Discovery and Mechanistic Study of a Small Molecule Inhibitor for

Jul 29, 2013 - ABSTRACT: Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability ...
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Discovery and Mechanistic Study of a Small Molecule Inhibitor for Motor Protein KIFC1 Jiaquan Wu,†,§,∥ Keith Mikule,‡,∥ Wenxian Wang,‡ Nancy Su,† Philip Petteruti,‡ Farzin Gharahdaghi,‡ Erin Code,† Xiahui Zhu,† Kelly Jacques,‡ Zhongwu Lai,‡ Bin Yang,‡ Michelle L. Lamb,‡ Claudio Chuaqui,‡ Nicholas Keen,‡ and Huawei Chen*,‡ †

Discovery Sciences and ‡Oncology Innovative Medicine Unit, AstraZeneca R&D Boston, Waltham, Massachusetts 02451, United States S Supporting Information *

ABSTRACT: Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability and tumorigenesis. Cancer cells have evolved mechanisms to bundle multiple centrosomes into two spindle poles to avoid multipolar mitosis that can lead to chromosomal segregation defects and eventually cell death. KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. To this end, we have identified the first reported small molecule inhibitor AZ82 for KIFC1. AZ82 bound specifically to the KIFC1/microtubule (MT) binary complex and inhibited the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a Ki of 0.043 μM. AZ82 effectively engaged with the minus end-directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5. Treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Consistent with genetic studies, our data confirmed that KIFC1 inhibition by a small molecule holds promise for targeting cancer cells with amplified centrosomes and provided evidence that functional suppression of KIFC1 by inhibiting its enzymatic activity could be an effective means for developing cancer therapeutics.

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impairing normal cells.9 Centrosome clustering mechanisms were investigated via an RNAi screen in multicentrosomal cells. Genes linked to centrosome clustering included chromosomal passenger complex (CPC) components, proteins involved in the organization and regulation of the cytoskeleton, and the minus end-directed motor protein KIFC1.10,11 Among these genes, KIFC1 stands out as an attractive candidate for cancer therapeutic development because it is not required for diploid cell division and small molecule inhibitors for other motor domain proteins have been demonstrated. KIFC1 belongs to the kinesin-14 family of motor proteins.12 Similar to other motor proteins, KIFC1 contains a MT binding domain, a stalk domain, and a motor domain,13 but the arrangement of these domains is reversed compared to that of kinesin-5 proteins. The motor domain of kinesin-5 is located at the N-terminus, whereas KIFC1 and other kinesin-14 proteins have their motor domains at the C-terminus. Functionally, during mitosis KIFC1 slides and cross-links MTs from the

entrosomes are the main microtubule-organizing centers and play an important role in accurate chromosome segregation during mitosis.1 Like chromosomes, centrosomes duplicate once per cell cycle, and normal diploid cells have two centrosomes that organize a bipolar mitotic spindle that functions to ensure equal chromosome segregation to daughter cells following mitosis.2 In contrast, many cancer cells carry amplified (more than two) centrosomes, and accumulating evidence indicates that centrosome amplification has a causal role in tumorigenesis.3−5 Centrosomes act dominantly to organize spindle poles, and therefore, unless bundled at the poles or inactivated, centrosome amplified cells would be expected to result in multipolar spindle formation. Multipolar metaphase arrangements can result in mitotic catastrophe,5 multipolar cell divisions, or whole chromosome loss or gains caused by merotelic kinetochore attachments. Each of these outcomes is incompatible with cell viability. To overcome this paradox, cancer cells have evolved mechanisms to cluster amplified centrosomes into two groups for bipolar mitosis.6−8 Thus inhibition of centrosome clustering may selectively drive cancer cells with amplified centrosomes to undergo multipolar mitosis and subsequent apoptosis without © 2013 American Chemical Society

Received: March 17, 2013 Accepted: July 29, 2013 Published: July 29, 2013 2201

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minus-end of spindles,14,15 thus opposing the function of plus end-directed kinesin-5 motor protein. Interestingly, it seems that in normal cells KIFC1 does not play an essential role in mitosis in that ablation of KIFC1 by a KIFC1-specific antibody in cultured cells did not change the MT architecture.16 In addition, even though KIFC1 RNAi affected the spindle length in HeLa cells during mitosis, spindle bipolarity was maintained.17 In contrast, in cells with supernumerary centrosomes KIFC1 functions as the main force to cluster amplified centrosomes for cells to pass mitosis. This was evidenced by the observation that KIFC1 knockdown by siRNA in centrosome-amplified cells (MDA-MB-231 and BT549) caused multipolar spindles and eventually cell death, but not in cancer cells without centrosome amplification (MCF7).10 These data suggest that small molecule inhibitors of KIFC1 may be effective in selectively targeting centrosomeamplified tumor cells. To this end, we have identified a small molecule KIFC1 inhibitor, AZ82, via a combination of HTS and iterative medicinal chemistry. We show in this report that AZ82 is a KIFC1-selective inhibitor that bound specifically to the KIFC1/ MT binary complex and inhibited MT-stimulated KIFC1 ATPase activity. We found that AZ82 enhanced the interaction of KIFC1 and MTs and inhibited the binding of ATP and release of ADP. AZ82 effectively inhibited the minus enddirected KIFC1 motor inside the cells and reversed the monopolar spindle phenotype induced by the inhibition of plus end-directed motor Eg5. Furthermore, the inhibition of KIFC1 motor domain by AZ82 caused centrosome declustering in cancer cells that harbor centrosome amplification. Together, these data suggest a potential therapeutic strategy by targeting the motor domain of KIFC1 for cancers with supernumerary centrosomes.

Figure 1. Structure of AZ82 and its inhibitory effect. (A) Chemical structure of AZ82. (B) Dose-dependent inhibition of ATPase activity of the KIFC1/MT complex (open circle) and KIFC1 (closed circle) by AZ82. Solid lines represent the fitting of percentage inhibition data to a 4-parameter logistic non-linear regression model using GraFit 5.13. (C, D) AZ82 is competitive to ATP (C) and noncompetitive to MTs (D). Solid lines represent the global fitting of velocities of reactions to non-linear regression models for competitive inhibition (C) and noncompetitive inhibition (D). Reactions were set with a fixed concentration of either MTs (C) or ATP (B), varied concentrations of either ATP (C) or MTs (D), and several concentrations of AZ82 (▼, no inhibitor; ∇, 0.05 μM AZ82; ▲, 0.1 μM AZ82; Δ, 0.19 μM AZ82; ■, 0.38 μM AZ82; □, 0.75 μM AZ82; ●, 1.5 μM AZ82).

RESULTS AND DISCUSSION Discovery of AZ82 as a KIFC1-Specific Inhibitor. Using genetic tools, we and others have demonstrated that KIFC1 is an interesting target for cancer therapeutic discovery.10 Even though several kinesin inhibitors including the kinesin Eg5 inhibitor ispinesib18 and the CENP-E (belonging to kinesin 7 family) inhibitor GSK92329519 are in different stages of clinical trials, no small molecular inhibitors of KIFC1 kinesin had been reported before we started this study. We screened a library of over 800,000 small molecule compounds with diverse structures and identified several chemical series as initial starting points. Iterative cycles of medicinal chemistry aimed at optimizing a phenylalanine-containing chemical series gave rise to a collection of improved compounds that effectively inhibited KIFC1 motor domain ATPase activity, with AZ82 being one of the most potent examples (Figure 1A and Supporting Information 1). AZ82 inhibited MT-stimulated KIFC1 ATPase activity with an IC50 of 0.3 μM but had a minimal inhibitory effect against the basal ATPase activity of KIFC1 even at 100 μM (Figure 1B). AZ82 is KIFC1-selective and showed no detectable activity against a panel of nine kinesin motor proteins including CENP-E, chromokinesin, Eg5, BimC, KiC3, Kif3C, kinesin heavy chain, MCAK, and MKLP1 when tested at 5 μM (Supporting Information 2), while at this concentration it inhibited KIFC1 activity by 95%. The selectivity over CENP-E is rather remarkable considering the shared phenylalanine core with the CENP-E inhibitor.19 An inhibition kinetic study performed under steady-state conditions revealed that AZ82 inhibited MT-stimulated ATPase

activity of KIFC1 in an ATP-competitive (Ki of 0.043 ± 0.003 μM) and MT-noncompetitive manner (Figure 1C and D). AZ82 Binds to the MT/KIFC1 Complex and Inhibits Binding of ATP and Release of ADP. Microtubuledependent kinesin motor proteins undergo dramatic conformational changes during an ATP hydrolysis reaction cycle, and an inhibitor could bind to any discrete conformation of its targeting kinesin to exert inhibitory effect.20 To address which state(s) of KIFC1 AZ82 specifically binds, we first conducted equilibrium dialysis/mass spectrometry (ED/MS) analysis. This assay can not only report to which component(s) in a protein complex a small molecule binds but also estimate the affinity of the binding. AZ82 in one side of a chamber (used at 1 μM) was dialyzed against several analytes including buffer, MT, KIFC1, or KIFC1/MT complex, respectively. The partitioning concentration of AZ82 at both sides of the chamber was quantified after equilibrium. After an overnight incubation, AZ82 was found to be evenly distributed to both sides of the dialysis membrane for buffer, MT, or KIFC1 but disproportionally enriched to the KIFC1/MT complex (Figure 2A), suggesting that AZ82 binds specifically to KIFC1/MT complex but not to KIFC1 or MT alone. Furthermore, the enrichment was KIFC1 concentration-dependent, and the data fit to a hyperbolic binding isotherm with an apparent KD of 0.69 ± 0.16 μM (Figure 2B), providing further evidence for the specific binding nature of this interaction. When the experiment was repeated with an inactive structural analogue of AZ82, none of the four conditions caused any compound enrichment (data not shown).

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samples were subsequently subjected to ultracentrifugation. The resulting pellets and supernatant were separated and analyzed by SDS-PAGE. Similar to the observation made on other kinesin proteins including Ncd,22 Eg5,21,23 and CENPE,19 ADP promoted the dissociation of KIFC1 from MTs, resulting in more free KIFC1 in supernatant when 1 mM ADP was included in the buffer (Figure 2C, lanes 1 and 3), whereas 1 mM AMP-PNP enhanced the KIFC1/MT interaction as all KIFC1 became associated with MT and pulled down to the pellet fraction (Figure 2C, lane 5). Inclusion of 20 μM AZ82 promoted association of KIFC1 to MTs, effectively antagonizing the endogenous ADP that was co-purified with KIFC1 (Figure 2C, lane 2). However, this antagonistic effect was attenuated in the presence of high concentration of ADP (Figure 2C, lane 4 vs lane 2), indicating that AZ82 was competing with ADP to stabilize a different KIFC1 state that has high affinity for MTs. We further characterized the KIFC1 inhibition by AZ82 using fluorescent nucleotide exchange experiments. When KIFC1, which was purified with bound ADP, was mixed with mant-ATP, a gradual increase in fluorescent signal was observed (Figure 2D), indicating the dissociation of the bound ADP with concomitant association of mant-ATP. MTs accelerated the rate of this process by almost 10-fold from 0.17 to 1.7 min−1 (Figure 2D). The steady-state hydrolytic rates of mant-ATP and ATP were similar (data not shown). After the fluorescent signal reached a plateau, the mixture was chased with an excess amount of ATP, and the fluorescent signal decreased following a first-order exponential decay (Figure 2E). MTs also enhanced the rate of ATP-promoted dissociation of mant-ADP to a similar extent (Figure 2E). AZ82 inhibited both processes with an IC50 of 0.90 ± 0.09 μM for mant-ATP binding and 1.26 ± 0.51 μM for mant-ADP releasing (Figure 2F). Interestingly, it appears that AZ82 was only able to offset the enhanced rate stimulated by MTs, as the rate of either mant-ATP binding or mant-ADP releasing under high concentrations of AZ82 was reduced to the basal level when KIFC1 was used alone. This data is consistent with the notion that AZ82 binds specifically to the KIFC1/MT complex and does not interact with KIFC1 protein directly when it is not associated with MTs. The requirement for binding to the KIFC1/MT complex perhaps explains the difficulty we experienced in obtaining a crystal structure of AZ82 with this kinesin. In lieu of an experimental structure, modeling of the AZ82-KIFC1 complex was carried out, making use of other reported kinesin-inhibitor structural data, to position the inhibitor within the most commonly observed inhibitor binding site between the α2 and α3 helices and the L5 loop (Figure 3 and Supporting Information 3). This binding mode was particularly guided by a model of the structurally similar GSK923295 bound to CENP-E.19 The thiophene ring of AZ82 is buried quite deeply in a hydrophobic pocket lined by the aromatic rings of Tyr 461 and Phe 542 and the aliphatic portion of the Glu 421 side chain, with the trifluoromethoxy group of the biaryl system positioned in a pocket formed by the L5 loop and the pyrrolidine tail directed toward the ADP binding site. AZ82 Suppresses the Phenotype Induced by Eg5 Inhibition in Cells. To test whether a small molecule KIFC1 inhibitor could block the monopolar spindles resulting from Eg5 inhibition, we treated HeLa cells with the Eg5-specific inhibitor AZD4877 followed by inhibition of KIFC1 motor activity by either AZ82 or KIFC1 siRNA. After 16 h of treatment, the majority of AZD4877-treated mitotic cells

Figure 2. AZ82 binds to the KIFC1/MT complex. (A, B) ED/MS showed that AZ82 was specifically enriched by KIFC1/MT complex. (C) Equilibrium binding and co-sedimentation of KIFC1 (3 μM) with MTs (6 μM) in the presence of the indicated nucleotides and AZ82 revealed inhibitor-induced loss of KIFC1 from the supernatant and increase in the MT pellet. (D, E) Fluorescent traces of binding (D) and chasing (E) of mant-ATP to KIFC1 or KIFC1/MT complex. mant-ATP at 1 μM concentration was mixed with 1 μM KIFC1 (○), KIFC1/MT complex (1 μM KIFC1 and 0.5 μM MTs, ●), or buffer (data not shown), and time courses of mant-ATP fluorescence enhancement were recorded (D). After fluorescent signals reached equilibrium, the mixtures were chased with 100 μM ATP and the traces were recorded (E). Smooth lines represent the best fitting of the traces to an exponential growth model (D) or an exponential decay model (E). (F) AZ82 inhibits the binding of mant-ATP to KIFC1/MT complex (●) and the dissociation of mant-ATP/ADP from KIFC1/ MT complex (■) in a dose-dependent manner. Experiments were conducted similar to those in panels D and E, except that AZ82 was included with several concentrations. Observed rate (kobs) of binding (●) or dissociation (■) were calculated by fitting raw data to an exponential growth model or an exponential decay model. IC50 values were calculated by fitting the kobs data to a 4-parameter logistic equation. AZ82 inhibited the binding and dissociation of mant-ATP with IC50’s of 0.90 ± 0.09 μM and 1.26 ± 0.51 μM, respectively.

Having demonstrated that AZ82 binds specifically to the MT/KIFC1 complex, next we set out to determine how AZ82 affects the interaction of KIFC1 with MT by co-sedimentation analysis, a robust tool for characterizing interactions between proteins and polymers that has been applied successfully to the study of several other motor proteins including Eg521 and CENP-E.19 The isolated KIFC1 carries tightly bound ADP, and attempts to prepare apo-KIFC1 under several conditions all resulted in significant protein precipitation, preventing us from using apoprotein in such experiments. KIFC1 was preincubated with MTs under different conditions as described, and the 2203

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AZ82 or KIFC1 siRNA in a pair of cancer cell lines that have a high or low percentage of cells with extra centrosomes. The BT-549 cell line (∼30% cells with extra centrosomes) was chosen as a representative of cancer cell lines with amplified centrosomes, while HeLa and MCF7 cells (4 centrosomes were counted as multipolar, and error bars represent standard deviation.

indicating that cells completed cell division. As expected, no increase in the mitotic population was observed with either KIFC1 depletion or AZ82 treatment, suggesting that loss of KIFC1 activity does not activate the spindle checkpoint nor trigger mitotic arrest. These findings are corroborated by our phase contrast time-lapsed video microscopy study where cotreatment of HeLa cells with AZ82 normalized the prolonged transition time from metaphase to anaphase caused by Eg5 inhibition (Figure 4B, Supporting Information 4). Taken together, the ability of AZ82 to mimic genetic KIFC1 depletion and reverse the Eg5 inhibition phenotype suggests that AZ82 specifically inhibited KIFC1 motor activity in cells. To further investigate the phenotypic effects, we compared the effects of KIFC1 inhibition by AZ82 with siRNA-mediated KIFC1 depletion in cancer cells with and without amplified centrosomes. It was hypothesized that aneuploid cells with unbundled centrosomes will eventually become apoptotic.9,27,36 Indeed, when BT-549 cells were treated with either AZ82 or KIFC1 siRNA, we observed robust induction of multipolar spindle formation, whereas minimal effects were seen in HeLa cells. In experiments where mitotic catastrophe was monitored in BT-549 and MCF7 cells for 44 h following AZ82 treatment in serum-containing media, evidence of increased metaphase to anaphase time and mitotic catastrophe was observed specifically in BT-549 cells (Supporting Information 7). These data

confirmed previous results obtained with KIFC1 knockdown10,17 but also supported the hypothesis that inhibition of the enzymatic activity of KIFC1 motor domain could specifically impact the survival of cancer cells with amplified centrosomes. Kinesin motor proteins are promising anti-cancer drug targets, and several inhibitors developed for Eg5 and CENP-E are being tested in the clinic for variety of human cancers (for a complete review, see ref 37). However, emerging anti-tumor activities in patients have been overshadowed by confounding toxicities that prevent further evaluation of some drug candidates in clinic.37 Both Eg5 and CENP-E are essential mitotic kinesins, and inhibition of their motor activity in normal cells will cause mitotic delay or arrest, leading to the undesired anti-proliferative effects in tissues with high proliferative rate including bone marrow.37 Consistent with previous molecular studies, our findings suggest that KIFC1 is dispensable for cell division in diploid cells and that functional inhibition of KIFC1 may spare normal tissues but specifically induce mitotic catastrophe in tumor cells with amplified centrosomes.10 In addition to its essential role in clustering extra centrosomes for bipolar mitosis, KIFC1 has been suggested to play other crucial roles in tumor cells. KIFC1 was found to have a direct effect in drug resistance and tumor metastasis.38,39 For example, KIFC1 was identified as one of the proteins 2206

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and the reactions were chased after 30 min by the addition of 100 μM ATP. Fluorescent traces were recorded on a Tecan M1000 fluorometer with excitation at 335 nm and emission at 448 nm. Other detailed experimental procedures can be found in Supporting Information.

whose overexpression conferred resistance to docetaxel in MDA-MB-231 and MDA-MB-468 cells,38 both of which are triple-negative breast cancer cells. This is consistent with the finding from our internal bioinformatic analysis that the triplenegative segment, which has the poorest prognosis among all breast cancers, has the highest KIFC1 expression level (Supporting Information 8). Recent data examining the effects of KIFC1 depletion in DNA damage repair (DDR)-deficient cancer cells suggested that KIFC1 inhibitors may also have synthetic lethal effects in cells in the absence of amplified centrosomes.40 Interestingly, similar to the actions of KIFC1 in centrosome-amplified cells, DDR-deficient cells were found to require KIFC1 for bundling acentrosomal spindle poles into a bipolar spindle. These findings further underscore the essential roles that KIFC1 plays in cancers and suggest that a selective KIFC1 drug could be broadly applicable to a variety of human cancers. In summary, we have discovered a small molecule inhibitor, AZ82, which specifically binds to the KIFC1/MT complex and shuts down its enzymatic activity. AZ82 can effectively inhibit KIFC1 function in cells. It is anticipated that KIFC1inhibitors may have a greater therapeutic margin than inhibitors for other kinesins. To the best of our knowledge, this represents the first reported small molecule to inhibit the enzymatic activity of KIFC1. In addition to demonstrating the potential for KIFC1 inhibition in synthetic lethal targeting of cancer cells, AZ82 should prove to be a useful tool for uncovering the fine details of KIFC1 motor function in mitosis.

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Vmax[S] (K m(1 + [I]/K is)) + [S](1 + [I]/K ii)

Corresponding Author

*E-mail: [email protected]. Present Address §

E. J. Corey Institute of Biomedical Research, Jiangyin, Jiangsu 214437, PR China. Author Contributions ∥

Vmax[S] K m + [S](1 + [I]/K ii)

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank C. Grande for her contribution in biochemical assay development and compound screening and A. Rosen and O. Seltser for their automation assistance.

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REFERENCES

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(1)

(2)

For uncompetitive inhibition:

v=

AUTHOR INFORMATION

Detailed experimental procedures and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

For noncompetitive inhibition: v=

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* Supporting Information

HTS Identification of KIFC1 Inhibitor. A library of structurally diverse compounds were screened using the MG assays to identify inhibitors of MT-stimulated KIFC1 motor domain ATPase activity. The primary screening generated a 0.3% hit rate and identified ∼2500 compounds with >40% inhibition at 10 μM of compound concentration. After a series of hit confirmation and evaluation activities, several unique chemical series were nominated for further SAR development. Among those, a scaffold with a phenylalanine core emerged as the leading series where AZ82 was developed as one of the leading compounds. KIFC1 Steady-State Inhibition. The mode of inhibition of AZ82 was evaluated using the PK/LDH coupled assay. Initial reaction velocities were determined for KIFC1 reactions at several fixed concentrations of AZ82, with varied concentrations of ATP at a fixed concentration of MT, or vice versa, in triplicates. Data were then fitted to one of the following equations by the GraFit 5.0 program. For competitive inhibition:

Vmax[S] [S] + K m(1 + [I]/K is)

ASSOCIATED CONTENT

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METHODS

v=

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(3)

Non-linear least-squares regression analysis was used to determine which equation fit the experimental data best. Fluorescent Nucleotide Exchange by KIFC1 Motor Domain. Mant-ATP binding and mant-ADP chasing were performed at the same PIPES buffer used in activity assays. Mant-ATP was mixed with either KIFC1 or KIFC1/MT complex (all components at 1 μM except for MT 0.5 μM) and DMSO or compound at different concentrations, 2207

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