Design of BTB-1 Analogs - Kif18A and its Inhibitor BTB-1

3. Results and Discussion

3.2 Kif18A and its Inhibitor BTB-1

3.2.2 Design of BTB-1 Analogs

A subset of closely related analogs of BTB-1 were synthesized to get a better understanding, which structural motifs of BTB-1 are necessary for its inhibitory activity against its target Kif18A, and to potentially find more effective inhibitors. To this end, the unsymmetrically substituted diphenylsulphone BTB-1 was divided into three scaffolds: the two phenyl moieties I, II and a linker between them (Figure 10).

To enhance lipophilicity and cell membrane permeability of BTB-1, lipophilic groups like fluorine and trifluoromethyl were introduced at phenyl moiety I in para position to the linker. Additionally, the electron withdrawing group NO2 was introduced to elucidate how altered electronegativity and sterical demand at this position affect the inhibitory activity towards Kif18A. In order to investigate the importance of the NO2 group in ortho position at phenyl moiety I for Kif18A inhibition, it was shifted to the meta position to the linker.The phenyl moiety II was isosterically exchanged to thiophene and the aromatic system was expanded to naphthalene. Various substituents (Cl, OMe, Me) were introduced in para position to the linker (phenyl moiety II). Finally, the sulfone linker was changed to an ether or sulfoxide to analyze the effect of the geometry of the linker on the inhibition of Kif18A.

Figure 10: Structure of BTB-1 and its division into scaffolds I, II and linker.

31 3.2.3 Synthesis of BTB-1 Analogs

The synthetic approach for BTB-1, as described in 3.2.1, was used in order to synthesize differently substituted sulfones. Starting with a nucleophilic aromatic substitution under basic conditions followed by H2O2 oxidation the sulfones 52-63 were received in low to good yields (Table 8).

Table 8: Reaction scheme for the synthesis of substituted diphenylsulfones. The substitution pattern and yields over two steps are given in the table. For BTB-1, the literature yield is given for comparison. Compounds marked with * were synthesized using sodium thiophenolate and 0.3 equiv. NaOH.

Cpd. R¹ R² Overall yield

BTB-1 Cl Ph 74%¹⁰⁶

52* NO2 Ph 25%

53* F Ph 12%

54* CF3 Ph 26%

55 Cl 2-thiophene 68%

56 Cl 2-naphthalene 6%

57 Cl 4-methylbenzene 7%

58 Cl 4-methoxybenzene 12%

59 H Ph 3%

60 H 4-methoxybenzene 5%

61 NO2 4-methylbenzene 18%

62 Cl 4-chlorobenzene 55%

63 NO2 4-chlorobenzene 77%

The racemic sulfoxides 64-69 were synthesized by changing the oxidant to meta-chloroperoxybenzoic acid (m-CPBA) and using it in stoichiometric quantities to receive a single oxidation process to the sulfoxides (Table 9).

Table 9: Synthetic route to the racemic sulfoxides. The yields and substitution patterns are given in the table.

Cpd. R¹ R² Yield

64 NO2 Ph 38%

65 Cl 2-naphthalene 66%

66 Cl 4-methoxybenzene 72%

67 H Ph 13%

68 H 4-methoxybenzene 2%

69 NO2 4-methylbenzene 20%

The NO2 substituent in ortho position to the linker was shifted to meta position.

Sulfone 70 was synthesized according to Moore et al. by an AlCl3 mediated reaction of benzene and the corresponding sulfonyl chloride in 46% yield (Scheme 8).¹⁵³

Scheme 8: AlCl3 mediated reaction of benzene and 4-chloro-3-nitrobenzenesulfonyl chloride to 70.

Further, the linker was changed from sulfone or sulfoxide to an ether by synthesizing 71 in 96% yield applying a SNAr of 1,4-dichloro-2-nitrobenzene with phenol (Scheme 9).

Scheme 9: Synthesis of the biphenylether 71 by nucleophilic aromatic substitution under basic conditions.

33 Another effort to diversify the linker was to introduce a phosphinic acid residue. 4-chloro-2-nitroaniline was converted into the iodide 72 using a diazetation-iodination as described in literature (Scheme 10).¹⁵⁴ The following copper mediated reaction of the iodide with phenylphosphinic acid did not yield the desired small molecule 73 (Scheme 10).¹⁵⁵ Only decomposition of the starting material was observed.

Scheme 10: Synthesis of iodide 72 in a one-pot procedure and the subsequent copper mediated reaction to 73.

In another attempt to synthesize 73 according to literature procedures 4-chloro-2-nitroaniline was first converted into its diazonium salt and afterwards the copper mediated reaction with dichloro(phenyl)phosphane was carried out (Scheme 11).^156-157 As well as the before mentioned approach this attempt did not lead to the desired product but only to decomposition of the starting material. Since at this time point in vitro studies revealed that the sulfone linker is essential for Kif18A inhibition, as discussed later on (chapter 3.2.4 and 3.2.11), no further attempts to synthesize 73 were carried out.

Scheme 11: Proposed synthetic rout to 73. The diazonium compound 74 was readily available through diazetation, but the copper mediated reaction to 73 lead only to decomposition of the starting material.

The main focus for the synthesis was not to optimize the synthetic procedures, but to yield pure substances in a fast manner for evaluation of their biological activity.

Therefore, all compounds were purified by column chromatography and/ or successive recrystallization resulting in lower yields.

3.2.4 Screening for Kif18A Inhibition and IC50 Determination

With the synthesized BTB-1 analogs 52-71 in hand the next step was to evaluate their ability to inhibit the ATPase activity of Kif18A. The motor-domain of Kif18A fused to a His-Tag (Kif18A^MD) was used in an enzyme coupled assay to evaluate the potency of the respective analogs. The ECA conditions were adjusted to obtain 50% inhibition at 5 µM BTB-1 and the ATPase activity in the presence of DMSO was normalized to 100%

(Figure 11). All compounds that showed higher inhibition than 25% were considered as active. This selection criteria was chosen in order to identify inhibitors with comparable properties like BTB-1.

35 Figure 11: Quantification of the screening results of BTB-1 and its derivatives tested at 5 µM. Green bars indicate small molecules that were considered as active, since they showed an inhibitory activity above 25%. Average of three independent experiments and standard deviations are shown. In the lower panel the structure of the identified hits are shown.

Five out of 20 compounds were considered as active (52, 53, 54, 55, and 59, Figure 11 green bars) and selected for further analysis. Next, the IC50 values of the five small molecules against Kif18A^MD were determined using the ECA. All compounds showed a concentration dependent inhibition in the low micromolar range (Figure 12).

Unfortunately, none of them showed a higher potency than BTB-1 (1.7 ± 0.2 μM).

-5%

10%

25%

40%

55%

70%

Inhibitiion in %

Cpd tested at 5 µM

10^-7 10^-6 10^-5 10^-4

0 20 40 60 80 100

BTB-1 52 53 54 55 59

% ATPase activity

Figure 12: Dose-response curves of BTB-1, 52, 53, 54, 55 and 59. All molecules showed a dose dependent inhibition in the low micromolar range, with BTB-1 being the most potent. Average of three independent experiments and standard deviations are shown.

With IC50 values of 3.0 ± 0.2 μM and 4.8 ± 0.4 μM for 59 and 53 respectively, they were the most potent inhibitors, indicating that the replacement of the halogen atom at phenyl moiety I is tolerated. The introduction of more bulky substituents at this position like NO2 (52) or CF3 (54) as well as the isosterical exchange of the phenyl moiety R² to a thiophene (55) resulted in reduced potency (IC50 values:

(52) 10.2 ± 2.0 μM, (54) 10.3 ± 1.9 μM, and (55) 6.4 ± 0.9 μM).

3.2.5 Mode of Inhibition and Selectivity

In order to clarify if the newly found inhibitors, like BTB-1, act in an ATP competitive manner¹⁰⁶, ECA analysis of Kif18A^MD activity in the absence of microtubules was performed. The inhibitors BTB-1, 59 or 53 were applied at 50 µM concentration.

37 Figure 13: In the upper panel the raw data of the ECA with Kif18A^MD and 50 µM BTB-1, 53 and 59 in the absence of microtubules are shown. The lower panel shows the decrease of absorbance at 340 nm relative to DMSO control. Both graphs show no inhibition of the tested small molecules for the basal ATPase activity of Kif18A^MD compared to the DMSO control. Average of two independent experiments and standard deviations are shown.

Comparable to BTB-1, both small molecules did not show any inhibitory effect on the basal, microtubule-independent ATPase activity of Kif18A (Figure 13), suggesting that the newly found inhibitors behave like BTB-1. Additionally, ATP titration experiments indicate that 59 acts in an ATP competitive manner like BTB-1 (Figure 14). These data suggest, that the identified small molecules are ATP-competitive inhibitors, which are only able to inhibit Kif18A when it is bound to microtubules.

0,5

ΔA340nm/min rel to DMSO control

38 were fitted to competitive inhibition mode using GraphPad Prism. Average of three independent experiments and standard deviations are shown.

In order to analyze compound selectivity towards Kif18A, the two most potent compounds 53 and 59 were tested in vitro for their effects on the ATPase activity of other kinesins related to Kif18A (Figure 15). Therefore, ECA was performed in the presence of 100 μM BTB-1, 53 or 59 and His-tagged motor domains of the mitotic kinesins Kif3A, Kif4A, Kif5A, Mklp1, Eg5, MPP1 or MCAK. 53 and 59 showed reduced inhibitory activity towards Kif3A and Kif5A compared to BTB-1 and comparable inhibitory activity towards the other tested kinesins (Figure 15). These findings suggest, that the substitution of chloride with hydrogen and fluoride atoms has a selectivity increasing effect.

Kif18A Kif5A Kif4A Kif3A Mklp1 Eg5 MPP1 MCAK 0

50 100

% Inhibition

BTB-1 53 59

Figure 15: The inhibitory effect of BTB-1, 53 and 59 at 100 µM on different mitotic kinesins are shown. Compared to BTB-1 53 and 59 show reduced inhibition of Kif5A and Kif3A and comparable inhibitory activity towards the other tested kinesins. Average of three independent experiments and standard deviations are shown.

3.2.6 Cellular Toxicity Studies

With the first in vitro results in hand, the next step was to analyze, if the identified novel Kif18A inhibitors are active in cells. Therefore, the whole small molecule collection was tested for their cytotoxicity on HeLa cells using an alamar blue assay (Figure 16 upper panel).¹⁵⁸ In this assay the percentage of viable cells are detected through the conversion of Resazurin in living cells into the fluorescent dye Resorufin.

The fluorescence serves as readout and is directly linked to the number of viable cells.

Interestingly, determination of the half maximal effective concentration (EC50 values) showed that all synthesized sulfoxides 64-69 had EC50 values in the low micromolar range (Table 10). The dose response curves of the five most potent compounds for Kif18A inhibition 52, 53, 54, 55, and 59 as well as BTB-1 are shown in Figure 16. Out of this set of inhibitors (Figure 16 lower panel) compound 52 showed the highest cytotoxicity with 1.1 ± 0.3 μM followed by 54 with 2.6 ± 0.4 μM. BTB-1 and 55 were less toxic with EC50 values of 35.8 ± 9.0 μM and 23.1 ± 7.3 μM, respectively. For 53

the lowest cytotoxicity was observed with EC50 values above 50 μM and 59 did not show a significant cytotoxicity at all.

10^-8 10^-6 10^-4 10^-2

0 50 100

BTB-1 52 53 54 55 59

% Cell viability

Figure 16: The upper panel shows a schematic representation of the alamar blue assay. Viable cells reduce the nonfluorescent dye Resazurin into the fluorescent Resorufin. The fluorescence serves as readout. The lower panel shows the dose response curves of BTB-1, 52-55 and 59 received from HeLa cells treated with different concentrations of inhibitors. Average of three independent experiments and standard deviations are shown. The structures of the tested compounds are shown in the lower panel.

41 Table 10: EC50 values in µM of the BTB-1 analogs received from the alamar blue assay. No cytotoxicity or values above 60 µM are described with “ “.

Cpd. EC50 (µM) BTB-1 35.8 ± 9

52 1.1 ± 0.3 53 54.7 ± 7.8 54 2.6 ± 0.4 55 23.1 ± 7.3 56 13.8 ± 2.9

57 -

58 36.2 ± 9.3

59 -

60 -

61 2.6 ± 0.4 62 20.8 ± 6.3

63 -

64 1.9 ± 0.2 65 5.5 ± 1.2 66 9.3 ± 2.1 67 20.8 ± 3.0 68 30.1 ± 4.6 69 1.8 ± 0.3 70 9.6 ± 1.3

71 -

3.2.7 Live Cell Imaging

Another attempt to investigate the cellular effects of the newly identified inhibitors was to use live cell imaging. A HeLa cell line that stably expresses green fluorescent protein (GFP) tagged Kif18A⁴⁹ was used in order to detect the localization of Kif18A and additionally the time in mitosis (time from NEBD to anaphase onset) was determined (Figure 17). In a first experiment cells synchronized in S-phase with thymidine were treated with 0.5% DMSO or 25 µM of the three most potent newly identified inhibitors 53, 55, and 59 or BTB-1. Small molecule 58 was used as toxicity control since its EC50 value in the alamar blue assay is comparable to BTB-1. 52 and 54 displayed a high toxicity at 25 µM and, were therefore used at different concentrations (10 µM, 5 µM and 1 µM, experiments are not shown).

0.5% DM

Figure 17: Schematic representation of live cell assay. Cells were synchronized in S-phase using thymidine. 6 Hours after release 0.5% DMSO or 5/25/50 µM inhibitors were added. No effect on the time in mitosis was observed. Average and standard deviation of two independent experiments are shown for 25 µM concentration. For 50 µM and 5 µM concentrations only one experiment was carried out and the standard deviation is shown.

43 Nevertheless, none of the small molecules interfered with the localization of Kif18A and no significant increase in time in mitosis compared to the DMSO control was detected (Figure 17). The experiment was repeated using 50 µM inhibitor concentration, except for 54 and 52, where 5 µM was applied. Due to toxic effects of 55 at 50 µM and 52 at 5 µM concentration both molecules were excluded from analysis. Even the higher concentrations of the inhibitors did not result in a detectable delocalization of Kif18A. Despite that, 54 led to an increased time in mitosis at 5 µM concentration compared to the DMSO control (Figure 17). BTB-1 showed a slight increase in mitotic timing (Figure 17). Since no effect on the Kif18A localization was observed, this experiment was only carried out once. In summary, the inhibitors are not efficient enough to inhibit Kif18A in cells, since the localization of Kif18A was not altered upon compound treatment.

3.2.8 Cellular Thermal Shift Assay

As another approach to elucidate the cellular effects of BTB-1 and the most promising inhibitor 53 a cellular thermal shift assay (CETSA) was performed. In this assay cell lysate is applied and therefore the small molecules do not have to pass the cell membrane.¹⁵⁹ This assays relies on the thermal stabilization of the target protein upon Inhibitor binding. Mitotic HeLa cell extract was prepared and treated with 0.5%

DMSO as solvent control or compounds. After incubation and thermal treatment the samples were analyzed by SDS-PAGE followed by western blot analysis. First, the temperature at which Kif18A denatures was determined in the presence of 100 µM BTB-1, 53 or 0.5% DMSO. Unfortunately, no stabilization of Kif18A due to inhibitor addition was observed. No intensity differences of the samples were detectable (Figure 18). The DMSO control revealed that Kif18A is destabilized at temperatures between 50°-54°C (Intensity decrease, Figure 18) and therefore this temperature range was used to evaluate if higher inhibitor concentrations are able to stabilize Kif18A.

Figure 18: Western blot analysis of temperature gradient from 46°C to 62°C in the presence of 0.5% DMSO, 100 µM BTB-1 or 53. In line 10 (DMSO control 52°C) was less lysate loaded. No stabilization of Kif18A was observed. CycB, Cdc27 and tubulin served as mitotic markers and loading controls.

BTB-1 and 53 were applied in 500 µM, 200 µM, 100 µM and 50 µM concentration, but still no stabilization of Kif18A was observed (Figure 19). Since BTB-1 as well as 53 are not able to stabilize Kif18A even at concentrations up to 500 µM, it is possible that these inhibitors are not able to bind efficiently to Kif18A in a cellular environment.

Figure 19: Western blot analysis of compound titration of BTB-1 and 53 from 50°C to 54°C. The first and last line was loaded with random lysate. No stabilization of Kif18A was observed. CycB, Cdc27 and tubulin served as mitotic markers and loading control.

45 3.2.9 Immunofluorescence Imaging for Kif18A Localization

In order to analyze in detail the effect of BTB-1 and derivatives 53, 54, and 59 on cells and the architecture of the mitotic spindle, immunofluorescence imaging was performed. The same HeLa cell line as described for live cell imaging (chapter 3.2.7) was used.⁴⁹ Cells synchronized in S-phase using a high concentration of thymidine were released after nine hours and then treated with the solvent control DMSO or 30/50 µM of BTB-1 or derivatives 53, 54, and 59 (Figure 20). Thirty minutes after compound addition, cells were chemically fixed and tubulin (red) and DNA (blue) structures were visualized by immunostaining and treatment with the dye Hoechst 33342, respectively. Since initial immunofluorescence analysis of derivative 52 revealed that this small molecule caused unspecific cytotoxic effects on both dividing and non-dividing cells, it was excluded from further microscopic studies. The immunofluorescence images revealed that neither 53 nor 59 significantly affected spindle structures or alignment of chromosomes at 30/50 µM concentration (Figure 20, only 50 µM is shown). Additionally, the plus-end accumulation of GFP-Kif18A at microtubules – which as shown previously depends on the motor activity of Kif18A^44-45 – was not affected by the treatment with 53 or 59.

47 Figure 20: The upper panel shows the assay scheme for cell synchronization and compound treatment. First, cells were synchronized in S-phase using thymidine.

Nine hours after the release, cells were treated with DMSO as solvent control, 50 nM Nocodazole (Noc) or 30/50 µM BTB-1, 53, 54, 59 followed by formalin fixation and immunostaining. Representative immunofluorescence images of cells treated with DMSO, 50 nM Nocodazole (Noc) or 30/50 µM compounds BTB-1, 53, 54, 59 are shown. For each condition, merged images with DNA (blue), GFP-Kif18A (green) and microtubules (red) are shown on the right. In grey is shown from left to right DAPI (DNA), Kif18A, and tubulin (scale bar = 5 µm).

These, taken together with the normal spindle structures, suggest that treatment with derivative 53 or 59 does not result in efficient Kif18A inhibition in cells. HeLa cells treated with 50 µM BTB-1 revealed severe defects in spindle morphology and chromosome alignment, while 30 µM BTB-1 showed a mild effect (Figure 20), consistent with previous reports¹⁰⁶. Similarly, HeLa cells treated with 30 µM 54 showed multiple spindle poles, highly disorganized and fragmented microtubule structures and no detectable alignment of chromosomes comparable to the effect of 50 nM Nocodazole^160-161 (Figure 20). Since the observed phenotypes did not correlate with Kif18A depletion (elongated spindles with hyperstable microtubules) ^{42, 46}, it can be speculated that Kif18A is not the relevant binding partner of BTB-1 or 54 in cells.

3.2.10 Tubulin Polymerization Assay and IC50 Determination

The spindle phenotype induced by 54 was reminiscent of the phenotype caused by low doses of Nocodazole, a microtubule destabilizing compound (Figure 20). To analyze if the small molecule collection or BTB-1 target microtubules, a turbidity-based in vitro microtubule polymerization assay was performed.^162-163 The ability of tubulin to polymerize in the presence of GTP at elevated temperatures in a glutamate buffer and a turbidity based readout was used (Figure 21).

Figure 21: Schematic representation of tubulin polymerization. α-(light green) and β-(dark green or brown) tubulin heterodimers polymerize to microtubules.

After GTP hydrolysis the destabilized plus-end depolymerizes. Upon exchange of GDP to GTP in the tubulin dimers repolymerization occurs.

α- and β- tubulin heterodimers are able to polymerize in the presence of GTP. The GTP cap consists of tubulin dimers in the GTP bound state and stabilizes the highly dynamic plus-ends of microtubules.^164-166 After GTP hydrolysis, depolymerization, often called catastrophe, takes place. This process can be stopped by addition of GTP-bound tubulin dimers. After a certain time, the polymerization and depolymerization processes reach a dynamic equilibrium as long as enough GTP is present. The microtubule polymerization efficiency in the presence of the solvent

49 control DMSO was normalized to 100% and 1 µM Nocodazole was used as positive control. A small molecule selection was used at 50 µM concentration (Figure 22).

Figure 22: Screening results of the tubulin polymerization assay. DMSO was used as solvent control and its activity was set to 100%. 1 µM Nocodazole was used as positive control and the small molecules as well as BTB-1 were tested at 50 µM concentration. Average of three independent experiments and standard deviations are shown.

Notably, 52 and 61 showed the highest inhibitory effect on microtubule polymerization at 50 µM followed by BTB-1, 54, 60, 63 and 64 (Figure 22). BTB-1, 53, 54, and 59 were selected for further analysis due to their high potency against Kif18A (chapter 3.2.4) and 52 due to its high cytotoxicity (chapter 3.2.6).

Determination of the IC50 values showed that 52 has an IC50 value of 1.2 ± 0.1 µM, indicating that the high cytotoxicity of the compound might be due to its potent inhibitory effect on microtubule polymerization (Figure 23). BTB-1 and 54 showed a moderate effect on microtubule polymerization with IC50 values of 27.5 ± 3.3 µM and 9.2 ± 1.0 µM, respectively. 59 showed an IC50 value of 209.4 ± 37.3 µM on microtubule polymerization. Small molecule 53 did not show any significant effect on the tubulin polymerization up to a concentration of 1 mM. Thus, these studies revealed that the introduction of bulky substituents in phenyl moiety I at para position to the linker (54), especially the introduction of a nitro group (52), drastically increased the undesired inhibitory effect on microtubule polymerization.

20%

40%

60%

80%

100%

120%

10^-7 10^-6 10^-5 10^-4 10^-3 0

20 40 60 80

100 BTB-1

52 53 54 59

% Tubulin polymerization

Figure 23: Dose-response curves of BTB-1, 52, 54, and 59 on microtubule polymerization in vitro with 52 being the most potent inhibitor. Derivative 53 did not show an effect on the tubulin polymerization up to 1 mM Average of three independent experiments and standard deviations are shown.

3.2.11 Structure Activity Relationships and Outlook

A small compound selection 52-71 was synthesized in one to two steps using BTB-1 as lead compound. The described synthesis route started from commercially available building blocks and allowed the robust and cost-efficient assembly of the small molecule collection. Several BTB-1 analogs, which inhibit the ATPase activity of

Im Dokument Novel Small Molecule Inhibitors for the Human Kinesins Mklp2 and Kif18A (Seite 44-0)