Induction of apoptosis in Ehrlich ascites tumour cells via p53 activation by a novel small-molecule MDM2 inhibitor

Research Paper

Induction of apoptosis in Ehrlich ascites tumour cells via

p53 activation by a novel small-molecule MDM2

inhibitor – LQFM030

Mariana F. da Mota^a,b, Alane P. Cortez^a, Polyana L. Benﬁca^a, Bruna dos S. Rodrigues^a, Thalyta F.

a

c

d

Castro , Larissa M. Macedo , Carlos H. Castro , Luciano M. Liao , Flavio S. de Carvalho^d,f, Luiz A. S.

~

ꢀ

Romeiro^e, Ricardo Menegatti^f, Hugo Verli^g, Bianca Villavicencio^gand Marize C. Valadares^a*

a

ꢀ

^

Laboratorio de Farmacologia e Toxicologia Celular, FarmaTec, Faculdade de Farmacia, Universidade Federal de Goias, UFG, Goiania, GO, Brazil

b

c

ꢀ

^

Lab. de Biologia e DNA Forense da Polıcia Tecnico-Cientıﬁca de Goias, GO - Brazil, Universidade Federal de Goias, UFG, Goiania, GO, Brazil,

Departamento de Fisiologia e Farmacologia, Instituto de Ciencias Biologicas, Universidade Federal de Goias, UFG, Goiania, GO, Brazil, ^dInstituto

^

ꢀ

^

e

ꢀ

^

ꢀ

de Quımica, Universidade Federal de Goias, UFG, Goiania, GO, Brazil, Faculdade de Ciencias da Saude, Universidade de Brasılia, UNB, Brasılia, DF,

f

ꢀ

^

ꢀ

^

Brazil, Laboratorio de Quımica Farmaceutica Medicinal (LQFM), Faculdade de Farmacia, Universidade Federal de Goias, UFG, Goiania, GO, Brazil

and ^gCentro de Biotecnologia, Universidade Federal de Rio Grande do Sul, UFRS, Porto Alegre, RS, Brazil

Keywords

Abstract

Ehrlich ascites tumor; LQFM030; MDM2;

Nutlins; p53

Objective The activation of the p53 pathway through the inhibition of MDM2

has been proposed as a novel therapeutic strategy against tumours. A series of cis-

imidazoline analogues, termed nutlins, were reported to displace the recombinant

p53 protein from its complex with MDM2 by binding to MDM2 in the p53

pocket, and exhibited an antitumour activity both in vitro and in vivo. Thus, the

purpose of this study was to evaluate the antitumour properties of LQFM030 (2),

a nutlin analogue created by employing the strategy of molecular simpliﬁcation.

Methods LQFM030 (2) cytotoxicity was evaluated in Ehrlich ascites tumour

(EAT) cells, p53 wild type, by the trypan blue exclusion test, and the mechanisms

involved in EAT cell death were investigated by light and ﬂuorescence micro-

scopy, ﬂow cytometry, real-time PCR and Western blotting.

Correspondence

ꢀ

Marize C. Valadares, Laboratorio de

Farmacologia e Toxicologia Celular,

ꢀ

Faculdade de Farmacia, Universidade Federal

ꢀ

de Goias, Praßca Universitaria 1166, 74605

^

220 Goiania, GO, Brazil.

E-mail: marizecv@ufg.br

Received October 1, 2015

Accepted April 30, 2016

Key ﬁndings Our results demonstrate that LQFM030 has dose-dependent

antiproliferative activity and cytotoxic activity on EAT cells, induces the accumu-

lation of p53 protein and promotes cell cycle arrest and apoptosis. p53 gene tran-

scription was unaffected by LQFM030 (2); however, MDM2 mRNA increased

and MDM2 protein decreased.

doi: 10.1111/jphp.12573

Conclusions These results suggest that the small-molecule p53 activator

LQFM030 (2) has the potential for further development as a novel cancer

therapeutic agent.

potent tumour suppressor role, the p53 pathway is usually

altered in tumour cells. Indeed, inactivating mutations in

Introduction

The activation of the p53 pathway via the inhibition of

MDM2 has been proposed as a novel therapeutic strategy. In

fact, studies have shown that the disruption of the p53–

MDM2 interaction by different macromolecular approaches

or by the suppression of MDM2 expression can lead to the

activation of p53 and tumour growth inhibition.^[1–3]

p53 gene are found in approximately 50% of all human

cancers; in the remaining cancers in which the p53 is not

mutated, the function of the p53 pathway is often inhibited

via other mechanisms, including the increased expression

of MDM2 (a negative regulator of p53). The fact that the

p53 signalling pathway is inactivated in virtually all cancers

has drawn great attention from the worldwide cancer

research community for targeting the p53 pathway for the

development of improved cancer therapies.^[2]Thus,

p53 is an important defence against cancer because it

controls the transition of cells from the G1 to S phase and

also induces cell cycle arrest and apoptosis.^[4,5]Due to its

1

LQFM030 a novel MDM2 inhibitor

Mariana F. da Mota et al.

drugging p53 pathway through the disruption of the

p53–MDM2 interaction using non-peptide small-molecule

inhibitors has recently appeared as an effective therapeutic

strategy for different tumours. Actually, several MDM2

inhibitors are described in the literature presenting differ-

ent structures, dihydroimidazothiazoles, oxopiperidine,

imidazoles and oxomorpholins, and many of them reached

already clinical trials.^[3]

was obtained from Life Technologies (Carlsbad, CA, USA).

The RNeasy mini kit, QuantiTect Reverse Transcription kit

and Qiagen Rotor-Gene SYBR Green PCR kit were

acquired from Qiagen (Valencia, CA, USA). NP-40 lysis

buffer and protease inhibitor cocktail were from Amresco

(Solon, OH, USA). All antibodies used were purchased

from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).

Nutlins (a series of cis-imidazoline analogues) are cur-

rently considered among the most promising activators of

p53 to induce p53-dependent apoptosis in several tumour

cells.^[6–8]Nutlins bind tightly into the p53 pocket of

MDM2, activate p53 pathway and trigger apoptosis in

wild-type p53 tumour cells.^[6,9]

LQFM030 (2) synthesis

Synthesis of 1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)

methyl) piperazin-1-yl) ethanone (2)

To a 500-ml round-bottomed ﬂask, 9.67 mmol of 1-(4-

chlorophenyl)-1H-pyrazole-4-carbaldehyde (5), 10.64

mmol of acethylpiperazine (6), 100 mg of Pd/C 10%,

70 ml of methanol and 60 psi of H₂were added. The result-

ing solution was ﬁltered, and compound (2) was obtained

as a beige solid; the quantitative yield was mp 92°C,

Rf = 0.56 (CH₂Cl₂:MeOH – 95:5). IR cm^ꢀ1: 3010, 2947,

1639 and 830. ¹HNMR: (500 MHz, CDCl₃)d 7.83 (d,

J = 0.6 Hz, H-5), 7.64 (d, J = 0.6 Hz, H-3), 7.62 (dd,

J = 9.8, 2.2 Hz, H-2⁰), 7.62 (dd, J = 9.0, 2.2 Hz, H-6⁰),

7.41 (dd, J = 9.0, 3.0 Hz, H-3⁰), 7.41 (dd, J = 9.0, 2.9 Hz,

H-5⁰), 3.63 (dd, J = 6.0, 4.0 Hz, H-11), 3.50 (s, H-6), 3.47

(dd, J = 6.0, 4.0 Hz, H-9), 2.47 (dd, J = 6.0, 4.0 Hz,

H-12), 2.45 (dd, J = 6.0, 4.0 Hz, H-8) ppm and 2.08 (s,

H-14). ¹³CNMR (125 MHz, CDCl₃) d 168.8 (C-13), 141.9

(C-3), 138.6 (C-1⁰), 131.9 (C-4⁰), 129.6 (C-3⁰/C-5⁰), 126.4

(C-5), 119.9 (C-2⁰/C-6⁰), 119.6 (C-4), 52.7 (C-12), 52.3 (C-8),

52.2 (C-6), 46.2 (C-9), 41.5 (C-11) and 21.3 (C-14) ppm.

LC-MS with electrospray ionization: m/z 319.16 [M + H]⁺.

A colourless needle-shaped single crystal of (I) with

dimensions 0.075 9 0.16 9 0.47 mm was mounted on a

Bruker Apex II Duo diffractometer, operating with Mo-Ka

radiation and at room temperature. Data collection was per-

formed using φ/x scans of 0.5° steps and exposure times of

20 s. Data reduction was carried out using SAINT and

SADABS,^[13]employing the multiscan absorption correction

method. The structure solution was accomplished with the

direct methods followed by the reﬁnement of structural

parameters using F²with the full-matrix least-squares

method; both methods are implemented in the Shelx soft-

ware package.^[14]Non-hydrogen atoms were reﬁned with

anisotropic atomic displacement parameters, and the hydro-

gen atoms were treated as isotropic and were allo-

In view of the potential antitumour property of nutlins

and its complex, analogues of this compound have been

designed to optimize the complex production and maintain

the antitumour activity and apoptosis-inducing activity.

One such analogue is LQFM030 (2), a heterocyclic com-

pound originally based on the prototype nutlin-1 using

molecular simpliﬁcation (Figure 1a). This compound was

designed and synthesized by our group, and previous stud-

ies demonstrated that LQFM030 (2) has a signiﬁcant cyto-

toxicity and apoptosis-inducing potential against a human

leukaemic cell line. Changes in cell cycle progression,

increasing Bax and decreasing Bcl-2 expression, cyto-

chrome c release and loss of the mitochondrial membrane

potential were observed with this compound. Furthermore,

the compound increased the survival of tumour-bearing

mice and showed low toxicity (unpublished data).

Considering that Ehrlich ascites tumour (EAT) is origi-

nally a carcinoma with wild-type p53 cell,^[10–12]the aim of

the current study was to examine the antitumoral activity

and the cell death mechanisms triggered by the novel syn-

thetic MDM2 inhibitor LQFM030 (2) on EAT cells through

the disruption of the p53–MDM2 complex.

Materials and Methods

Chemicals and antibodies

RPMI-1640 medium, fetal bovine serum, streptomycin,

penicillin G, propidium iodide and RNAse were purchased

from Sigma Aldrich (St. Louis, MO, USA). Ethanol, trypan

blue and Tween 20 were obtained from Vetec (Rio de

Janeiro, RJ, Brazil). Hoechst 33342 and the BCIP/NBT sub-

strate kit were acquired from Invitrogen/Life Technologies

(Carlsbad, CA, USA). Cytoﬁx/Cytoperm was obtained from

BD Biosciences (San Jose, CA, USA). The Annexin V apop-

tosis detection FITC kit was obtained from eBioscience

(San Diego, CA, USA). The CaspaTag Caspase In Situ Assay

Fluorescein kit (caspases-3/7, -8 and -9) was purchased

from Millipore Corporation (Billerica, MA, USA). TRIzol

wed to depend on their parent atoms with U_eq

=

1.2 U_eq(C_aromatic, CH₂) and 1.5 U_eq(C_methyl, O_water). Hydro-

gen atoms were located in calculated positions, and the

hydrogen atoms of the structural water molecule were

located in the Fourier difference map and restrained with O–

ꢁ

H distance of 0.92 A. Infrared (IR) spectra were obtained

with a Nicolet-55a Magna spectrophotometer using potas-

sium bromide plates. The assays were carried out using LC-

2

Mariana F. da Mota et al.

LQFM030 a novel MDM2 inhibitor

Figure 1 (a) Design of LQFM030 (2) from nutlin (1). (b) Ortep representation of LQFM030 (2) with atomic displacement parameters drawn at

30% of probability level. Hydrogens are represented by spheres of arbitrary radii. (c) Synthetic route of LQFM030 (2).

ESI-MS/MS mass spectrometer equipped with electrospray

ionization source in the positive mode, ESI (Agilent Tech-

nologies 1200 Series/Applied Biosystems MDS Sciex API

3200 Triple Quadrupole, MS/MS, Santa Clara, CA, USA).

The progress of all the reactions was monitored by thin-

layer chromatography (TLC), which was performed on 2.0-

to 6.0-cm aluminium sheets precoated with silica gel 7 60

(Merck, Kenilworth, NJ, USA) to a thickness of 0.25 mm.

The developed chromatograms were viewed under ultravio-

let light (254–265 nm) and treated with iodine vapour. For

column chromatography, we used Merck silica gel (70–

230 mesh). Reagents and solvents were purchased from

commercial suppliers.

obtained free of charge via http://www.ccdc.-

cam.ac.uk/conts/retrieving.html or from the Cambridge

Crystallographic Data Centre, 12 Union Road, Cambridge

CB21EZ, UK; fax: (+44) 1223-336-033; or e-mail: de-

posit@ccdc.cam.ac.uk.

The compound LQFM030 (2) was stored in a freezer

at ꢀ20°C and prepared in the form of an oil/water

emulsion at the time of testing, due to it hydrophobic-

ity. The emulsion was prepared as follows. A sample of

5 mg/ml of LQFM030 (2) was added to 100 ll sun-

ﬂower oil, 100 lg soya phosphatidylcholine and 50 ll

ethanol. The mixture was vortexed for 3 min, and the

ﬁnal volume was completed with ultrapure water. For

the control group, the emulsion was prepared without

LQFM030 (2).

CCDC number 963584 contains the supplementary crys-

tallographic data for compound (2). These data can be

3

LQFM030 a novel MDM2 inhibitor

Mariana F. da Mota et al.

incubated with or without eight concentrations (0.033–4.320

m^M) of LQFM030 in sextuplicate for 24 or 48 h. The cell

suspension and a trypan blue solution (0.2% in phosphate-

buffered saline (PBS)) were mixed 1 : 10, and the viability of

the cells was estimated using a haemocytometer (Boeco,

Hamburg, Germany); the stained cells were scored as dead.

Molecular docking

Based on the crystallographic structure of MDM2 com-

plexed with nutlin-3a (PDB ID 4HG7),^[15]we built

LQFM030 in Avogadro^[16]and calculated partial charges

with CHELPG^[17]using GAMESS.^[18,19]The receptor

MDM2 and the ligands nutlin-3a and LQFM030 were pre-

pared for docking using AutoDockTools4^[20,21]and then

submitted for docking using DockThor^[22,23]and Auto-

dock.^[24]The best resulting pose of each docking program

was compared with nutlin-3a and P53 complexed with

MDM2 using PyMol (The PyMOL Molecular Graphics

System, version 1.7.4, Schrodinger Inc., New York, NY,

USA), and contacts were detected with LigPlot+.^[25]

Morphological analysis by light and

ﬂuorescence microscopy

EAT cells were treated with LQFM030 (2) with 810 lM and

incubated for 24 h, cytospinned onto glass slides and

stained with HE. After staining, the slides were examined

using light microscopy (Axio Scope.A1 Carl Zeiss^â, Ober-

kochen, Germany), and the images were captured using a

1009 objective with the AxioVs40 software, 4.7.2.0, Carl

Zeiss, Oberkochen, Germany. The nuclear alterations were

assessed using the DNA-binding dye Hoechst 33342, as

described,^[26]using a 209 objective.

Mice

Male Swiss albino mice (25–30 g) were obtained from

ꢀ

Industria Quımica do Estado de Goias , Goiania, GO,

Brazil. All of the animals were kept under constant environ-

mental conditions, with a 12-h/12-h light–dark cycle and

temperature of 23 ꢁ 2°C, fed standard granulated chow

and given drinking water ad libitum. The animal experi-

ments were performed in accordance with the Institutional

Protocols of Animal Care. The experimental protocol

(CEUA – PRPPG/UFG no. 018/12) was approved by Insti-

tutional Ethic Committee of this university.

DNA cell cycle analysis

For the determination of the cell cycle-phase distribution

and cell proliferation, 5 9 10⁵EAT cells/ml were incubated

with or without 810 l^MLQFM030 (2) for 24 h. The cells

were washed twice with PBS at 1500 rpm for 10 min, and

the cell pellets were resuspended in ice-cold 70% ethanol

overnight at 4°C. The suspension was centrifuged, and the

cell pellet was resuspended in 500 ll PBS solution contain-

ing 0.2 mg/ml RNAse and 0.05 mg/ml PI and incubated at

4°C for 2 h. Fluorescence was measured with the FACS-

Canto II ﬂow cytometer (BD Biosciences) using PE x FL2

channels. The assay was performed in triplicate, and 10 000

events were acquired. The analysis of the data was performed

using ModFit software, BD Bioscience, San Jose, CA, USA.

Cell culture, growth conditions and

treatment

EAT was maintained in Swiss albino mice in ascites form

by successive transplantations. Ascitic tumour cell counts

were made in a Neubauer haemocytometer using the try-

pan blue dye exclusion method. Cell viability was always

found to be 95% or more. Tumour cell suspensions were

prepared in phosphate-buffered saline solution (PBS) at

pH 7.4 to ﬁnal concentrations of 6 9 10⁷viable cells/ml.

Ten days after the inoculation of EAT cells in the abdomi-

nal cavity of mice, the cells were isolated by needle aspira-

tion, washed in saline and cultured in RPMI 1640

supplemented with HEPES (25 m^M), L-glutamine (2 mM),

sodium bicarbonate (25 m^M), 10% FBS, 2-mercaptoethanol

(50 lM) and antibiotics (100 U/ml penicillin and 100 lg/

ml streptomycin). The cells were incubated in culture ﬂasks

at 37°C in a humidity atmosphere containing 5% of CO₂.

Detection of apoptosis by an Annexin V

binding assay

PS externalization was analysed by ﬂow cytometry using

the Annexin V apoptosis detection FITC kit. Cells

(1 9 10⁶cells/ml) untreated and treated with 810 lM

LQFM030 (2) for 24 h were washed with PBS and resus-

pended in 0.1 ml of the binding buffer. The cells were

stained with an anti-Annexin V-FITC antibody and propid-

ium iodide (PI), following the manufacturer’s instructions,

and scanned in the FL-1 (FITC) vs. FL-2 (PI) channels.

Assessment of cell viability by the trypan

blue exclusion method

Flow cytometric determination of cell

membrane integrity

EAT cells (5 9 10⁵cells/ml) were seeded into 96-well ﬂat-

bottomed microtitre plates (TPP, Trasadingen, Switzerland)

in RPMI-1640 medium supplemented with 10% FBS and

For the determination of cell membrane integrity, EAT cells

(1 9 10⁶cells/ml) untreated and treated with 810 lM

4

Mariana F. da Mota et al.

LQFM030 a novel MDM2 inhibitor

LQFM030 (2) for 24 h were washed with PBS and incu-

bated for 5 min in a solution of PI (2 lg/ml) in PBS. A

total of 10 000 events were acquired for the analysis using

the FacsDiva software, BD Bioscience, San Jose, CA, USA

and a histogram plot of the PE ﬂuorescence (x-axis) versus

counts (y-axis) was shown as the logarithmic ﬂuorescence

intensity.

cDNA diluted 1/10 and 200 n^Mof primers using Rotor-

Gene SYBR Green PCR kit (Qiagen). All experiments were

run with controls NTC and -RT. PCR speciﬁcity was deter-

mined using both a melting curve analysis and gel elec-

trophoresis.

The sequences of primers were described for p53 and

p21,^[27]caspase-3, -8, -9^[28]and PGK1 (phosphoglycerate

kinase 1).^[29]MDM2 primers were constructed using Pri-

mer3 and BLAST (NCBI): forward 5⁰-TCCCCTTATCGTC

TGGAAGC-3⁰; reverse: 5⁰-GAGATTTCCTTAGCTGACTA

TTGG-3⁰. PCR conditions were as follows: 95°C for 5 min,

followed by 40 cycles of 5 s for 95°C and 10 s at 60°C using

Rotor-Gene (Qiagen) equipment. The endogenous gene

used for normalization was PGK1. The relative expression

Flow cytometric analysis of DNA

fragmentation

The amount of fragmented DNA was measured by ﬂow

cytometry. EAT cells (1 9 10⁶cells/ml) untreated and trea-

ted with 810 l^MLQFM030 (2) for 24 h were washed with

PBS and incubated with 0.2 ml lysis buffer (0.1% sodium

citrate, 0.1% Triton X-100 and 2 lg/ml PI) for 15 min at

4°C. A total of 10 000 events were acquired for the analysis

using the FacsDiva software, and a histogram plot of the PE

ﬂuorescence (x-axis) versus counts (y-axis) was shown as

the logarithmic ﬂuorescence intensity.

was calculated using the 2^ꢀDDCmethod.^[30]The results

t

were obtained from two independent experiments; each

was carried out in duplicate and repeated three times.

Western blot analysis of MDM2

For the Western blot analysis of the MDM2 protein, EAT

cells (5 9 10⁵cells/ml) were treated or untreated with

810 l^MLQFM030 (2), and the cell lysates were prepared

with NP-40 lysis buffer (containing a protease inhibitor

cocktail) for 30 min on ice. The cell debris was pelleted by

centrifugation at 2200 g and 4°C for 10 min, and the

supernatant was recovered. Approximately 40 lg of pro-

tein, as estimated by the Bradford method, was separated

on a 10% SDS-polyacrylamide gel and electrotransferred

to nitrocellulose membranes (Millipore). After blocking

with 5% non-fat skim milk powder in BSA, the mem-

branes were incubated with an antibody against MDM2

(1 : 250). The blots were washed three times with TBST

and incubated with the secondary antibody tagged with

alkaline phosphatase (1 : 1000) for 1.5 h. After washing

with TBST for 30 min, the MDM2 protein was detected

using the chromogenic substrate BCIP/NBT. Equal protein

loading in each lane was conﬁrmed by probing with an

anti-GAPDH antibody (1 : 1000). Quantiﬁcation of the

band density was performed using ImageJ software and

calculated as the ratio of GAPDH expressed in the same

sample.

Flow cytometric analysis of the protein

expression of p53, p21 and p27

For the determination of the expression of the pro-apopto-

tic proteins p53, p21 and p27, EAT cells (1 9 10⁶cells/ml)

untreated and treated with 810 l^MLQFM030 (2) for 24 h

were washed with PBS, suspended in 0.25 ml of a ﬁxation

and permeabilization solution (Cytoﬁx/Cytoperm) and

incubated for 20 min at 4°C. The cells were then washed

twice in PBS-T20 and incubated with speciﬁc antibodies

(anti-p53, anti-p21 or anti-p27) for 15 min at room tem-

perature. The cells were washed in PBS-T20. A total of

10 000 events were acquired for the analysis using the Facs-

Diva software, and a histogram plot of the FITC or PE ﬂuo-

rescence (x-axis) versus counts (y-axis) was shown as the

logarithmic ﬂuorescence intensity.

Quantitative RT-PCR

Transcriptional proﬁle of p53, MDM2, p21, caspases-3, -8

and -9 was analysed after the treatment of EAT cells with

405 l^MLQFM030 (2). Total RNA from EAT cells was

obtained using the TRIzol reagent or the RNAeasy mini kit,

following the manufacturers’ instructions. Concentration

and the purity of the isolated RNA were determined by

measuring the optical density at 260 and 280 nm (Nano-

Drop 8000 – Thermo Scientiﬁc, Waltham, MA, USA). The

RNA integrity was veriﬁed by electrophoresis through 1.5%

agarose gels stained with ethidium bromide. QuantiTect

Reverse Transcription Kit (Qiagen) was used for cDNA

synthesis with 1 lg of RNA, following the manufacturer’s

instructions. Real-time PCR was performed with 2 ll of

Flow cytometric analysis of caspases-3/7, -8

and -9 expression

The activity of caspases-3/7, -8 and -9 was measured using

the CaspaTag Caspase In Situ Assay Fluorescein kit follow-

ing the manufacturer’s instructions. A total of 10 000

events were acquired for the analysis using the FacsDiva

software, and a histogram plot of the FITC ﬂuorescence

(x-axis) versus counts (y-axis) was shown as the logarith-

mic ﬂuorescence intensity.

5

LQFM030 a novel MDM2 inhibitor

Mariana F. da Mota et al.

with (I). As suggested by the short donor acceptor (DA)

ꢁ

Statistical analysis

distance of 2.917 (2) A and the linearity of the O2–H. . .N4

The data were represented as the mean ꢁ SD, and the val-

ues were obtained in at least two independent experiments

repeated tree times each (n). The statistical analyses were

performed using Student’s t-test with the GraphPad Prism

program (GraphPad Software version 5.0; San Diego, CA,

USA). Differences were considered signiﬁcant when P-

values were <0.05.

H-bond with an angle of 169 (3)°, this water molecule con-

nects neighbour molecules (symmetry operation i: x ꢀ 1/2,

ꢀy + 1/2, ꢀz + 1) with a H-bond of type O2–H. . .O1ⁱ, a

ꢁ

DA distance of 2.816 (2) A and a OHO angle of 171 (2)°

(Table 1). The structure of LQFM030 (2) determined using

X-ray diffraction is illustrated in Figure 1b.

Molecular Docking studies

Results

Docking analysis revealed that the chlorophenyl group of

LQFM030 (2) calculated by Autodock is located in proxim-

ity of the same group of nutlin-3a, and so is Trp23 of P53

(Figure 2). The same group of LQFM030 (2) calculated by

DockThor is close to the other chlorophenyl group of nut-

lin-3a, being close to Leu26 of P53, which is also an impor-

tant residue in interaction with MDM2.^[32]

LQFM030 (2) synthesis

LQFM030 (2) was produced via the synthetic route shown

in Figure 1, beginning with synthesis of the 1-(4-chloro-

phenyl)-1H-pyrazole (4) compound using the classical

method described by,^[13]with a yield of 92.0%. The 1-(4-

chlorophenyl)-1H-pyrazole-4-carbaldehyde (4) compound

was produced (with a yield of 77.9%) via chemospeciﬁc

and region-speciﬁc formulation of 1-(4-chlorophenyl)-1H-

pyrazole (5), which was performed under Duff’s condi-

tions.^[14]LQFM030 (2) was obtained under catalytic

hydrogenation conditions with a quantitative yield. After

three steps, LQFM030 (2) was produced with a global yield

of 71.6%, which was higher than that of the compound

nutlin 1 (1), which was produced with a global yield of

6.4% in eight synthetic.^[31]

LQFM030 (2) induced cytotoxicity in EAT

cells

The effects of LQFM030 (2) on the viability of EAT cells

using the trypan blue exclusion test were investigated. EAT

cells were treated with LQFM030 (2) at increasing concen-

trations (0.033–4.32 m^M). A marked concentration-depen-

dent growth inhibition was observed when the cells were

treated with LQFM030 (2) for 24 and 48 h. LQFM030 (2)

showed 50% growth inhibition after 24 h of treatment at

810 l^M; the IC₅₀value was 100 lM after 48 h of treatment

(Figure 3a). Using the IC₅₀value for 24 h (810 lM), we

The structure of the compounds was investigated and

conﬁrmed by infrared spectroscopy and NMR spec-

troscopy, combining the ¹H, HSQC and HMBC correlation

spectra, as well as mass spectrometry.

detected

a loss of plasma membrane integrity in

27.25 ꢁ 2.1% of the EAT cells by ﬂow cytometry after

incubation with PI (Figure 3b).

X-ray analysis

Table 1 Data collection and reﬁnement statistics

An Ortep representation of LQFM030 (2) is given in Fig-

ure 1b and shows a staggered conformation in which the

chlorophenyl ring is at a dihedral angle of 10.5 (1)° to the

LQFM030 (I)

Data collection

ꢁ

pyrazole ring. The C4 atom is located 0.133 (3) A from the

best plane of the pyrazole. The piperazine presents a chair

Space group

P2₁2₁2₁

Cell dimensions

ꢁ

conformation with the best plane (of r.m.s. of 0.004 A) of

ꢁ

a, b, c (A)

6.0320 (8), 8.9792 (13), 31.306 (5)

the carbon atoms C5/C6/C7/C8 rotated 70.14 (7)° from the

pyrazole plane. The N4 atom has sp³hybridization, while

the N3 atom is sp2, with a N3–C9 bond distance of 1.345

ꢁ

Resolution (A)/h interval (°)

0.8/1.30–26.28

0.022/0.017

1.86

R

_symm/R_int

I/rI

ꢁ

Completeness (%)

Redundancy

Reﬁnement

99.7

(3) A and the other N–C bond lengths varying from 1.459

3

4.36

ꢁ

(2) to 1.473 (2) A. As a consequence of the N3sp

hybridization, the formyl group C9/C10/O1 is in a plane

ꢁ

(r.m.s. 0.005 A) with the atoms C6/N3/C8.

ꢁ

Resolution (A)

0.8

No. reﬂections total/I > 2rI

R1/wR2 (all data)

12 724/3406

0.044/0.087

217/3

LQFM030 (2) was obtained as a monohydrate chiral

crystalline solid, indicating that the present conformation is

stable under normal conditions and showing the strong

afﬁnity of the piperazine N atom for a proton donor. The

water molecule participates in two strong hydrogen bonds

No. parameters/restraints

R.m.s. deviations

ꢁ

Bond lengths (A)

0.003

0.20

Bond angles (°)

6

Mariana F. da Mota et al.

LQFM030 a novel MDM2 inhibitor

Figure 2 Interaction of ligands with MDM2. (a) General view of MDM2 (teal) complexed with P53 (grey) and nutlin-3a (green). (b) Detail of the

superposition of an aromatic ring with chlorine from nutlin-3a and a tryptophan of P53, which is a key residue in the interaction of P53 with

MDM2. (c) Interaction between P53 and LQFM030 as calculated by DockThor. (d) Interaction between P53 and LQFM030 as calculated by Auto-

dock (notice the superposition of the aromatic ring with chlorine of LQFM030 and the tryptophan of P53. (e) Hydrophobic contacts of MDM2

with nutlin-3a and with LQFM030 by DockThor and Autodock, respectively. Notice the recurrence of Gly58, Ile61, Gln72 and Val93, which also

interact with P53 (not shown), and Leu54, which interacts with tryptophan from P53.

phase. There was a meaningful increase in the cells in G1

LQFM030 promoted morphological and

phase (74.79 ꢁ 8.9%) compared with the untreated cells

nuclear changes in EAT cells

(45.80 ꢁ 6.6%; P < 0.05), while cells in

S

phase

The morphological analysis of EAT cells stained with HE

clearly revealed the presence of apoptotic cells after

the treatment with LQFM030 (2). As can be observed in

Figure 4a, the control cells exhibited nuclei with dispersed

chromatin and an organized plasma membrane. In con-

trast, the cells treated with LQFM030 (2) for 24 h presented

membrane blebbing, vacuolization (Figure 4b) and nuclear

fragmentation (Figure 4c). After nuclear staining with

Hoechst 33342, the nuclei in the viable cells displayed a

regular, oval shape, with homogeneous chromatin

(Figure 4d), whereas shrinking and fragmentation of the

nuclei, chromatin condensation and low ﬂuorescence were

observed in the apoptotic cells (Figure 4e).

(15.15 ꢁ 5.9%) decreased. Although there was no statisti-

cal relevance, G2/M phase arrest also could be detected

(Figure 5). These data suggest that LQFM030 (2) could

arrest the cell cycle at G1 and G2/M and prevent EAT cell

proliferation.

LQFM030 (2) induced phosphatidylserine

externalization and DNA fragmentation on

EAT cells

To further explore the EAT cell death mechanisms triggered

by LQFM030 (2) and whether it elicits similar apoptosis,

phosphatidylserine (PS) exposure was assessed using

Annexin V/PI double staining (Figure 6a–c). Although

phosphatidylserine exposure does not occur in all apoptosis

cases,^[33,34]this process is frequently used to evaluate the

apoptotic cell death. As determined by the cytometric anal-

ysis, a reduction in viable cells was observed after LQFM030

(2) treatment (90.45 to 54.20 ꢁ 1.1%; P < 0.05), with an

increase of 15.35 ꢁ 0.9% of early apoptotic cells (Annexin

V +/PI ꢀ; P < 0.05) and 12.80 ꢁ 0.28% of late apoptotic

LQFM030 promoted cell cycle arrest in EAT

cells

To evaluate whether LQFM030 (2) treatment of EAT cells

would interfere in cell cycle dynamics, cytometry analyses

were performed. The results revealed that LQFM030 (2)

treatment signiﬁcantly arrested the EAT cells at the G1

7

LQFM030 a novel MDM2 inhibitor

Mariana F. da Mota et al.

Figure 3 (a) Cytotoxic effects of LQFM030 on EAT cells after 24 and 48 h of treatment (0.033–4.32 mM). Cell viability was examined with try-

pan blue exclusion method and expressed as a percentage of the control. Experiments were performed in sextuplicate. Each bar presents

means ꢁ SD of three independent experiments. The IC₅₀values were 0.81 and 0.10 mM, respectively. (b) Effects of LQFM030 on plasma mem-

brane integrity. EAT cells were treated for 24 h with 0.81 m^MLQFM030 and analysed on a ﬂow cytometer. Histograms displaying PE ﬂuorescence

(x-axis) versus counts (y-axis) have been shown in logarithmic ﬂuorescence intensity. The bar presents mean ꢁ SD of two independent experiments

(*P < 0.05 compared with control).

cells (Annexin V +/PI +; P < 0.05). Furthermore, the num-

ber of necrotic or secondary necrotic cells (Annexin V ꢀ/PI

+) also increased to 19.65 ꢁ 0.49% (P < 0.05). These data

suggest that EAT cell death mechanism triggered by

LQFM030 (2) is apoptosis.

known that p53 activation promotes cycle arrest via

p21WAF1/CIP1, which is potent cyclin-dependent

a

kinase (CDK) inhibitor that can effectively block the cell

cycle progression in G1 and G2 phases. In addition, p27

is another CDK inhibitor also promoting cell cycle

arrest.^[35]Thus, the role of these proteins was evaluated in

EAT cells after LQFM030 (2) treatment (Figure 7a–i).

Our results showed an increase in p21 (81.25 ꢁ 2.89%),

p27 (68.20 ꢁ 3.67%) and p53 (33.80 ꢁ 5.76%) protein

levels in the treated EAT cells compared with the control

(34.10 ꢁ 3.81%, 26.80 ꢁ 1.55% and 12.03 ꢁ 2.32%,

respectively; P < 0.05). Similar to protein level, p21

mRNA also increased; however, p53 mRNA levels did not

show signiﬁcant changes, suggesting that the increase in

p53 protein reﬂects its decreased degradation rather

than the induction of p53 mRNA transcription level

(Figure 7j–l).

Analysis of DNA fragmentation was determined after the

treatment of EAT cells with LQFM030 (2). As observed in

Figure 6d–f, treatment with 810 l^MLQFM030 (2) for 24 h

increased DNA fragmentation by 38.1 ꢁ 9.6% compared

with the control cells (2.1 ꢁ 0.07%, P < 0.05).

LQFM030 (2) modulated p21, p27, p53 mRNA

and protein levels in EAT cells

Because the obtained data suggest that LQFM030 (2) trig-

gers cell cycle arrest and apoptosis, the mechanisms

involved in these processes were explored. It is well

8

Mariana F. da Mota et al.

LQFM030 a novel MDM2 inhibitor

Figure 4 Apoptotic morphology of EAT cells upon LQFM030 treatment. The cells were treated with 0.81 mM LQFM030 for 24 h and stained

with HE. Images were taken using a 1009 objective with the AxioVs40 software. (a) Control cells showing nuclei with dispersed chromatin and

organized plasma membrane. (b) LQFM030-treated cells presenting membrane blebbing and vacuolization (arrows) (c) Treated cells presenting

nuclear fragmentation and vacuolization (arrows). Changes in the nuclear morphology of EAT cells. Nuclear staining was performed with Hoechst

33342. Images were taken using a 209 objective with LAS-AF software. (d) Control cells. (e) LQFM030-treated cells presenting chromatin conden-

sation, low ﬂuorescence and shrinking nuclei.

of treatment with LQFM030 (2) (0.06 ꢁ 0.05), with a

LQFM030 (2) treatment changes MDM2

signiﬁcant reduction in the band density in relation to

mRNA and protein levels in EAT cells

the control cells (0.37 ꢁ 0.13) (Figure 8a,b). Strikingly,

Once the LQFM030 (2) increased p53 protein expression,

the next step was to analyse the mRNA and protein levels

of MDM2. A Western blot analysis of the p53 inhibitor

protein MDM2 showed that MDM2 decreased after 24 h

unlike protein analysis that revealed MDM2 protein

decreases after LQFM030 (2) treatment in EAT cells,

the MDM2 mRNA levels were found to be increased

(Figure 8c).

9

LQFM030 a novel MDM2 inhibitor

Mariana F. da Mota et al.

Figure 5 Analysis of the effects of LQFM030 on EAT cell cycle. For the determination of cell cycle-phase distribution, EAT cells were permeabi-

lized and nuclear DNA was labelled with propidium iodide (PI). The analysis was carried out by ﬂow cytometry. The histograms show (a) untreated

cells, (b) cells treated with 0.81 m^MLQFM030 for 24 h, (c) percentual of EAT cells in G1, S and G2 phases before and after LQFM030 treatment.

Each bar presents mean ꢁ SD of three independent experiments (*P < 0.05 compared with control).

caspase-8, 3.7-fold increase in caspase-9 and 4-fold

LQFM030 (2) induced caspase-mediated

increase in caspase-3 compared with the control cells

apoptosis in EAT cells

(P < 0.05) (Figure 9a–i). Similar to protein, caspase-9

To further investigate the death mechanisms elicited by

this compound, the activity of caspases-3/7, -8 and -9

mRNA presented high levels, while caspase-8 mRNA

slightly increased (Figure 9j–l). In contrast, caspase 3

mRNA levels decreased (Figure 9m). Although apparently

was measured. We observed

a 2.8-fold increase in

Figure 6 Flow cytometry analysis of phosphatidylserine externalization in LQFM030-treated EAT cells. Cells were stained with Annexin V-FITC

and PI. (a) Untreated cells and (b) cells treated with 0.81 m^MLQFM030 for 24 h. Left inferior squares (Annexin V ꢀ/PI ꢀ) = % viable cells. Right

inferior squares (Annexin V +/PI ꢀ) = % early apoptotic cells. Left superior squares (Annexin V +/PI +) = % late apoptotic cells. Right superior

squares (Annexin V ꢀ/PI +) = % necrotic cells. (c) Comparison of ﬂuorescence intensity of Annexin V-FITC and PI between untreated and

LQFM030-treated cells. The bar presents mean ꢁ SD of two independent experiments (*P < 0.05 compared with control). (d–f) Percentage of

DNA fragmentation in LQFM030-treated EAT cells. EAT cells were treated for 24 h with 0.81 m^MLQFM030 and analysed on a ﬂow cytometer.

Histograms displaying PE ﬂuorescence (x-axis) versus counts (y-axis) have been shown in logarithmic ﬂuorescence intensity. (d) Untreated cells, (e)

cells treated with 0.81 m^MLQFM030 for 24 h, (f) comparison of ﬂuorescence intensity of PI between untreated and LQFM030-treated cells. The

bar presents mean ꢁ SD of two independent experiments (*P < 0.05 compared with control).

10

Mariana F. da Mota et al.

LQFM030 a novel MDM2 inhibitor

11

LQFM030 a novel MDM2 inhibitor

Mariana F. da Mota et al.

Figure 7 Effect of LQFM030 on the expression of p21, p27 and p53. EAT cells were treated for 24 h with 0.81 mM LQFM030 and analysed on

a ﬂowcytometer. Histograms displaying PE/FITC ﬂuorescence (x-axis) versus counts (y-axis) have been shown in logarithmic ﬂuorescence intensity.

(a,d,g) Untreated cells. (b,e,h) Cells treated with 0.81 m^MLQFM030 for 24 h. (c,f,i) Comparison of ﬂuorescence intensity of PE/FITC between

untreated and LQFM030-treated cells. The bar presents mean ꢁ SD of two independent experiments (*P < 0.05 compared with control). (j,l) Real-

time PCR analysis of p21 and p53 mRNA after exposure to 0.405 m^MLQFM030 for 24 h. The normalized gene was PGK1. The data were

obtained from two independent experiments, repeated three times, and standard error bars (ꢁSD) represent intra-experimental error.

counterintuitive, this result suggests that the effector cas-

pase activated by LQFM030 (2) treatment was caspase-7,

instead of caspase-3.

described to date, including benzodiazepinediones, nutlins,

spiro-oxindoles, quinolinols, isoindolinones, sulfonamides,

chalcones, terphenyls and piperazine-4-phenyl derivatives,

many of which show relatively weak bioactivity. Within this

context, only the nutlins, spiro-oxindoles and benzodi-

azepinediones are considered to be particularly valu-

able.^[3,9,36]

Discussion

Considering the crucial role of p53 in tumour suppression,

the reactivation of the p53 function by disrupting the p53–

MDM2 interaction using inhibitors agents is now recog-

nized as a promising strategy for anticancer drug design.

Many series of small-compound inhibitors have been

Several studies revealed the potential of nutlins in

inhibiting the p53–MDM2 interaction, promoting cell cycle

arrest and triggering apoptosis in different cancer cell

lines.^{[6,9,37–44]}

12

Mariana F. da Mota et al.

LQFM030 a novel MDM2 inhibitor

Figure 8 Effect of LQFM030 on the expression of MDM2. (a) Western blot analysis after the treatment of EAT cells for 24 h with 0.81 mM

LQFM030. Blot of two independent experiments (C1 and T1 – control and treated cells, respectively, of experiment 1; C2 and T2 – control and

treated cells, respectively, of experiment 2). GAPDH is shown as a loading control. (b) Band density of untreated and LQFM030-treated cells calcu-

lated as the ratio of GAPDH expressed in the same sample. The bar presents mean ꢁ SD of two independent experiments (*P < 0.05 compared

with control). (c) Real-time PCR analysis of MDM2 mRNA after exposure to 0.405 m^MLQFM030 for 24 h. The normalized gene was PGK1. The

data were obtained from two independent experiments, repeated three times, and standard error bars (ꢁSD) represent intra-experimental error.

In this work, we report the synthesis, characterization

and biological properties of LQFM030 (2). This compound

was derived from molecular simpliﬁcation of the lead com-

pound nutlin 1 (1), where subunits A, B and C of the nut-

lin-1 (1) were preserved in LQFM030 (2) and the chiral

centres were removed, making the synthesis easier and

cheaper. The data showed that LQFM030 (2) can interfere

on the p53–MDM2 interaction that effectively activates the

p53 pathway and induces cell cycle arrest or apoptosis in

wild-type p53 EAT cells. The identiﬁed cell growth inhibi-

tion was concentration dependent with the loss of plasma

membrane integrity and DNA fragmentation.

because the relative impact of apoptotic activity and other

activity of p53 could either enhance or prevent the thera-

peutic response.^[48]

In relation to MDM2, LQFM030 (2) increased MDM2

mRNA, but reduced MDM2 protein, probably associated

with an increase in its degradation. In most studies, nutlin

compounds promote MDM2 increase in protein and

mRNA levels in almost all wild-type p53 tumour

cells.^{[6,41,43–45]}Furthermore, the pharmacological strategy

based on nutlin-3a tends to have transient effects and is

counterbalanced by the strong feedback loop regulating the

reciprocal interactions between MDM2 and p53.^[1]How-

ever, the nutlin 3-s treatment of B-cell chronic lymphocytic

leukaemia (B-CLL), in which low level expression of p53

transcripts was found, does not upregulate MDM2 despite

the progressive stabilization and the accumulation of p53

and its targets.^[46]The variability in MDM2 expression in

tumour cell responses after nutlin-3 treatment indicates

that under some circumstances and tumour cell types, this

compound can induce the different cell death mechanisms.

Concerning speciﬁcally MDM2 protein and mRNA expres-

sion, our results are similar to those obtained with MI-219,

a spiro-oxindole MDM2 antagonist, which promoted an

increase in MDM2 transcripts, but downregulated the pro-

tein level. The authors suggested that MI-219 post-transla-

tional process regulates HDM2 protein by inducing

autoubiquitination and the degradation of itself.^[46,49]This

could be, at least in part, an explanation for the decrease in

MDM2 protein after LQFM030 (2) treatment. Moreover,

nutlin-3 stabilizes MDM2⁰s conformation and protects

MDM2 from degradation.^[46]Our results in vitro are in

accordance with the in-silico study.

P53 pathway activation by nutlin 1 occurs due to the

decreased degradation of p53 rather than the elevated

expression of the p53 gene, once p53 transcript level was

not affected by nutlin treatment.^[6]Similar results were

obtained with LQFM030 (2) compound; an increased

amount of p53 was detected by cytometry, but p53 tran-

script level was not altered by LQFM030 (2). In addition,

p21 and p27 protein and mRNA increased after the treat-

ment with LQFM030 (2).

Other small-molecule inhibitors of p53–MDM2 interac-

tions, such as nutlins, RG7112 (nutlin derivative), RG7388

and M-219, also present a similar pattern in wild-type p53

cells, as expected.^[6,37,45,46]Indeed, RG-7112, another nutlin

derivative, advanced into clinical development.^[47]Strik-

ingly, RITA, another inhibitor of p53–MDM2 interactions

in wt-p53 cells, does not depend on p21 activity in growth

suppression. One probable explanation is that RITA dis-

rupts p53–MDM2 interaction; RITA binds to p53, not to

MDM2 protein.^[48]It is essential to know that p53–MDM2

disruption can trigger different cell death mechanisms,

13

LQFM030 a novel MDM2 inhibitor

Mariana F. da Mota et al.

Figure 9 Effect of LQFM030 on the activity of caspase-8, -9 and -3/7. (a,d,g) Untreated cells. (b,e,h) Cells treated with 0.81 mM LQFM030 for

24 h. (c,i,f) Comparison of ﬂuorescence intensity of FITC between untreated and LQFM030-treated cells. Each bar presents mean ꢁ SD of two

independent experiments (*P < 0.05 compared with control). (j,l,m) Real-time PCR analysis of caspases-8, -9 and -3 mRNA after exposure to

0.405 m^MLQFM030 for 24 h. The normalized gene was PGK1. The data were obtained from two independent experiments, repeated three times,

and standard error bars (ꢁSD) represent intra-experimental error.

Even though at least in vitro, caspase activation is not a

strict requirement for multiple cases of apoptosis,^[34]the

above ﬁndings prompted us to further investigate whether

caspases would play a role in cell death after LQFM030 (2)

treatment, because it is well established that caspase activa-

tion is involved in the nutlin-induced apoptotic

programme.^[6,46,50,51]Our results revealed that similar to

nutlins, mRNA level and activity of all caspases analysed

showed an increased activity after the treatment. These

results suggest the activation of both intrinsic and extrinsic

apoptotic pathways.

Previous studies revealed that p53 has a direct apopto-

genic role in the mitochondria in response to multiple

death stimuli. A rapid p53 translocation to mitochondria,

after death stimuli, triggers a ﬁrst wave of apoptosis that is

transcription independent and may precede a second

slower wave that is transcription dependent.^[52,53]Later

studies showed that the direct mitochondrial programme is

14

Mariana F. da Mota et al.

LQFM030 a novel MDM2 inhibitor

a major mechanism in nutlin-induced p53-mediated apop-

tosis in tumour cells; apoptosis occurred through the cas-

pase activation.^[53]p53 translocation to the mitochondria

promotes its interaction with Bcl2 family members and

induces mitochondrial outer membrane permeabilization

(MOMP). The characteristic of caspase-dependent intrinsic

apoptosis relies on MOMP induction with the subsequent

activation of caspase-9 initiator and caspase-3 effector.^[34]

This seems to be the most common mechanism of intrinsic

apoptosis because caspase-3 is the best well-characterized

effector caspase.^[54,55]Our results with LQFM030 (2)

showed that both caspase-9 mRNA and protein increased,

leading to the effector caspase-3/7 activation. Nevertheless,

despite the increase in effector caspase-3/7 activity, the cas-

pase-3 mRNA decreased after the treatment. This result

suggests that the effector caspase activated by LQFM030 (2)

is caspase-7. Interestingly, Devarajan et al.^[57]described

that approximately 75% of the breast tumour cells analysed

in their study revealed the lack of caspase-3 transcripts and

caspase-3 protein expression, while the remaining samples

showed substantial decreases in caspase-3 expression.

Because EAT cells are derived from murine breast carci-

noma, our data are in agreement with these results.

Furthermore, studies conducted in CASP-3-deﬁcient

MCF-7 cells revealed that caspase-7 is indeed responsible

for the proteolysis during the demolition phase of apopto-

sis.^[57]Regardless of whether caspase-8 mRNA did not alter

after the LQFM030 (2) treatment, its activity increased sig-

niﬁcantly, indicating that our compound also induces

extrinsic pathway, similar to nutlins.^[51,58]Apparently, the

extrinsic and intrinsic apoptotic pathways show a key role

in response to p53 activation.^[3]In sum, the in-vitro results

were in agreement with in-silico results.

the assumption that they can be used at lower doses (‘the

nanomolar rule’). However, critical parameters such as the

balance between efﬁcacy and toxicity are ignored, rejecting

the efﬁcacious compounds or selecting the ineffective or

toxic compounds. Moreover, there are efﬁcacious anti-

cancer agents lacking the nanomolar potency.^[59,60]Accord-

ing to Secchiero et al.,^[1]it will be unlikely that nutlin-3

will be used as a monotherapy, but combined with innova-

tive drugs, such as TRAIL or bortezomib, might be an effec-

tive approach in cancer therapy. In a similar way,

LQFM030 (2) could be combined with other therapeutic

agents to increase the treatment efﬁcacy. Another approach

would be LQFM030 (2) optimization, which is under way.

Conclusion

Our results suggested that a simpliﬁed analogue of nutlin-

1, LQFM030 (2), could have an inhibitory effect on the

p53–MDM2 interaction, reactivating p53 and its main

functions, cell cycle arrest and apoptosis. These results sug-

gest that the small-molecule p53 activator LQFM030 (2)

could be of value in the development of a novel cancer

therapeutic agent.

Declarations

Funding

This study was supported by Fundaßc~ao de Apoio ꢂa Pesquisa

(FUNAPE)-UFG, Goi^ania-GO, Conselho Nacional de

ꢀ

Desenvolvimento Cientıﬁco e Tecnologico (CNPq), Finan-

ciadora de Estudo e Projetos (FINEP), Coordenaßc~ao de

ꢀ

Aperfeißcoamento de Pessoal de Nıvel Superior (CAPES)

and Fundaßc~ao de Apoio a Pesquisa do Estado de Goiꢀas

(FAPEG).

The current paradigm of anticancer drug therapy is the

development compounds with nanomolar in-vitro potency,

showing that they would be efﬁcacious and safer based on

4. Chattopadhyay S et al. Protein A-acti-

vated macrophages induce apoptosis

in Ehrlich’s ascites carcinoma through

nitric oxide-dependent pathway.

7. Mir R et al. Mdm2 antagonists induce

apoptosis and synergize with cisplatin

overcoming chemoresistance in TP53

wild-type ovarian cancer cells. Int J

Cancer 2013; 132: 1525–1536.

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17

Article Doi

Induction of apoptosis in Ehrlich ascites tumour cells via p53 activation by a novel small-molecule MDM2 inhibitor – LQFM030

DOI: 10.1111/jphp.12573

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