Selective Aurora kinase inhibitors identified using a Taxol-induced checkpoint sensitivity screen
The members of the Aurora kinase family play critical roles in the regulation of the cell cycle and mitotic spindle assembly and have been intensively investigated as potential targets for a new class of anti-cancer drugs. We describe a new highly potent and selective class of Aurora kinase inhibitors discovered using a phenotypic cellular screen. Optimized inhibitors display many of the hallmarks of Aurora inhibition including endoreduplication, polyploidy, and loss of cell viability in cancer cells. Structure-activity relationships with respect to kinome-wide selectivity and guided by an Aurora B co-crystal structure resulted in the identification of key selectivity determinants and discovery of a sub-series with selectivity towards Aurora A. A direct comparison of biochemical and cellular profile with respect to published Aurora inhibitors including VX-680, AZD1152, MLN8054, and a pyrimidine-based compound from Genentech demonstrates that compounds 1 and 3 will become valuable additional pharmacological probes of Aurora dependent functions.
INTRODUCTION<!>Phenotypic Screen Identifies SAC Inhibitor<!>Compound 1 Potently Inhibits Aurora Kinases<!>Crystal structure of Aurora B: INCENP in complex with compound 1<!>Summary of Structure-Activity Relationships<!>In vitro Selectivity of Compound 1<!>Compound 1 Inhibits Aurora-dependent spindle assembly processes<!>Antiproliferative activity<!>SIGNIFICANCE<!>Chemistry<!>Cell Culture<!>In vitro kinase assay (Mitotic Kinase Panel)<!>Immunofluorescence<!>Mitotic Checkpoint Assays<!>Fluorescence-Activated Cell Sorting Analysis (FACS)<!>Proliferation, Colony Outgrowth
<p>The inhibition of critical regulatory mitotic kinases using ATP-competitive small molecules is an active area of research in the quest for a new class of anti-cancer therapeutics. Numerous compounds targeting key cell cycle kinases including Cyclin-dependent kinases (Cdk), Aurora (Aur), Polo-like kinases (Plk) and the kinesin-5 molecular motor have been advanced into clinical testing. The clinical rationale for targeting mitosis to treat cancer is provided by Taxol, a highly successful anti-cancer agent that arrests cell division by stabilizing microtubule polymers thereby disrupting the cellular machinery required for mitotic spindle assembly. Unfortunately, to date most of the small molecules targeting cell cycle kinases have displayed limited clinical efficacy and have suffered from dose-limiting bone marrow toxicity. We hypothesized that there might exist small molecule kinase inhibitors that synergize with Taxol augmenting the anti-proliferative and apoptotic response.</p><p>Previous reports have demonstrated that the cell death response to Taxol treatment is dependent upon the ability of cells to maintain a mitotic arrest (1–3). This phenomenon has been attributed, in part, to post-translational modification and inactivation of anti-apoptotic proteins during mitosis allowing for engagement of a productive apoptotic response (4–6). This post-translational modification is lost when cells exit mitosis leading to stabilization of anti-apoptotic proteins and concomitant decrease in Taxol-mediated cell death. Therefore, we hypothesized that the identification of a small molecule that maintained a mitotic arrest independent of the spindle assembly checkpoint (SAC) status could potentiate the apoptotic response to Taxol. Conversely, a small molecule that inhibits the SAC would be expected to weaken the apoptotic response to Taxol. We performed a medium throughput proliferation assay of approximately 1000 known and novel small molecule kinase inhibitors alone and in combination with Taxol to find compounds that could agonize or antagonize the anti-proliferative effects of Taxol. One class of compounds that emerged as antagonists of Taxol-induced growth inhibition from this screening effort was a series of pyrimido benzodiazepines exemplified by 1 and 3. A candidate-based approach combined with extensive chemical proteomic and kinase binding panel-based profiling effort established that these compounds are potent Aurora A/B kinase inhibitors.</p><p>Aurora A and B share significant sequence similarity, particularly within their kinase domains, however each kinase exhibits unique precise temporal and spatial control by dynamic association with accessory proteins (7–19). These interactions allow Aurora A and B to independently regulate many important mitotic processes. Aurora A regulates the separation of centrosomes in S phase/early G2 (20–22) and contributes to bipolar spindle formation in mitosis by regulating microtubule (MT) nucleation, bundling, and stabilization (23–25). Aurora B facilitates proper bipolar end-on MT-kinetochore attachment (26–28), participates in SAC signaling (29–31), and mediates chromosome condensation and cohesion (32). Aurora B re-localizes to the central spindle during late anaphase and to the mid-body during telophase thereby facilitating cytokinesis (33). Chemical perturbation of Aurora kinases has proven invaluable in parsing the temporal and spatial functions of each isoform and assessing the therapeutic potential in inhibiting kinase activity in the context of cancer.</p><p>Detailed biochemical and cellular mechanism of action studies demonstrated that these inhibitors potently inhibited the Aurora kinases at low nanomolar concentration in cells. Compound treatment faithfully recapitulated phenotypes associated with RNAi and chemical inhibition of Aurora A (20–22, 32) and B (16, 26–28, 30, 33) kinases including monopolar spindle formation, cytokinesis failure, and polyploidy. Additionally, compound 1 efficiently disables the SAC which is consistent with the known requirement for a Taxol induced arrest requiring a functional checkpoint. We co-crystallized 1 with the Xenopus laevis Aurora B/INCEP complex and determined the structure at 1.85 Å resolution. We used this structure in conjunction with kinome-wide selectivity profiling to guide chemical modifications that allowed the identification of key selectivity determinants and the generation of Aurora A-selective agents. We compared the anti-proliferative effects of these new Aurora kinase inhibitors to 4 literature compounds: VX680 (compound 32), a pan-Aurora inhibitor; AZD1152 (compound 33), an Aurora B selective agent, and two Aurora A selective compounds MLN8054 (compound 34) and a pyrimidine-based compound from Genentech (compound 35) (Figure 1) (34–37). Consistent with previous studies, the comparison of these compounds to existing Aurora inhibitors demonstrates that much of their anti-proliferative activity is derived from inhibition of Aurora B (38).</p><!><p>In order to identify compounds that could modulate the spindle assembly checkpoint we performed a proliferation screen for compounds that could synergize or antagonize with Taxol. We screened a diverse library (~1000 compounds) of heterocyclic ATP-site directed kinase scaffolds in dose response format to identify new small molecule modulators of Taxol-induced mitotic arrest and cell death. We conducted the screen in Hela and MCF7 cell lines that are sensitive and resistant to Taxol-induced cell death, respectively (3). Compounds eliciting a differential effect in combination with Taxol when compared to Taxol alone were then studied for their effect on cell cycle progression. We identified a broad class of pyrimidodiazepinone compounds which represent a privileged ATP-site directed pharmacophore which has been successfully elaborated to generate selective inhibitors of Erk5, LRRK2 and Mps1 (39–41). For example, the benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-ones, represented by compound 1 (Figure 2a) emerged as a particular promising 'hit' compound. When cells were co-treated with Taxol (100 nM) and 1 (50 nM) there was a significant reduction in cell survival compared to Taxol as a single agent over a 48 hour time period (Figure 2b). Consistent with the notion that abrogation of mitotic arrest mitigates cell death caused by microtubule stabilizers, compound 1 treatment resulted in a premature exit from a Taxol-induced mitotic arrest (Figure 2c). These results suggested that 1 was capable of functioning as an inhibitor of the spindle assembly checkpoint.</p><!><p>In order to identify the molecular target(s) of 1, we pursued a candidate-based approach and investigated its ability to inhibit the enzymatic activity of known spindle-assembly checkpoint regulating kinases such as Aurora A, Aurora B, Bub1, CDK1/cycB, Plk1, Mps1 and Nek2A (Figure 3a). Compound 1 potently and selectively inhibited the enzymatic Aurora A, B and C kinases with IC50s of 5.6 nM, 18.4 nM, and 24.6 nM respectively (Figure 3b). The selectivity of these inhibitors was surprising considering the structural similarity of 1 to previously reported kinase inhibitors (39–42). For example, the slight modification from a two-ring system, exemplified by Mps1-IN-2 and BI2536, to a three ring system, exemplified by 1, was sufficient to abrogate the ability of 1 to inhibit Mps1 and Plk1 activity. The structural basis for this result will be discussed in the following section.</p><!><p>We determined the crystal structure of the Aurora B:INCENP complex from Xenopus laevis (21) in complex with compound 1. The crystals contain a tight complex of residues 60–361 Aurora B (Aurora B60–361) and residues 790–847 of INCENP (INCENP790–847), the so-called IN-box (Figure 3c). Crystals of the Aurora B:INCENP complex with compound 1 diffracted X-rays to a resolution of 1.85 Å (Table S1). Electron density "difference" maps with phases calculated from the model of the Aurora B60–361:INCENP790–847 complex revealed additional density in the Aurora B ATP-binding pocket (Figure 3c). The density could be fitted unequivocally with an atomic model of compound 1. As expected, the inhibitor bound to the ATP binding pocket of Aurora B, hydrogen bonded to the carbonyl of Ala173 within the hinge backbone. No significant structural rearrangements of the kinase backbone are observed in the presence of the compound (Figure S1). Binding is stabilized by a number of hydrophobic interactions involving Leu170 (the gatekeeper residue), Phe172, Leu223, and the P-loop residues Leu99 and Val107 (Figure 3d).</p><p>To rationalize the observed selectivity of 1 for Aurora–family kinases, we superimposed the models of Aurora A, Mps1 and Plk1 onto the Aurora:INCENP:compound 1 structure. We then assessed if 1 could at least in principle be fitted without major steric clashes in these additional active sites (Figure S2). As expected based on the high sequence conservation relating Aurora A and Aurora B, compound 1 could be nicely modeled in the Aurora A catalytic cleft, probably accounting for the 5.6 nM IC50 (Figure S2a–b). On the other hand, a predicted steric clash between large hydrophobic residues in the active sites of Mps1 and Plk1 (Ile663 and Phe169, respectively) and the additional anthranilic ring of compound 1 is probably the cause of the poor inhibitory activity of compound 1 against these kinases (Figure S2). Interestingly, a cis-peptide bond between Ile663 and Asp664 in the Mps1 active site causes a potential clash against the third ring of the pyrimido benzodiazepine moiety (Figure S2).</p><!><p>Having assessed the intracellular activity and in vitro selectivity of 1 we sought to evaluate the potential of this scaffold to generate selective Aurora A or Aurora B agents. For these SAR studies, we desired to efficiently modify the 2-amino and fused anthranilic acid moieties of the core benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one scaffold. To do so, we developed a concise four-step synthetic route to complete the synthesis of a comprehensive analog library of our initial screening 'hit' 1 (40). Details of the synthetic methods and compound characterization are described in the Supplementary Materials.</p><p>Approximately 30 benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one analogs were evaluated for their ability to inhibit Aurora A and B kinase activity (IC50 data - Tables 1 and 2, structure-activity relationship (SAR) data - Figure 4). Comparison with our previously developed ERK5 (40) and LRRK2 (39) inhibitors revealed that substitution of the ortho-position of the 2-anilino moiety of 1 is not tolerated for potent Aurora inhibition (compound 2 vs. 3), likely due to steric clash with Phe172 (Figure 3d). We first explored the 2-amino moiety of this scaffold by replacing the aniline substituent of 1 with various 3-substituted, 4-substituted, and 3,4-disubstituted anilines (Table 1). These modifications all yielded compounds possessing similar activity against Aurora A and B. These results indicated that diverse substitutions at 3- or 4-position of 2-aniline moiety were well tolerated, but did not impart significant selectivity between Aurora kinases. Interestingly, when 3,4-disubstituted anilines were introduced, these analogs (12–14) showed much better in vitro activity against Aurora A compared with Aurora B (compounds 12 vs 7, 13 vs 3, and 14 vs 9). This result suggested that additional substituents at the 3-position will affect the selectivity of Aurora A vs B. Compound 18 containing an unsubstituted lactam amide exhibited a 10-fold decrease in activities for both Aurora kinases, which indicated the methyl substitution of the lactam is preferred.</p><p>We next investigated the effects of additional substituents at the 3-position of aniline with respect to Aurora A vs B selectivity (19–23, Table 2). Compound 23 having the combination of 3-trifluoromethyl and 4-(4-ethyl-piperazin-1-yl)-methyl substituents gives the best in vitro selectivity with an IC50 ratio of 12. We ten fixed 3-trifluoromethyl-4-(4-ethyl-piperazin-1-yl)-methyl aniline at the 2-position of benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one, and studied the effects of modification to the N-substituent (X) of the anthranilic acid and to the aryl ring (R3) (24–31, Table 2). Modification of the N-substituent (X) from methyl, to isopropyl, to cyclopentyl, exemplified by 23, 24 and 25 respectively, led to gradual decreases in enzyme activity and selectivity. Linkages using oxy (26) or sulfur (27) resulted in a dramatic loss of activity. These results suggest that modifications to the N-substituent (X) are not well tolerated because the linkage groups are the determinant of the dihedral angle of 7-membered ring. There appears to be a great effect of substituent position for selectivity as the 3-methyl, 4-methyl, 3-fluoro, and 4-fluoro analogs (28 vs 30, 29 vs 31) exhibited marked difference in selectivity of Aurora A vs B. Among them, compound 29 represented the best in vitro selectivity (36-fold) and activity of 13.4 nM against Aurora A. Facing the 3-position of the aniline group in Aurora A and Aurora B are distinct residues (Thr217 and Glu177, respectively), suggesting that this position of the Aurora kinase scaffold acts as a selectivity filter in binding to these derivatives (Figure S2b).</p><p>The SAR exploration of the benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one scaffold revealed that no substitution at 2-position of 2-amino moiety, the combination of 3-trifluoromethyl and 4-(4-ethyl-piperazin-1-yl)-methyl substituents of 2-amino moiety, N-methyl substitution at lactam position, linkage group (X) of N-methyl substituent, and 3-fluoro substituted anthranilic acid were key structure determinants to achieve potent in vitro activity and selectivity of Aurora A vs B. The most selective compound 29 inhibited Aurora A with an in vitro IC50 of 13.4 nM exhibited 36-fold selectivity of Aurora A vs B. Despite showing superior in vitro selectivity against Aurora B these trifluoromethyl-functionalized compounds suffered from decreased intracellular selectivity and potency (Figure S3–S5). The reason for this discrepancy is not immediately apparent but it does not appear to be due to the usual explanation of poor cell penetration as demonstrated below.</p><!><p>To comprehensively investigate the selectivity of this new class of Aurora inhibitors compounds 1, 23, and 29 were profiled at a concentration of 1 μM using the KINOMEscan methodology across a panel of 442 kinases (43). This analysis revealed that these new Aurora inhibitors possessed highly selective profiles with KINOMEscan selectivity score S10 of 0.09, 0.1 and 0.04 for compound 1, 23 and 29, respectively (Table S2). This broad spectrum approach offers excellent coverage of the kinome and helped identify a number of potential off-target kinases including (Table S2). Confirmatory dose-response analysis in biochemical kinase assays for compound 1 revealed sub-100 nM IC50 against DCAMKL2, LRRK2, NUAK1 and TAOK1 (Table S3). A second complimentary chemical proteomics approach using a desthiobiotin-ATP reagent as an ATP-site directed covalent probe was used to study the selectivity of 1 in the context of Hela cell lysates (44). In this assay format a compound is tested for its ability to protect labeling of the catalytic and/or activation loop lysine by the desthiobiotin-ATP probe (Table S4). At 100 nM and 1 μM, concentrations at which the SAC silencing phenotype is fully penetrant, compound 1 displays good selectivity for Aurora kinases. These unbiased approaches demonstrate that up to concentration of 1 μM compound 1 is highly specific inhibitor for Aurora A, B, and C however further analysis is required to investigate whether the additional targets mentioned above are indeed bonafide targets at higher concentrations in cells. Compound 1 is a potent biochemical inhibitor of LRRK2 (IC50= 30.5 nM) and this should be considered if it is being employed in scenarios where LRRK2 is expressed.</p><!><p>To further characterize whether the cellular effects of 1 were derived from direct inhibition of Aurora kinases, we performed a battery of cellular assays that report on intracellular Aurora activity which we compared directly with established Aurora inhibitors. Both Aurora A and B demonstrate increase in kinase activity during mitosis as a result of association with accessory proteins that generate fully competent kinase complexes (7–19) and by autophosphorylation of critical residues (T288 AurA and T232 AurB) in the kinase activation (T) loop (7–9, 12, 26, 45). To assess the effect of 1 on the autophosphorylation status of Aurora A and B, Hela S3 cells were synchronized in mitosis in the presence of DMSO or 1 (Figure 5a). DMSO-treated cells displayed strong Aurora kinase activity, while introduction of 1 led to a dose-dependent decrease in kinase activity with complete inhibition of both Aurora A and B by 250 nM (Figure 5b). Stable cyclin B levels demonstrate that loss of kinase activity was not caused by mitotic exit. Comparison of 1 with 32, a pan-Aurora kinase inhibitor, revealed similar activities against the activation of Aurora A and B; while 33, an Aurora B selective agent, displayed moderate intracellular activity against Aurora A (Figure S3). All three compounds resulted in a decrease of Ser10 phosphorylation on Histone H3, a known Aurora B substrate (Figure 5c, 5d, and S4). Consistent with the known dependence of MT-kinetochore error correction on Aurora B kinase activity, treatment with compound 1 prevented the de-stabilization of improper connections manifesting significant chromosome congression defects in metaphase cells (Figure 5e and S5).</p><p>Aurora A localized at the centrosome is responsible for maturation and separation of centrosomes (32, 46–49). Decreased Aurora A kinase activity leads to shortened bipolar spindles as a result of the inability to generate the force necessary to separate centrosomes (20–22, 32). Complete inhibition of Aurora A results in the formation of monopolar spindles. To determine if 1 elicits spindle phenotypes consistent with inhibition of Aurora A, we examined spindle morphology in the presence 1. To do so, cells were arrested in metaphase in the presence of DMSO or 1 and the spindle morphology was analyzed by indirect immunofluorescence. Cells treated with 1 exhibited a dose-dependent increase in the frequency of shortened and monopolar spindles with visually complete monopolarity at roughly 800 nM (Figure 5d, 5f). Similar results were found with 32, whereas the Aurora B selective 33 did not result in significant monopolarity up to a concentration of 500 nM (Figure S5). Together these results indicate that 1 inhibits both Aurora A and B in cells at roughly similar concentrations.</p><!><p>To evaluate the antiproliferative activity of these new Aurora inhibitors, select compounds were assayed in dose response format against the human colorectal cancer cell lines HCT116 and HT29 and the human cervical cancer cell line HeLa S3 (Figure 6a, S6; Table 1 and 2). The results suggested that the antiproliferative activity of a compound correlated closely with its ability to inhibit Aurora B kinase activity. Indeed, the pan-Aurora inhibitors exemplified by 1 and 3 clustered closely with 32 and 33 based on common anti-Aurora B activity, while the Aurora A selective compounds 23 and 29 clustered with the Aurora A selective inhibitors 34 and 35 (Figure S6, Tables 1–3). These results are consistent with previous reports demonstrating that the antiproliferative activity of an Aurora inhibitor appears to correlate with its ability to inhibit Aurora B (38).</p><p>FACS analysis allowed us to follow the relative genomic integrity of cells as they progressed through the time course of the anti-proliferative assay. Congruent with the essential roles of Aurora A and B in the faithful execution of cytokinesis, treatment with 1 caused cells to fail cytokinesis and become polyploid (Figure 6b). Over the course of 96 hours cell size increased, cells underwent endoreduplication, and reached DNA contents of 16c. As cells continued to undergo errant rounds of cell division in the presence of 1, a sub-G1 population, typically indicative of cell death/debris, began to form starting at 48 hours. Propidium iodide (PI) staining to differentiate live vs. dead cells indicated that 72–96 hours following treatment with 1 significant cell death was observed in the population (Figure 6c, 6d). Loss of cell viability was also reflected in an inability of 1-treated cells to form viable progeny in a colony formation assay (Figure 6e, 6f). Similar levels of ploidy and cell death were witnessed with comparable concentrations of 32 and 33 (Figure S7 and S8).</p><p>Despite showing superior in vitro selectivity for Aurora A 23 and 29 suffered from decreased intracellular potency (Figures S3–S8). For example, while compound 29 inhibited Aurora A with a biochemical IC50 of 13.4 nM, significant intracellular Aurora A inhibition was not seen until approximately 500 - 1 μM (Figures S3–S5). One plausible explanation for this discrepancy is poor cell penetrability. To test this hypothesis we used a two-pronged approach utilizing modified versions of the desthiobiotin-ATP proteomics assay, which allowed us to identify compounds with poor cell penetrability. In brief, cells were pre-treated with test compounds or DMSO, lysed, and the lysate probed with desthiobiotin-ATP reagent. As 23 and 29 exhibit strong activity against Aurora A in vitro it would be expected that these compounds when treated to live cells would remain bound to Aurora A in the lysate and compete with desthiobiotin-ATP for the Aurora A active site. Secondly, to assess whether these compounds can indeed compete directly for Aurora A, in the absence of intact cellular membrane, lysates were co-treated with 23 (or 29) and desthiobiotin-ATP. Compounds that can compete with desthiobiotin-ATP directly in lysates but not when pre-treated to intact cells are deemed to have poor cell penetrability. Compound 1, which has demonstrated intracellular activities on Aurora A consistent with its enzyme IC50, served as a positive control. It displayed significant competition against the desthiobiotin-ATP reagent for Aurora A in both lysates and live cell treatment in a dose dependent manner (Figure S9). Despite a diminished dose dependency compounds 23 and 29 demonstrated comparable ability to compete with the desthiobiotin-ATP probe in the context of lysate and live cell treatment. Therefore, this result cannot support the assertion that poor cell penetrability is the cause of the reduced penetrance of Aurora A-associated phenotypes.</p><!><p>These studies demonstrate that it is possible to obtain highly potent and selective inhibitors of Aurora kinases directly from a phenotypic cellular screen. Compounds 1 and 3 represent a novel class of pan-Aurora inhibitors with the ability to fully inhibit intracellular Aurora A and B activity at 500 nM concentrations. The phenotypic consequences of treatment of cells with 1 and 3, include cytokinesis failure, endoreduplication, and eventual loss of cell viability consistent with cellular effects established with the pan-Aurora inhibitor 32 and the Aurora B selective 33. Previous reports demonstrated that inhibition of Aurora B is the primary source of the anti-proliferative effects elicited by Aurora inhibitors (38). The results obtained here are consistent with those reports with 1 and 3 clustering with inhibitors displaying strong anti-Aurora B activity in anti-proliferative assays. When co-treated with Taxol cells exhibited a decrease in Taxol-induced cell death supporting the notion that mitotic arrest is crucial to facilitating Taxol toxicity.</p><p>The structures described here resulted from the synthetic evolution of predecessor compounds displaying activities against distinct kinases including Plk1, Mps1, Erk5, and LRRK2 (39–42). These compounds serve as prime examples of how a pyrimidine-based core scaffold can be diversified by ring addition and expansion to generate compounds with differential activities. Subtle modifications to these existing compounds, guided by activity-based profiling have culminated in potent pan-Aurora inhibitors and Aurora A selective agents. A co-crystal structure of 1 with X. laevis Aurora B illustrated the structural basis for the binding affinity of compound 1 revealing that it binds in a puckered conformation in the ATP binding site. This co-crystal structure provided a platform for systematic modification of the scaffold to investigate selectivity with respect to other kinases. In addition to selectivity determinants this SAR study enumerated many structural features that are critical for anti-Aurora activity. Furthermore, an extensive structure-activity relationship study, guided by this structural information, resulted in the identification of compounds with 12 and 36-fold biochemical selectivity for Aurora A which possessed poor cellular activity. The poor cellular activity of the Aurora A selective inhibitors 23 and 29 do not appear to be a result of poor cell penetration. The weak Aurora A-dependent phenotypes could come as a consequence of the need for a more complete inhibition of Aurora A activity in order to elicit an Aurora A-dependent phenotype relative to the amount of inhibition of Aurora B required for an Aurora B-dependent phenotype. However, the strong Aurora A-dependent phenotypes elicited by the pan-Aurora inhibitors 1 and 3 do not support this explanation.</p><p>Compounds from this scaffold class have demonstrated a high degree of selectivity for Aurora kinases. The comparison of these new compounds with currently available inhibitors each with different off-target profiles will further the investigation into the therapeutic value of inhibiting this family of kinases. Furthermore, continued chemical interrogation of this privileged scaffold has potential to further our understanding of its SAR and to identify new kinase targets. Compounds of this scaffold class have shown favorable pharmacokinetic properties (40) suggesting that these compounds may facilitate inhibition of kinases at physiologically and therapeutically relevant concentrations.</p><!><p>Compounds were synthesized by modification of existing procedures (WO/2010/080712). See online Supplementary Material for characterization data.</p><!><p>Hela, Hela S3, and MCF7 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Sigma), while HCT116 and HT29 cells were grown in McCoy's 5A (Invitrogen). All cell lines were supplemented with 10% FBS (Sigma) and 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin (Invitrogen) and cultured at 37°C in a humidified chamber in the presence of 5% CO2, unless otherwise noted.</p><!><p>The conditions for the kinase assays shown in Figure 3a have been previously described (29).</p><!><p>Protocols for immunofluorescence have been previously described (41).</p><!><p>Hela S3 were plated at roughly 40% cell density. The following day the cells were treated with medium containing thymidine for 24 hours.</p><p>After thymidine block, the cells were washed with PBS and replaced with fresh medium for 8 hours. After which time medium containing test compound was added for 1 hour followed by co-treatment with test compound and Taxol. After 4 hours, the cells were harvested and the mitotic index was determined by immunoblotting for cyclin B. For experiments examining phosphorylation of Aurora A and B cells were treated as above but were co-treated with Taxol and MG132 for 2 hours prior to harvesting and immunoblotting.</p><!><p>Cells were treated with compound for various periods of time. Cells were trypsinized, washed once in phosphate-buffered saline (PBS), and fixed overnight at −20°C with 80% ethanol in PBS. Cells were washed three times with PBS. Finally, cells were resuspended in PBS containing 0.1% Triton X-100, 25 μg· ml−1 propidium iodide (PI, Molecular Probes), and 0.2 mg·ml−1 RNase A (Sigma) and incubated for 45 minutes at 37°C. For discrimination of live and dead cells by PI staining cells were treated with compound for various periods of time. Cells were trypsinized, washed with PBS, and treated with PBS containing 25 μg· ml−1 PI. Cell cycle distribution or PI stain for viability was determined on a BD FACScan (BD Biosciences) and analyzed on FlowJo (Treestar).</p><!><p>Taxol sensitivity proliferation assays and colony formation assays were performed as previously described (40), (41). Proliferation assays testing compounds alone were conducted using Cell Titer Glo as described in product manual.</p>