Clin Transl Oncol DOI 10.1007/s12094-014-1260-0

RESEARCH ARTICLE

A new family of choline kinase inhibitors with antiproliferative and antitumor activity derived from natural products A. Este´vez-Braun • A. G. Ravelo • E. Pe´rez-Sacau J. C. Lacal

•

Received: 16 September 2014 / Accepted: 21 November 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014

Abstract Background Choline kinase alpha (ChoKa) is a critical enzyme in the synthesis of phosphatidylcholine, a major structural component of eukaryotic cell membranes. ChoKa is overexpressed in a large variety of tumor cells and has been proposed as a target for personalized medicine, both in cancer therapy and rheumatoid arthritis. Materials and methods Triterpene quinone methides (TPQ) bioactive compounds isolated from plants of the Celastraceae family and a set of their semisynthetic derivatives were tested against the recombinant human ChoKa. Those found active as potent enzymatic inhibitors were tested in vitro for antiproliferative activity against HT29 colorectal adenocarcinoma cells, and one of the

Electronic supplementary material The online version of this article (doi:10.1007/s12094-014-1260-0) contains supplementary material, which is available to authorized users. A. Este´vez-Braun  A. G. Ravelo  E. Pe´rez-Sacau Instituto Universitario de Bio-Orga´nica ‘‘Antonio Gonza´lez’’, Universidad de La Laguna, Avda. Astrofı´sico Fco. Sa´nchez 2, 38206 La Laguna, Tenerife, Spain A. Este´vez-Braun  A. G. Ravelo  E. Pe´rez-Sacau Instituto Canario de Investigacio´n del Ca´ncer (ICIC), Las Palmas, Spain URL: http://www.icic.es E. Pe´rez-Sacau Centro Atla´ntico del Medicamento (CEAMED), Parque Cientı´fico Tecnolo´gico de Tenerife (PCTT), Avda. de la Trinidad s/n 38204, San Cristo´bal de La Laguna, S/C Tenerife, Spain J. C. Lacal (&) Traslational Oncology, IdiPaz, Paseo de la Castellana 261, 28046 Madrid, Spain e-mail: [email protected]

active compounds was tested for in vivo antitumoral activity in mice xenographs of HT29 cells. Results Among 59 natural and semisynthetic TPQs tested in an ex vivo system, 14 were highly active as inhibitors of the enzyme ChoKa with IC50 \10 lM. Nine of these were potent antiproliferative agents (IC50 \10 lM) against tumor cells. At least one compound was identified as a new antitumoral drug based on its in vivo activity against xenographs of human HT-29 colon adenocarcinoma cells. Conclusions The identification of a new family of natural and semisynthetic compounds with potent inhibitory activity against ChoKa and both in vitro antiproliferative and in vivo antitumoral activity supports further research on these inhibitors as potential anticancer agents. Their likely role as antiproliferative drugs deserves further studies in models of rheumatoid arthritis. Keywords Triterpene quinone methides  Natural products  Choline kinase  Choline kinase inhibitors  Antitumoral drugs  Rheumatoid arthritis

Introduction Choline kinase alpha (ChoKa) is involved in the synthesis of phosphatidylcholine, the major phospholipid of all eukaryotic membranes [1]. Two genes have been identified in humans, CHKA and CHKB, which generate three polypeptides, ChoKa1, ChoKa2 and ChoKb with in vitro choline kinase activity [2]. While ChoKa1 and ChoKa2 derive from the same gene by differential splicing and are almost identical except that ChoKa1 contains an extra stretch of 18 amino acids, ChoKb is 60 % homologous to ChoKa1 and ChoKa2.

123

Clin Transl Oncol

Abundant evidence has been provided supporting the use of ChoKa as a drug target in oncology [3]. Thus, in addition to its metabolic role, ChoKa1 has been described to be oncogenic when overexpressed in mammalian cells [4] and a new prognostic marker for predicting the outcome of early-stage non-small cell lung cancer (NSCLC) patients and hepatocarcinomas [5, 6]. In keeping with this, ChoKa has been found to be increased in several human tumors such as lung, breast, colorectal, prostate, bladder and ovary [7–11], an indication of its potential clinical relevance. On the other hand, ChoKa inhibitors have been designed and synthesized with demonstrated in vitro antiproliferative activity toward a large panel of tumor-derived cells lines including breast, lung, colon, bladder, liver, ovary, bone, cervix, kidney, pancreas, melanoma and brain tumors, and show efficient in vivo antitumoral activity against human xenografts derived from colon, epidermoid, breast and bladder tumors [12–16]. The mechanism of action of ChoK inhibitors has been elucidated and is based on the production of ceramides, specifically in tumor cells [17, 18]. Finally, microarray analysis of transcriptome of human cells overexpressing ChoKa or treated with ChoK inhibitors reveals its effects on cell cycle regulation and interference with apoptosis signaling, a phenomenon that explains its pro-mitogenic and oncogenic activity [19]. Genetic inhibition of ChoKa further supports its potential use as a molecular target in oncology. Thus, an siRNA designed against both ChoKa and ChoKb has been demonstrated to interfere with the proliferation of breast cancer cells inducing their differentiation to milk-producing cells [20]. Also, an siRNA designed specifically against ChoKa shows antiproliferative and proapoptotic activity toward breast, urinary bladder, cervix and non-small cell lung cancer cells, a similar result to that obtained with pharmacological inhibition of ChoKa [1]. Furthermore, different strategies have reached the conclusion that ChoKa, but not ChoKb should be the target for a specific and successful cancer therapy at least in some cellular systems [22, 23]. ChoKa is a bona fide new target for the development of anticancer drugs, and several types of molecules are defined as inhibitors of its activity. As the enzyme requires ATP for its catalytic action, structural analogous of choline and ATP have been reported as ChoK inhibitors [3, 12–16, 24–30]. Also, the presence of Mg2? is an absolute requirement for enzyme activity; therefore, a number of ions have been described to be able to displace this cation and affect the catalytic action. Also, choline analogs can interfere with the enzyme and show inhibitory effects due to their structural homology to the natural substrate. In particular, hemicholinium-3 (HC-3) derivatives have been described as highly specific ChoK inhibitors [3, 12–16]. These compounds have two ammonium cations wearing a

123

choline-like moiety in each extreme of the molecule and a bis-phenyl rigid spacer being a key requirement for inhibitory activity the presence of a hydrophobic part in the molecule [31]. New ChoK inhibitors have been described recently, demonstrating that efficient inhibition can be achieved by alternative and complementary mechanisms involving both the choline and the ATP-binding pockets. At least two alternative mechanisms for inhibition of the monomeric form of the P. falciparum ChoK have been demonstrated involving competitive and non-competitive interactions with choline and ATP [32]. An intermediate phosphorylated form of the enzyme has been identified, common to the human dimeric enzyme [26, 32]. These new inhibitors and a better knowledge of the enzymatic reaction of this relevant kinase related to cancer development may provide alternative sources for a successful design of novel cancer drugs. Thus, the identification of new structures that efficiently interfere with ChoKa activity will provide new tools for the design of more potent anticancer drugs. A recent study has shown that ChoKa is expressed in rheumatoid arthritis (RA) synovial tissue and in cultured fibroblast-like synoviocytes (FLS), and that its expression is increased after pro-inflammatory cytokine stimulation [33]. In keeping with its potential role in the setup of RA disease, selective inhibition of ChoKa efficiently suppressed RA in mice as well as the aggressive behavior of cultured RA FLS, suggesting that ChoKa inhibition could be an effective strategy for the treatment of arthritis. Here, we report the ChoKa inhibitory activity of a set of natural and semisynthetic TPQ derivatives. These compounds present a pentacyclic D:A-friedo-nor-oleanane-type skeleton with two hydrophilic and electron-deficient electronic regions located on the A and E rings, in each extreme of the molecule. They also have a hydrophobic zone located on the B, C and D rings. Some of them showed potent antiproliferative activity against tumorderived cell lines and possessed in vivo activity as potent tumor growth inhibitors. Preliminary docking studies are provided to explore the binding mode between the ligands and the receptor.

Materials and methods Ex vivo ChoK activity assays TPQ derivatives were tested against partially purified recombinant human ChoKa1 expressed in Escherichia Coli as previously described [16, 22]. Ex vivo ChoK activity assays were performed in buffer containing 100 mM Tris– HCl pH 8.0, 100 mM MgCl2 and 10 mM ATP. Physiological choline concentration (200 mM) was used as

Clin Transl Oncol

substrate in the presence of methyl[14C]-choline chloride (50 ± 60 Ci/mmol, Amersham International), and reactions were performed at 37 °C for 30 min. Hydrophylic derivatives of choline (i.e., choline, phosphorylcholine) were resolved by thin-layer chromatography (TLC) plates (60 A silica gel, Whatman, Clifton, NJ, USA) using as liquid phase 0.9 % NaCl: methanol: ammonium hydroxide (50:75:5; v/v/v). Radioactivity corresponding to PCho was automatically quantified by an electronic radiography system (Instantimager; Packard, Meriden, CT, USA). Cell proliferation assays Human HT-29 (colorectal adenocarcinoma) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) under standard conditions of temperature (37 °C), humidity (95 %) and CO2 (5 %). Cells were seeded on 24-well plates and incubated for 24 h in growth medium. Then, cells were incubated in fresh medium containing increasing concentrations (quadruplicates for each concentration) of the compounds for 72 h. Cell proliferation assays were determined using the crystal violet method as previously described [16]. Maximum tolerated dose The MTD experiments were performed using five Swiss immunocompetent mice for each dose. Groups of animals were treated with each substance at 1, 5, 10 or 15 mg/kg. All compounds were diluted at the same concentration as a stock solution (5 mM) and successive dilutions were done in phosphate buffer saline (PBS, 1X) for injections. The treatment consists of two cycles of a daily i.p. injection during 5 days, followed by 9 days of rest. Mice were observed daily and death cases during and at the end of the treatment recorded as survivors/total treated mice. In vivo antitumoral activity assays All experiments performed with mice were carried out following the Spanish and EU legislation after approval by the corresponding ethics committee at the Centro Nacional de Biotecnologı´a, Madrid, Spain. Human tumor xenografts were established by s.c. injection of HT-29 tumor-derived cells in athymic CD1 nude mice. When tumors reached a volume of 0.1 cm3, mice were randomized to control and treated groups (8 mice each). Treatments were performed i.p. with a schedule of daily consecutive doses of 7.5 mg/ kg for 5 days, separated by 9 days. Tumors were monitored at least twice a week by measuring the major (D) and minor (d) diameters, and tumor volume was calculated as V = (Dx d2)/2. Differences between control and treated

mice were analyzed with the Mann–Whitney test using SPSS software. Molecular docking AutoDock 3.0.5 [34] was the software used to carry out all the docking analysis. The crystal structure of human ChoKa was retrieved from the Protein Data Bank (PDB entry 3g15). The hydrogen atoms of the protein were added using the software package ADT Tools. Protein atom types and potentials were assigned according to the Amber 4.0 force field with Kollman united-atom charges. The initial structures of TPQs were optimized using Gaussian 03 at level HF/3-21G with RESP charges. The minimized geometry was employed to prepare the ligands for their use in docking program AutoDock 3.0.5 (http://www.scripps. edu/mb/olson/doc/autodock/) following the standard procedure. The 3-D grid with 60 9 60 9 60 points and a ˚ was created by the AutoGrid algorithm spacing of 0.375 A to evaluate the binding energies between the ligands and the proteins. Default docking parameters were used except number of generations, population size and docking runs, which are fixed at 27 000, 100 and 250, respectively. The Lamarckian genetic algorithm was applied to analyze protein–ligand interactions. The docked structures of the ligands were generated after a reasonable number of evaluations. Figures for best scoring dockings were generated by PyMOL [35].

Results and discussion Chemistry TPQs are bioactive compounds isolated from plants of the Celastraceae family [36, 37]. 22b-Hydroxytingenone (1), tingenone (2) and pristimerine (3) are three representative examples of natural occurring TPQs (Fig. 1). They have a D:A-friedo-nor-oleanane skeleton, with the main chemical features of these types of compounds being the presence of a methylene quinoid moiety in the AB rings, a rigid transdecalin system in the CD rings and the presence of oxygenated substituents in the E ring. We have previously reported the cytotoxic activity of several natural and semisynthethic-related compounds against several tumoral cell lines (1–29) (see Fig. 1) [38–43]. Based on previous structure–cytotoxic activity relationships [38], we obtained a new set of TPQ derivatives (30–61) by carrying out several modifications such as bromination, oxime formation and esterifications. Synthesis procedures of all derivatives are provided elsewhere (Supplementary Data 1) as well as their 1H-NMR and 13C-NMR spectra (Supplementary Data 2).

123

Clin Transl Oncol

R3

O E C

O

R2

D

O

R2 R1

H

R1O

O

OR3

A R1O

H3CO R2OCH2

HO 13 R1=H; R2= =O; R3=OH 14 R1=H; R2=OH; R3=H 15 R1=R2=OH; R3=H

1 R1=H; R2=OH 2 R1=R2=H 4 R1=MOM; R2=H 5 R1=Ac; R2=H 6 R1=m,m,p(OMe)3Bz; R2=H 7 R1=COCH2CH3; R2=H 8 R1=COCH(CH3)2; R2=H 9 R1=pNO2Bz; R2=H 10 R1=H; R2=OCO(CH2)2COOH 11 R1=H; R2=OCO(CH2)2COOEt 12 R1=H; R2= O O

16 R1=m,m,p(OMe)3Bz; R2=R3=H 17 R1=R2=R3=m,m,p(OMe)3Bz

COOMe R2 3 18 19 20 21

O

N

O HN

R1O

OH O

HO

H

COOMe

H

HO R3O

O

O

R1=R2=H R1=MOM; R2=H R1=m,m,p(OMe)3Bz; R2=H R1=COCH2CH3; R2=H R1=H; R2=OH

COOMe

COOMe

O

O

OH

22

R2 R1

23

O

24 R1=H; R2=OH; R3=H 25 R1=CHO; R2=R3=H 26 R1=Me; R2=H; R3=CH2O(CH2)2OMe COOMe

COOMe

H

HO HO

COOMe

O CHO CHO 27

O

O

O 28

29

O

Fig. 1 Structures of natural and semisynthetic TPQ derivatives

With the aim of introducing bromine atoms into the TPQ skeleton, we used several brominating agents such as boron tribromide, N-bromo succinimide and Br2/CHCl3. The treatment of compound 1 with 1.5 equiv of BBr3 in dry dichloromethane at 0 °C for 30 min afforded two compounds: 30 (27 %) brominated at the position C-19

123

and the unexpected a,b-unsaturated ketone 31(40 %), which does not have the hydroxyl group at C-22 (Fig. 2). The orientation a for the bromine at C-19 in 30 was established on the basis of the NOE effect detected between the doublet at d 4.03 (J = 5.6 Hz) corresponding to H-19 and the singlet at d 0.65 attributable to Me-28.

Clin Transl Oncol

O

Br

30 min

OH

H

O

O

O

+

H

O HO H

O

HO

OH

31 (40%)

30 (27%) O

HO 24 h

1

H

HO HO

32 (20%) O

O 24 h H

O

H

HO HO

HO

33 (28%)

2 COOMe

COOMe H

O

30 min HO HO

HO 3

34 (40%)

Reagents and conditions. BBr3 (1.5 equiv), dry CH2Cl2, 0ºC, N2 atm.

Fig. 2 Reaction of TPQs (1–3) with BBr3

When the reaction mixture was left at 0 °C for 24 h, only one compound (32) was formed in low yield (20 %). This compound shows the same a,b-unsaturated carbonyl moiety in the E ring to that in compound 31, and it also shows aromatization of the A and B rings with lack of the methyl 25. Probably, this compound is formed from 31 by the attack of a bromide to Me-25 and the consequent evolution toward the phenolic triterpene 32. A similar result was obtained when the same reaction conditions were applied to tingenone (2) and compound 33 was formed, while a different rearrangement was detected when pristimerine (3) was employed as substrate (Fig. 2). In this case, only one compound 34 was also formed in 40 % yield. This compound was a tetracyclic derivative with A and B rings aromatized and open C ring. The same product was previously obtained by Nakanishi et al. [44] on treatment of pristimerine with sulfuric acid.

When compound 1 was reacted with N-bromosuccinimide (NBS), two brominated derivatives (35) and (36) were obtained (Fig. 3). Compound (35) has a bromine at C-11, while compound (36) presents a more complex structure which includes the presence of a C-11-C-12 double bond, migration of the methyl Me-25 from C-9 to C-10 and a dicarbonyl moiety at the ring A. The structures of both compounds were determined by the thorough analysis of HMBC and ROESY correlations. Similar compounds (37–40) were obtained in the bromination of tingenone (2) and pristimerine (3). We also treated compounds 1 and 2 with 1 equiv of Br2/CH2Cl2 as brominating agent. In these cases only the brominated derivatives at C-11 (35 and 37) were obtained in low yield. Several transformations were carried out to modify the number and the nature of hydrogen bond donors and acceptors in rings E and A. Thus, the carbonyl group at

123

Clin Transl Oncol

Br H

O

OH

O

O

O 11

O

OH

H

+

O

OH

H

1

O

HO

HO

Br

36 (5%)

35 (11%)

1

O

O O

Br 11

H

O

Br

H

O

H

O 1

+ O

HO HO

38 (9%)

37 (21%) 2

COOMe

COOMe COOMe Br

Br H

O

O

11

H

+

O

H

1

O

HO HO 3

40 (14%)

39 (3%)

Reagents and conditions. NBS (2 equiv), dry CH2Cl2, rt, 2 h. Fig. 3 Reactions of TPQs with NBS

C-21 in the E ring of the compounds 1, 2 and 31 was converted into the corresponding oximes 41–43 by treatment with hydroxylamine hydrochloride (NH2OHHCl) in the presence of sodium acetate (Fig. 4). We also prepared several acyl derivatives at C-2. The best reaction conditions to achieve this type of derivatives were obtained by using 3 equiv of triethylamine (Et3N), catalytic amounts of DMAP and 1.5 equiv of the corresponding acyl chloride. In a previous work, we observed that when pyridine was used, derivatives having ring A aromatized with a catechol system were formed [38]. To study the influence on the activity of several groups of different nature, size, lipophilicity and stereoelectronic properties, we employed a variety of acylating agents such as acetyl, p-bromobenzoyl, nicotinoyl, lauroyl and N,Ndimethyl carbamoyl chlorides, and compounds (44–60) were obtained (Fig. 5). When derivative (50) was reacted with nicotinoyl chloride in the presence of 3 equiv of Et3N and catalytic amount of DMAP, compound (61) was obtained instead of the expected acyl derivative at C-22. 61 presents aromatized ring A, the nicotinoyl moiety at C-2 and a hydroxyl group at C-6 (Fig. 6).

123

NOH

O

H

R

1 R=OH 2 R=H

H

41 R=OH (96%) 42 R=H (66%) NOH

O

H

31

R

H

43 (52%)

Reagents and conditions. NH2OH·HCl (3 equiv), ethanol, NaOAc (2 equiv), reflux, 24 h. Fig. 4 Formation of oxime derivatives (41–43)

Clin Transl Oncol Fig. 5 TPQ derivatives at C-3

R2

O R1

H

O R3O

COOMe

H

O

44 45 46 47 48 49 50 51 52 53 54

R1=R2=H; R3=CO(CH2)10CH3(48%) R1=R2=H; R3=COCH3 (54%) R1=R2=H; R3=nicotinoyl (46%) R1=R2=H; R3=N,N-dimethyl carbamoyl (25%) R1=OH; R2=H; R3=CO(CH2)10CH3(32%) R1=OH; R2=H; R3=nicotinoyl (45%) R1=OH; R2=H; R3=N,N-dimethyl carbamoyl (72%) R1=OH; R2=H; R3=pBrBz (57%) R1=OH; R2=Br; R3=pBrBz (52%) R1=OH; R2=Br; R3=CO(CH2)10CH3 (19%) R1=OH; R2=Br R3=N,N-dimethyl carbamoyl (58%)

55 R=CO(CH2)10CH3(73%) 56 R=N,N-dimethyl carbamoyl (48%) 57 R=pBrBz (20%)

RO X

H

O

58 R=CO(CH2)10CH3; X=O (37%) 59 R=N,N-dimethyl carbamoyl; X=O (70%) 60 R=N,N-dimethyl carbamoyl; X=NOH (33%)

RO

Reagents and conditions. Et3N (3 equiv), CH2Cl2, RCOCl (1.5 equiv), cat DMAP, N2 atm, 0 ºC, 1 h.

O

H

O N

N

O O

OH O N

O

H

O

OH

O OH

O 50

61 (39%)

Reagents and conditons: nicotinoyl chloride (1.5 equiv), Et3N (3 equiv), cat DMAP, rt, 24 h Fig. 6 Formation of derivative 61

Ex vivo inhibition of ChoKa Since our set of compounds have potent cytotoxic activities and fit the structural requirements for being ChoKa inhibitors (hydrophobic zone and two electron-deficient regions separated by a rigid spacer), we assayed the previously obtained (1–29) and the new TPQ derivatives (30–61) for ChoKa inhibition. TPQ derivatives were tested against the ChoKa enzymatic activity using partially purified recombinant human ChoKa1 expressed in E. coli that lacks ChoK activity; therefore the activity that could be observed was exclusively from the human recombinant expressed

isoform. The partially purified ChoKa enzyme was incubated as described under ‘‘Materials and methods’’ in the presence of increasing concentrations of each compound. Out of the 59 compounds tested, 1, 5, 10, 11, 21, 30, 31, 45, 46, 48, 49, 51, 52 and 56 were highly active against ChoKa with an IC50 between 0.6 and 8.6 lM. An analysis of the IC50 values for the set of TPQs (Table 1) led us to establish several preliminary structure–activity relationships. The presence of the quinoid system in the AB rings seems to be necessary for the activity. The esterification of the hydroxyl group at C-3 increases or decreases the activity depending on the nature of the acyl group. With

123

Clin Transl Oncol Table 1 Ex vivo ChoKa inhibitory activity of TPQ derivatives (1–61) Compound

IC50 (lM)

Compound

IC50 (lM)

Compound

IC50 (lM)

5

4.8

11.1

10

4.7

11.3

11

2.1

4.1

13

11.2

25.9

21

7.3

3.8

30 31

3.3 0.6

8.8 5.9

35

10.8

12.1

45

5.3

26.4

[25/ND

46

8.8

13.3

17.5

43

[25/ND

2

[25/ND

23

43.4

44

48.0

3

14.9

24

25.5

45

5.3

4

39.9

25

40.8

46

8.8

5

4.8

26

162.7

47

33.4

6

31.8

27

30.9

48

5.2

7

20.3

28

987.0

49

8.6

8

25.3

29

153.0

50

14.2

9

31.5

30

3.3

51

2.6

0.6

52

IC50 in vitro (lM) (HT-29) 6.5

22

31

IC50 ex vivo (lM) (ChoK) 4.9

4.9

4.7

Compound

1 1

10

Table 2 Antiproliferative activity of selected ChoKa inhibitors

0.6

11

2.1

32

[25/ND

53

12

73.5

33

[25/ND

54

19.5

48

5.2

9.6

13

11.2

34

36.1

55

110.6

49

8.6

7.2

14

30.0

35

10.8

56

7.1

51

2.6

9.4

[25/ND

52

0.6

8.0

325.0

56

7.1

11.3

15

12.3

36

NT

57

16

181.1

37

17

151.0

38

NT

59

34.9

18

26.6

39

[25/ND

60

[25/ND

19

65.5

40

[25/ND

61

130.0

20

84.3

41

27.7

HC-33

45–57

21

7.3

42

[25/ND

13.6

58

The enzymatic ex vivo reaction takes place in the presence of increasing concentrations of the compound. The concentration of each compound that produces 50 % inhibition is calculated and expressed as IC50 (lM) ND not determined, NT not tested

respect to the effect of E-ring substitution pattern on the inhibitory activity, in general the compounds derived from 22-b-hydroxy-tingenone (1) are more active than those derived from tingenone (2), and these are more active than those derived from pristimerine (3). There are some exceptions, for example compound (56) belonging to the pristimerine series showed higher inhibitory activity (IC50 = 7.1 lM) than 47 (IC50 = 33.4 lM) and 50 (IC50 = 14.2 lM). Antiproliferative and antitumoral assays Fourteen of the compounds tested were highly active against ChoKa with IC50 values lower than 10 lM. Since ChoKa has been recently described as a new oncogene with a relevant role in human cancer [4–11], and the specific inhibition of this enzyme has been described as a promising new target for anticancer therapy [3], all compounds with IC50 values in the range of lower or around 10 lM (1, 5, 10, 11, 13, 21, 30, 31, 35, 45, 46, 48, 49, 51, 52 and 56) were analyzed for their antiproliferative activity against the human colon adenocarcinoma-derived HT-29

123

The enzymatic ex vivo data represent the concentration of each compound rendering 50 % inhibition as IC50 (lM) (data from Table 1). For the in vitro antiproliferative assay, compounds were tested against human colon adenocarcinoma cells (HT-29) at different concentrations for 144 h. The IC50 (50 % inhibitory concentration) is quantified by plotting the log OD (optical density) versus log drug concentration

cells (Table 2). Several compounds were found also to have potent antiproliferative activity with less than or around IC50 = 5 lM, with the most remarkable compounds being 1, 11, 21 and 31. Compound 31 was found to be unstable and not considered for further experiments. Compound 1 was further investigated as a potential candidate for a new antitumoral drug. Preliminary toxicity studies were performed to find an appropriate dose to test in antitumor experiments with nude mice. LD50 (Lethal Doses) experiments were carried out using 5 CD1 nude mice (Harlan Sprague–Dawley, Inc.) for each dose in four increasing doses. The treatment consisted of a daily intraperitoneal injection for 5 days, followed by 9 days of rest, and after that another cycle of 5 days of treatment. Deaths during and at the end of the treatment were registered. An LD50 = 12.5 mg/kg was determined for compound 1. Compound 1 was then analyzed for its in vivo antitumoral activity in nude mice. Tumors were generated by subcutaneous injection of the HT-29 tumor-derived cell line in the back of immunosuppressed CD1 nude mice. The compound was administered by intraperitoneal injection, dissolved in sterile 0.9 % NaCl. Control mice received an equivalent volume of vehicle alone, following an identical schedule. Treatment was initiated when the tumors reached a mean volume of 0.1 cm3, and tumors were monitored at

Clin Transl Oncol

Fig. 7 Antitumoral activity in vivo of compound (1)

least twice a week. Treatment with 7.5 mg/kg of 22-bhydroxy-tingenone (1) significantly inhibited tumor growth to a 75 % reduction (p = 0.03, n = 15) (Fig. 7). Docking studies for TPQs into ChoK

and only one side of the planar HC-3 molecule contributed to the hydrophobic interaction with the groove. The ChoKa1 model showed that ADP and two magnesium ions were located at the nucleotide-binding site of the N-terminal lobes. Docking analysis of several TPQ ligands into ChoKa1 protein (3g15.pdb) was carried out. Figure 8 shows compound 1 and 46 as representative TPQ ligands, and HC-3 was used as control to evaluate the predictive power of the model by comparison to the X-ray structure. Compound 1 shows the ligand docking into the same place and in similar manner to HC-3 (Fig. 8). The model predicts that TPQ ligands establish the same interactions with key residues such as Tyr-354 and Tyr-440. In the case of compound 1, interactions with Arg-146 and Gln-308 seem to be also important. More interestingly, there is an important cluster of poses only for the active compounds with an efficient pi stacking interaction between A ring of the TPQs and Mg2? which is shown for compound 46 as an example (Fig. 8b).

Conclusion

To better understand the key molecular interactions between ChoKa and TPQ inhibitors, docking studies were performed. The human ChoKa complex with HC-3 and ADP has been crystallized [45] revealing that HC-3 binds to a groove on the C terminal lobe near the interlobe cleft in a manner where one oxazinium ring occupied the choline-binding pocket, and the other oxazinium ring was partially exposed to the solvent. The HC-3-binding groove was lined by hydrophobic residues (Tyr-354, Phe-361, Trp420, Trp-423, Ile-433, Phe-435, Tyr-437, and Tyr-440),

Based on TPQ skeleton of natural products from plants of the Celastraceae family, we have identified a new family of natural and semisynthetic products that are potent inhibitors of the human ChoKa1, some of them in the submicromolar range. Several of these compounds show also potent antiproliferative activity against human tumorderived cells in the low, single-digit micromolar range. Finally, at least one compound was identified as a new antitumoral drug based on its in vivo activity against xenographs of human HT-29 colon adenocarcinoma cells. Docking analysis were carried out into ChoKa protein (3g15.pdb), and all active compounds showed an important

Fig. 8 Docking model of a compound 1 (gray) and HC-3 (magenta). All active compounds show an important cluster of poses with ligands docking into the same place as HC-3. In the case of compound 1, interactions with Arg-146 and Gln-308 seem to be important.

b Another important cluster of poses for the active compounds was detected with an efficient pi stacking interaction between the A ring of the TPQs and Mg2? (gray balls) and it is shown for compound 46 as an example (green); HC-3 (magenta) is shown as reference

123

Clin Transl Oncol

cluster of poses docked into the same place and in a similar manner to HC-3. The active compounds showed also an efficient pi stacking interaction between A ring of the TPQs and Mg2?. The identification of new structures that efficiently interfere with ChoKa activity will provide new sources for the design of more potent inhibitors that along with a better knowledge of the enzymatic reaction involved in their interaction with this relevant cancer-related kinase may provide alternative tools for a successful design of novel efficient cancer drugs. Acknowledgments The authors appreciate the contribution to experimental performance of Dulce Mesa Siverio and Haydee Cha´vez. This work has been supported by grants to JCL from Ministerio de Ciencia e Innovacio´n (SAF2008-03750, SAF2011-29699, RD060020-0016 and RD12/0036/0019) and by grants to AEB and AGR from Ministerio de Ciencia e Innovacio´n (SAF 2012-37344-C03-01 and SAF 2009-13296-C02-01) and Instituto Canario de Investigacio´n del Ca´ncer (ICIC). Conflict of interest JCL is a stockholder of TCD Pharma SL. All the remaining authors declare no competing financial interest.

References 1. Lykidis A, Jackowski S. Regulation of mammalian cell membrane biosynthesis. Prog Nucleic Acid Res Mol Biol. 2001;65:361–93. 2. Aoyama C, Liao H, Ishidate K. Structure and function of choline kinase isoforms in mammalian cells. Progr Lipid Res. 2004;43:266–81. 3. Lacal JC. Choline kinase: a novel target for antitumor drugs. IDrugs. 2001;4:419–26. 4. Ramirez de Molina A, Gallego-Ortega D, Sarmentero J, Ban˜ez-Coronel M, Martin-Cantalejo Y, Lacal JC. Choline kinase is a novel oncogene that potentiates RhoA-induced carcinogenesis. Cancer Res. 2005;65:5647–53. 5. Ramirez de Molina A, Sarmentero-Estrada J, Belda-Iniesta C, Taron M, Ramirez de Molina V, Cejas P, et al. Expression of ChoKa to predict outcome in patients with early-stage non-small-cell lung cancer: a retrospective study. Lancet Oncol. 2007;8:889–97. 6. Kwee SA, Hernandez B, Chan O, Wong L. Choline kinase alpha and hexokinase-2 protein expression in hepatocellular carcinoma: association with survival. PLoS One. 2012;7(10):e46591. 7. Ramirez de Molina A, Gutierrez R, Ramos MA, Silva JM, Silva J, Bonilla F, et al. Increased choline kinase activity in human breast carcinomas: clinical evidence for a potential novel antitumor strategy. Oncogene. 2002;21:4317–22. 8. Eliyahu G, Kreizman T, Degani H. Phosphocholine as a biomarker of breast cancer: molecular and biochemical studies. Int J Cancer. 2007;120:1721–30. 9. Iorio E, Mezzanzanica D, Alberti P, Spadaro F, Ramoni C, D’Ascenzo S, et al. Alterations of choline phospholipid metabolism in ovarian tumor progression. Cancer Res. 2005;65:9369–76. 10. Hernando E, Sarmentero-Estrada J, Koppie T, Belda-Iniesta C, Ramirez de Molina V, Cejas P, et al. A critical role for choline kinase-alpha in the aggressiveness of bladder carcinomas. Oncogene. 2009;28:2425–35. 11. Iorio E, Ricci A, Bagnoli M, Pisanu ME, Castellano G, Di Vito M, et al. Activation of phosphatidylcholine cycle enzymes in human epithelial ovarian cancer cells. Cancer Res. 2010;70:2126–35. 12. Hernandez-Alcoceba R, Saniger L, Campos J, Nunez MC, Khaless F, Gallo MA, et al. Choline kinase inhibitors as a novel approach for antiproliferative drug design. Oncogene. 1997;15:2289–301. 13. Rubio-Ruı´z B, Conejo-Garcı´a A, Rios-Marco P, Carrasco-Jime´nez P, Segovia J, Marco C, et al. Design, synthesis, theorical calculations and biological evaluation of new no-symmetrical choline kinase inhibitors. Eur J Med Chem. 2012;50:154–62. 14. Martı´n-Cantalejo Y, Sa´ez B, Monterde MI, Murillo MT, Bran˜a MF. Synthesis and biological activity of new bispyridinium salts of 4,40 -bispyridil-5,50 -perfluoroalkyl-2,20 -bisoxazoles. Eur J Med Chem. 2011;46:5662–7. 15. Hernandez-Alcoceba R, Fernandez F, Lacal JC. In vivo antitumor activity of choline kinase inhibitors: a novel target for anticancer drug discovery. Cancer Res. 1999;59:3112–8.

123

16. Lacal JC, Campos JM Preclinical characterization of RSM-932A, a novel anticancer drug targeting the human choline kinase alpha, an enzyme involved in increased lipid metabolism of cancer cells. Mol Cancer Therap. doi:10.1158/ 1535-7163.MCT-14-0531. 17. Rodriguez-Gonzalez A, Ramirez de Molina A, Fernandez F, Ramos MA, Nunez MC, Campos J, et al. Inhibition of choline kinase as a specific cytotoxic strategy in oncogene-transformed cells. Oncogene. 2003;22:8803–12. 18. Rodriguez-Gonzalez A, Ramirez de Molina A, Fernandez F, Lacal JC. Choline kinase inhibition induces the increase in ceramides resulting in a highly specific and selective cytotoxic antitumoral strategy as a potential mechanism of action. Oncogene. 2004;23:8247–59. 19. Ramirez de Molina A, Gallego-Ortega D, Sarmentero-Estrada J, Lagares D, Gomez del Pulgar T, Bandres E, et al. Choline kinase as a link connecting phospholipid metabolism and cell cycle regulation: implications in cancer therapy. Int J Biochem Cell Biol. 2008;40:1753–63. 20. Glunde K, Raman V, Mori N, Bhujwalla ZM. RNA interference-mediated choline kinase suppression in breast cancer cells induces differentiation and reduces proliferation. Cancer Res. 2005;65:11034–43. 21. Ban˜ez-Coronel M, Ramirez de Molina A, Rodriguez-Gonzalez A, Sarmentero J, Ramos MA, Garcia-Cabezas MA, et al. Choline kinase alpha depletion selectively kills tumoral cells. Curr Cancer Drug Targets. 2008;8:709–19. 22. Gallego-Ortega D, Ramirez de Molina A, Ramos MA, Valdes-Mora F, Barderas MG, Sarmentero-Estrada J, et al. Differential role of choline kinase alpha and beta isoforms in human carcinogenesis. PLoS One. 2009;4:e7819. 23. Gruber J, See Too WC, Wong MT, Lavie A, McSorley T, Konrad M. Balance of human choline kinase isoforms is critical for cell cycle regulation. Implications for the development of choline kinase-targeted cancer therapy. FEBS J. 2012;279:1915–28. 24. Clem BF, Clem AL, Yalcin A, Goswami U, Arumugam S, Telang S, et al. A novel small molecule antagonist of choline kinase-a that simultaneously suppresses MAPK and PI3K/AKT signaling. Oncogene. 2011;30:3370–80. 25. Hudson CS, Knegtel RM, Brown K, Charlton PA. Kinetic and mechanistic characterisation of choline kinase-a. Biochim Biophys Acta. 2013;1834:1107–16. 26. Trousil S, Carroll L, Kalusa A, Aberg O, Kaliszczak M, Aboagye EO. Design of symmetrical and nonsymmetrical N, N-dimethylamino pyridine derivatives as highly potent choline kinase alpha inhibitors. Med Chem Commun. 2013;4:693–6. 27. Sa´nchez Martı´n R, Campos JM, Conejo-Garcı´a A, Cruz-Lo´pez L, Ba´n˜ez-Coronel M, Rodrı´guez-Gonza´lez A, et al. Symmetrical bis-quinolinium compounds: new human choline kinase inhibitors with antiproliferative activity against the HT-29 cell line. J Med Chem. 2005;48:3354–63. 28. Milanese L, Espinosa A, Campos JM, Gallo MA, Entrena A. Insight into the inhibition of human choline kinase: homology modeling and molecular dynamics simulations. Chem Med Chem. 2006;1:1216–28. 29. Sahu´n-Roncero M, Rubio-Ruiz B, Saladino G, Conejo-Garcı´a A, Espinosa A, Vela´zquez Campoy A, et al. The mechanism of allosteric coupling in choline kinase a1 revealed by the action of a rationally designed inhibitor. Angew Chem Int Ed Engl. 2013;52:4582–6. 30. Schiaffino-Ortega S, Lo´pez-Cara LC, Rı´os-Marco P, Carrasco-Jimenez MP, Gallo MA, Espinosa A, et al. New non-symmetrical choline kinase inhibitors. Bioorg Med Chem. 2013;22:7146–54. 31. Conejo-Garcia A, Ban˜ez-Coronel M, Sanchez-Martin RM, Rodriguez-Gonzalez A, Ramos A, Ramirez de Molina A, et al. Influence of the linker in bispyridium compounds on the inhibition of human choline kinase. J Med Chem. 2004;47:5433–40. 32. Zimmerman T, Moneriz C, Diez A, Bautista JM, Go´mez del Pulgar T, Cebria´n A, et al. RSM-932A is a promising synergistic inhibitor of the choline kinase of plasmodium falciparum. Antimicrob Agents Chemother. 2013;57:5878–88. 33. Guma M, Sanchez-Lopez E, Lodi A, Garcia-Carbonell R, Tiziani S, Karin M, et al. Choline kinase inhibition in rheumatoid arthritis. Ann Rheum Dis. 2014;. doi:10.1136/annrheumdis-2014-205696. 34. Morris GM, Goodsell DS, Hallyday RS, Huey R, Hart WE, Belew RK, et al. Automated docking using a Lamarckian genetic algorithm and a empirical binding free energy function. J Comput Chem. 1998;19:1639–62. 35. DeLano W PyMOL, version 0.99. DeLano Scientific LLC, South San Francisco. http://www.pymol.sourceforge.net/. 36. Alvarenga N, Ferro E. Bioactive triterpenes and related compounds from Celastraceae. Stud Nat Prod Chem. 2005;30:635–702 [bioactive natural products (part K)]. 37. Gutierrez F, Estevez-Braun A, Ravelo AG, Astudillo L, Zarate R. Terpenoids from the Medicinal Plant Maytenus ilicifolia. J Nat Prod. 2007;70:1049–52. 38. Ravelo A, Este´vez-Braun A, Cha´vez H, Pe´rez-Sacau E, Mesa-Siverio D. Recent studies on natural products as anticancer agents. Curr Top Med Chem. 2004;4:241–65. 39. Chavez H, Rodriguez G, Estevez-Braun A, Ravelo AG, Estevez-Reyes R, Gonzalez AG, et al. Macrocarpins A-D, new cytotoxic nor-triterpenes from Maytenus macrocarpa. Bioorg Med Chem Lett. 2000;10:759–62. 40. Chavez H, Estevez-Braun A, Ravelo AG, Gonzalez AG. New phenolic and quinone-methide triterpenes from maytenus amazonica. J Nat Prod. 1999;62:434–6. 41. Chavez H, Valdivia E, Estevez-Braun A, Ravelo AG. Structure of new bioactive triterpenes related to 22b-hydroxytingenone. Tetrahedron. 1998;54:13579–90.

Clin Transl Oncol 42. Mesa-Siverio D, Chavez H, Estevez-Braun A, Ravelo AG, Cheiloclines A-I. First examples of octacyclic sesquiterpene-triterpene hetero-Diels-Alder adducts. Tetrahedron. 2005;61:429–36. 43. Oramas-Royo S, Cha´vez H, Martı´n-Rodrı´guez P, Ferna´ndez-Pe´rez L, Ravelo AG, Este´vez-Braun A. Cytotoxic Triterpenoids from Maytenus retusa. J Nat Prod. 2010;73:2029–34.

44. Nakanishi K, Takahashi Y, Budzikiewicz H. Pristimerine. Spectroscopic properties of the dienone-phenol-type rearrangement products and other derivatives. J Org Chem. 1965;30:1729–34. 45. Hong BS, Allali-Hassani A, Tempel W, Finerty PJ, MacKenzie F, Dimov S, et al. Crystal structures of human choline kinase isoforms in complex with hemicholinium-3. J Biol Chem. 2010;285:16330–4.

123