Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs as potential IκB kinase β inhibitors for the treatment of pancreatic cancer

Drug Design, Development and erapy

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O r i g i n a l r e s e a r c h

Design, synthesis, and biological evaluation of novel

EF24 and EF31 analogs as potential iκB kinase β

inhibitors for the treatment of pancreatic cancer

This article was published in the following Dove Press journal:

Drug Design, Development andTherapy

12 May 2017

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Xuemeng Xie

Jinfu Tu

heyi You

Abstract: Given the important role that inhibitory kappa B (IκB) kinase β (IKKβ) plays in

pancreatic cancer (PC) development and progression, inhibitors targeting IKKβ are believed to

be increasingly popular as novel anti-PC therapies. Two synthetic molecules, named EF24 and

EF31, exhibited favorable potential in terms of inhibition of both IKKβ activity and PC cell

proliferation. Aiming to enhance their cellular efﬁcacy and to analyze their structure–activity

relationship, four series of EF24 and EF31 analogs were designed and synthesized. Through

kinase activity and vitality screening of cancer cells, D6 displayed excellent inhibition of both

IKKβ activity and PC cell proliferation. Additionally, multiple biological evaluations showed

that D6 was directly bound to IKKβ and signiﬁcantly suppressed the activation of the IKKβ/

nuclear factor κB pathway induced by tumor necrosis factor-α, as well as effectively inducing

cancer cell apoptosis. Moreover, molecular docking and molecular dynamics simulation analysis

indicated that the dominant force between D6 and IKKβ comprised hydrophobic interactions.

In conclusion, D6 may be a promising therapeutic agent for PC treatment and it also provides

a structural lead for the design of novel IKKβ inhibitors.

Bingren hu

Department of laparoscopic surgery,

First affiliated hospital of Wenzhou

Medical University,Wenzhou, Zhejiang,

People’s republic of china

Keywords: anti-pancreatic cancer activity, IκB kinase β, molecular docking, molecular dynam-

ics simulation

Abbreviations

ATCC, American Type Culture Collection; CUR, curcumin; DCM/MeOH, dichlo-

romethane/methanol; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDC/NHS,

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide; EDTA,

ethylenediaminetetraacetic acid; EGFR, epidermal growth factor receptor;

ESI-MS, electron-spray ionization mass spectrometry; FBS, fetal bovine serum; FDA,

US Food and Drug Administration; GAPDH, glyceraldehyde 3-phosphate dehydro-

genase; GEM, gemcitabine; G_SA, surface area energy; GSK-3α/β, glycogen synthase

kinase-3α/β; HEPES, [2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid];

HPLC, high-performance liquid chromatography; HTS, high-throughput screening;

IC₅₀, half-maximal inhibitory concentration; IκB, inhibitory kappa B; IKKβ, IκB

kinase β; IR, inhibitory ratio; KD, kinase domain; K_d, equilibrium dissociation con-

stant; LCPO, linear combinations of pairwise overlaps; LGA, Lamarckian genetic

algorithm; MD, molecular dynamics; MM/GBSA, molecular mechanics/generalized

Born surface area; MS, mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide; NF-κB, nuclear factor κB; NMR, nuclear magnetic

resonance; PBST, phosphate buffered saline plus Tween 20; PC, pancreatic cancer;

correspondence: Bingren hu

Department of laparoscopic surgery,

First Afﬁliated Hospital of Wenzhou

Medical University, Wenzhou, Zhejiang

325000, People’s republic of china

Tel/fax +86 577 5557 9422

email wzmuhbr@163.com

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Drug Design, Development andTherapy 2017:11 1439–1451

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http://dx.doi.org/10.2147/DDD T .S133172

Xie et al

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PDAC, pancreatic ductal adenocarcinoma; PDB, Protein ubiquitination, and separation of the IκB/NF-κB complex,

Data Bank; PE/EA, petroleum ether/ethyl acetate; pIKKβ, allowing the release and translocation of NF-κB into the

phosphorylated IKKβ; PKC, protein kinase C; PVDF, poly- nucleus to regulate multiple key genes involved in cell

vinylidene diﬂuoride; RMSD, root-mean-square deviation; oncogenesis, inﬂammation, and apoptosis.^18–21In recent

RU, response units; SASA, solvent accessible surface area; years, numerous studies have documented that constitutive

SDD, scaffold/dimerization domain; SDS-PAGE, sodium activation of NF-κB triggered by deregulation of IKKβ

dodecyl sulfate polyacrylamide gel electrophoresis; siRNA, occurs in almost all PC specimens, as well as in various PC

small interfering RNA; SPR, surface plasmon resonance; cell lines.^14,22,23For example, Ling et al²⁴noted that IKKβ-

STAT-3, signal transducer and activator of transcription-3; mediated NF-κB activation is highly required for develop-

TBST, tris-buffered saline plus Tween 20; TLC, thin layer ment of PDAC, and pancreas-targeted IKKβ inactivation

chromatography; TLR4, toll-like receptor 4; TNF, tumor inhibited both NF-κB activation and PDAC tumorigenesis.

necrosis factor; ULD, ubiquitin-like domain.

Accumulating evidence suggests that aberrant IKKβ

activity intensively contributes to pancreatic tumorigenesis

by triggering the production of many antiapoptotic and

Introduction

PC is currently one of the most devastating malignant proliferation-related molecules that NF-κB regulates, such

neoplasms.^1,2According to statistics, it has been estimated as Bcl-xL, cIAP, cyclin D1, KU70, and KU80.^25,26Moreover,

that the number of new cases of Americans diagnosed with the IKKβ/NF-κB pathway is a versatile and complicated

PC was 53,070 in 2016, and 41,780 people had died of this pathway that is widely associated with other pivotal signal

disease.³The overall 5-year survival rate of PC patients proteins (Ras, MAPKs, GSK-3β, and Notch), which may

is ~7%.³For patients with advanced disease, this ﬁgure activate IKKβ/NF-κB in a bypass manner and promote car-

is worse, as low as 1%.⁴Due to the highly nonspeciﬁc cinogenesis as an accomplice.^22,24,27Wilson and Baldwin²²

incipient symptoms, 80%–85% of patients diagnosed reported that GSK-3α/β upregulated the activity of IKKβ to

with PC already were in the advanced and metastatic promote the survival of PANC-1 and MiaPaCa-2 cells (two

stages and not suited for surgical resection.⁵Thus, che- human PC cell lines). All these reports reveal that exploiting

motherapy becomes the principal remedy for patients with effective IKKβ inhibitors could be an advisable approach for

advanced PC. As the only standard ﬁrst-line therapy for the treatment of PC.

advanced PC patients, GEM, a conventional cytotoxic drug,

Given the important therapeutic potential of IKKβ inhibi-

exerts favorable anticancer action by breaking down the tors for treating PC, numerous selective IKKβ inhibitors have

deoxynucleotide metabolism of PC cells.^6,7However, the been discovered, including BMS-345541, ML-120B, and

increasing occurrence of acquired resistance by PC cells TPCA-1, the IC₅₀values of which were 300, 45, and 18 nM,

and the nonselective cytotoxicity to normal cells led to the respectively (Figure 1).^28–30Nonetheless, no small-molecule

outcomes of GEM monotherapy being extremely poor.^8–10IKKβ inhibitor has been approved for clinical applications so

Currently, various combination therapeutic regimens have far.³¹Part of the reason for these inhibitors possessing excel-

been assessed in clinical or preclinical practice in the hope lent enzymatic inhibitory activity but failing in the preclinical

of overcoming the intricate mechanism of resistance.^11,12trials is their poor metabolism and severe toxicity to normal

Unfortunately, this approach is also more likely to increase cells. CUR, a well-known anticancer compound derived from

the incidence of toxic effects, such as irreversible myelo- turmeric, has become the research hotspot of natural prod-

suppression and gastrointestinal adverse reactions.¹³Thus, ucts and drug candidates in recent years due to its inhibition

development of new targeted therapies for PC is highly of multiple targets highly related to cancer, such as IKKβ,

urgent and crucial. Of the proteins closely related to the STAT-3, and PKC, as well as its nontoxicity (Figure 1).^32,33

formation and progress of PC, IKKβ is considered to be However, the low biostability of CUR dramatically restrains

an important potential target.^14,15

its clinical application. By screening a large number of CUR

The IKK family consists of a series of four enzymes analogs, two more stable and effective analogs of CUR,

(IKKα, IKKβ, IKKγ, and IKKε).¹⁶IKKβ is the predomi- named as EF24 and EF31 (Figure 1), were discovered suc-

nant catalytic subunit mediating the classic IKK/NF-κB cessively, the IC₅₀values of which against IKKβ were 1.9

pathway, and it contains an N-terminal KD, a central ULD, and 1.92 μM, respectively.^34,35In addition, EF24 and EF31

and a C-terminal SDD structurally.¹⁷Following extracellular also exhibited striking inhibition of PC cells.^36,37Although the

stimulations (stress, cytokines, and free radicals), IKKβ KD favorable suppression effect of EF24 and EF31 against IKKβ

becomes phosphorylated, resulting in the phosphorylation, and PC cells attracted much researcher interest, surprisingly,

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Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs

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Figure 1 chemical structures of some reported iKKβ inhibitors.

Abbreviation: iKKβ, inhibitory kappa B kinase β.

there is still no systematic medicinal chemistry research on column (4.6 ×150 mm, 5 μM; Agilent) was used. The spectral

EF24 and EF31 analogs. characterization information (NMR, MS, and HPLC data)

In this research, aiming to screen more potent anti-PC and parts of representative original spectrums are displayed

agents, and to build a valid pharmacophore model of EF24- in the Supplementary materials.

and EF31-based inhibitors, four (A–D) series of analogs

Target compounds were obtained through a one-step

with EF24 and EF31 as the lead compounds were designed Claisen-Schmidt condensation reaction taking 4-piperidone

and synthesized. Through kinase activity screening and SPR monohydrate hydrochloride and various substituted aro-

analysis, D6 was found to be a direct inhibitor of IKKβ. matic aldehydes as starting materials. The experimental

Molecular docking and MD stimulation studies were per- procedure is summarized in brief. The corresponding

formed to estimate the key interactions between D6 and IKKβ benzaldehyde (4 mmol) and 4-piperidone monohydrate

KD. Further, the biological evaluation results indicated that hydrochloride (2 mmol) were dissolved in a mixture of

D6 has the potential to be developed as an anti-PC drug.

absolute ethanol (18 mL) and distilled water (2 mL). Aque-

ous NaOH (40%, 1 mL) was added dropwise with stirring

in an ice water bath (0–4°C). The reaction temperature was

gradually elevated to room temperature. After completion of

the reaction as monitored by TLC (up to 4–24 h), distilled

water (20 mL) was poured into it, resulting in the product

being precipitated totally. After ﬁltration, the residues were

puriﬁed by column chromatography using appropriate ratio

of PE/EA or DCM/MeOH as eluent.

Materials and methods

general procedure for the synthesis of

compounds

All chemical reagents were purchased from Sigma-Aldrich

Co., St Louis, MO, USA, Aladdin, or Alfa Aesar and used

without puriﬁcation. Silica gel (GF254) for TLC and col-

umn chromatography (200–300 mesh) was obtained from

Aladdin. Melting points were determined on an uncorrected

X4 microscopic melting point meter (Shanghai Instrument screening for iKKβ inhibition

Physical Optics Instrument Co, Shanghai, China). ¹H-NMR The kinase activity of IKKβ was determined by mobility shift

spectra were recorded on a Bruker 500 MHz Avance DRX assay on a Caliper Life Sciences Labchip EZ Reader. The

spectrometer (UC Davis NMR Facility, 4303 Tupper Hall, enzyme solution was prepared in a 1.25× kinase base buffer

Davis, CA, USA). The chemical shifts are presented in terms (62.5 mM HEPES, 0.001875% Brij-35, 12.5 mM MgCl₂,

of parts per million with tetramethylsilane as the internal ref- and 2.5 mM DTT). Stop buffer (100 mM HEPES, 0.015%

erence. Data of ESI-MS in positive mode were recorded on a Brij-35, 0.2% coating reagent No 3, and 50 mM EDTA) was

Waters TQ-S micro-mass spectrometer (Waters Corporation, prepared. Then, 5 μL of the test compounds at 100 μM in

Milford, MA, USA). All the synthesized compounds have at 10% DMSO was diluted with 10 μL of 2.5× enzyme solution

least 95% purity as assessed by HPLC, which was performed in a 384-well assay plate and incubated at room temperature

on an Agilent 1200 device (Agilent Technologies Co. Ltd, for 10 min. Moreover, 10 mM EDTA was used as the low

Wang Jing Bei Road, Beijing, China) with a ﬂow rate of control. Then, 10 μL of peptide solution was added into each

1 mL/min and a gradient of 50%–60% (v/v) MeOH in H₂O well. After incubation at 28°C for 1 h, 25 μL of stop buffer

using a diode array detector. A ZORBAX Eclipse XDB-C18 was added into the mixture to stop the reaction. Conversion

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Xie et al

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data were then collected on the EZ reader automatically, and 0.375 Å. The docking parameters were as follows: 200 con-

the percentage inhibition was calculated relative to blank formations were generated, which were clustered according

wells (containing no enzyme and 1%, v/v, DMSO) and total to the RMSD tolerance of 1.5 Å, population size of 300,

wells (containing all reagents and 1%, v/v, DMSO). The maximum number of evaluations 25,000,000, and other set-

conversion formula used is as follows:

tings were set at the default parameters. A reasonable pose

with best-predicted binding afﬁnity of D6 was selected for

detailed analysis and further studies.

Inhibition% = (Max − conversion)/(Max − Min) × 100%

(1)

MD simulation

“Max” stands for the DMSO control, “Min” stands for

low control, and “conversion” means the average of three

experimental values given by the EZ reader.

Preparation of structures

The reasonable pose was used as the initial structure for MD

simulations. Prior to MD simulations, the electrostatic poten-

tials of D6 were computed by the HF/6-31G* level of theory

in Gaussian09 program. Then the atomic partial charges were

obtained by ﬁtting the electrostatic potentials using the RESP

ﬁtting technique. The generation of the partial charges and

the force ﬁeld parameters for D6 was accomplished using the

antechamberprograminAssistedModelBuildingwithEnergy

Reﬁnement (AMBER; USA)-14 simulation package.⁴⁰In the

MM optimizations, ff99SB force ﬁeld and gaff force ﬁeld

were used for IKKβ and D6, respectively. The complex was

solvated in a box of TIP3P water molecules with a 10 Å distance

between the protein surface and the box boundary. Moreover,

the counterions of Na⁺were added to neutralize the systems.

To determine the IC₅₀of the test compounds relative to

kinase activity, ten gradient concentrations of the compounds

(100, 33.330, 11.110, 3.700, 1.230, 0.410, 0.140, 0.046, 0.015,

and 0.005 μM) were set up. The inhibition ratios for differ-

ent concentrations were determined and calculated, and the

concentration−inhibition rate curve was ﬁt using the GraphPad

Prism software (GraphPad, San Diego, CA, USA).

sPr analysis

SPR experiments were performed on a ProteOn XPR36

Protein Interaction Array system (Bio-Rad Laboratories,

Hercules, CA, USA). All solutions used in the experi-

ment were prepared with ultrapure water, ﬁltered with a

0.22-μM membrane ﬁlter before use. IKKβ solution in

PBST (5 mM, pH 7.4) at a concentration of 1 mg/mL was

diluted to 30 μg/mL with sodium acetate buffer (pH 4.5).

The chip was activated with EDC/NHS (10 μL/min for

600 s). Then, IKKβ was loaded (5 μL/min for 400 s) and

immobilized covalently. Approximately 8,000 RU of IKKβ

was immobilized on the chip. Any excess of unbound IKKβ

was removed by ﬂowing PBS solution (5 mM, pH 7.4, with

5%, w/v, DMSO). D6 was prepared as 20–100 μM solution

in PBS solution (5 mM, pH 7.4, with 5%, w/v, DMSO), and

injected (10 μL/min for 100 s). After each loading, data were

collected and analyzed with the ProteOn manager software

(Bio-Rad Laboratories).

Molecular minimizations and conventional MD

simulations

Before the MD productive simulation, we carried out an

equilibration protocol. The geometry of the system was

minimized in two steps before MD simulation. First, the

water molecules were reﬁned through 2,500 steps of steep-

est descent, followed by 2,500 steps of conjugate gradi-

ent, keeping the protein ﬁxed with harmonic restraints of

5.0 kcal mol^-1Å^-2. Second, the complexes were relaxed

by 5,000 cycles of minimization procedure (2,500 cycles

of steepest descent and 2,500 cycles of conjugate gradient

minimization). Then, the whole system was heated from

0 to 300K running 100 ps MD with position restraints at

constant volume. Subsequent isothermal–isobaric ensemble

(NPT)-MD was performed for 100 ps to adjust the solvent

Molecular docking

Molecular docking analysis was carried out by the latest density with a time constant of 1.0 ps for pressure relaxation.

version of AutoDock 4.2.6 package.³⁸AutoDock is a ﬂex- Harmonic restraints with force constants of 1 kcal mol^-1Å^-2

ible docking program, which is based on the fundamental were applied to IKKβ and D6. An additional 100 ps of uncon-

principle of LGA. The coordinates of human IKKβ (PDB ID: strained NPT-MD at 300K with a time constant of 2.0 ps for

4KIK) were downloaded from the PDB.³⁹In the preliminary pressure relaxation was performed to relax the system without

step of the protein preparation, AutoDock4 atomic radii and constraints. Finally, the system was submitted to 60 ns MD

Gasteiger partial charges were assigned to the protein and simulation. During simulation, the pressure and temperature

ligands. The scoring grid dimensions of 60×60×60 Å were were maintained using the Langevin temperature scalings.⁴¹

assigned using the AutoGrid module with grid spacing of SHAKE constraints were applied to all bonds involving

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Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs

hydrogen.⁴²In addition, periodic boundary conditions and were treated with different concentrations (0, 0.080, 0.400,

electrostatic interactions were treated using the particle mesh 2.000, 10.000, and 50.000 μM) of the test compounds. After

Ewald algorithm with cutoff of 10 Å.⁴³Conformational snap- 72 h incubation, 25 μL of MTT (5 mg/mL; Sigma) was added

shots were saved every 10 ps for further analysis.

to each well for 4 h at 37°C in the dark. Next, 150 μL of

DMSO was added to each well to solubilize the MTT meta-

bolic product and the absorbance was read at 490 nm.

MM/gBsa binding free energy calculations

The MM/GBSA method has been widely used for free energy

calculation and to investigate the energy contributions of each

residue to binding.^32,45The basic theory of the MM/GBSA

approach is that the free energy of binding can be obtained

through calculating only the end points of the thermodynamic

Western blot analysis

PANC-1 and BxPC-3 cells (treated with the test compound

after 24 h starvation, and then stimulated with 1 ng/mL TNF-α

for 30 min) were lysed in a lysis buffer for 15 min on ice. The

cell lysates were centrifuged at 4°C to remove insoluble com-

ponents. Protein (80 μg) from each sample was subjected to

SDS-PAGE and transferred onto PVDF membranes (Bio-Rad

Laboratories). After incubation with 5% skim milk in TBST

for 1.5 h, the membranes were incubated with primary anti-

bodies (pIKKβ, IκB, cleaved PARP, bcl-2, and GAPDH;

Santa Cruz) at 4°C overnight and then with the horseradish

peroxidase-labeled secondary antibody at room temperature

for 1 h. The results were visualized using enhanced chemi-

luminescence reagents (Bio-Rad Laboratories), and the band

densities were quantiﬁed using ImageJ analysis software

(National Institutes of Health, Bethesda, MD, USA).

cycle of ligand binding. The binding free energies (∆G_bind

)

were calculated based on the 200 snapshots evenly extracted

from 50 ns to 60 ns according to the following equations:

∆G_bind= ∆G_comp− (∆G_rec+ ∆G_lig)

∆G_bind= ∆E_MM+ ∆G_solv− T∆S

∆E_MM= ∆E_int+ ∆E_vdW+ ∆E_ele

∆G_solv= ∆G_GB+ ∆G_SA

(2)

(3)

(4)

(5)

In Equation (2), the free binding energy ∆G_bindwas cal-

culated as a sum of the three terms in Equation (3). ∆E_MM

and ∆G_solvrepresent the MM interaction energy and solva-

tion energy, respectively. T∆S represents the change of the

conformational entropy upon ligand binding at temperature

T. For Equation (4), ∆E_MMcan be decomposed into three

components: intermolecular interaction energy (∆E_int), van

der Waals energy (∆E_vdW), and electrostatic energy (∆E_ele);

for Equation (5), the value of the solute’s dielectric constant

was used as the default (ε=1) for both the polar solvation

energies (∆G_GB), and we used a value of 80 for the exterior

dielectric constants. The nonpolar part (∆G_SA) was estimated

by the LCPO method: ∆G_SA=0.0072×∆SASA.

hoechst staining

For qualitative measurement of apoptosis, PANC-1 cells

were seeded in six-well plates and incubated with the test

compounds for 12 h. Furthermore, cells were stained with

Hoechst 33342 according to the protocol provided with the

kit (Beyotime Biotech, Nantong, China). Micrographs of all

groups were taken using a ﬂuorescence microscope (Nikon,

Tokyo, Japan) with a 200× objective lens.

statistical analysis

The results are presented as mean values ± standard devia-

tion. Student’s t-test and a two-way analysis of variance

(ANOVA) were used to analyze the differences between

sets of data. Statistics were analyzed using GraphPad Prism

cell culture

Three pancreatic adenocarcinoma cell lines, PANC-1, software (GraphPad). A P-value ,0.05 was considered sta-

MiaPaCa-2, and BxPC-3 (ATCC, Manassas, VA, USA), tistically signiﬁcant. All in vitro experiments were repeated

were cultured in RPMI-1640 medium (Sigma-Aldrich at least three times.

Canada, Mississauga, ON, Canada) supplemented with

Results

10% (v/v) FBS standard (Thermo Scientific, Waltham,

MA, USA) and 10 mg/mL gentamicin (Gibco BRL, VWR, chemistry

Mississauga, ON, Canada).

We observed that EF24 and EF31 shared the same

4-piperidone middle linker, which may have an impor-

tant influence on their inhibitory effects. Hence, we

MTT cell proliferation assay

Three pancreatic cancer cell lines (5,000 cells per well) were designed four series of compounds with the framework of

seeded on a 96-well plate. After incubation overnight, cells diarylidenepiperidin-4-one. The “A” series of compounds

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Xie et al

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Scheme 1 The design of (A–D) series of derivatives of EF24 and EF31.

is obtained by changing the pyridine of EF31 into different (IC₅₀=0.8 μM, Figure 3), while both the other compounds

ﬁve-membered heterocycles (Scheme 1). The strategy for “B” had lower values than EF31 and EF24.

and “C” series of compounds was altering the 2-F phenyl of

According to the screening results, several preliminary

EF24 into other monosubstituted (B) or bis-substituted (C) points of structure–activity relationship can be summarized.

phenyl groups. Considering that the EF24 and EF31 mol- First, altering the pyridine ring into ﬁve-membered hetero-

ecules were much smaller relative to the ATP-binding pocket cycles resulted in the original activity almost diminishing

of IKKβ, we hypothesized that introducing some big steric in the “A” series compounds, suggesting that the pyridinyl,

hindrance groups may effectively utilize the spare residues or at least, the hexatomic ring of EF31 may be a pivotal

in the deep pocket to form more helpful interactions, which moiety for the maintenance of its inhibitory conformation.

generated the “D” series of compounds.

In addition, in the “B” and “C” series compounds, the results

Through a classical Claisen-Schmidt condensation reac- showed that halogen-substituted analogs of EF24 indeed are

tion, the A–D series compounds were synthesized (yield more effective than the methoxyl-substituted ones, indicating

varied from 30% to 80%). The synthesis route is shown in that introducing an electron-withdrawing group may be more

Figure 2, and their molecular structures, which were con- useful to enhance activity. In the last series, it is difﬁcult to

ﬁrmed by MS and proton NMR (¹H-NMR) spectroscopy, conclude the structure–activity relationship, which needs to

are shown in Figure 3. As the purity is important to the be investigated after more compounds are synthesized.

structural character and also to the bioactivity, all the syn-

Direct binding demonstration, molecular

thetic compounds were analyzed by HPLC to ensure that

they were adequately pure for use in biological experiments docking, and MD simulation of D6

(purity $95%).

SPR experiments were performed to evaluate the direct

binding between D6 and IKKβ. As shown in Figure 4, the

SPR response value was observed to increase gradually with

elevated ZCL278 concentrations and a low equilibrium dis-

sociation constant (K_d) of 22.2 μM was obtained. Combined

screening for inhibition of iKKβ kinase

activity and preliminary analysis of the

structure–activity relationship

The four series of analogs of EF24 and EF31 were initially with its excellent kinase inhibition, it could be concluded

screened for inhibition of IKKβ kinase activity at a con- that D6 was very likely to be a direct, high-afﬁnity binding

centration of 20 μM by a mobility shift assay. As shown in inhibitor of IKKβ.

Figure 3, compounds B1, B2, B4, C6, D3, and D6 exhibited

To further clarify the interactions between D6 and

considerable kinase inhibition (IR .70%). Based on our IKKβ KD, molecular docking and MD simulation studies

past experience, these six compounds with IR .70% were of IKKβ/D6 were carried out. Initially, we docked D6 into

expected to be active inhibitors of IKKβ kinase. By deter- the IKKβ KD based on a human IKKβ crystal structure for

mination of IC₅₀, D6 showed the strongest IKKβ inhibition reference (PDB code: 4KIK). The optimum docking results

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Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs

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were chosen as the initial structures for MD simulation. change to accommodate each other and reach the optimal

The detailed RMSD of the receptor in the IKKβ/D6 complex binding mode (induced-ﬁt phenomenon). To further validate

was converged after 20–30 ns in 60 ns MD simulations with our simulated result, the cocrystallization assay of IKKβ/D6

the backbone RMSD of ~1.7 Å for IKKβ and ~0.4–0.5 Å is still under way. In short, a critical hydrophobic interaction

for D6, suggesting that the IKKβ/D6 complex became suf- was clariﬁed as the underlying mechanism of D6 and IKKβ

ﬁciently stable through 60 ns MD simulations (Figure 5A). KD interaction.

To illuminate the roles of the individual residues in

D6 suppresses the growth of Pc

determining the interactions between IKKβ and D6, the

MM/GBSA method was used to analyze the contributions cell lines

of each residue. It can be observed that the most contribut- Numerous studies support the concept that the constitutive

ing residues are Ile-165, Tyr-98, Leu-21, Cys-99, Gly-102, activation of the IKK/NF-κB pathway promotes growth and

Met-65, Met-96, Val-74, Val-152, and Gly-101 (Figure 5B). development in a number of different cancers, including

Except for Tyr-98, the other nine residues are hydrophobic PC.^26,46The antiproliferative effect of the four IKKβ kinase

amino acids. The MD simulation results (Figure 5C) intui- inhibitors (IKKβ IC₅₀,10 μM; B1, B2, B4, and D6) on the

tively exhibit that the backbone of D6 was interposed into a three PC cell lines was determined using the MTT assay.

hydrophobic cleft developed by the above nine hydrophobic The PC cell lines were treated with varying concentrations

amino acids. The left phenyl ring of D6 formed p–p stack- of four inhibitors, their leads EF24 and EF31, or CUR for

ing with the benzene side chain of Tyr-98 to stabilize the 72 h. Among all the compounds tested, D6 exhibited the

binding conformation, and another phenyl ring generated greatest inhibitory activity in the PANC-1 and MiaPaCa-2

hydrophobic contacts with the residues Met-96, Met-65, cell lines, the IC₅₀values being lower than those for EF24

Ile-165, and Val-74. Moreover, the bilateral dimethylamino and EF31 (Table 1). The growth inhibition by D6 in BxPC-3

ethoxy long chain of D6 was oriented toward the outside was slightly weaker than that by EF31, but much better than

solvent areas of the protein, which can be explained by the that by EF24. This result denoted that D6 is a promising

fact that the ligand and receptor underwent a conformational anti-PC agent in vitro.

A

B

C

D

O

Y

1

2

5

3

X

5

R′

1

6

Y

R′′′

2

3

R′′

R′

4

N

H

N

H

N

H

N

H

4

Compound

X

N

O

S

N

O

R′

IR^a(%)

42.08

23.09

22.88

16.01

13.95

Compound

Y

IR^a(%)

77.11

80.92

39.27

85.13

Compound

Y

F

R′′

IR^a(%) Compound

R′′′

IR^a(%)

–9.60

28.85

72.80

17.40

46.59

A1

A2

A3

A4

A5

R = H

B1

B2

B3

B4

B5

3-F

4-F

C1

C2

C3

C4

C5

5-F

65.63

44.19

39.46

38.93

68.13

D1

D2

D3

D4

D5

5-Bromo-1H-indolyl

2-OMe styryl

4-Br styryl

4-OMe

6-OMe

2-CI

2-Br

R = 5-Me

Br 5-OMe

Naphthyl

2-OMe 51.13

Br

6-F

Triphenyl amino

N,N-Dimethyl-2-

A6

S

R = 5-Me

18.29

B6

B7

4-CI

3-Br

30.15

29.85

C6

C7

Br

5-F

74.47

34.42

D6

D7

phenoxyethanamine

93.61

5-CI

N,N-Dibutylphenylamino 44.87

B8

4-Br

48.84

C8

C9

Br

CI

5-Br

6-CI

31.20

15.32

B9

3-OMe 16.49

4-OMe 9.99

B10

Figure 3 inhibition of iKKβ kinase by the (A–D) series of analogs of EF24 and EF31 at a concentration of 20 μM was screened through a caliper mobility shift assay.

Note: ir^arefers to the inhibitory ratio of the measured compounds at a concentration of 20 μM.

Abbreviations: iKKβ, inhibitory kappa B kinase β; ir, inhibitory ratio; Me, methyl; OMe, methoxy.

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Drug Design, Development andTherapy 2017:11

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of IKKβ, which leads to the downstream IκB/NF-κB

complex getting detached and the subsequent degradation

of IκB. IκB is a crucial inhibitory factor deciding whether

the NF-κB target genes are activated or not. Thus, we deter-

mined the inﬂuence of D6 on the phosphorylation of IKKβ

and the expression of intracellular IκB in PANC-1 cells by

Western blot analysis. The results (Figure 6) showed that D6

signiﬁcantly suppressed the TNF-α-induced phosphorylation

of IKKβ and increased the level of IκB in a dose-dependent

manner with a similar effect as for EF31.

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Figure 4 Direct-binding afﬁnity between D6 and iKKβ demonstrated by sPr.

Note: K_a, K_d, equilibrium dissociation constant.

Abbreviations: iKKβ, inhibitory kappa B kinase β; KD, kinase domain; rU, response

units; sPr, surface plasmon resonance.

D6 induces apoptosis of Pc cell lines

NF-κB, as a nuclear transcription factor, regulates the expres-

sion of many antiapoptotic genes. So, we further evaluated

the apoptosis induction effect of D6. The morphological

D6 inhibits the phosphorylation of iKKβ

and the downstream signaling

TNF-α binding to TLR4 (the receptor of the NF-κB pathway changes in the PC cell nuclei were determined by Hoechst

present on the cell membrane) induces the phosphorylation staining, and the expression of apoptosis-related molecules

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Figure 5 Molecular docking and MD simulation analysis of binding of D6 to the activity cavity of iKKβ.

Notes: (A) Backbone rMsDs are shown as a function of time for iKKβ/D6 in 60 ns MD simulation. (B) Per-residue value of the top ten contributions to the binding effective

energy of D6. Per-residue contributions were calculated by the MM-gBsa decomposition method. (C) last snapshot of iKKβ/D6 in 60 ns MD simulation.

Abbreviations: iKKβ, inhibitory kappa B kinase β; MD, molecular dynamics; MM-gBsa, molecular mechanics/generalized Born surface area; rMsD, root-mean-square

deviation.

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Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs

Table 1 six compounds (B1, B2, B4, C6, D3, D6, and CUR)

were selected for determining the ic₅₀values of iKKβ kinase

activity, and the active compounds B1, B2, B4, and D6 (iKKβ

ic₅₀,10 μM) were further measured for the antiproliferative

effects in three Pc cell lines (Panc-1, MiaPaca-2, and BxPc-3)

by the well-established MTT assay

the enhanced IKKβ activity in PC cells led to their excessive

proliferation and protection from cell death.^15,26Conversely,

downregulation of IKKβ/NF-κB using small inhibitors or

siRNA successfully suppressed the growth, angiogenesis,

and metastasis of PC.^15,50Thus, the exploitation of IKKβ

inhibitors as a novel therapeutic strategy against PC has

attracted much attention in the past decades.

Compound

IC₅₀(µM)

IKKβ

PANC-1

MiaPaCa-2

BxPC-3

Several favorable selective IKKβ inhibitors were dis-

covered through HTS of synthetic small molecules or by

recognizing active natural products. Through HTS strate-

gies, BMS-345541, ML-120B, and TPCA-1 (Bristol-Myers

Squibb Company, Millennium Pharmaceuticals and Glaxo-

SmithKline Pharmaceuticals Ltd.) were screened and evalu-

ated in rodent models for their antitumor effect. Nevertheless,

the emergence of chronic toxicity of ML-120B and TPCA-1

in in vivo experiments greatly restricts the possibility of their

further clinical application.^21,51Apart from the inhibitors

identiﬁed by HTS, naturally sourced IKKβ inhibitors, such

as curcumin, apigenin, genistein, and wedelolactone, are

believed to be effective and of low toxicity in the treatment

of PC.^52–54Of particular note is that a phase II clinical trial

of curcumin in patients with advanced PC has been carried

B1

8.9±1.1

5.6±0.5

3.4±1.4

12.4±5.6

.20

12.8±6.9

9.6±2.7

8.2±0.9

nD^a

11.8±1.8

10.6±4.3

6.3±1.3

nD^a

21.5±13.3

12.0±0.9

5.2±1.1

nD^a

B2

B4

C6

D3

nD^a

D6

0.8±0.3

1.92^b

1.9^b

0.7±1.1

1.7±1.8

2.8±3.0

1.3±0.4

5.0±0.5

4.2±1.5

24.8±7.4

1.9±1.3

2.1±0.9

5.9±5.9

17.9±2.3

EF31

EF24

CUR

20.5±1.5

21.5±17.3

Notes: The test results at both enzyme and cell levels are shown. The lead compounds

EF31, EF24, and CUR are used for comparison. 1.92^aand 1.9^awere the ic₅₀values

of EF31 and EF24, respectively, against iKKβ.^31,32all data are presented as the mean

value of three to ﬁve independent tests. ND^ameans not determined; 1.92^band 1.9^b

are the ic₅₀values in the previous reported reference. The bold values represent the

favorable inhibitory activity of two most active derivatives of EF24 and EF31.

Abbreviations: CUR, curcumin; ic₅₀, half-maximal inhibitory concentration;

iKKβ, inhibitory kappa

diphenyltetrazolium bromide; nD, not determined.

B kinase β; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-

(cleaved PARP and anti-apoptotic factor Bcl-2) was detected out (NCT00094445).⁵⁵But unfortunately, curcumin failed in

by Western blot analysis. PANC-1 cells were treated with D6 the clinical test due to poor stability and bioavailability.

(2.5 and 5 μM) or EF31 (5 μM) for 12 h. Hoechst staining

Herein, we started the analysis of two more potent and

showed chromatin condensation (strong blue ﬂuorescence) stable curcumin analogs, EF24 and EF31, and designed

and nucleus fragmentation (Figure 7A). For Western blot a series of derivatives of EF24 and EF31. The two major

analysis, PANC-1 and BxPC-3 cells were incubated with innovative points that can be concluded are as follows: 1) this

D6 (2.5 and 5 μM) or EF31 (5 μM). Results (Figure 7B–F) study presents a systematic medicinal chemistry modiﬁcation

showed that the expression of cleaved PARP was increased work based on the structural character of EF24 and EF31 for

and was accompanied by decreased expression of Bcl-2, both the ﬁrst time; 2) by kinase screening and SPR assay, a novel

in a dose-dependent manner. These results indicated that D6 IKKβ inhibitor, numbered as D6, displaying commendable

is an effective inducer of PC cell apoptosis.

efﬁciency both at the enzyme and cellular levels has been

identiﬁed.

Discussion

Our results (Table 1) demonstrated that D6 is a direct

PC is a strong aggressive malignancy with poor prognosis.⁴⁷powerful IKKβ inhibitor with a desirable small IC₅₀value

Despite massive efforts being taken to improve the clinical (0.8 μM) that is less than half the values of EF24 (1.90 μM)

efﬁcacy of chemotherapy, the outcomes of current remedies and EF31 (1.92 μM) and 25-fold less than that of curcumin

remain far from inadequate.⁴⁸GEM was approved by the FDA (20.5 μM). However, except for D6, the other synthetic com-

as the ﬁrst-line chemotherapy drug for patients with locally pounds showed relatively lower activity, as well as cellular

advanced or metastatic PC. However, the acquired resistance inhibition, when compared with EF24 and EF31. To clarify

to GEM chemotherapy treatment immensely restricted the the underlying reason for the difference, we performed

clinical application of GEM.⁴⁹Accompanied with the rapid molecular docking and MD simulation studies on the D6/

development of targeted therapy, an increasing number of IKKβ complex. The corresponding results (Figure 5) revealed

molecular targets that are closely related to the tumorigenesis that the bilateral 4-(dimethylamino)ethoxy substituents of D6

and progress of PC were discovered.⁴⁸Among these, IKKβ is not only formed crucial interactions with the adjacent hydro-

considered to be a promising target.²⁶A number of investiga- phobic amino acid residues, but also prolonged its N,N-dim-

tions reported that constitutive NF-κB activity resulting from ethylamino group into the outside solvent region. Comparing

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Drug Design, Development andTherapy 2017:11

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Xie et al

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Figure 6 compound D6 suppressed TnF-α induced phosphorylation of iKKβ and degradation of the downstream signal molecule iκB in a dose-dependent manner.

Notes: (A) Panc-1 cells were incubated with the compounds D6 (1, 2.5, and 5 μM), EF31 (5 μM), or DMsO. after stimulation with TnF-α (1 ng/ml) for 30 min, the

protein levels of piKKβ and iκB were detected by Western blotting with the corresponding total iKKβ protein or gaPDh as loading control. (B and C) The columns show

the normalized optical density as a percentage of the control. Bars represent the mean ± seM of three to four independent experiments. **P,0.01; ***P,0.001; “ns” means

no signiﬁcance.

Abbreviations: DMsO, dimethyl sulfoxide; gaPDh, glyceraldehyde 3-phosphate dehydrogenase; iKKβ, inhibitory kappa B kinase β; piKKβ, phosphorylated iKKβ; seM,

standard error of the mean; TnF, tumor necrosis factor.

the bilateral substituents in the “C” series compounds with in the 6- and/or 7-position extending toward the solvent, to

the 4-(dimethylamino)ethoxy group in D6, it is not difﬁcult increase the overall solubility of the molecule and stabilize

to ﬁnd that the chance of developing reliable hydrophobic the inhibitory conformation.^56,57

interaction between the bilateral substituents, as well as the

In this study, our ﬁndings identiﬁed that D6 effectively

interaction with the solvent region, was dramatically reduced. suppresses the activation of the IKKβ/NF-κB pathway and

Therefore, based on the excellent activity of D6, which is far the proliferation of PC cells (Table 1 and Figure 6). On the

superior to that of the others, we speculated that introducing a other hand, D6 could induce the apoptosis of PC cells by

long-chain hydrophobic moiety in the bilateral aromatic rings intensifying the expression of proapoptotic molecules and

may effectively contribute to elevating the inhibitory activity downregulating the relevant antiapoptotic proteins in a dose-

of EF24- and EF31-based IKKβ inhibitors. In addition, if dependent manner (Figure 7). In continuing research, other

there is a hydrophilic group at the end of a long chain, it may impacts of D6 on PC progress need to be evaluated, such as

lead to an unexpected increase in activity. It can be explained invasion, migration, and cell cycle, as well as the antitumor

by the fact that almost all the FDA-approved kinase inhibi- growth effect in vivo. However, all the existing evidence

tors possessed a hydrophilic motif oriented outward toward suggests that D6 should be considered to be an efﬁcient

the solvent region.⁵⁶For example, geﬁtinib, erlotinib, and IKKβ inhibitor for potential anti-PC therapy and a useful

afatinib, the representative medicines of the EGFR inhibitors lead compound for further pharmaceutical development of

being used in current clinical practice, all possess a long chain novel IKKβ inhibitors.

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Drug Design, Development andTherapy 2017:11

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Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs

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Figure 7 compound D6 induced Pc cell apoptosis.

Notes: (A) hoechst staining was performed in Panc-1 cells cultured with (a) DMsO, (b and c) D6 (2.5 and 5 μM) or (d) EF31 (5 μM) for 12 h (200×). (B) effects of D6

on the activation of ParP and inhibition of Bcl-2 in Panc-1 and BxPc-3 cells. (C–F) The quantitative analysis of cleaved ParP and Bcl-2 band was performed using imageJ

image processing program. Data are presented as the mean ± sD of three independent experiments performed in triplicate: *P,0.05; **P,0.01; ***P,0.001; “ns” means

no signiﬁcance.

Abbreviations: DMsO, dimethyl sulfoxide; gaPDh, glyceraldehyde 3-phosphate dehydrogenase; sD, standard deviation.

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Drug Design, Development andTherapy 2017:11

1449

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Xie et al

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15. Radhakrishnan P, Bryant VC, Blowers EC, et al. Targeting the NF-kappa

B and mTOR pathways with a quinoxaline urea analog that inhibits

IKK beta for pancreas cancer therapy. Clin Cancer Res. 2013;19(8):

2025–2035.

16. Gamble C, McIntosh K, Scott R, et al. Inhibitory kappa B kinases as

targets for pharmacological regulation. Br J Pharmacol. 2012;165(4):

802–819.

Conclusion

With EF24 and EF31 as lead compounds, A–D series of deriva-

tives were designed and synthesized. Among them, D6 was

screened by kinase inhibition assay and identiﬁed as a direct-

binding inhibitor of IKKβ KD by SPR. Molecular docking and

MD simulation analyses demonstrated that the binding mode

of IKKβ/D6 was most likely to be hydrophobic interactions.

Moreover, biological assays of anti-PC efﬁcacy of D6 indicated

effective inhibition of proliferation, as well as the activation of

IKKβ/NF-κB pathway, and induction of apoptosis of PC cells.

Based on these ﬁndings, we conclude that D6 is a potential

agent for treatment of PC. This work provides a reference for

the rational discovery of novel IKKβ inhibitors.

17. Xu G, Lo YC, Li Q, et al. Crystal structure of inhibitor of kappaB kinase

beta. Nature. 2011;472(7343):325–330.

18. Wu HW, Wang GH, Wang ZW, et al. A negative feedback loop between

miR-200b and the nuclear factor-kappa B pathway via IKBKB/IKK-beta

in breast cancer cells. FEBS J. 2016;283(12):2259–2271.

19. Sakamoto K, Hikiba Y, Nakagawa H, et al. Inhibitor of kappaB kinase

beta regulates gastric carcinogenesis via interleukin-1alpha expression.

Gastroenterology. 2010;139(1):226–238.

20. Koppe C, Gautheron J, Vucur M, et al. A new molecular interaction

between IKK alpha/beta and RIPK1 regulates biliary homeostasis and

hepatocarcinogenesis. Hepatology. 2016;63(1 suppl):257a–258a.

21. Greten FR, Arkan MC, Bollrath J, et al. NF-kappa B is a negative regu-

lator of IL-1 beta secretion as revealed by genetic and pharmacological

inhibition of IKK beta. Cell. 2007;130(5):918–931.

22. Wilson W, Baldwin AS. Maintenance of constitutive I kappa B kinase

activity by glycogen synthase kinase-3 alpha/beta in pancreatic cancer.

Cancer Res. 2008;68(19):8156–8163.

23. Weichert W, Boehm M, Gekeler V, et al. High expression of RelA/

p65 is associated with activation of nuclear factor-kappa B-dependent

signaling in pancreatic cancer and marks a patient population with poor

prognosis. Br J Cancer. 2007;97(4):523–530.

Disclosure

The authors report no conﬂicts of interest in this work.

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Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs as potential IκB kinase β inhibitors for the treatment of pancreatic cancer

DOI: 10.2147/DDDT.S133172

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