The First Pentacyclic Triterpenoid Gypsogenin Derivative Exhibiting Anti-ABL1 Kinase and Anti-chronic Myelogenous Leukemia Activities

Abstract

The discovery of the chimeric tyrosine kinase breakpoint cluster region kinase-Abelson kinase (BCR-ABL)-targeted drug imatinib conceptually changed the treatment of chronic myelogenous leukemia (CML). However, some CML patients show drug resistance to imatinib. To address this issue, some artificial heterocyclic compounds have been identified as BCR-ABL inhibitors. Here we examined whether plant-derived pentacyclic triterpenoid gypsogenin and/or their derivatives show inhibitory activity against BCR-ABL. Among the three derivatives, benzyl 3-hydroxy-23-oxoolean-12-en-28-oate (1c) was found to be the most effective anticancer agent on the CML cell line K562, with an IC₅₀ value of 9.3 µM. In contrast, the IC₅₀ against normal peripheral blood mononuclear cells was 276.0 µM, showing better selectivity than imatinib. Compound 1c had in vitro inhibitory activity against Abelson kinase 1 (ABL1) (IC₅₀=8.7 µM), the kinase component of BCR-ABL. In addition, compound 1c showed a different inhibitory profile against eight kinases compared with imatinib. The interaction between ATP binding site of ABL and 1c was examined by molecular docking study, and the binding mode was different from imatinib and newer generation inhibitors. Furthermore, 1c suppressed signaling downstream of BCR-ABL. This study suggests the possibility that plant extracts may be a source for CML treatment and offer a strategy to overcome drug resistance to known BCR-ABL inhibitors.

Cancer is a worldwide health problem, and despite intensive research efforts over the last several decades, an effective cure has not been identified.¹⁾ However, the recent discovery of molecular targeted drugs against cancer at the end of the 20th century has led to promising results.^1,2) Imatinib (Fig. 1), which targets the chimeric tyrosine kinase breakpoint cluster region kinase-Abelson kinase (BCR-ABL),^3–6) was discovered based on the findings that chronic myelogenous leukemia (CML) is caused by a chromosomal translocation that results in the constitutively expressed and active BCR-ABL.^7,8) While imatinib can be initially effective in treating CML, some patients have shown drug resistance to imatinib.^9–11) Several new generation drugs with a different binding mode to Abelson kinase (ABL) have thus been developed.^6,12–16) These drugs are artificial heterocyclic compounds, however, if widely distributed natural products and/or their derivatives show the inhibitory activity against BCR-ABL, it would be more convenient to get.

Fig. 1. Structures of Gypsogenin Derivatives 1c, 1e and 1f, and Imatinib

Pentacyclic triterpenoids, a category of natural compounds, can be extracted from a variety of plants and show various activities.^17,18) Some of these triterpenoids were reported to exhibit anti-tyrosine kinase activity.^19,20) Here we focused on a pentacyclic triterpenoid gypsogenin,^21,22) extracted from Gypsophila species,^23,24) which is widely distributed in the Eurasian continent. Previous studies have demonstrated anti-cancer activities of gypsogenin derivatives 1c, 1e and 1f (Fig. 1) using several cell lines.²⁵⁾ However, the cytotoxicity of gypsogenin derivatives against a CML cell line has not been reported. In this study, we examined the anti-CML and anti-tyrosine kinase activities of gypsogenin derivatives, recently synthesized by one of the authors,²⁵⁾ using cellular and in vitro assays, respectively.

MATERIALS AND METHODS

Cell Culture and Drug Treatment

The K562 human CML cell line²⁶⁾ was cultured in RPMI 1640 purchased from Wako Pure Chemical Industries, Ltd. and supplemented with 10% fetal bovine serum (FBS) from Biosera. Peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640 supplemented with 10% FBS. In experiments, cells were plated at 1×10⁵ cells/mL into 24-well tissue culture plates and cultured for 24 h before the addition of the drugs (the optimal cell number for cytotoxicity assays was determined in preliminary experiments). Stock solutions (0.3, 1, 3 and 10 mM) of compounds 1c, 1e and 1f,²⁵⁾ and imatinib (from Wako Pure Chemical Industries, Ltd.) were prepared in dimethyl sulfoxide (DMSO), and the solutions were added to culture medium. The concentration of DMSO in the final culture medium was 1%.

Cytotoxicity Assay

The level of cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (from Dojindo Molecular Technologies) was quantified as previously described in the literature with small modifications.^27,28) Cells were cultured in the presence of various concentrations of the tested compounds for 24 h. Cells were then stained with MTT solution and incubated for an additional 4 h at 37°C. The solution was removed and the formazan crystals were solubilized by adding 100 µL DMSO. The absorbance of the converted dye in living cells was measured at a wavelength of 550 nm using a microplate spectrophotometer Infinitive M1000 purchased from Tecan. Cell viability was calculated as the percentage of the viable control cells. All experiments were performed in triplicate and IC₅₀ values were defined as the drug concentrations that reduced absorbance to 50% of control values.

Kinase Inhibitory Activity

Kinase selectivity profiling system assays from Promega were performed according to the manufacturer’s instructions. Multipoint dose-response experiments were performed using 8 kinases, namely ABL1, breast tumor kinase (BRK), Burton’s tyrosine kinase (BTK), C-terminal Srk kinase (CSK), Fgr/Yes novel protein A (FYN A), T-lymphocyte specific kinase (LCK), v-yes-1 Yamaguchi sarcoma viral related oncogene homolog B (LYN B) and v-src sarcoma viral oncogene homolog (SRC). Briefly, the kinase and substrate strips were diluted with 95 µL 2.5x Kinase Buffer and 15 µL of 100 µM ATP solutions, respectively. Kinase reactions were performed using 1 µL of compound solution at varying concentrations (3, 10, 30 and 100 µM), 2 µL of kinase working stock and 2 µL of ATP/substrate working stock. After 1 h incubation at room temperature, kinase activity was quantified using the ADP-Glo Kinase Assay from Promega. Kinase inhibition was quantified using a luminescence microplate spectrophotometer Infinitive M1000. The concentration of the test compounds required to decrease the kinase activity by 50% was determined using ImageJ software and identified as the IC₅₀.

Molecular Docking Study

The Molecular Operating Environment (MOE 2015.10) was used for molecular docking and visualization procedures. The co-crystal structure of imatinib with ABL tyrosine kinase was retrieved from the Brookhaven Protein data bank (PDB ID code: 1IEP).²⁹⁾ Before docking simulations, ligands and the target protein were prepared with the standard protocol of MOE 2015.10, as previously reported.³⁰⁾ All docking calculations were performed using the default MOE settings. The pose of the best docking energy was considered.

Immunoblot Analysis

Cells were cultured in the presence of 20 µM of the compounds for 3 or 6 h, and lysed in phosphate buffered saline (PBS)/Laemmli sample buffer (1 : 1) as described previously.³¹⁾ Immunoblot analysis was conducted using phospho-specific-p44/42 mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit monoclonal antibody (mAb) (1 : 1000) (from Cell Signaling Technology) or anti-β-actin clone AC-15 (from Sigma-Aldrich). Immunoreactivity was detected by chemiluminescence using ImmunoStar LD (from Wako Pure Chemical Industries, Ltd.).

RESULTS

We first performed assay to use general CML cell line K562.²⁶⁾ We performed MTT assays in K562 human CML cells treated with compounds 1c, 1e and 1f at various concentrations (3–100 µM) for 24 h (Fig. 2A). Imatinib was used as a positive control. All compounds showed concentration-dependent inhibitory activity, and their IC₅₀ values were lower than 25 µM. Compound 1c was found to be the most cytotoxic agent, with an IC₅₀ value of 9.3 µM, followed by compound 1e with an IC₅₀ value of 15.0 µM. Compound 1f had an IC₅₀ value of 21.3 µM. The control drug imatinib showed an IC₅₀ value of 4.2 µM.

Fig. 2. Cytotoxicity of Compounds on K562 Cells (A) and Peripheral Blood Mononuclear Cells (B) after 24 h Treatment as Determined by MTT Assay

The experiments were repeated for three times, and their standard deviations are shown as error bars.

We next examined the cytotoxicity of 1c on PBMCs compared with imatinib. As shown in Fig. 2B, Compound 1c exhibited ca. 10 times lower cytotoxicity (IC₅₀=276.0 µM) than imatinib (IC₅₀=26.6 µM). These results suggest that compound 1c has a significant degree of selective toxicity towards the K562 cell line.

We speculated that the cytotoxic activity of 1c may be due to its potential inhibitory activity against BCR-ABL. We thus next examined the kinase inhibitory potency of 1c against ABL1, since ABL1 is the kinase portion of BCR-ABL. We also examined a panel of other kinases, including BRK, BTK, CSK, FYN A, LCK, LYN B and SRC. Imatinib was included for comparison. The results are shown in Table 1. Compound 1c inhibited ABL1 with an IC₅₀ value of 8.7 µM, although the activity was weaker than that of imatinib. Furthermore, the inhibitory profile of 1c on the eight kinases was different from that of imatinib. For example, 1c showed a stronger inhibitory effect against CSK and LYN B than ABL1, while imatinib showed strong inhibition of ABL1.

Table 1. Inhibition of Protein Kinases by 1c and Imatinib

Kinase	IC50 (µM)
	1c	Imatinib
ABL1	8.71±1.14	0.21±0.23
BRK	13.8±1.2	19.0±2.9
BTK	17.8±3.7	>100
CSK	1.54±1.52	16.4±3.8
FYN A	>100	10.0±2.9
LCK	9.12±2.92	0.30±0.24
LYN B	2.92±1.52	5.82±1.98
SRC	27.4±4.6	99.2±6.0

The experiments were repeated for three times, and their standard deviations are shown.

To rationalize BCR-ABL tyrosine kinase inhibition by compound 1c, we examined the interaction of 1c within the ATP binding site residues of BCR-ABL using molecular docking simulation based on the co-crystal structure of imatinib with ABL.²⁹⁾ As shown in Fig. 3, the binding mode of 1c is different from that of imatinib and newer generation BCR-ABL inhibitors.⁶⁾ The binding mode of compound 1c showed the formation of two H-bonds. The first H-bond is settled between the benzyl ester carbonyl group and Asp381 NH. The second one is established with the Met290 sulfur atom (Fig. 3A). On the other hand, imatinib binds not only Asp381 and Met290, but also Glu286, Thr315, Met318 and Ile360 of ABL (Fig. 3B). Notably, the bulkiness of the triterpene skeleton has sterically hindered the deep adoption of compound 1c by the pocket, unlike imatinib, which is completely buried in the active site forming the six interactions (Fig. 3B). This incomplete interaction of compound 1c could explain the weaker kinase inhibition activity against ABL compared with imatinib.

Fig. 3. Binding Interactions of 1c into the ABL Tyrosine Kinase ATP Binding Site Compared with Imatinib as Determined by MOE 2015.10

A Ribbon diagram of 1c (neutral sticks) into ABL (1IEP) showing two H-bonds (dashed black lines) with Asp381 and Met290 (green sticks). B Overlay of binding conformations of 1c (neutral sticks) and imatinib (blue sticks) with 1IEP.

BCR-ABL is known to activate its various downstream signaling.³²⁾ Among the signaling pathways, rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/ERK is verified to be important for leukemogenesis by a fact that this pathway is activated in BCR-ABL inhibitor resistant cells.^33–35) We then examined phosphorylation of ERK in K562 cells treated with 1c or imatinib. The cells were treated with 20 µM of the drug, which affects on the cell viability after 24 h treatment (Fig. 2A), and lysed at earlier time to conduct immunoblot analysis using anti-phoshpho-ERK1/2 antibody. As shown in Fig. 4, 1c showed inhibitory effect of phosphorylation of ERK like imatinib, demonstrating that 1c suppresses signaling downstream of BCR-ABL.

Fig. 4. Effect of Compounds on ERK Signaling

K562 cells were treated with 1c or Imatinib (20 µM) for 3 or 6 h, and immunoblot analysis was conducted.

DISCUSSION

In this work, we demonstrate that the gypsogenin derivative 1c exhibits inhibitory activity against ABL and found that its binding mode may be different from that of imatinib and newer generation BCR-ABL inhibitors. Specially, Thr315 of ABL is critical to bind to imatinib, and newer inhibitors dasatinib and nilotinib⁶⁾ unlike 1c. In clinical use, drug resistance by generation of point mutation of this amino acid changing to Ile315 (T315I) has been a serious problem.^36,37) We can expect that 1c would be a lead of the latest inhibitors which are effective against drug resistant BCR-ABL.

IC₅₀ of 1c against ABL1 in vitro is 8.7 µM, and that against K562 cells is 9.3 µM. In costrast, the in vitro and cellular IC₅₀ of imatinib is 0.21 and 4.2 µM, respectively. The smaller difference between in vitro and cellular experiments is seen on 1c, and this may be due to more efficient inhibition of BCR-ABL protein in a cell and/or inhibition of the other tyrosine kinases related to cell viability. Although a ratio of IC₅₀ of imatinib against PBMC and K562 is 6.3 (26.6/4.2 µM), that of 1c is 29.7 (276.0/9.3 µM), showing that 1c is more selective than imatinib. This is thought to be caused by less inhibitory activity of 1c against the other tyrosine kinases in PBMC and/or its less off-target effect. Imatinib is known to inhibit not only ABL1 but also the other some tyrosine kinases.³⁸⁾ The detail is under the progress.

To the best of our knowledge, this is the first study to show the inhibitory activity of pentacyclic triterpenoids and their derivatives on ABL. These results suggest the possibility of plant extracts used for treating CML instead of the known BCR-ABL inhibitors. Further investigation is in progress.

Acknowledgment

This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (17H03999) (to M. O.).

Conflict of Interest

The authors declare no conflict of interest.

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