“

Inhibition of the regulatory interaction between p53-HDM2

proteins, as anti-cancer agents "

A dissertation presented by

SAI WOON MERNG TIP

For the BSc (Hons) in Biomedical Sciences

In the
University of Bradford

Division of Biomedical Sciences

School of Life Sciences
University of Bradford
Bradford

Submission Date : 5th January 2015

Word Count : 5000

Table of Contents

Abstract ........................................................................................................................................................ 3

Introduction ................................................................................................................................................ 3

Properties of p53 and HDM2 proteins ............................................................................................... 7

Regulation of p53-HDM2 proteins complex .................................................................................. 11

Inhibitors of p53-HDM2 proteins interaction .............................................................................. 15

1. Small molecule inhibitors of p53-HDM2 proteins interaction ...................................................... 15

1.1 Nutlins ............................................................................................................................................................................. 17

1.2 Spiro-oxindole .............................................................................................................................................................. 22

2. Natural Product Inhibitors of p53-HDM2 proteins interaction .................................................... 25

2.1 Chalcones ....................................................................................................................................................................... 26

2.2 Chlorofusin .................................................................................................................................................................... 26

2.3 Hexylitaconic acid ....................................................................................................................................................... 27

3. Proteomimetic inhibitor of p53-MDM2 proteins interaction ........................................................ 27

3.1 β-Hairpin Inhibitor ..................................................................................................................................................... 28

Conclusion ................................................................................................................................................ 29

References ................................................................................................................................................ 30

Abstract

The p53 tumor suppressor protein’s function is frequently attenuated or

deleted in most of the human cancer tumors, making it a popular therapeutic target of

cancers. Designing small molecule inhibitor, which can interfere the p53-HDM2

proteins interaction and reactivation of the p53 protein function, is a favorable

therapeutic approach in many human cancers holding wild-type p53. In addition, many

potent and selective small molecule inhibitors of p53-HDM2 proteins interaction have

been identified using high-resolution X-ray crystallography, computational and

structure-based design device. So far, seven small molecule inhibitors are going under

clinical development as an anti-cancer agent.

Introduction

The tumor suppressor p53 protein, encoded by TP53 gene, was first discovered

as a 53 kDa protein present in human and mouse cells by six groups of researchers in

1979 through its co-immunoprecipitation with antibodies against SV40 viral protein

(Deleo et al., 1979; Kress et al., 1979; Lane and Crawford, 1979; Linzer and Levine,

1979; Melero et al., 1979; Smith et al., 1979). At first, TP53 gene was believed to be an

oncogene because of the high level of p53 protein expression in most cancers.

Afterward, TP53 gene was identified as a tumor suppressor gene encoding p53 protein

and the gene was found on the short arm of chromosome 17 (Baker et al., 1989).

Tumor suppressor p53 protein plays a pivotal role in many major cellular processes

and in maintaining stability of genome by means of transcriptional and non-

transcriptional activities. TP53 gene is the most frequently mutated gene in human

malignant tumors than other genes in human genome (Kandoth et al., 2013). In
3


addition, there have been identified more than 25,000 mutations in TP53 gene

(Petitjean et al., 2007). HDM2 (human double minute 2, MDM2 in mouse), a negative

regulator protein of p53 tumor suppressor protein encoded by HDM2 gene, was

discovered in 1992 (Fakharzadeh et al., 1993). HDM2 protein binds to and negatively

regulates p53 protein activities whereas HDM2 gene is a transcriptional target gene of

p53 protein forming negative feedback loop (figure 1) (Momand et al., 1992; Picksley &

Lane, 1993). There are approximately 14.1 million of new malignancy cases as well as

8.2 million deaths worldwide due to malignancy in 2012 (Bray et al., 2013; Ferlay et al.,

2013). TP53 gene was inactivated in half of human cancer cases (Hollstein et al., 1991),

as it is the most important tumor suppressor gene in cancers. Most of the inactivation

of p53 protein is due to missense mutation, a single base pair change that results in

substitution of different amino acid in the resulting protein, in TP53 gene.

HDM2
HDMX

Double-stranded
DNA

Figure 1. Autoregulatory feedback loop inhibition of p53 by HDM2. HDM2 directly binds to p53 and
inhibits its transcriptional activity, causes ubiquitinization and proteasomal degradation of p53, and
exports p53 out of the nucleus. HDMX, a homolog of HDM2, also directly binds to the transactivation
domain of p53 and inhibits p53 activity, but does not cause degradation of p53. Tumor suppressor ARF
binds to HDM2 and sequesters HDM2 into the nucleolus, leading to stabilization of p53.

Taken from (Shangary and Wang, 2009)

The germline mutation of TP53 gene was associated with Li-Fraumeni

syndrome, an autosomal dominant hereditary disorder (Varley, 2003). Li-Fraumeni

syndrome is a rare cancer predisposition disorder and showed loss of heterozygosity in

TP53 gene causing accumulation of mutant p53 protein in cancer tumors (Sedlacek et

al., 1998). In addition, HDM2 gene amplification and overexpression of HDM2 protein

were seen in more than one third of sarcomas with wild-type p53 that lead the cell to

escape from p53-regulated growth control (Oliner et al., 1992). Despite the status of

p53, the increased level of HDM2 protein result in genome instability and delays in

p53-dependent DNA double-strand break repair. Dysregulation of p53 signaling

pathways are needed for development of human malignant tumors, so restoration or

reactivation of endogenous p53 protein functions may be valuable in cancer treatment

as well as applied research. Currently, many difference approaches (figure 2) have

been attempted to restore wild-type p53 protein function and activation of p53

pathway as anti-cancer drugs (Table 1).

Mutant p53 p53-HDM2 interaction

reactivators inhibitors

HDM2

HDM2 E2 ligase
inhibitors

Blocking HDM2
Gene Therapy expression

Figure 2. Showing several strategies targeting p53-HDM2 pathway for cancer therapy.

Taken from (Wang and Hu, 2012)

Table&1.8list8of8compunds8and8small8molecules8that8activate8p538pathway8through8different88888888888
meachanism8of8action

Small8molecules8targeting8wild-type8p53-HDM28axes Small8molecules8reactivaing8mutant8p53
Nutlins PRIMA-1MET
Sulfonamide8I CP31398
NSC66811 PhiKan083
Terphenyl814 MIRA-3
TDP665759 STIMA-1
TDP521252 SCH529074
MI-219/-319 Ellipticine
PXn727/822 RETRA8(reactivation8of8transcriptional8reporter8activity)
Isoindolinone874a p53R3
SJ-172550 WR1065
XI-006
RITA8 (reactivation8of8p538and8induction8of8tumor8cells8apoptosis)
JNJ-26854165
(stapled)8peptides

Vaccination Genetherapy
P53-SLP8vaccine Advexin
INGN-2258 (a8dendritic8cell-based8p538vaccine) Gendicine
ALT-801 SCH-585008 (recombinant8adenovirus-p53)
ONYX-015

Antisense
GEM2408(Hdm28mRNA)

Taken from (Cheok et al., 2011)

Properties of p53 and HDM2 proteins

p53 protein is well-known potent tumor suppressor protein encoded by TP53

gene. The p53 protein has 393 amino acids with four major functional domains (figure

3). They are (1) N-terminus transactivation domain which is responsible for binding

with transcription factors, (2) DNA binding domain responsible for binding with DNA

sequence which is in or near promoters of its target gene, (3) C-terminus regulatory

domain which contains both six lysines for ubiquitination by HDM2 and nuclear export

signal and (4) tetramerization domain subjects to formation of tetramer as p53 protein

function transcriptionally.

Transactivation Domain / Tetramerization

proline-rich region Domain Regulatory Domain
1 92 102 DNA-binding Domain
355 363 393
p53
HDM2
DNA-binding NLS NES 464 471 489
1 Domain 301 488
Zn-finger RING-finger

Figure 3. Schematic of the functional domain of p53 protein (top) and HDM2 protein (bottom). The
transactivation/proline-rich domain of p53 responds to stress signals of DNA damage/radiation/UV by site-
specific phosphorylation. The C-terminus(regulatory domain) of p53 contains six lysine residues which
respond to stress by acetylation, inhibiting HDM2-mediated ubiquination, carried out within the RING-finger
motif of HDM2. Zinc fingers generally act as motifs for protein-protein or protein-nucleic acid interaction , but
the exact role in HDM2 is unclear. The acidic domain of HDM2 contains residues essential for phosphorylation
and regulation, and residues 464-471 contain a nucleolar localization signal.
Abbreviations: NLS, nuclear localization signal; NES, nuclear export signal; Zn-finger, Zinc finger

Taken from (Peirce and Findley, 2010)

The p53 protein resides at low level of concentration in normal cellular

conditions and it behaves as a potent transcription factor settles in the nucleus in

order to easily access the DNA (Deoxyribonucleic acid). Its activation is initiated by

cellular stress conditions such as DNA damage, UV or ionizing radiation, cellular

hypoxia, oncogenic stimuli, some viruses and various metabolic changes. After its

activation, the p53 protein transcriptionally activates many downstream target genes

through p53 binding sites in their regulatory region to begin the specific functions

(figure 4). They are (1) cell circle arrest through a p21 tumor suppressor protein, (2)

senescence, (3) apoptosis through the pro-apoptotic proteins PUMA (p53 up-regulated

modulator of apoptosis) and Noxa (Nakano and Vousden, 2001; Oda et al., 2000), (4)

genetic stability and (5) inhibit angiogenesis. Moreover, p53 protein can also induces

apoptosis through transcriptional-independent mechanism after its activation by

cellular stress (Speidel, 2010). Accumulation p53 protein in the cytosol and

mitochondria is due to stress-induced stimuli and results in activation of pro-apoptotic

proteins, Bax and/or Bak, (figure 5) (Youle and Strasser, 2008). Once the p53 protein is

at the mitochondrion, it induces mitochondrial outer membrane permeabilization

(MOMP) that leads to the release of cytochrome C, EndoG and Smac/DIABLO (pro-

apoptotic factors), causing apoptosis (figure 5) (Moll et al., 2006). In addition to its

pivotal role as a cellular gatekeeper, the TP53 gene is the most frequent target for

genetic variations in most cancers. Roughly fifty per cent of all human malignant

tumors express mutant p53, which especially having mutations in DNA binding domain

result in lost of its transcriptional function (Hainaut and Hollstein, 2000).

The level of p53 is tightly control in normal cells due to its growth inhibitory

and pro-apoptotic effect, which can harm normal proliferating cells. The action of p53

protein is mainly controlled by HDM2 protein, encoded by HDM2 gene, and HDM2

protein was identified as an oncoprotein (Momand et al., 2000). In the structure of

HDM2 protein (figure 3), it has a p53 interaction domain, which binds to the terminal

amino acid residues at transactivation domain of p53 protein. Both a nuclear

8


localization signal (NLS) and a nuclear export signal (NES) help in transporting HDM2

protein back and forth between the cytoplasm and the nucleus, so that the activity of

p53 protein is strictly regulated by HDM2 protein (Freedman and Levine, 1998; Roth et

al., 1998). A central acidic domain of HDM2 protein interacts with ribosomal protein L5

and p300/CBP (CREB-binding protein) as well as contributing in degradation of p53

protein (Argentini et al., 2001; Zhu et al., 2001). The RING motif, downstream of acidic

domain, is liable for the E3 ligase activity of HDM2 protein. The HDM2 gene was

overexpressed in most of the human cancer tumors by mean of gene amplification.

The HDM2 amplification frequency in the human tumors is about 7%, high frequency

of 20-30% in soft tissue sarcomas, 16% in osteosarcomas and 13% in oesophageal

carcinomas respectively (Momand et al., 1998). Its main function is to regulate the p53

protein level within the cell through autoregulatory feedback loop mechanism (Figure

1). Since p53 protein activates transcription and up-regulation of HDM2 gene, which is

one of the target gene of p53 protein resulting the increase of the HDM2 protein level.

Conversely, HDM2 protein blocks p53 protein from its transcriptional activity and

causing ubiquitylation by E3 ligase activity and degradation in the proteasome by

transporting p53 protein out of the nucleus (Haupt et al., 1997; Honda et al., 1997;

Kubbutat et al., 1997). Hence, both p53 and HDM2 protein have short half-life and low

concentration levels in normal cellular conditions to get adequate function of cell

processes such as cell division (Freedman et al., 1999). Furthermore, the p53 protein

also binds to a dozen of other proteins (PTEN, ATM, CHK1/2) (Braithwaite et al., 2006)

and involves in a wide range of physiologic processes such as regulation of reactive

oxygen species (ROS) (Budanov, 2014).

Angiogenesis
Growth Arrest DNA repair

Apoptosis
Figure 4. Regulation of p53 by MDM2 showing the stress signals that activate the pathway, mediators
that detect the signals, and downstream transcriptional activators affected by the pathway and their
outcomes.

Taken from (Khoury and Domling, 2012)

Nucleus

Figure 5.Interplay of the nuclear and cytoplasmic functions of p53 in apoptosis. Nuclear p53 induces
the expression of MDM2, which acts to inhibit the protein through binding and ubiquitinylation. Cellular
stress signals interrupt this inhibition, allowing p53 to accumulate both in the nucleus and in the
cytoplasm. In the latter, p53 is sequestered by anti-apoptotic Bcl-2 proteins such as Bcl-XL. Another
target of nuclear p53, PUMA, functions to disrupt the Bcl-XL-p53 interaction. The released p53 can now
trigger MOMP and apoptosis through interaction with, for example, Bax.

Taken from (Green and Kroemer, 2009)

10


Regulation of p53-HDM2 proteins complex

The protein-protein interaction of p53-HDM2 plays a central role in many

cellular processes and pathways. The level of p53 protein and its activation was kept

control by two regulator proteins, HDM2 and HDMX, but mainly by HDM2 protein. The

HDM2 protein is crucial for development of cell, which is evidenced by genetic study.

In the experiment, the HDM2 gene was knocked-out in mice and was result death

during embryonic stage but it was recovered by simultaneous knocked-out of p53 gene

(Jones et al., 1995; Montes de Oca Luna et al., 1995). This indicates that the interaction

between p53-HDM2 proteins is very crucial for cell development, growth, survival,

death and may regulate many complex cellular pathways. In addition, the relation

between p53-HDM2 proteins was complicated and associated with many other

pathways and protein partners such as p14ARF and HDMX (figure 1). The p14ARF protein

is a tumor suppressor protein or an alternative reading frame, which inhibits HDM2

protein by blocking its E3 ubiquitin ligase activity. The tumor suppressor function of

p14ARF protein depends on wild type p53 (Honda and Yasuda, 1999). Accordingly, the

p14ARF protein interferes the p53 negative feedback inhibition by binding with HDM2

protein (figure 1). The HDMX protein (so called MDM4 or MDMX in mouse) is a

homologue of HDM2 protein with higher structural homology and encoded by HDMX

gene. It was first identified by the screening of p53-binding proteins (Shvarts et al.,

1996) and as HDM2 binding protein in a yeast two-hybrid screen (Sharp et al., 1999;

Tanimura et al., 1999). HDMX protein was also interacted with both p53 and HDM2

proteins identified by co-immunoprecipitation studies (Stad et al., 2000). Both HDMX

and HDM2 have shared and conserved similar domains in N-terminal p53-binding

domain, a zinc-binding motif and a C-terminal RING-finger (Shvarts et al., 1996) but

11


HDMX lacks in either functional nuclear localization signal (NLS) or nuclear export

signal (NES) (Gu et al., 2002; Migliorini et al., 2002). The MDMX protein is also

important for cellular growth, development and possibly in tumorigenicity. The

experiment shown lack of development in mice by HDMX gene knocked-out and

rescued by counter knocked-out of p53 gene (Parant et al., 2001). HDMX protein

functions as a negative regulator of p53 protein by blocking the transactivation activity

of p53 protein and increase the function of HDM2 but not involves in p53 protein

nuclear export and degradation by ubiquitination in proteasome due to lack of E3

ligase activities (Linares et al., 2003). The overexpression of HDMX is approximately

20% in human malignancies (Toledo and Wahl, 2006; Wade and Wahl, 2009).

The site of the p53-HDM2 proteins interaction is between the N-terminal p53

binding domain of HDM2 protein from residues 1-41 and the N-terminal HDM2 binding

domain of p53 protein from residues 1-118 so-called BOX1 domain (Chen et al., 1993;

Oliner et al., 1993; Picksley et al., 1994). The first identification of a region contributed

in p53-HDM2 proteins interaction was through a yeast two-hybrid screen (Oliner et al.,

1993) and with an immunoprecipitation methods (Chen et al., 1993). The study of

crystal structures (figure 6) on the N-terminal domains of HDM2 protein of Xenopus

laevis and human interact with p53 N-terminal domains showed the residues 15-29 on

N-terminal domain of p53 was the most responsible regions for the p53-HDM2

proteins interaction and it binds deep into the large crack on the surface of HDM2

protein (Kussie et al., 1996). Among the interface between p53-HDM2 proteins, the

three hydrophobic residues Phe19 (phenylalanine19), Trp23 (tryptophan23) and Leu26

(leucine26) from p53 transactivation domain form α-helix structure and protrude deep

into a huge hydrophobic pocket on the HDM2 protein surface (figure 6).

12


A B
p53

HDM2 HDM2
Figure 6. (A)Crystal structure of the p53 binding pocket on HDM2 protein (B)Crystal structure of the
p53-HDM2 proteins interaction. HDM2 rendered as a surface using MOLCAD (Sybyl) and colored
according to cavity depth, and the p53-peptide pictured as sticks.

Taken from (Murray and Gellman, 2007)

The discovery of this detail crystal structure can gives an understanding on how

the HDM2 protein binds and inhibits the p53 transcriptional activities and how to

interfere the p53-HDM2 interaction complex which is needed for developing the

cancer drugs. The p53-HDM2 interaction was in tight control under normal condition

in cell but it can be intervened during cellular stress conditions. Three crucial

mechanisms involve in this intervene between p53 and HDM2 proteins interaction; (1)

stress induces post-translational modifications of both proteins, (2) interacting

proteins, (3) transportation of both p53 and HDM2 cause wide separation. In addition,

there are many kinds of proteins involve in the regulation of this p53-HDM2 proteins

interaction and mostly depend on what sort of stress signal was incoming (Horn and

Vousden, 2007). In cancer, the overexpression of HDM2 and HDMX become

dysregulation of negative feedback inhibition and causing inadequate p53 activation

leading to inefficient in cell circle arrest and/or apoptosis in cancer cells. Hence,

interfering the p53-HDM2 proteins interaction by mean of HDM2 antagonists will

defeat the oncogenic overexpression of HDM2 and result in regain of endogenous p53

13


function lead to activation of downstream target genes causing apoptosis of the cancer

cells. Therefore, p53-HDM2 proteins interaction become a target in understanding of

the cancer biology and in prospective novel drugs development to treat cancer.

It have been discovered many small-molecule compounds with different

approaches to activate p53 pathway (Table 1) and interfering p53-HDM2 proteins

interaction was one of that approach. Conjointly, all of the experiments shown the

same result that increase activation of p53 pathway and expression by inhibition of

p53-HDM2 proteins complex results in cancer cells growth arrest and apoptosis.

Although, the studies have many limitations like the applicability to conduct or

approach in living cells or oncogenic mouse models. As an identification of a crystal

structure of interface between p53 and HDM2 proteins, the small-molecule HDM2

inhibitors were developed base on the three hydrogen bonds bridge and the interface

between p53-HDM2 proteins, which is relatively small. In protein-protein interaction,

the flatter the surface among proteins and the larger the tendency to be buried by one

of the two proteins. When considering on inhibitors of p53-HDM2 proteins interaction,

the inhibitors will have to (1) have amino acids that mimic the three important

residues of p53 N-terminal domain (Phe19, Trp23, Leu26) as it is crucial for p53-HDM2

proteins binding (2) relatively small-molecule inhibitors since the interfaces between

proteins are small (3) having a lipophilic groups in inhibitors owing to the fact that the

interfaces between p53 and HDM2 is hydrophobic (non-polar) as well as it can increase

the binding energy. In conventional cancer treatment therapy, the activities of the

tumor suppressor p53 protein is very importance in response to genotoxic stress as it

form a central role which combines many stress response pathways and making the

decision between cell cycle arrest, senescence, apoptosis and other physiological

processes (Vousden and Prives, 2009).

14


Inhibitors of p53-HDM2 proteins interaction

1. Small molecule inhibitors of p53-HDM2 proteins interaction

The mechanism of activation of p53 pathway or p53 accumulation by small

molecule HDM2 inhibitors are much distinct from that induced by traditional

chemotherapy and radiation. Both conventional methods induce p53 protein

accumulation by mean of post-translational modifications like phosphorylation of p53

protein causing non-specific and genotoxic effect to cells. In contrast to

chemotherapy/radiation, HDM2 inhibitors such as Nutlins (cis-imidazoline derivative)

or MI-219 (spiro-oxindole derivative) cause accumulation or activation of p53 protein

in cancer and normal cells without damaging the DNA or requiring p53

phosphorylation. Both Nutlins and MI-219 are specific and non-genotoxic HDM2

inhibitors for activating p53 pathway. Beside, there are many small molecule

compounds that have been gone through in preclinical and Phase 1 clinical trial (Table

2). To get the best result in inhibition of p53-HDM2 proteins interaction by small-

molecule inhibitors, it should match with this criteria (1) having a high binding affinity

(2) having specificity to HDM2 (3) effective cellular activity in malignant cells with wild-

type p53 (4) having sensible pharmacokinetic figure. Some small-molecule inhibitors

that do not bind HDM2 like RITA (Reactivation of p53 and Induction of Tumor cell

Apoptosis), which binds to p53 protein rather HDM2 protein and activates p53 target

genes follow by extensive apoptosis in many tumor cells with wild-type p53 (Issaeva et

al., 2004).

15

,Table.2,,Small(molecule,/,compound,targets,p53(HDM2,proteins,interaction,and,its,research,development*

Small(molecule,/, Mechanism,of, ClinicalTrials.gov,

No. Stage,of,Development Company
Compound Action,/,Target Identifiers

Phase,Ι,trial,in,advanced,solid,tumors,, NCT00559533,,,,,
Inhibit,p53(HDM2, solid,tumors,,haematological,neoplasms, NCT01164033,,,,,
1 Nutlin/RG7112 Roche
binding,/,HDM2 and,liposarcomas,(all,completed),,,,,,,,,,,,,,,,
NCT00623870,,,,,
(1**) NCT01143740

RG7112,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
inhibit,p53(HDM2,
2 Phase,I,in,AML,(completed) NCT01635296 Roche
with,cytarabine binding,,/,HDM2

RG7112,,,,,,,,,,,,,,,,,,,,,,,,,,,,
inhibit,p53(HDM2, Phase,I,in,soft,tissue,sarcoma,
3 NCT01605526 Roche
with,doxorubicin binding,/,HDM2 (completed)

Spiro(oxindoles,,,,,,,,,,,,,,,,,,,,,,,
inhibit,p53(HDM2, Preclinical,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Ascenta,
4 (
(MI(219) binding,/,HDM2 (2**) Therapeutics

inhibit,p53(HDM2, Phase,I,in,malignant,neoplasms,
5 MI(773,(SAR,405838) NCT01636479 Sanofi
binding,/,HDM2 (recruiting)

inhibit,p53,binding, Preclinical,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
6 RITA ( Aprea
/,p53 (3**)

Benzodiazepinediones, inhibit,p53(HDM2, Preclinical,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,

7 ( (
(TDP665759) binding,/,HDM2 (4**)

JNJ(26854165, inhibit,p53(HDM2, Phase,I,in,patient,with,hematologic, Johnson,&,

8 NCT00676910
(serdemetan) binding,/,HDM2 neoplasms,(5**) Johnson

inhibit,p53(HDM2,
9 PXN727,and,PXN822 Preclinical ( Priaxon
binding,/,HDM2

inhibit,p53(HDM2, Phase,I,in,advanced,malignancies,
10 RO5502781 NCT01462175 Roche
binding,/,HDM2 (recruiting)

RO5502781,with, inhibit,p53(HDM2,
11 Phase,I,in,AML,(recruiting) NCT01773408 Roche
cytarabine binding,/,HDM2

inhibit,p53(HDM2, Phase,I,in,advanced,solid,tumor, Daiichi,

12 DS(3032b NCT01877382
binding,/,HDM2 lymphoma,(recruiting) Sankyo

Phase,I,clinical,trial,in,adult,patients,
inhibit,p53(HDM2,
13 NVP(CGM097 with,selected,advanced,solid,tumors, NCT01760525 Novartis
binding,/,HDM2
(recruiting)

Phase,I,clinical,trials,in,advanced,solid,
inhibit,p53(HDM2, NCT01463696,
14 MK(8242 tumors,or,acute,myelogenous,leukemia, Roche
binding,/,HDM2 NCT01451437
(recruiting)
AML,(,acute,myeloid,leukemia
*Sourced from the ClinicalTrials.gov database, (Yu et al., 2014), (Khoo et al., 2014), 1** (Saha et al., 2013), 2** (Xue et al., 2007),
3** (Issaeva et al., 2004), 4** (Shangary and Wang, 2009), 5** (Chargari et al., 2011; Kojima et al., 2010)

16

1.1 Nutlins

The Nutlin is the first potent and selective small molecular compound HDM2

inhibitor from class of cis-imidazoline compound. The HDM2 inhibitor were discovered

by Vassilev and colleagues, using the conventional wide screening of chemical

compound libraries base on the crystal structure of p53-HDM2 proteins interaction at

Roche Research Center, Nutley, USA in 2004. So the name Nutlin was came from

‘Nutley’ plus ‘inhibitor’. Nutlins have a main structure of cis-imidazoline and gone

through a series of modifications on its chemical compound, finally the potent small

molecular HDM2 inhibitor was obtained (figure 7). In addition, there are three Nutlin

variants Nutlin-1, Nutlin-2 and Nutlin-3. Among them, Nutlin-3 is an active enantiomer

with IC50 values of 90nM more potent than others two Nutlins. By studying the crystal

structure of the interaction between HDM2 and Nutlin-2 (figure 8B), the Nutlin binds

to the surface of HDM2 in N-terminal p53 binding site as it imitates the crucial

peptides on p53 protein binds with HDM2 pockets at high degree of binding. There are

replacement of helical structure backbone of peptide with the imidazoline scaffold to

get frigid fashion and direct protruding of bromophenyl group into Trp23 pocket,

another bromophenyl moiety placing at Leu26 pocket, the ethyl ether side chain fixed

into Phe19 pocket respectively.

Figure 7. Structure-based design of Nutlin compounds as HDM2 inhibitors

Taken from (Zhao et al., 2013a)

17

A B C

HDM2 HDM2 HDM2

Figure 8.Crystal structural of binding between (A) p53 peptide, (B) the cis-imidazoline analog
Nutlin-2 and (C) the spiro-oxindole analog MI-219 to HDM2.
Side chains of p53 residues involved in the p53-HDM2 interaction are shown in stick
representation, whereas Nutlin-2 and MI-219 are shown in ball and stick representation. Nutlin-
2 is shown with carbons in cyan, nitrogen in blue, oxygen in red, fluorine in light blue, and
chlorine in green. The surface representation of HDM2 in each case is shown with carbons in
gray, nitrogen in blue, oxygen in red, and sulfur in yellow. Hydrogen bonds are depicted with
yellow lines and hydrogen atoms are excluded for clarity. The PyMOL program was used to
generate figures.

Taken from (Shangary and Wang, 2009)

As normally all three groups reside at Phe19, Trp23 and Leu26 of p53 peptides

deep into the three pockets on HDM2 surface protein (Vassilev et al., 2004). The mode

of action of Nutlin-3 is to inhibit the interaction between p53-HDM2 proteins by

binding with HDM2 protein as an antagonist. Therefore, p53 protein will free from

HDM2 protein binding and increase accumulation of wild-type p53 in the cells follow

by elevation of HDM2 and p21 tumor suppressor protein in the way that match with

p53 pathway activation. However, the p53 protein accumulation was not owing to the

increase of p53 expression or p53 mRNA in cell but because of lessen degradation of

p53 protein through E3 ubiquitinization ligase activity of HDM2. Nutlin-3 will stabilizes

the p53 and activates the p53 pathway by mean of non-genotoxic fashion in the cancer

cells that preserve the wild-type p53 and harmless to normal cells. So, the

phosphorylation of p53 through genotoxic way was not seen among the cancer cells

treated with Nutlin-3. Although, most of the anti-cancer drugs cause p53

phosphorylation at serine residues follow by activation of p53 pathway by mean of

genotoxic stress in cancer cells (Verma et al., 2010).

18

The concept of anti-cancer effect of Nutlin-3 through non-genotoxic way entice

the therapeutic point while most of conventional anti-cancer drugs induce toxic or

DNA damage which effect bystander normal tissue and potential to become secondary

malignancy later in life. Nutlin-3 causes cell cycle arrest at G1/S and G2/M borders

through p21-dependent (Tovar et al., 2006) and apoptosis through p73 protein

dependent follow by activation of PUMA and Noxa in wild-type p53 tumor cells (Lau et

al., 2008) (figure 9). Nutlin-3 also reduces the migration and invasion ability of p53

wild-type tumor cells by suppressing the activity of RhoA and Rac1 (figure 9) (Moran

and Maki, 2010). Rho A is Ras homolog gene family and regulates the actin

cytoskeleton in the formation of fibers within the cell. Rac1 is Ras-related C3

botulinum toxin substrate 1 protein in human cells regulate cells to cells adhesion,

motility and epithelial differentiation. Nutlin-3 has an anti-angiogenic effects through

inhibition of VEGF (vascular endothelium growth factor) by interfering HDM2 and HIF-

1α binding (hypoxia-inducible factor 1α) under hypoxic situation as HDM2 binds with

HIF-1α promotes the release of VEGF (LaRusch et al., 2007) (figure 9).

However, there are some issues for Nutlins needs to be considered. Some

studies shown Nutlin-3 exposures with wild-type p53 cancer cells or fibroblasts caused

senescence and cell cycle arrest but it was not a permanent arrest, then got recovery

after stopping exposure to Nutlin-3 and resume cycling. These findings showed it was

likely to have a major problem in Nutlin-3 based treatment (Efeyan et al., 2007). There

was a report that Nutlin resistant occur in HDM2 mutation cases such as mutation

either in or adjacent to the N-terminal domain of HDM2 protein. The mutant HDM2

(M62A and Q24R) can reduces the transcriptional activity of p53 protein and interferes

the Nutlins binding on HDM2 protein and becoming resistance (Wei et al., 2013a) but

one study showed a stapled peptides can inhibit the Nutlin-resistant mutant HDM2
19

cancer cells (Wei et al., 2013b). In addition, the interaction of HDMX and nutlin

happens away from the binding site with very short-lived (residence time) and less

direct interactions with the binding site when compared to HDM2. Therefore, the

activity of nutlin on HDMX protein was reduced because of the different topologies of

the electrostatic potentials of these two proteins (Elsawy et al., 2013).

HDM2

p53

Figure 9. Molecular Mechanisms of the actions of Nutlin-3

Nutlin-3 binds to the p53 binding pocket in HDM2 and inhibits the p53-HDM2 interaction, leading to
stabilization of p53 and induction of p53 and induction of p53-dependent cell cycle arrest or
apoptosis. Nutlin-3 can also inhibit cancer cell migration and invasion in a p53-dependent manner,
most likely through inhibition of RhoA and Rac 1 activity. In addition to p53, Nutlin-3 also blocks the
interaction of HDM2 with p73, E2F1, HIF-1α, resulting in increased apoptosis and decreased VEGF
production and decreased angiogenesis (HIF-1α). Nutlin-3 is predicted to block the interaction of
HDM2.

Taken from (Shen and Maki, 2011)

To date, most human cancer tumors are treated with conventional genotoxic

chemotherapy and radiation therapy. Majority of chemotherapeutic agents are

cytotoxic compounds with non-specific targets and the most at risk tissue are those

with high cell division either normal or cancer cells (Blagosklonny, 2004; Sikora et al.,

20

1999). The lack of selectivity becomes the side effect of chemotherapeutic agents

causing hair loss, appetite changes, fatigue, nausea, vomiting and pancytopenia

amongst cancer patients. In the development of selective anti-cancer agents, most of

the researches are focusing on the cure rate with the current available drugs by

optimizing drug combinations or refining drug delivery to malignant tumors. As the

cytotoxic drugs target the dividing or cycling cells either normal or cancer cells, the

chemoprotection can be attained by selectively causing temporary cell cycle arrest in

normal cells which have high cell division rate while cancer cells are dividing. This idea

was known as cyclotherapy (Blagosklonny and Darzynkiewicz, 2002; Blagosklonny and

Pardee, 2001). In this case, a p53 activator will stop normal high dividing cells from

proliferation (quiescence stage) while leaving the p53-deficient tumor vulnerable to

current traditional genotoxic chemotherapy (Carvajal et al., 2005; Kranz and

Dobbelstein, 2006; Van Leeuwen et al., 2012).

The cyclotherapy-chemotherapy combination becomes considerable choice in

cancer treatment to cure with higher protection on normal cells without reducing the

chemotherapy efficacy of the treatment. Nutlin-3 is also used as a small-molecule p53

activator in cyclotherapy regimes. Since, it has a reversible cytostatic effect leading to

the collection of cells in G1 and G2 phases of cell circle as well as protecting cells from

cytotoxicity tested by using S and M phase-specific poisons. Amongst p53 activators,

nutlin-3 becomes the most hopeful chemoprotectant in general with fine and highly

selective safety for normal cells from anticancer drugs tested (Van Leeuwen et al.,

2012; Vassilev et al., 2004). In addition, nutlin-3 can completely prevent from

neutropenia in mouse treated with an anticancer agent (Sur et al., 2009). Nutlin-like

compounds are now going through phase I clinical trial and have been experimented

21

with different chemotherapeutic drugs as combination therapy in many cancer types

(Table 3).

Table&3.((Summary(of(published(experimental(drug(combination(with(nutlin

No. Drugs(combinations Tumour(types((xenograft(models)

1 Nutlin(with(doxorubincin;(nutlin(with(cytarabine AML(and(B9CLL
2 Nutlin(with(vincristine Neuroblastoma,(rhabdomyosarcoma(and(melanoma
3 Nutlin(with(roscovitine Various(cancers
4 Nutlin(with(valproic(acid AML
5 Nutlin(with(Aurora(kinase(inhibitors Various(Cancers
6 Nutlin(with(1,2,59dihydroxyvitamin(D3 AML
7 Nutlin(with(XIAP(inhibitor Ovarian(cancer
8 Nutlin(with(cisplatin Prostate(cancer
9 Nutlin(with(androgen9depleting(agent Melanoma
10 Nutlin(with(CDK1(inhibitor;JNJ97706621 Haematological(malignancies
11 Nutlin(with(TRAIL Renal(cell(carcinoma
12 Nutlin(with(ABT9737 Various(cancers
13 Nutlin(with(selumetinib(AZD6244) AML
14 Nutlin(with(KPT9185 AML
15 Nutlin(with(sorafenib((independent(of(p53(status) AML
16 Nutlin(with(dasatinib((independent(of(p53(status) B9CLL
17 Nutlin(with(radiation Lung(and(prostate(cancer
Abbreviation (:( AML,acute(myeloid(leukaemia;(B9CLL,(B(cell(chronic(lymphocytic(leukaemia;(CDK1,(cyclin9dependent(kinase1;(p53,(tumour(
suppressor(p53;(TRAIL,(TNF9related(apoptosis9inducing(ligand;(XIAP,X9linked(inhibitor(of(apoptosis(protein .

Taken from (Khoo et al., 2014)

1.2 Spiro-oxindole

The spiro-oxindole derivatives are small molecule HDM2 inhibitors developed

outline structure by a computational modeling to activate the p53 pathway by

resembling the three major amino acid residues on p53 protein which interact with the

binding pocket on the surface of HDM2 protein (figure 8C). In the X-ray crystallography

of p53-HDM2 proteins interaction, the indole ring of p53 residue Trp23 looks the most

essential part for binding with HDM2 protein. It was deep within a hydrophobic pocket

on the surface of HDM2 protein and has hydrogen bond between amino group and

backbone carbonyl of the residue Leu54 of HDM2 protein. Therefore, an oxindole

compound could mimic this tryptophan residue of p53 protein. A new group of potent

small molecule that inhibits the p53-HDM2 proteins interaction was identified based

on spiro-oxindole core structure as a commencing point from many natural alkaloids.

22

The spiro-oxindole can be used as a scaffold structure for a designed compound which

may mimic the main three amino acid residues of p53 protein (Murray and Gellman,

2007). The fist compound named MI-5 was designed to interact with HDM2 (figure 10)

with the oxindole moiety fits into the Trp23 pocket, the phenyl group deep into the

Phe19 pocket and the isopropyl group takes the Leu26 pocket respectively.

Figure 10. Structure-based design of a new class of spiro-oxindole derivative
compounds as HDM2 inhibitors.
Abbreviation : PK - pharmacokinetic, Ki - inhibitory constant

Taken from (Zhao et al., 2013b)

In a fluorescence polarization binding assay, MI-5 was showed to bind

recombinant HDM2 with the inhibitory constant (Ki) value of 8.5 𝜇M (Ding et al., 2005).

The further computational modeling found that addition of a chorine atom at the

phenyl group of MI-5 and replacement of the isopropyl group with tert-butyl help in

23

the interactions with HDM2 protein. As a result, a MI-17 (figure 10) was discovered

with the inhibitory constant (Ki) value 86 nM, which is 100 times more potent than the

first compound MI-5 in binding with HDM2 protein. It was seen to activate p53 protein

in tumor cells with wild-type p53. When MI-17 had the half maximal inhibitory

concentration (IC50) of 830nM in LNCaP prostate cancer cell line in growth inhibition

assay and it was 27 fold less effect in PC-3 cancer cell line with deleted p53. In p53-

HDM2 X-ray structure and mutation analysis show that Leu22, fourth residue of p53

protein, also seem to take a major role in the interaction of p53-HDM2 proteins. Wang

and colleagues used a 2-morpholin-4-yl-ethylamine compound placing at the 5’-

carbonyl carbon of MI-17 and it will mimic the Leu22 of p53 protein as well as better

physiochemical properties. MI-63 (figure 10) was obtained from further optimization

by adding a fluorine substitution on phenyl ring of MI-17 structure to get more

metabolic stability of compound and it had inhibitory constant (Ki) value of 3nM in

binding with HDM2 protein.

Although MI-63 has fine solubility in water, it only has inferior oral

bioavailability about 10% in pharmacokinetic studies in mice (Ding et al., 2006). By

improving the bioavailability of MI-63, MI-219 (figure 10) was resulted. It binds to

HDM2 protein with inhibitory constant (Ki) value of 5nM, compare with 36nM of

nutlin-3 and has 65% of oral bioavailability tested in mice. The additional studies were

done to identify the therapeutic potential and molecular mechanism of action of MI-

219 (Yu et al., 2009). It has excellent specificity for inhibiting the p53-HDM2 proteins

interaction and excellent cell permeability, which is a critical value of small molecule as

an anti-cancer agent. Moreover, MI-219 has more than 10,000-fold selectivity for

HDM2 protein relative to HDMX even thought HDMX is a closely related homolog of

HDM2 and both using the similar binding pocket during interact with p53 protein. MI-
24

219 causes activation of p53 pathway in cells with wild-type p53 by inhibiting the

interaction between p53 and HDM2 proteins, but it failed to activate in the cell with

mutated p53. It also effectively inhibited the cell growth in many cancer cells (SJSA-1,

LNCaP, and 22Rv1) with wild-type p53 with the half maximal inhibitory concentration

(IC50) of approximately 1 𝜇M and has 10 fold more selective to cancer cells with

mutated or deleted p53 protein. Although MI-219 failed to cause tumor regression, it

can completely inhibit the tumor growth in many xenograft mouse models of human

cancer (Shangary et al., 2008). MI-219 caused p53 protein activation in normal murine

tissue head to cell cycle arrest without cell death and no tissue damage despite

repeated dosing but induced apoptosis in cancer cells. By extended studying in vivo

and vitro for MI-219, the data shown it has a potent and a specific inhibitor of HDM2

protein and reached into preclinical study. The further optimization of the

pharmacokinetic properties of MI-219 resulted in MI-888 (figure 10). It has inhibitory

constant (Ki) value of o.44 nM with high potency and selectivity in blocking growth of

tumor cells with wild-type p53 protein over mutated or deleted p53 protein. In

addition, MI-888 has wonderful oral bioavailability in mouse and causes tumor

regression in animal models of human cancer by daily oral route. The MI-888 analogue

has been reached into Phase 1 clinical development (Zhao et al., 2013b).

2. Natural Product Inhibitors of p53-HDM2 proteins interaction

Around 60 percent of many drugs that interfere p53-HDM2 proteins interaction

in the market are from natural line (Newman et al., 2003). There are only three

compounds from natural origin having inhibitory action against p53-HDM2 proteins

interaction; Chalcone, Chorofusin and Hexylitaconic acid (Murray and Gellman, 2007).

Among them, chalcone based inhibitor was the first and most extensive studies

25

(Bowman et al., 2004; Kumar et al., 2003; Stoll et al., 2001) while chlorofusin was the

second inhibitor (Duncan et al., 2001). In addition, Hexylitaconic acid was also

currently been recorded as one of inhibitor from natural origin (Tsukamoto et al.,

2006).

2.1 Chalcones

Holak and coworkers identified the interaction between p53-HDM2 proteins

interaction and chalcones due to its broad antitumor activities in 2001 (Stoll et al.,

2001). They found that chalcone was bound in the tryptophan pocket of HDM2 protein

and the second aromatic ring contacts with HDM2 residues Phe55 and Try56 outside

of the canonical p53 binding pocket. Although chalcone class compound was identified

as the inhibitor of p53-HDM2 proteins complex, it was not very specific for the target

protein. The p53 protein released from the p53-HDM2 proteins complex treated with

chalcone was unable to bind with DNA. Since it would effects the activity and

conformation of p53 protein. Isoliquiritigenin, a natural chalcone, can induces cell

cycle arrest and apoptosis in hepatic malignant cells through the activation of p53

pathway with the concentration of 10-20 𝜇g/ml except its binding to HDM2 protein

has not been identified (Hsu et al., 2005).

2.2 Chlorofusin

Chlorofusin, the inhibitor of p53-HDM2 proteins complex, was first identified

by Williams and colleagues from the fermentations of a multiple collection of

microorganisms (Duncan et al., 2001). It binds to the N-terminal residue of HDM2

protein with dissociation constant value of 4.7 𝜇 M. The future production of

chlorofusin analogues may yield more potent ligands for HDM2 than the other class of

natural products.

26

2.3 Hexylitaconic acid

Hexylitaconic acid was discovered from a fermentation culture of

marine-derived fungus, Arthrinium sp., using spectroscopic analysis. The inhibition of

p53-HDM2 proteins complex was tried out by ELISA (enzyme-linked immunosorbent

assay) method with IC50 of 50𝜇g/ml. Currently, many synthetic inhibitors of p53-HDM2

proteins complex have been appeared (Tsukamoto et al., 2006).

3. Proteomimetic inhibitor of p53-MDM2 proteins interaction

The proteomimetic inhibitors form an oligomeric scaffold, which mimic to α-helix

peptide of p53 protein. However, the structure of peptide is more bigger than the

small-molecule inhibitors, it permits the oligomers to bind more surface regions on

HDM2 protein and get better binding affinity. Many small-molecule inhibitors can not

bind to mutant HDM2, which have mutation on its N-terminal p53 binding domain and

it can be overcome by stapled peptides inhibitor which still have an affinity to bind

with above mutant HDM2 (Wei et al., 2013b). There is a limitation for proteomimetic

inhibitors such as inferior cell permeability and it not good for intracellular target sites

except protein-protein interaction present on the cell surface.

Although the present of potential advantages in proteomimetic inhibitors

relative to small-molecule inhibitors, most of the proteomimetic inhibitors for p53-

HDM2 proteins interaction are not as effective as some small-molecule inhibitors until

now. Among many peptide inhibitors, β-hairpin peptides have the ability of interfering

the p53-HDM2 proteins interaction like the Nutlins (Robinson, 2008). Currently, the

attempt to the development of proteomimetic inhibitors was notably diminished while

many small molecule inhibitors of p53-HDM2 proteins interaction were identified.

27

The following are some proteomimetic inhibitors;

1. ∝-peptide inhibitors

2. 𝛽-peptides

3. 𝛽-Oligobenzamides

4. Peptoids

5. 𝛽-Hairpin Protein Epitope Mimeitcs

3.1 𝜷-Hairpin Inhibitor

A cyclic 𝛽-hairpin that mimic the 𝛼-helix structure of p53 protein was identified

by Robinson and colleagues to get the effect of inhibition of p53-HDM2 proteins

interaction (Fasan et al., 2004). It was designed as a scaffold structure to keep the

amino acid residues of phenylalanine and tryptophan in the exact relative position in

order to interact with their respective binding pocket on the surface of HDM2 protein

and the first compound of cyclic 𝛽-hairpin was using D-Pro-L-Pro dipeptide turn unit to

stabilize the scaffold conformation (Jiang et al., 2000). The second compound of 𝛽-

hairpin peptide was identified by optimizing the first lead compound with a half

maximal inhibitory concentration (IC50) of 140 nM, about 900-fold more effective than

the lead compound (Fasan et al., 2006). This cyclic 𝛽-hairpin peptides were effectively

applied to many biomolecular targets and showing the advantage of these peptide

epitope mimetic (Descours et al., 2002; Dias et al., 2006; Shankaramma et al., 2003). It

needs to look whether it can applicable in vivo against the p53-HDM2 proteins

interaction, since 𝛽-hairpin peptides have poor cell permeability.

28


Conclusion

Targeting the protein-protein interaction of p53-HDM2 by a small molecule

HDM2 inhibitor shows much more complex and difficult than targeting the receptors

and enzymes on the cell surface. But the major improvement has been done in the last

decade in identifying many potent small molecule compounds and some have been

reached clinical trials. So far, seven small molecule inhibitors of p53-HDM2 proteins

interaction (Table 2) have been progressed into Phase I clinical trial and they may be

the most potential compounds for the future anti-cancer drugs. But there have been

needed to take an effort on development of small molecule inhibitors for p53-HDMX

proteins interaction as it contributed in most cancers development. Besides, whether

HDM2 inhibitors can be used as a single or combination agents in the treatment of

malignancies is still need to be verified (Muller and Vousden, 2013).

29


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