Discovery of orally active chalcones as histone lysine specific demethylase 1 inhibitors for the treatment of leukaemia

Abstract Histone lysine specific demethylase 1 (LSD1) has emerged as an attractive molecule target for the discovery of potently anticancer drugs to treat leukaemia. In this study, a series of novel chalcone derivatives were designed, synthesised and evaluated for their inhibitory activities against LSD1 in vitro. Among all these compounds, D6 displayed the best LSD1 inhibitory activity with an IC50 value of 0.14 μM. In the cellular level, compound D6 can induce the accumulation of H3K9me1/2 and inhibit cell proliferation by inactivating LSD1. It exhibited the potent antiproliferative activity with IC50 values of 1.10 μM, 3.64 μM, 3.85 μM, 1.87 μM, 0.87 μM and 2.73 μM against HAL-01, KE-37, P30-OHK, SUP-B15, MOLT-4 and LC4-1 cells, respectively. Importantly, compound D6 significantly suppressed MOLT-4 xenograft tumour growth in vivo, indicating its great potential as an orally bioavailable candidate for leukaemia therapy.


Introduction
Histone lysine specific demethylase 1 (LSD1) has been an epigenetic target for cancer therapy since its identification in 2004 1 . Aberrant over-expression of LSD1 is observed in various leukaemia cell lines and is closely associated with proliferation, migration and invasion [2][3][4] . These findings underscore the biological importance of LSD1 and therapeutic potential of LSD1 inhibitors for leukaemia therapy 5 . LSD1 inhibitors (Iadademstat, GSK2879552 and CC-90011) have entered the clinical stages and are used to treat leukaemia (Figure 1) 6 . Dithiocarbamate 26 and (Bis)urea 31 as potent LSD1 inhibitors also effectively reduce the tumour growth against different human cancer cells 7,8 .
Chalcones as 1,3-diaryl-2-propene-1-ones with the enone system between two aromatic rings possess a wide range of biological activities such as antibacterial, antioxidative, anticancer, antileishmanial, antiulcer, antiangiogenic, antiviral, immunosuppressive and anti-inflammatory activities [9][10][11] . More particularly, a number of synthetic and natural chalcones exhibited the potent anticancer activity against many cancer cell lines 12,13 . Chalcone 1 (Figure 2), a natural product isolated from the root of Glycyrrhiza inflata, could inhibit the drug transport function of ABCG2 and reverse ABCG2-mediated multidrug resistance in human multidrug-resistant cancer cell lines 14 . Chalcone 2 exhibited the reduction of tumour cell growth combined with inhibition of Notch1 intracellular domain 15 . Naphthalene-chalcone derivative 3 was found to induce significant cell cycle arrest at the G2/M phase and cell apoptosis against MCF-7 cell line 16 . Chalcone 4 displayed the potent antiproliferative activity against cancer cells by up-regulating the expression of P53 protein 17 .
Molecular hybridisation is a new concept in drug design and development based on the combination of bioactive moieties of different compounds to produce a new hybrid with the improved affinity and efficacy 18 . These above interesting findings about LSD1 inhibitors and our continuous quest to identify more potently anticancer agents led to the molecular hybridisation of a LSD1 scaffold and an antitumor fragment to generate a new LSD1 inhibitor with the potentially anticancer activity. As shown in Figure 3, a molecular hybridisation strategy based on the structures of the reported LSD1 inhibitor 26 and antitumor agent 4 produced a scaffold that has three parts: (i) chalcone as an anticancer pharmacophore; (ii) a dithiocarbamate unit as the potential LSD1 moiety; (iii) an amide linker between chalcone and dithiocarbamate to form the hydrogen bond with LSD1. To the best of our knowledge, there have been few literature reports regarding anticancer chalcone derivatives as potent LSD1 inhibitors so far.

General
Reagents and solvents were purchased (Innochem, Beijing, China). Melting points were determined on a micromelting apparatus (Tianjin XinZhou Science and Technology Co., Ltd, Tianjin, China). 1 H NMR and 13 C NMR spectra were recorded on a NMR spectrometer (DNP-NMR spectrometer, HuZhou Jingke Instrument Co., Ltd, FuZhou, China). High resolution mass spectra of all derivatives were recorded on a Waters Micromass Q-T of Micromass spectrometer by electrospray ionisation (Skyray Instrument, JiangShu, China).
General procedure for the synthesis of compounds C1$C4 Chalcone derivatives B were prepared by a condensation reaction from 1-(4-azidophenyl)ethan-1-one A and different benzaldehydes without purification. To a solution of triphenylphosphine (1 mmol), chalcone intermediates B (2 mmol), and tetrahydrofuran (12 ml) was added water (3 ml), the mixture was stirred for 4 h. Upon the completion, ethyl acetate and water were added. The organic layers were washed with water for several times to remove the tetrahydrofuran, and then evaporated to give the crude products. The crude product (1 mmol), chloroacetyl chloride (1.2 mmol), and triethylamine (0.5 mmol) were dissolved in acetone (10 ml) to stir for 8 h at room temperature. Upon completion, the system was purified with column chromatography (hexane: ethyl acetate ¼ 9:1) to obtain analogues C1~C4. Compound C4 was a reported chalcone intermediate from the previous reference [19].
(E)-2-Chloro-N-(4-cinnamoylphenyl)acetamide (C1) Yellow solid, yield: 65%; m.p.: 145-147 C. 1      General procedure for the synthesis of compounds D1$D7 To a solution of analogues C1~C4 (2 mmol) in acetone (20 ml) was added carbon disulphide (3 mmol), sodium phosphate dodecahydrate (1.5 mmol) and piperazine derivatives (2 mmol). The reaction mixture was stirred for 12 h. After the end of the reaction was established by TLC, the solvent was removed under vacuum, and excess saturated Na 2 CO 3 solution was added. The resulted mixture was extracted with ethyl acetate, dried over MgSO 4 , filtered, and concentrated under vacuum. The product was purified by a silica gel column using ethyl acetate and petroleum ether as eluent to afford compounds D1~D7. All the 1 H NMR and 13 C NMR spectra of compounds D1~D7 were listed in Supporting Information.    20 . MOLT-4&shLSD1 cells and MOLT-4&shControl cells were also established and cultured according to the published references [21,22]. After the incubation for 24 h, cancer cell lines were cultured with the chalcone D6 at different concentrations. Then, 20 lL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/ml) was added and the cells were incubated for 4 h. The absorbance was measured using a microplate reader (DeTie Technology Co., Ltd, NanJing, China).

Dialysis assay
In the dialysis experiment, after incubation of the recombinant LSD1 and chalcone derivatives for 1 h at 37 C, we dialysed the reaction system against 50 mmol/L HEPES buffer for 24 h at 4 C and the reversibility was evaluated based on the activity of LSD1 in the dialysis tube.

Dilution assay
LSD1 recombinant was incubated with the targeted compound, GSK-LSD1, or DMSO for 1 h. Then, the reaction system was diluted for 80 times. Finally, the above stated method was applied to detect the activity of LSD1 before and after dilution.

The ultrafiltration experiment
In the ultrafiltration experiment, LSD1 recombinant was incubated with a concentration of 20-fold IC 50 inhibitor. The mixture was then added to a 10 kDa cut-off ultrafiltration tube (Millipore, Darmstadt, Germany) for centrifugation to remove the unbound compound. Finally, reversibility of the compound was evaluated by LSD1 assay for the upper chamber reaction system.

Western blotting
Western blot was performed with the total lysates using RIPA buffer (Hyclone, Los Angeles, CA, USA). Same amounts of protein were subjected to SDS-PAGE, and then transferred to nitrocellulose membranes (PALL, Cortland, NY, USA). After blocking with 5% milk solution, the membranes were incubated at 4 C with respective antibody overnight, followed by the incubation with a secondary antibody. Finally, the blot was visualised by enhanced chemiluminescence kit (Thermo Fisher, Waltham, MA, USA).

Molecular docking studies
All molecular modelling studies were performed with the Autodock software (The Scripps Research Institute, San Diego, CA, USA). The crystal structure of LSD1 (PDB code: 5l3e) was downloaded from the RCSB protein database. The targeted compound was first generated using Pymol software. Following generation, the files were converted to the.pdbqt format using OpenBabel. It was then docked using AutoDockTools. The docked conformations and information were then docked and their resulting conformations were visualised using Pymol.

Results and discussion
Chemistry A typical synthetic route for chalcone based LSD1 inhibitors is described in Scheme 1. Chalcone B was prepared by the condensation reaction of 1-(4-azidophenyl)ethan-1-one with different benzaldehydes. Intermediates C1$C4 were formed by the reduction reaction and acylation reaction. Next, the intermediates C1$C4 were reacted with carbon disulphide and piperazines under the presence of triethylamine to form chalcones D1$D7.
In vitro LSD1-inhibitory activity of chalcones C1$C4 and D1$D7 The LSD1 inhibitory activity of all synthesised compounds C1$C4 and D1$D7 was examined according to reported references [24,27]. Their results of inhibitory activities against LSD1 in vitro were summarised in Table 1. In this work, Dithiocarbamate 26 and chalcone 4 were used as reference compounds. The replacement of the chlorine atom by the dithiocarbamate fragment resulted in a powerful improvement of LSD1 inhibitory activity for chalcone-dithiocarbamate derivatives D1$D7 compared with the corresponding chalcone analogues (C1$C4). Especially, compound D6 showed the potently inhibitory effect with an IC 50 value of 0.14 lM (> 100-fold more potent than C3). This result suggests that dithiocarbamate moiety may play a synergistic role in determining activity. As LSD1 belongs to the monoamine oxidase (MAO) family, the inhibitory effects of compounds C1$C4 and D1$D7 to its homologies MAO-A and MAO-B were also examined using commercially available kits 28,29 . From the results of Table 1, all synthesised compounds C1$C4 and D1$D7 had no significant effects on MAO-A and MAO-B. These findings indicated the high selectivity of chalcone-dithiocarbamate inhibitors D1$D7 on LSD1 in vitro. In addition, we found that the substitution on the phenyl ring was important for the activity showing an over 6-fold activity loss, when the fluorine atom was replaced with the hydrogen atom (compounds D3 vs. D5). Replacement of the ethyl group of compound D5 with a methyl group (D4) led to a loss of the activity. However, changing the benzyl group (compound D7) to a tertbutoxycarbonyl group (compound D6) led to a significant improvement of the activity against LSD1. All these results indicated that the substituent group at piperazine ring may play an important role for their inhibitory activity. The detailed illustration for preliminary structure activity relationship (SAR) of target derivatives was showed in Scheme 2.  dependent manner (Figure 6(B)). Importantly, the dialysis experiment ( Figure 6(C)) and dilution assay (Figure 6(D)) indicating that chalcone D6 was a reversible LSD1 inhibitor. To further confirm the potential binding manner of chalcone D6 against LSD1 recombinant, the centrifuge experiment was also carried out. With the aid of 10 kDa ultracentrifuge filter, reversible compound was  supposed to be removed from LSD1 by centrifuge. So, chalcone D6 was characterised as a reversible LSD1 inhibitor as split of chalcone D6 by ultracentrifuge may rescue the activity of LSD1 (Figure 6(E)). All these results showed that chalcone D6 could selectively inhibit LSD1 in a time dependent and reversible manner.
Antiproliferative effects of chalcone D6 against MOLT-4 LSD1 knockdown cells LSD1 was aberrantly over-expressed in leukaemia cells, and associated with tumorigenesis 32 . In view of the inhibitory potency against LSD1, chalcone D6 was chosen for further antiproliferative studies. In this work, we used the LSD1 knock-down MOLT-4 cells (MOLT-4&shLSD1) and control cells (MOLT-4&shControl) to investigate its antiproliferative activity. Firstly, the gene expression of LSD1 in MOLT-4&shLSD1 cells and MOLT-4&shControl cells was detected by quantitative real-time PCR, the results were shown in Figure 7(A). With these two cell lines in hand, we nextly used the MTT assay to examine the antiproliferative effects of chalcone D6 against MOLT-4&shLSD1 cells and MOLT-4&shControl cells. As shown in Figure 7   LSD1 inhibition, and also suggested that chalcone D6 was cellularly active against LSD1, excluding off-target effects.
Chalcone D6 regulated the expression of LSD1 substrates H3K9me1/2 To further determine the inhibitory effects of chalcone D6 against LSD1 in MOLT-4 cells, amount of two reported LSD1 substrates H3K9me1 and H3K9me2 were analysed by western blotting experiments. As shown in Figure 8, the amount of H3K9me1 and H3K9me2 showed a concentration dependent accumulation in the presence of chalcone D6. In addition, the treatment of D6 in MOLT-4 cells did not affect the expression level of LSD1. Collectively, these results suggested that chalcone D6 is a cellular active LSD1 inhibitor in leukaemia MOLT-4 cells.

Molecular docking of chalcone D6
Based on the above experiments, chalcone D6 has been identified as a novel LSD1 inhibitor. In the current work, molecular docking methodologies were also used to explore any molecular interaction exist between chalcone D6 and residues lies in the active  site cativity of LSD1. We have used Autodock as an automated tool to perform docking and selected PDB code 5l3e (Resolution: 2.80 Å). As shown in Figure 9, chalcone D6 formed three hydrogen bonds with residues His532, Asn535 and Asp556, respectively. In addition, chalcone D6 formed hydrophobic effects with residues Leu386, Phe382 and Phe538. These results explained that chalcone scaffold was a promising unit for targeting LSD1. Based on the reported reference [33], E11 as a reference compound was docked using the same protocol and compared with chalcone D6. In the Figure 9, the reference compound E11 (yellow structure) was docked into a similar pocket as chalcone D6 (magenta structure).

Xenograft study of chalcone D6
Since the potently antiproliferative activity of chalcone D6 against MOLT-4 cells, we also evaluated the anticancer effects of chalcone D6 on xenograft models bearing MOLT-4 cells. After the treatment of chalcone D6 (60 mg/kg and 100 mg/kg), the body weight of mice, the tumour weight and the tumour volume were measured and recorded. As shown in Figure 10, chalcone D6 inhibited tumour growth remarkably, while the body weight was almost unchanged, suggesting the antitumor efficacy and low global toxicity.

Conclusion
A series of chalcone derivatives were designed, synthesised and evaluated for LSD1 inhibitory activity. All chalcone-dithiocarbamate hybrids D1$D7 exhibited potentially inhibitory activity against LSD1. Especially, chalcone D6 showed the best LSD1 inhibitory activity with an IC 50 value of 0.14 lM. In addition, D6 inhibited cell proliferation with IC 50 values of 1.10 lM, 3.64 lM, 3.85 lM, 1.87 lM, 0.87 lM and 2.73 lM against HAL-01, KE-37, P30-OHK, SUP-B15, MOLT-4 and LC4-1 leukaemia cells. Further investigations demonstrated that compound D6 selectively inhibited LSD1 in a time dependent and reversible manner. It also up-regulated the expression levels of H3K9me1 and H3K9me2 against MOLT-4 cells. Importantly, chalcone D6 inhibited in vivo tumour growth in a xenograft model without apparent toxicity. Taken together, chalcone D6 could be a lead candidate for its further development in the treatment of leukaemia.