range. We also performed a multidrug resistance study in cell lines overexpressing P-glycoprotein and showed that some of these inhibitors may have the potential to overcome susceptibility to this efflux pump. Finally, we performed molecular docking of the compounds and determined the structures of two Eg5-inhibitor complexes to explain the structure-activity relationship of these compounds.

A good molecular target for prostate cancer chemotherapy

An exciting new basic medical research study shows that inhibition of the activity of the kinesin spindle protein Eg5 effectively blocks cell division and induces cell death in prostate cancer cells.1 The potent anticancer drug S-(methoxytrityl)-l-cysteine (S(MeO)TLC) specifically blocks activity of Eg5 in prostate cancer cells, arrests cell division, induces cell death during mitosis and inhibits prostate cancer cells in a mouse model of prostate cancer.1 Prostate cancer is a leading cause of cancer death in men in the United States with over 27 000 deaths due to castration-resistant prostate cancer in 2009.2 Androgen deprivation therapy is effective with most patients for 1 to 2 years. However, prostate cancer cells become resistant to androgen deprivation and progress to castration-resistant prostate cancer.3 Patients can then undergo a standard combination chemotherapy based upon mitotic spindle inhibition by taxanes such as docetaxel.4, 5, 6 However, taxanes cause serious side effects such as neurotoxicity,7 drug-binding site mutations on β-tubulin,8 and overexpression of P-glycoprotein9 and β-tubulin.10 Cancer cells thus develop taxane-related drug resistance. There are no good standard treatment options for patients with castration-resistant prostate cancer when the cancer advances and alternative treatment strategies are urgently needed. That is why this new study is so important. Eg5, a member of the kinesin-5 motor proteins,11 is critical for bipolar spindle formation and chromosome separation during mitosis.12, 13 Inhibition of Eg5 by S(MeO)TLC blocks cell division leading to cell death thus providing an option to microtubule-targeted cancer chemotherapy. Eg5 inhibitors overcome multidrug resistance, since Eg5 is not transported out of cells by P-glycoprotein.14, 15 In addition, kinesin spindle proteins such as Eg5 are absent in differentiated neurons.4 One potent kinesin spindle protein inhibitor ispinesib (SB-71599) is being tested in clinical trials where it appears to be generally well tolerated with mild hematologic and little other toxicity.4 In two recent studies involving Eg5 inhibition in prostate cancer, researchers found that docetaxel-resistant and non-resistant prostate cancer cell lines showed sensitivity to antisense oligonucleotide or S-trityl-l-cysteine.16, 17 In a previous study by some authors of the present study, the Eg5 inhibitor S(MeO)TLC was 10-fold more potent than the antisense oligonucleotide or S-trityl-l-cysteine for inhibition of cell proliferation and it was extremely specific toward Eg5.18 In another recent study, S(MeO)TLC was shown to have potent anticancer activity in bladder cancer in vitro and in vivo.19 In conclusion, more emphasis is now being placed on therapies that aim at specific molecular targets in tumor cells.4 In clinical studies of these antimitotics, neurotoxicity has not been observed to a significant degree and neutropenia is the main toxicity. Unfortunately, early studies in their use have been disappointing in terms of efficacy. However, it is hoped that new agents may improve the therapeutic value of this class of drug and that these newer agents may continue to lengthen the survival of cancer patients and improve upon toxicities in the years ahead.

A potent chemotherapeutic strategy in prostate cancer: S-(methoxytrityl)-L-cysteine, a novel Eg5 inhibitor

Docetaxel-based combination chemotherapy remains the predominant treatment for castration-resistant prostate cancer. However, taxane-related drug resistance and neurotoxicity have prompted us to develop substitute treatment strategies. Eg5 (kinesin spindle protein), which is crucial for bipolar spindle formation and duplicated chromosome separation during the early phase of mitosis, has emerged as an attractive target for cancer chemotherapy. The aim of this study was to investigate the anticancer efficacy of S-(methoxytrityl)-L-cysteine (S(MeO)TLC), a novel Eg5 inhibitor in prostate cancer. Eg5 expression was examined in human prostate cancer cell lines and tissue microarrays were constructed from clinical specimens. Antiproliferative activity of S(MeO)TLC in prostate cancer cells was assessed by a cell viability assay. The anticancer effect and inhibitory mechanism of S(MeO)TLC in prostate cancer cells was further explored by Hoechst staining, flow cytometry and immunofluorescence. In addition, the antitumor effect of S(MeO)TLC on subcutaneous xenograft models was assessed. Eg5 expression was identified in PC3, DU145 and LNCaP cells. More than half of prostate cancer clinical specimens displayed Eg5 expression. S(MeO)TLC exhibited more powerful anticancer activity in prostate cancer cells compared with the other four Eg5 inhibitors tested. S(MeO)TLC induced cell death after arresting dividing cells at mitosis with distinct monopolar spindle formation. S(MeO)TLC exhibited its significant inhibitory activity (P<0.05) on subcutaneous xenograft models also through induction of mitotic arrest. We conclude that Eg5 is a good target for prostate cancer chemotherapy, and S(MeO)TLC is a potent promising anticancer agent in prostate cancer.

Microtubule Stabilization Triggers the Plus-End Accumulation of Kif18A/kinesin-8

The precise control of spindle microtubule (MT) dynamics is essential for chromosome capture and alignment. Kif18A/kinesin-8, an essential regulator of kinetochore MT dynamics, accumulates at its plus-ends in metaphase but not prometaphase cells. The underlying mechanism of time-dependent and kinetochore MT-specific plus-end accumulation of Kif18A is unknown. Here, we examined the factors required for the MT plus-end accumulation of Kif18A. In Eg5 inhibitor-treated cells, Kif18A localized along the MTs in the monopolar spindle and rarely accumulated at their plus-ends, indicating that MT-kinetochore association was not sufficient to induce Kif18A accumulation. In contrast, taxol treatment triggered the rapid MT plus-end accumulation of Kif18A regardless of kinetochore association. Furthermore, Aurora B inhibitor-induced stabilization of the plus-ends of kinetochore MTs promoted the plus-end accumulation of Kif18A. In the absence of Kif18A, treatment with taxol but not Eg5 inhibitor causes highly elongated mitotic MTs, suggesting the importance of plus-end accumulation for the MT length-controlling activity of Kif18A. Taken together, we propose that there is a mutual regulation of kinetochore MT plus-end dynamics and Kif18A accumulation, which may contribute to the highly regulated and ordered changes in kinetochore MT dynamics during chromosome congression and oscillation.

Exploring the Intermediate States of ADP−ATP Exchange: A Simulation Study on Eg5

While mitotic kinesins have attracted significant attention in recent years as new anticancer drug targets, the underlying mechanism of kinesin-catalyzed ATP hydrolysis is still under investigation. Crystal structures of Eg5, one of the best-studied kinesins, have been solved in both ADP-bound and ATP-bound states. However, it is still extremely challenging to experimentally obtain structural information on the functionally important intermediate states, such as the nucleotide free (apo) and the initial ATP-kinesin collision state. Systematic molecular dynamics simulations were performed in this study to mimic different nucleotide binding states and explore the critical structural and dynamic variations during ADP-ATP exchange. Clear conformational changes from "ADP-like" toward "ATP-like" were observed from the simulation results. A highly conserved residue Arg(234) was found to play a key role during the nucleotide exchange. This positively charged residue acted as the "hub" of a hydrogen-bond network that extended the effect of γ-phosphoryl group to both SW-I and SW-II regions. Comparison among the results of different nucleotide binding states indicated that the existence of γ-phosphoryl was immediately sensed at the initial ATP collision state by residue Ser(233), and this initial interaction induced the "back-door" opening and the "front-door" closing of the nucleotide binding pocket. In addition, several potential allosteric binding sites were identified through combination of correlation analysis and binding site mapping approaches based on the simulated apo ensemble, which provided additional targeting sites for novel allosteric Eg5 inhibition. These molecular simulation results provided not only a better understanding of Eg5-catalyzed ATP hydrolysis but also the structural basis for design of novel specific Eg5 inhibitors as anticancer therapeutic agents.

Asymmetric Synthesis and in vivo Biological Inactivity of the Right‐Hand Terpenoid Fragment of Terpendole E

AbstractSynthesis of the DEF‐ring terpenoid fragment of terpendole E, an Eg5 inhibitor, is described. The DE‐ring was constructed by a modification of Barrero's radical cyclization. The F‐ring tetrahydropyran was then constructed by acid‐induced cyclization of an epoxy alcohol, which was prepared by cross‐metathesis followed by Shi's epoxidation. Cell‐based assays indicated that the DEF‐ring fragment is not capable of inhibiting cell growth and cell cycle progression of human cancer cell lines, indicating that the DEF‐ring fragment alone is not sufficient for the biological activity.

Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction

During interphase, centrosomes are held together by a proteinaceous linker that connects the proximal ends of the mother and daughter centriole. This linker is disassembled at the onset of mitosis in a process known as centrosome disjunction, thereby facilitating centrosome separation and bipolar spindle formation. The NIMA (never in mitosis A)-related kinase Nek2A is implicated in disconnecting the centrosomes through disjoining the linker proteins C-Nap1 and rootletin. However, the mechanisms controlling centrosome disjunction remain poorly understood. Here, we report that two Hippo pathway components, the mammalian sterile 20-like kinase 2 (Mst2) and the scaffold protein Salvador (hSav1), directly interact with Nek2A and regulate its ability to localize to centrosomes, and phosphorylate C-Nap1 and rootletin. Furthermore, we demonstrate that the hSav1-Mst2-Nek2A centrosome disjunction pathway becomes essential for bipolar spindle formation on partial inhibition of the kinesin-5 Eg5. We propose that hSav1-Mst2-Nek2A and Eg5 have distinct, but complementary functions, in centrosome disjunction.

1LB Development and validation of robust immunohistochemical assays for phospho-histone-H3 and Eg5 as pharmacodynamic biomarkers to support Eg5 inhibitor (LY2523355) clinical trials in patients with advanced malignancies

A. Nasir, R.D. Van Horn, B. Domazet, X.S. Ye, K.M. Credille, A. Kyshtoobayeva, J.T. Brandt, E.H. Westin, A.E. McGlothlin, T.R. Holzer. Eli Lilly and Co., Diagnostic and Experimental Medicine, Indianapolis IN, USA; Eli Lilly and Co., Lilly Research Labs, Indianapolis IN, USA; Clarient, Immunopathology, Aliso Viejo CA, USA; Eli Lilly and Co., Oncology Business Unit, Indianapolis IN, USA; Eli Lilly and Co., Statistics, Indianapolis IN, USA

1LB Development and validation of robust immunohistochemical assays for phospho-histone-H3 and Eg5 as pharmacodynamic biomarkers to support Eg5 inhibitor (LY2523355) clinical trials in patients with advanced malignancies

1LB Development and validation of robust immunohistochemical assays for phospho-histone-H3 and Eg5 as pharmacodynamic biomarkers to support Eg5 inhibitor (LY2523355) clinical trials in patients with advanced malignancies

Lamin B Counteracts the Kinesin Eg5 to Restrain Spindle Pole Separation during Spindle Assembly

Lamin B is a component of the membranous spindle matrix isolated from Xenopus egg extracts, and it is required for proper spindle morphogenesis. Besides lamin B, the spindle matrix contains spindle assembly factors (SAFs) such as Eg5 and dynein which are known to regulate microtubule organization and SAFs known to promote microtubule assembly such as Maskin and XMAP215. Because lamin B does not bind directly to microtubules, it must affect spindle morphogenesis indirectly by influencing the function of spindle matrix-associated SAFs. Using different assays in Xenopus egg extracts, we found that depleting lamin B caused formation of elongated and multipolar spindles, which could be reversed by partially inhibiting the kinesin Eg5, revealing an antagonistic relationship between Eg5 and lamin B. However, lamin B only very weakly antagonizes Eg5 in mediating poleward microtubule-flux based on fluorescence speckle microscopy. Depleting lamin B led to a very small but statistically significant increase in flux. Furthermore, flux reduction caused by partial Eg5 inhibition is only slightly reversed by removing lamin B. Because lamin B does not bind to Eg5, our studies suggest two nonexclusive mechanisms by which lamin B can indirectly antagonize Eg5. It could function in a network that restricts Eg5-driven microtubule sliding only when microtubules come into transient contact with the network. Lamin B could also function to sequester microtubule polymerization activities within the spindle. Without lamin B, increased microtubule assembly caused by the released SAFs would lead to excessive microtubule sliding that results in formation of elongated and multipolar spindles.

The Effect of Adjuvant Dimethylenastron, a Mitotic Kinesin Eg5 Inhibitor, in Experimental Glaucoma Filtration Surgery

Background: The aim of the study was to analyze the effect of Dimethylenaston, a mitotic kinesin 5 (Eg5) inhibitor, in an experimental setting of glaucoma filtration surgery.Methods: On 37 chinchilla rabbits (ChBBCH), glaucoma filtration surgery similar to clinical practice, was performed. The animals received either no adjuvant, one unilateral subconjunctival injection of Dimethylenastron (1.0 µmol, 3.0 µmol), or the vehicle alone at baseline and in two further groups additionally at days 3 and 7 thereafter (1.0 µmol, 3.0 µmol). The evaluation of antifibrotic efficacy was performed by clinical response, histological examination, and immunohistochemical staining for smooth muscle actin (SMA) and CD 31. The animals were sacrificed on day 14, and the eyes processed for histology.Results: The vehicle was well tolerated. Except for two cases of transient fibrinous reaction after the injection of 3.0 µmol Dimethylenastron, no adverse effects, such as inflammation or blurring of the optical media, were observed. A bleb scarring occurred in the group that received surgery only, adjuvant DMSO, or Dimethylenastron 3.0 µmol. Dimethylenastron (1.0 µmol) induced a milder scarring compared with the control group but the length of bleb survival was not significantly prolonged (p = 0.053, Kaplan-Meier log rank test). In all groups, the intraocular pressure correlated with the fibrotic process and reached normal levels within 14 days after surgery. Those groups injected with 1.0 µmol Dimethylenastron revealed a significantly reduced ratio of intraocular pressure and a milder, but not sufficiently reduced, subconjunctival fibrotic reaction according to the histological and immunohistochemical analysis.Conclusions: The subconjunctival administration of Dimethylenastron 1.0 µmol induced a milder conjunctival scarring. The applied concentrations of Dimethylenastron did not improve the surgical outcome of glaucoma filtration treatments in rabbits sufficiently.

Structure–Activity Relationships and Molecular Docking of Novel Dihydropyrimidine‐Based Mitotic Eg5 Inhibitors

Dihydropyrimidine-based compounds belong to the first discovered inhibitors of the human mitotic kinesin Eg5. Although they are used by many research groups as model compounds for chemical genetics, considerably less emphasis has been placed on the improvement of this type of inhibitor, with the exception of two recent studies. Dihydropyrimidines can be divided into class I (analogues that bind in the S configuration) and class II type inhibitors, which bind in the R configuration. Herein we report the synthesis and optimization of novel class II type dihydropyrimidines using a combination of in vitro and docking techniques.

A Potent Chemotherapeutic Strategy for Bladder Cancer: ( S)-Methoxy-Trityl-L-Cystein, a Novel Eg5 Inhibitor

Eg5, which has an essential role in the formation and maintenance of a bipolar mitotic spindle, was recently identified as an attractive target in cancer chemotherapy. We examined the anticancer activity of a novel Eg5 inhibitor for bladder cancer with particular reference to metastatic disease. We examined bladder cancer cell lines and clinical tissue samples for Eg5 expression and analyzed the antiproliferative activity of 5 Eg5 inhibitors in cell lines by cell viability assay. The anticancer efficacy of the most potent Eg5 inhibitor was investigated in vitro by apoptosis assay with Hoechst nuclear staining and flow cytometry. Immunofluorescence and immunostaining were used to elucidate the inhibitory mechanism. We evaluated the inhibitory effect in vivo in subcutaneous xenograft and metastatic cancer models. Eg5 expression was increased in bladder cancer samples vs that in normal bladder epithelium samples. (S)-methoxy-trityl-L-cystein showed the strongest antiproliferative activity of the 5 Eg5 inhibitors and induced cell death after mitotic arrest via the caspase dependent apoptotic pathway. In vivo (S)-methoxy-trityl-L-cystein effectively suppressed tumor growth in subcutaneous and metastatic xenograft models. Survival time in (S)-methoxy-trityl-L-cystein treated nude mice was significantly longer than in untreated mice (p <0.001). (S)-methoxy-trityl-L-cystein is a promising, novel anticancer agent for bladder cancer. Our data indicates its potential as effective therapy for metastatic bladder cancer.

Structural Basis for Inhibition of Eg5 by Dihydropyrimidines: Stereoselectivity of Antimitotic Inhibitors Enastron, Dimethylenastron and Fluorastrol

Human kinesin Eg5, which plays an essential role in mitosis by establishing the bipolar spindle, has proven to be an interesting drug target for the development of cancer chemotherapeutics. Here, we report the crystal structures of the Eg5 motor domain complexed with enastron, dimethylenastron, and fluorastrol. By comparing these structures to that of monastrol and mon-97, we identified the main reasons for increased potency of these new inhibitors, namely the better fit of the ligand to the allosteric binding site and the addition of fluorine atoms. We also noticed preferential binding of the S-enantiomer of enastron and dimethylenastron to Eg5, while the R-enantiomer of fluorastrol binds preferentially to Eg5. In addition, we performed a multidrug resistance (MDR) study in cell lines overexpressing P-glycoprotein (Pgp). We showed that one of these inhibitors may have the potential to overcome susceptibility to this efflux pump and hence overcome common resistance associated with tubulin-targeting drugs.

The Conserved L5 Loop Establishes the Pre-Powerstroke Conformation of the Kinesin-5 Motor, Eg5

Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.

Docetaxel-Resistant Prostate Cancer Cells Remain Sensitive toS-Trityl-l-Cysteine–Mediated Eg5 Inhibition

Castrate-resistant prostate cancer remains a major clinical challenge. Due to the toxicity profile of taxane-based chemotherapy and treatment failure in some patients, novel agents with improved efficacy to side effect profiles are urgently needed. Eg5, a member of the kinesin-5 family, controls the formation of the bipolar spindle during cell division, and suppressed Eg5 function leads to mitotic arrest. S-Trityl-L-cysteine (STLC) is a novel Eg5-specific small-molecule inhibitor. Here, we report the first study to evaluate its use in prostate cancer. In a panel of prostate cancer cells, LNCaP and PC3 cells were the most and least sensitive to STLC treatment, with a 7.2-fold difference in their respective GI(50) values: 250 nmol/L and 1.8 micromol/L. In LNCaP cells, treatment with either STLC or docetaxel resulted in transient G(2)-M arrest and subsequent caspase-mediated cell death. However, STLC- and docetaxel-treated PC3M cells have distinct fates: STLC induced a transient G(2)-M arrest, followed by polyploidy; in contrast, docetaxel-treated PC3M cells progressed to apoptosis after a transient G(2)-M arrest. Docetaxel-resistant LNCaP-derived (LDocR) cells respond to STLC in a similar manner to the parental cells. Although the docetaxel-resistant PC3M-derived (PDocR) cell line and its parental PC3M cells have similar GI(50) to STLC treatment, PDocR cells showed significantly more G(2)-M arrest and less apoptosis. Hence, although docetaxel-resistant prostate cancer cells remain responsive to Eg5 inhibition with STLC, there are key differences at the cell cycle level, which may have implication in future development.

Abstract 4430: A novel mitotic kinesin Eg5 inhibitor exerts the growth-inhibitory effect in cancer cells in a manner independent of induction of monoastral formation but dependent on KRAS mutation status

Abstract Mitotic kinesin Eg5 plays an important role in mitosis, as it is critical for proper bipolar spindle assembly. After the discovery of the first Eg5 inhibitor monastrol, a number of Eg5 inhibitors have been developed as anticancer drugs. We have previously synthesized a series of S-trityl-L-cysteine (STLC) derivatives as Eg5 inhibitors and demonstrated potent inhibitory effect on Eg5 ATPase activity and induction of mitotic arrest with characteristic monoastral spindles in HeLa cells. In this study, we evaluated the growth-inhibitory acitivities of novel STLC derivatives, Compound1, Compound2, and Compound3 by MTT assay in a wide range of human cancer cell lines derived from gastric (MKN1, MKN45, MKN74, NUGC3, NUGC4, NCI-N87), colon (C170, DLD1, HCT15, COLO205), pancreatic (AsPC1, BxPC3, SUIT2) and lung (PC-9, PC-14, H1975, H441, A549) cancers and investigated potential predictive biomarkers. Among the derivatives, Compound3 showed the most potent growth-inhibitory effect and sensitivity between cell lines showed marked differences, with IC50 values ranging from 0.134 to 7.14 μM. Pancreatic cancer cell line AsPC1 and non-small cell lung cancer (NSCLC) cell lines H441 and A549 which harbor KRAS mutation showed relatively high IC50 values compared to other cell lines without KRAS mutation. Compound3 induced G2/M arrest in all cell lines as early as 8 hours after the treatment by flowcytometry, while the increase of the apoptotic sub-G1 fraction was not observed in these AsPC1, H441 and A549 cells harboring KRAS mutation. These results suggested that differential sensitivity to Compound3 and attenuated induction of apoptosis by Compound3 were attributed to KRAS mutation. Induction of monoastral spindles formation by Compound3 was observed as early as 8 hours after the treatment not only in sensitive cell lines but in refractory cell lines such as AsPC1 and H441 cells by immunocytochemistry, suggesting the induction of monoastral spindles formation does not work as a predictive biomarker for clinical development. There was no correlation observed between the growth-inhibitory effect and the basal Eg5 expression level of each cell line by western blot analysis. No significant difference was observed in the induction of a spindle checkpoint kinase BubR1 phosphorylation among the cells, a potential predictive biomarker in response to the Eg5 inhibitor treatment by western blot analysis. Taken together, a novel mitotic kinesin Eg5 inhibitor Compound3 should be considered for further exploration and development and KRAS mutation status was suggested to be a candidate for a predictive biomarker of poor response to Eg5 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4430.

Abstract 4427: Efficacy of a novel mitotic kinesin Eg5 inhibitor in docetaxel-resistant non-small cell lung cancer cells

Abstract Lung cancer is the leading cause of cancer-related deaths in Japan and worldwide. Non-small cell lung cancer (NSCLC) accounts for a majority of lung cancer and docetaxel is one of the key drugs in the treatment of metastatic NSCLC in combination with platinum agents as first-line therapy and is also given as second-line monotherapy. However, most of the patients acquire resistance and fail to respond not long after the initial treatment. Development of novel therapeutics which is active in docetaxel-resistant NSCLC is necessary. Mitotic kinesin Eg5 is critical for proper bipolar spindle assembly during mitosis. It also plays an important role in cancer cell proliferation and has been thought to be a good target for cancer therapeutics. We have synthesized an S-trityl-L-cysteine (STLC) derivative Compound3 as a novel Eg5 inhibitor and previously demonstrated potent growth-inhibitory effect in a wide variety of cancer cell lines. In this study, we evaluated the efficacy of Compound3 in docetaxel-resistant NSCLC cell line PC-14/TXT. Compound3 showed potent growth-inhibitory effect in PC-14/TXT cells by MTT assay without cross-resistance to docetaxel. In order to study the mechanism of growth inhibition by Compound3, we performed flow cytometry analysis and evaluated the effects of docetaxel and Compound3 on cell cycle progression in PC-14 and PC-14/TXT cells. Docetaxel induced G2/M cell cycle arrest only in PC-14 cells but it did not in PC-14/TXT cells. Compound3 induced G2/M cell cycle arrest not only in PC-14 cells but also in PC-14/TXT cells. Compound3 induced increase of sub-G1 fraction in PC-14 cells, suggesting Compound3 induced apoptosis in PC-14 cells. On the other hand, we did not observe increase of sub-G1 fraction by Compouond3 treatment in PC-14/TXT cells. These results suggest Compound3 exerts growth-inhibitory effect in PC-14/TXT cells by cell cycle arrest and it has a differential growth-inhibitory property between PC-14 and PC-14/TXT cells. Effect of docetaxel and Compound3 on spindles formation was evaluated by immunocytochemistry. Docetaxel induced multipolar spindles formation in PC-14 cells but did not show any effect in PC-14/TXT cells. On the other hand, Compound3 induced distinctive monoastral spindles formation in both PC-14 and PC-14/TXT cells as early as 8 hours after the treatment. We performed immunoblot analyses to further investigate the effect of Compound3 on molecules relevant to Eg5. There was no correlation observed between the growth-inhibitory effect and the basal Eg5 expression level between two cell lines. No significant difference was observed between the cells in the status of spindle checkpoint kinases such as MAD2 and BubR1. Taken together, a novel mitotic kinesin Eg5 inhibitor Compound3 should be considered for further exploration and development as a novel therapeutic option for the patients with non-small cell lung cancer who failed docetaxel treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4427.

Abstract 4429: Characterization of the kinesin spindle protein inhibitor AZD4877

Abstract The process of mitosis is a validated point of intervention in cancer therapy and a variety of anti-mitotic drugs are successfully being used in the clinic. To date, all approved antimitotics target the spindle microtubules, thus interfering with spindle dynamics, leading to mitotic arrest and apoptosis. While effective, these drugs are also associated with a variety of side effects, including neurotoxicity. In recent years, mitotic kinesins have attracted significant attention in the search for novel, alternative mitotic drug targets. In addition, kinesin inhibitors may overcome resistance to microtubule targeting drugs. Here we describe the pharmacological evaluation of AZD4877, a selective Eg5 inhibitor. AZD4877 exhibits potent cell growth inhibition in vitro against a broad panel of both solid and hematological tumor cell lines. Efficacy is associated with formation of monoastral spindles, mitotic block and the induction of apoptosis. AZD4877 also demonstrates activity in multiple tumor xenograft models, including primary bladder tumor explants and a Rituximab-insensitive non-Hodgkin's Lymphoma model (DoHH2T53). Combination studies with a taxane agent (Docetaxel) demonstrate additive efficacy of tumor growth inhibition, with notable tumor regrowth delay in the combination arm. In summary, AZD4877 is an active Eg5 inhibitor with potent anti-tumor activities in vitro and in vivo. The efficacy observed in combination studies and in a rituximab-resistant tumor model suggests possible clinical applications for this agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4429.