University of Groningen Development of novel anticancer agents for protein targets Estrada Ortiz, Natalia

Development of novel anticancer agents for protein targets

Estrada Ortiz, Natalia

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from

it. Please check the document version below.

Document Version

Publisher's PDF, also known as Version of record

Publication date:

2017

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

Estrada Ortiz, N. (2017). Development of novel anticancer agents for protein targets. University of

Groningen.

Copyright

Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

DEVELOPMENT OF NOVEL ANTICANCER AGENTS

FOR PROTEIN TARGETS

N

ATALIA

E

STRADA

O

RTIZ

2017

Constantinos Neochoritis Viktoriia Starokozhko

Cover design: Felipe Uribe Morales Layout design: Natalia Estrada Ortiz Printed by: Ipskamp printing

The research presented in this thesis was financially supported by the Department of Science, Technology and Innovation of the Colombian Government (Colciencias). Printing of this thesis was supported by the University of Groningen, Faculty of Science and Engineering and the University Library.

ISBN (printed version): 978-94-034-0142-3 ISBN (digital version): 978-94-034-0141-6

No parts of this thesis may be reproduced or transmitted in any form or any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system, without permission of the author

Agents for Protein Targets

PhD thesis

to obtain the degree of doctor at the University of Groningen under the authority of the rector Magnificus Prof. Dr. E. Sterken

and in accordance with the decision by the College of Deans. The public defense will take place on Friday 20 October 2017 at 16.15 hours

by

Natalia Estrada Ortiz

born August 17, 1985 in Medellin, Colombia

Prof. G.M.M. Groothuis Prof. A. Casini Assessment Committee Prof. F.J. Dekker Prof. P. Olinga Prof. R.J. Pieters

,WdZϭ

'›Ä›Ùƒ½/ÄãÙʗç‘ã®ÊÄ

ϭ

,WdZϮ

®Ã݃ėÊç㽮ěÊ¥㫛㫛ݮÝ

ϭϵ



WZd

/Ä«®®ãÊÙÝÊ¥WϱϯͬD—ÃϮ/Äã›Ùƒ‘ã®ÊÄ

,WdZϯ

,ÊóãÊ—›Ý®¦ÄƒÝ瑑›ÝÝ¥ç½ÖϱϯͲ×ÃϮͬø®Ä«®®ãÊÙ͗

Ϯϱ



ƒã«ÊÙÊ禫Êò›Ùò®›óƒÝ›—ÊÄ‘ÙùÝヽÝãÙç‘ãçٛÝ

,WdZϰ

Ù㮥®‘®ƒ½ÑÙʑù‘½›ÝƒÝÖÊã›ÄãÖϱϯͲ×ÃϮ®Ä«®®ãÊÙÝ ϱϱ

,WdZϱ

Ϯ͕ϯ͛Ͳ®Ý;ϭ͛«Ͳ®Ä—ʽ›Ϳ,›ã›Ùʑù‘½›Ý͗

ϭϬϵ



E›óÖϱϯͬ×ÃϮͬ×ÃøÄユÊÄ®ÝãÝ

WZd

®Ê½Ê¦®‘ƒ½‘ã®ò®ãùÊ¥sƒÙ®ÊçÝ&ƒÃ®½®›ÝÊ¥D›ãƒ½



ÊÃÖ½›ø›Ý

,WdZϲ

'ʽ—;®Ϳ‘ÊÃÖ½›ø›Ýó®ã«½ƒÄÝÊÖكþʽ›Ͳãù֛½®¦ƒÄ—Ý͗

ϭϰϱ



›øò®òÊãÊø®‘ʽʦ®‘ƒ½›òƒ½çƒã®ÊÄ

,WdZϳ

E›ó/ÄÝ®¦«ãÝ®ÄãÊ㫛dÊø®‘®ãùƒÄ—dكÄÝÖÊÙã

ϭϲϭ



D›‘«ƒÄ®ÝÃÝÊ¥®ÝÖ½ƒã®Ä®Ä»®—Ä›ù®Ä‘ÊÃփٮÝÊÄ



ãʃ¦Ê½—ͲƒÝ›—ƒÄ㮑ƒÄ‘›Ùƒ¦›Äã

,WdZϴ

Ä㮑ƒÄ‘›Ù¦Ê½—ÄͲ«›ã›Ùʑù‘½®‘‘ƒÙ›Ä›‘ÊÃÖ½›ø›Ý͗ ϭϴϯ



ƒ‘ÊÃփكã®ò›®Äò®ãÙʃė›øò®òÊÝãç—ù

,WdZϵ

^çÃÃÙùƒÄ—®Ý‘çÝÝ®ÊÄ

Ϯϭϱ



;E›—›Ù½ƒÄ—Ý›^ƒÃ›Äòƒãã®Ä¦Ϳ

ϮϮϱ

WWE/y



‘»ÄÊ󽛗¦›Ã›ÄãÝ

Ϯϯϵ



Êçã㫛çã«ÊÙ

Ϯϰϭ

CHAPTER

1

C

ANCER

Cancer is a group of diseases involving abnormal cell growth with the potential to spread to other parts of the body, affecting populations in all countries and all regions. In 2012 there were an estimated 14.1 million new cases of cancer diagnosed worldwide (excluding non-melanoma skin

cancer) and 8.2 million estimated deaths from neoplastic conditions.1,2 _{Specifically, cancer is a disease}

of abnormal gene expression, comprising more than 100 types of malignant neoplasms affecting humans and its increased incidence is often associated with genetic predisposition, factors as gender, ethnicity and age. Additionally, lifestyle choices as smoking, reduced physical activity, poor diet, and environmental exposure to toxicants, are known to be linked to the occurrence of a multitude of

neoplastic diseases (Figure 1).3

Figure 1. Risk factors for development of cancer

In the last decades the main interest of various pharmaceutical companies is the development and

discovery of potential anticancer drugs and treatments.4,5 For several years the research was focused

on the discovery and development of conventional cytotoxic compounds. However, this approach showed a major liability, adverse effects due to general toxicity, due to poor selectivity of the drug candidates.

In fact, in the last 20 years, a great number of new experimental chemotherapeutic compounds have not been approved by the FDA (U.S Food and Drug Administration) and EMA (European Medicines

Agency) due to their undesirable toxicity and cross reactivity observed in clinical trials.6 Recently,

thanks to the advances in genomics and proteomics, our understanding of cancer has grown considerably, and enables the design of targeted therapies, leading to changes in the paradigms of

anticancer drug discovery towards molecularly targeted therapeutics.7

The hallmarks of the events leading to neoplastic growth shared by most of the human cancers are depicted in Figure 2 and include: self-sufficiency in growth signals, insensitivity to anti-growth signals,

1

2

3

4

5

6

7

8

9

can be targeted to develop anticancer drugs (small molecules, antibodies, etc.), hopefully with reduced side effects.

Figure 2. The hallmarks of cancer and therapeutic targeting strategies. Adapted from: hallmarks of cancer new generation, Hanahan, D.; Weinberg, R. A, 2011.

C

HEMOTHERAPY STRATEGIES

:

Most of the anticancer chemotherapeutic agents have severe side effects due to possible interactions with various biological targets, low tissue and cell specificity (i.e. no discrimination between healthy and cancer cells). Another drawback is the development of drug resistance particularly in very

advanced tumors.10 In general, the mechanism of action of the classical anticancer drugs involves

interference with pathways of cellular division, synthesis of nucleic acids and/or cell cycle proteins. This alteration can affect rapidly dividing healthy cells, leading to adverse responses, including

induction of new tumors as a response to the genotoxicity of the treatment itself.11

The main targets of most of the new molecular-targeting drugs can be listed as receptors, membrane phospholipids, integrins, adhesion molecules, membrane-anchored receptors, enzymes, signaling proteins, RNA, microRNA, and DNA. They are typically dysregulated in tumor cells, compared to

normal tissue and the novel anti-cancer drugs are designed to specifically modulate these targets.7,12

This dysregulation can occur as specific mutations or changes in the expression profile of certain proteins or transcription factors, to allow the tumor cells to grow rapidly avoiding the cell cycle check points that in normal conditions should lead to apoptosis or cell death. Therefore, targeting the specific dysregulated pathways should reduce the side effects observed in the traditional anticancer drugs.

About 60% of the compounds available for chemotherapy are from natural origin 13–15 _{including taxol}

obtained from T. brevifolia, vincristine derived from the plant V. rosea, doxorubicin and bleomycin

(figure 3), fermentation products of bacteria from the Streptomyces genus, among others.4,15,16

O O O O O O NH O HO OH HO O O O O O O O OH OH O OH OH O O OH NH2 N N H2N HN O H N NH2 O NH2 NH2 O O O N H NH N O HO OH OH O O OH OH O NH2 O HO HN O NH O HO H H H H H N S S N N H O S H 1.Taxol 3.Doxorubicin 4.Bleomycine N H N O O OH N N O O O_O H OH H O O 2.Vincristine

Figure 3. Examples of anticancer natural drugs

As an alternative to small molecules as anticancer agents, antibody-based therapy is one of the most

valuable strategy to treat hematological malignancies and solid tumors.17,18 _{When the T cells of the}

immune system recognize cancer cells as abnormal, they generate a population of cytotoxic T lymphocytes (CTLs), that are able to locate and infiltrate malignancies, binding with them and subsequently killing the cancer tissue. CTLs, under normal physiological conditions require a balance

to avoid destroying healthy tissue surrounding the malignancies.17,19–22

However, this response, is often inefficient, since tumors can actively highjack immunity, upregulating

negative signals through cell surfaces, thereby inhibiting T-cell activation.18,19,22,23 _{The most common}

antibody therapies include targeting epidermal growth factor receptor (EGFR), vascular endothelial

growth factor (VEGF), lymphocyte-associated antigens CTLA4, CD20, CD30 and CD52.18 Recently, an

extra target came to play an important role in immunotherapy against cancer, programmed death-1 (PD-1) protein, several antibodies based drugs targeting the PD-1 pathway are currently in clinical

trials21 _{and a few had been approved by the FDA and the EMA.}24,25 _{Additionally, the antibody-targeted}

therapy includes antibody drug conjugates, using antibodies and cytotoxic drugs (cisplatin, doxorubicin, etc.) in a synergistic manner to boost the cancer elimination and survival rate in the

clinic.26 _{Some examples of the most successful antibodies based drugs in the market to treat cancer}

are cetuximab, bevacizumab and rituximab targeting EGFR, VEGF and CD20 antigens, respectively 27,28

Small molecules (organic and metal-based) are an essential element in the arsenal of drugs to fight cancer. Their main targets include: DNA, tyrosine kinases, protein-protein interactions (p53/mdm2,

Bcl-xL/BH3, XIAP/Smac, etc), and modulation of protein synthesis.7 In the following section, the two

different approaches with organic and metal containing compounds that were studied in this thesis are described.

1

2

3

4

5

6

7

8

9

1.

T

ARGETING

P

ROTEIN

-

PROTEIN INTERACTIONS WITH SMALL MOLECULES

In human cells, there are more than 300,000 Protein-Protein Interactions (PPIs)29 _{and this number is}

greatly larger than the number of single proteins. PPIs are of utmost importance and are implicated in almost all biological processes. Proteins fulfill their roles by interacting with many other cellular components, and the various interaction patterns of a protein are at least as important as the biochemical activity of the protein itself. Therefore, specific and potent modulation of PPIs by small,

drug-like molecules would facilitate novel ways of drug discovery. 29–32 Although the biological

relevance of PPIs is evident, this target class has been problematic in the design of new drugs.33,34

Crystal structures produced during the last 30 years showed that PPI interfaces are generally flat and

large (roughly 1,000–2,000 Å2_{), making the design and development of small molecules that could}

target those regions troublesome.33,35 However, in the last decades, a huge progress has been made in

developing synthetic compounds that modulate PPIs, using several strategies, including peptide mimetics, virtual screening, structural based design or screening of natural products or synthetic

libraries.31 _{Currently, they are about 40 PPIs described as potential targets and several of these PPI}

inhibitors are currently in clinical trials.34,36 Herein, we present some examples of the most relevant

PPIs as oncology targets for small molecules.

1.1.

P53/M

DM

2(X)

The protein p53, described for the first time in 1979, is considered to be the cellular gatekeeper or

guardian for cell division and growth37,38 and was the first tumor-suppressor gene to be identified.39

P53 stimulates the expression of a set of downstream target genes that can induce apoptosis, facilitate DNA repair or activate cell cycle arrest upon cellular stress signals induced by DNA damage,

oncogene activation and hypoxia.40–43 P53 is mutated in over 50% of human cancers and these

mutations are related to the DNA-binding domain and preclude p53 from acting as a transcription

factor.44 _{In other cases, the activity of p53 is inhibited either by binding to viral proteins or by}

alterations in other genes that code for proteins interacting with or linked to the function of p53 such

as mouse double minute MDM2 and MDMX.45

The level of p53 in cells is subject to tight control, and its main non-redundant regulators are MDM2 and MDMX (MDM2 and MDM4 in mice, HDM2 and HDMX in humans), which cooperate with each

other as part of a feed-back loop to regulate p53 activity in a different fashion .46 _{Therefore, blocking}

the interaction between wild-type p53 and its negative regulators MDM2 and MDMX has become an important approach in oncology to restore p53s antitumor activity. Interestingly, based on the multiple disclosed compound classes and co-crystal structures, p53–MDM2 is perhaps the most

studied and targeted protein–protein interaction in the last decade.47–49 _{Furthermore, several}

compounds have proceeded into phase I, II and III clinical trials (Figure 4).30,49–51 Additional information

about the current state of development of inhibitors of this important protein-protein interaction can be found in chapter 3.

Figure 4. Main pathways with interesting PPIs to target with novel anticancer therapeutics. Adapted from: Protein– protein interactions and cancer: small Molecules going in for the kill, M. Arkin, 2005

1.2.

B

CL

-

X

L/BH3

Evasion of apoptosis is one of the hallmarks of cancer cells, and the Bcl-2 family proteins play a crucial

part.52,53 _{The Bcl-2 family of proteins consists of three main classes: the pro-apoptotic members Bax}

and Bak stimulate apoptosis by releasing cytochrome c from the mitochondria, while the anti-apoptotic members Bcl-2 and Bcl-xL inhibit Bax and Bak, and the BH3-only proteins (Bcl-2 homologue)

promote apoptosis by inhibiting Bcl-2 and Bcl-xL proteins.54 _{Up-regulation of Bcl-2 or Bcl-xL is a}

common phenomenon in cancer and is usually related to drug resistance. Therefore, inhibitors of the of Bcl-2 or Bcl-xL, by using BH3 analogues to mimic Bcl-2/BH3 or Bcl-xL/BH3 protein-protein

interaction, could induce apoptosis or work together with other know anticancer therapies.32,55 _{So far}

no dual inhibitor for both Bcl-2 and Bcl-xL made it to the clinic, but in 2016 a Bcl2 inhibitor, venetoclax (Figure 4), was approved by the FDA and the EMA for the treatment of chronic lymphocytic

leukaemia.56

1.3.

XIAP/S

MAC

Inhibitors-of-apoptosis proteins (IAPs) are negative regulators of apoptosis. X-linked inhibitor of apoptosis protein (XIAP), also known as inhibitor of apoptosis protein 3 (IAP3), is a protein that stops apoptotic cell death. XIAP is up-regulated in many cancers, therefore it has been an interesting

oncological target for drug discovery. IAPs bind and inactivate caspases 3, 7 and 9.57–59 The

pro-apoptotic protein Smac, a mitochondrial protein and negative regulator of XIAP, binds to IAPs, and

induces the activation of caspases thereby re-activating the apoptosis cascade.7,31 _Several

compounds have been developed to inhibit XIAP to modulate its activity, and currently there are

1

2

3

4

5

6

7

8

9

N O O O Cl N N N O 5.NVP-CGM097 O N N S O O N N Cl Cl O 6. (RG7112) N O O S O H N O H N O _O N NH N N Cl O 7.Venetoclax 8.ASTX-660 9.LCL-161 O N N H O N N F H Cl H Cl O H N N H O N S _N O F H

Figure 5. Small molecules in clinical trials as modulators of p53/MDM2 (5-6), BCL-XL/BH3 (7) and XIAP/SMAC (8-9) protein-protein interactions.

2. M

ETAL

-

BASED COMPOUNDS

Metals have been used in medicine for 5000 years:65 in 3000 BC the Egyptians were using copper to

sterilize water, to treat headaches, burns, and itching.66,67 _{Thereafter, in 2500 BC the Chinese were}

treating furuncles, smallpox and skin ulcers with pure gold.68 _{During the Renaissance mercurous}

chloride was used as diuretic agent and at the beginning of last century, arsenic (arsphenamine) was

used to treat syphilis and gold cyanide was used for tuberculosis treatment.69 In 1912, antimony

compounds were introduced to treat leishmaniasis, and in 1929 gold compounds were officially

introduced as a therapeutic option for rheumatoid arthritis treatment.65,67,69

In the late 60’s, platinum drugs emerged as an alternative to treat cancer, and nowadays, they still

appear in more chemotherapy regimens than any other class of anticancer agents.67,70,71 Since then, a

multitude of experimental metal complexes have been developed showing different mechanisms of

action and applications.65,67,72–74

2.1.

C

ISPLATIN AND ANALOGUES

Like numerous cytotoxic anticancer compounds, cisplatin was accidentally discovered by Barnett

Rosenberg while he was studying the effect of electric fields on bacterial growth.75,76 E. coli cells were

grown in medium containing ammonium chloride buffer, and an electrical current was applied to the bacteria through platinum electrodes submerged in the solution. and inhibition of the bacterial cell

division was observed in the treated cultures.76,77 After a thorough investigation, it was found that the inhibition was induced by the hydrolysis products from the platinum electrodes which behave as

cytostatic agents.78 _{Following these initial experiments, cisplatin was tested against panels of human}

cancer cell lines and found to be a potent cytotoxic compound. The mechanism of action of cisplatin is generally thought to be its interaction with DNA to form DNA-cisplatin adducts, primarily 1,2-intrastrand cross-links with purine bases (Figure 6). The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis through several signal transduction pathways, including ATR, p53,

p73, and MAPK, leading finally to cell death.79–81

Pt H₃N Cl H₃N Cl Pt H₃N Cl H₃N OH2 Pt H₃N

H₃N Repair not posible

Apoptosis!

H₂O

Figure 6. Molecular mechanism of cisplatin toxicity.

The potency of cisplatin was demonstrated in clinical studies, and it is currently used to treat different

types of cancers including sarcomas, cancers of soft tissue, bones, and muscles.80 _{Unfortunately the}

side effects associated with cisplatin treatment in the clinic include severe toxicity to the kidney and the nervous system, hearing difficulties, nausea, and vomiting, among others. Subsequently, several studies were performed to optimize the properties of cisplatin, and to reduce its side effects, leading to the development and approval of carboplatin and oxaliplatin (Figure 7). Both these compounds became alternatives for clinical use for cancer types with acquired resistance to cisplatin and they are

included along with cisplatin in the list of essential drugs of the world health organization.27,81–83

Pt H3N Cl H₃N Cl 10. Cisplatin Pt H3N O H₃N O O O Pt HN O N H O O O 11. Carboplatin 12. Oxaliplatin Figure 7. Cisplatin and derivatives.

2.2.

O

THER METALS

The discovery of cisplatin and its platinum (II) analogues, suggested that other metal complexes might have similar antitumor activity, and probably could display a diverse pattern of cancer cells toxicity and

selectivity.65 _{Starting from the 80’s, numerous metal containing compounds have been studied as}

potential anticancer agents, including ruthenium, palladium, copper, tin, and gold, among others.70

Metal-based compounds are known to bind and interact with a diversity of proteins with different

roles, including transporters, antioxidants, electron transfer proteins, and DNA-repair proteins. 70,84–87

Among the possible pharmacological targets, several proteases are inhibited by Pt(II), Ru(II), Re(IV),

Cu(II) and Co(III) complexes74,88–90_{. Several studies reported on the proteasome inhibition by}

1

2

3

4

5

6

7

8

9

trans-tetrachloro-(dimethylsulfoxide)imidazole-ruthenate(III), and KP1019 were the first Ruthenium

compounds to enter into clinical trials for anticancer therapy.67 Ruthenium complexes showed a

different mechanism of action compared to the known platinum drugs, with reduced cytotoxicity and without inhibition of primary tumor growth, nonetheless, the complex was able to decrease the

spread of metastasis.95,96 Ru DMSO NH Cl Cl Cl Cl HN H N 14.NAMI-A O OV O O O O 13. Bis(maltolato)oxovanadium(IV)

Figure 8. Examples of metal containing drugs studied for the treatment of diabetes and cancer

2.3.

G

OLD COMPLEXES

Gold-based complexes are particularly interesting due to their different possible oxidation states (e.g. Au(I) and Au(III)), stability and ligand exchange reactions, which confer them different mechanisms of

activity compared to cisplatin.97–99 _{Preliminary studies on the anticancer activity of the Au(I) complex}

auranofin ([Au(I)(2,3,4,6-tetra-O-acetyl-1-(thio-NS)-E-D-glucopyranosato)(triethylphosphine)]),

presently used in the clinic to treat severe rheumatoid arthritis, revealed cytotoxic activity levels

similar to cisplatin on cancer cells in vitro (Figure 9).100 _{Auranofin is currently undergoing clinical trials}

to treat cancer, HIV, amoebiasis and tuberculosis.101 _{These findings subsequently led to a large}

number of Au(I) complexes being currently evaluated and developed as cytotoxic anticancer agents.98

O S OAc AcO AcO OAc Au P 15.Auranofin N N Au Cl 16 N N N N Au ) O O N N N N ( O O BF4 17

Figure 9. Gold complexes with interesting anticancer activity

Among the possible targets for cytotoxic Au(I) complexes, the seleno-enzyme thioredoxin reductase

(TrxR) is among the most studied protein targets.70,74 TrxR belongs to the thioredoxin system involved

in H2O2 detoxification and it is overexpressed in a number of cancer types.70,102 The thioredoxin system

comprises the small redox protein thioredoxin (Trx), reduced nicotinamide adenine dinucleotide phosphate (NADPH), thioredoxin reductase (TrxR) and peroxiredoxin (Prx) (Figure 10). Thioredoxins are proteins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange, they are found in nearly all known organisms. Mitochondria are considered the most important cellular sources of reactive oxygen species (ROS). Superoxide anion can originate from the respiratory chain and is converted by superoxide dismutase to hydrogen peroxide, which is subsequently inactivated by the glutathione and thioredoxin systems leading to a steady state

between formation and consumption of hydrogen peroxide.70,102

Gold is known for its high affinity towards thiol groups and consequently it is hypothesized that Cys-Se-Cys moiety present in C-terminal

Figure 10. Thioredoxin system scheme leading to intracellular redox balance by conversion of H2O2. From: Thioredoxin

reductase: A target for gold compounds acting as potential anticancer drugs. Adapted from: A. Bindolini, et al., 2009.

The inhibition of thioredoxin reductase by gold(I/III) complexes, makes it unable to reduce back oxidized thioredoxin that accumulates together with hydrogen peroxide and both act on different intramitochondrial targets leading to the opening of the mitochondrial permeability transition pore and/or to an increase of the permeability of the outer membrane. Hydrogen peroxide is released to the cytosol where causes oxidation of Trx1, that, similarly, to mitochondrial thioredoxin (Trx2), cannot be reduced back by the gold(I/III) complexes-inhibited thioredoxin reductase. Oxidized thioredoxin stimulates the MAP kinases pathways leading to cell death. (Figure 11).

Figure 11. Mechanism of thioredoxin system by gold(I/III) complexes leading to cellular death. Adapted from: Thioredoxin reductase: A target for gold compounds acting as potential anticancer drugs, A. Bindolini, et al., 2009.

One of the most representative family of gold(I) compounds tested for their anticancer effects, is the organometallic gold(I) N-heterocyclic carbene (NHC) complexes featuring anticancer activity in the

micromolar or sub-micromolar range in vitro.104–109 _{For example, compound 16 (Figure 9) and its}

derivatives displayed cytotoxic activity in the low micromolar range towards a small panel of cancer cell lines, comparable with cisplatin activity, and showed no interactions with model proteins such as lysozyme and cytochrome c. Conversely, they formed adducts with Atox-1 and it may imply that these

1

2

3

4

5

6

7

8

9

accumulate selectively inside the mitochondria of cancer cells due to their higher mitochondrial

membrane potential),109,112–114 . Furthermore, inhibition of protein tyrosine phosphatases (PTP) was

also reported for certain anticancer Au(I) NHC complexes,115 and even stabilization of DNA

G-quadruplexes (Figure 9, compound 17).98,116,117

C

ONCLUDING REMARKS

Over the last decades, the discovery and development of cancer therapeutics has been a rapidly growing area, with different approaches and applications to improve the current therapies used in the clinic nowadays.

However, adverse side effects are still a main concern in the drug development process. Therefore, it is important to improve the methodologies and the screening systems to determine potential adverse effects of drug candidates. Targeted therapies are designed to tackle cancer cells on their weak spots, specific miss-regulated pathways, but it is still necessary to carefully study possible off-targets to avoid side effects.

There is a growing need to improve the efficiency of developing new drug-like compounds. A new

compound entering phase 1 clinical trial for any indication has about 8%4 _{chance to make it to the}

next clinical phase.

Drugs with different chemical profile, such us metal complexes, besides cisplatin, offer a new opportunity to discover new targets and ways to modulate toxicity directed to cancer cells due to the specific redox balance and microenvironment of such cellular group. Additionally, classic organic compounds, can be produced in mass to discover new lead hits. A way to reduce time and optimize the chemical synthesis is the use of multicomponent reactions (MCRs) to produced libraries of compounds that can be tested for biological activity on High Throughput Screening (HTS) systems to validated Structure Activity Relationship (SAR) and select lead compounds for further optimization. In this thesis, we present the design and evaluation of new p53-MDM2/X inhibitors based on previously described compounds, to increase their potency and explore a different region on the p53-MDM2/X interphase as drug target. Additionally, a series of gold complexes were studied to understand their possible mechanism of action compared with cisplatin, including cancer cell based studies and healthy tissue toxicity evaluation using rat Precision Cut Tissue Slices (PCTS) to unravel uptake, pathways involved in the toxicity and possible selectivity of the compounds towards cancer cells.

R

EFERENCES

(1) Ferlay, J.; Soerjomataram, I.; Ervik, M.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.; Forman, D.; Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC. Lyon, France: International Agency for Research on Cancer. 2013.

(2) World Cancer Report 2014; Stewart, B.,

Wild, C., Eds.; International Agency for Research on Cancer: Lyon, France, 2014. (3) Jemal, A.; Bray, F.; Center, M. M.; Ferlay,

J.; Ward, E.; Forman, D. Global Cancer Statistics. CA. Cancer J. Clin. 2011, 61 (2), 69–90.

(4) Narang, A. S.; Desai, D. S. Anticancer Drug Development. In Pharmaceutical

Perspectives of Cancer Therapeutics; Lu, Y.,

Mahato, R. I., Eds.; Springer US, 2009; pp 49–92.

(5) Kummar, S.; Gutierrez, M.; Doroshow, J. H.; Murgo, A. J. Drug Development in Oncology: Classical Cytotoxics and Molecularly Targeted Agents. Br. J. Clin.

Pharmacol. 2006, 62 (1), 15–26.

(6) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand Efficiency: A Useful Metric for Lead Selection. Drug Discov. Today 2004, 9 (10), 430–431.

(7) Turkson, J. Cancer Drug Discovery and Anticancer Drug Development. In The

Molecular Basis of Human Cancer;

Coleman, W. B., Tsongalis, G. J., Eds.; Springer New York, 2017; pp 695–707. (8) Hanahan, D.; Weinberg, R. A. The

Hallmarks of Cancer. Cell 2000, 100 (1), 57–70.

(9) Hanahan, D.; Weinberg, R. A. Hallmarks of Cancer: The Next Generation. Cell 2011,

144 (5), 646–674.

(10) Li, Y. T.; Chua, M. J.; Kunnath, A. P.; Chowdhury, E. H. Reversing Multidrug Resistance in Breast Cancer Cells by Silencing ABC Transporter Genes with Nanoparticle-Facilitated Delivery of Target SiRNAs. Int. J. Nanomedicine 2012, 7, 2473–2481.

(11) Valerio, L. G.; Cross, K. P. Characterization and Validation of an in Silico Toxicology Model to Predict the Mutagenic Potential of Drug Impurities. Toxicol. Appl.

(13) Gordaliza, M. Natural Products as Leads to Anticancer Drugs. Clin. Transl. Oncol. 2007, 9 (12), 767–776.

(14) Guilford, J. M.; Pezzuto, J. M. Natural Products as Inhibitors of Carcinogenesis.

Expert Opin. Investig. Drugs 2008, 17 (9),

1341–1352.

(15) Cragg, G. M.; Newman, D. J.; Yang, S. S. Natural Product Extracts of Plant and Marine Origin Having Antileukemia Potential. The NCI Experience٣. J Nat Prod 2006, 69 (3), 488–498.

(16) Newman, D. J.; Cragg, G. M. Natural Products as Sources of New Drugs over the Last 25 Years. J. Nat. Prod. 2007, 70 (3), 461–477.

(17) Carter, P. Improving the Efficacy of Antibody-Based Cancer Therapies. Nat.

Rev. Cancer 2001, 1 (2), 118–129.

(18) Scott, A. M.; Wolchok, J. D.; Old, L. J. Antibody Therapy of Cancer. Nat. Rev.

Cancer 2012, 12 (4), 278–287.

(19) He, J.; Hu, Y.; Hu, M.; Li, B. Development of PD-1/PD-L1 Pathway in Tumor Immune Microenvironment and Treatment for Non-Small Cell Lung Cancer. Sci. Rep. 2015, 5, 13110.

(20) Zak, K. M.; Kitel, R.; Przetocka, S.; Golik, P.; Guzik, K.; Musielak, B.; Dömling, A.; Dubin, G.; Holak, T. A. Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1. Structure 2015, 23, 2341–2348.

(21) Dömling, A.; Holak, T. A. Programmed Death-1: Therapeutic Success after More than 100 Years of Cancer Immunotherapy.

Angew. Chem. Int. Ed. 2014, 53 (9), 2286–

2288.

(22) Tumeh, P. C.; Harview, C. L.; Yearley, J. H.; Shintaku, I. P.; Taylor, E. J. M.; Robert, L.; Chmielowski, B.; Spasic, M.; Henry, G.; Ciobanu, V.; West, A. N.; Carmona, M.; Kivork, C.; Seja, E.; Cherry, G.; Gutierrez, A. J.; Grogan, T. R.; Mateus, C.; Tomasic, G.; Glaspy, J. A.; Emerson, R. O.; Robins, H.; Pierce, R. H.; Elashoff, D. A.; Robert, C.; Ribas, A. PD-1 Blockade Induces Responses by Inhibiting Adaptive Immune Resistance. Nature 2014, 515 (7528), 568– 571.

1

2

3

4

5

6

7

8

9

(24) FDA PD-1 PD-L1 https://google2.fda.gov/search?q=pd- 1%20pd-l1%20&client=FDAgov&proxystylesheet=F DAgov&output=xml_no_dtd&site=FDAgov &requiredfields=-archive:Yes&sort=date:D:L:d1&filter=1 (accessed May 17, 2017). (25) EMA PD-1 PD-L1 http://www.ema.europa.eu/ema/index.jsp ?curl=search.jsp&site=pfoi_collection&ent sp=0&sort=date:D:L:d1&client=pfoi_front end&curl=search.jsp&btnG=Search&entqr =0&oe=UTF-8&proxyreload=1&q=pd1+pd- l1&ie=UTF-8&ud=1&mid=&output=xml_no_dtd&prox ystylesheet=pfoi_frontend&filter=0&ulang =&ip=172.16.80.221&access=p&entqrm=0 &wc=200&wc_mc=1&start=70 (accessed May 17, 2017).

(26) Polakis, P. Antibody Drug Conjugates for Cancer Therapy. Pharmacol. Rev. 2016, 68 (1), 3–19.

(27) WHO Model List of Essential Medicines. 2015.

(28) Hendriks, D.; Choi, G.; de Bruyn, M.; Wiersma, V. R.; Bremer, E. Chapter Seven - Antibody-Based Cancer Therapy: Successful Agents and Novel Approaches. In International Review of Cell and

Molecular Biology; Galluzzi, L., Ed.;

Academic Press, 2017; Vol. 331, pp 289– 383.

(29) Bier, D.; Thiel, P.; Briels, J.; Ottmann, C. Stabilization of Protein–Protein

Interactions in Chemical Biology and Drug Discovery. Prog. Biophys. Mol. Biol. (30) Estrada-Ortiz, N.; Neochoritis, C. G.;

Dömling, A. How To Design a Successful P53-MDM2/X Interaction Inhibitor: A Thorough Overview Based on Crystal Structures. ChemMedChem 2016, 11 (8), 757–772.

(31) Arkin, M. Protein–protein Interactions and Cancer: Small Molecules Going in for the Kill. Curr. Opin. Chem. Biol. 2005, 9 (3), 317–324.

(32) Ivanov, A. A.; Khuri, F. R.; Fu, H. Targeting Protein-Protein Interactions as an Anticancer Strategy. Trends Pharmacol.

Sci. 2013, 34 (7), 393–400.

(33) Arkin, M. R.; Tang, Y.; Wells, J. A. Small-Molecule Inhibitors of Protein-Protein Interactions: Progressing toward the Reality. Chem. Biol. 2014, 21 (9), 1102– 1114.

(34) Scott, D. E.; Bayly, A. R.; Abell, C.; Skidmore, J. Small Molecules, Big Targets: Drug Discovery Faces the Protein-Protein Interaction Challenge. Nat. Rev. Drug

Discov. 2016, 15 (8), 533–550.

(35) Hwang, H.; Vreven, T.; Janin, J.; Weng, Z. Protein–protein Docking Benchmark Version 4.0. Proteins Struct. Funct.

Bioinforma. 2010, 78 (15), 3111–3114.

(36) Basse, M. J.; Betzi, S.; Bourgeas, R.; Bouzidi, S.; Chetrit, B.; Hamon, V.; Morelli, X.; Roche, P. 2P2Idb: A Structural Database Dedicated to Orthosteric Modulation of Protein-Protein Interactions. Nucleic Acids

Res. 2013, 41 (Database issue), D824-827.

(37) Lane, D. P. P53, Guardian of the Genome.

Nature 1992, 358 (6381), 15–16.

(38) Levine, A. J. P53, the Cellular Gatekeeper for Growth and Division. Cell 1997, 88 (3), 323–331.

(39) Finlay, C. A.; Hinds, P. W.; Levine, A. J. The P53 Proto-Oncogene Can Act as a Suppressor of Transformation. Cell 1989,

57 (7), 1083–1093.

(40) Vazquez, A.; Bond, E. E.; Levine, A. J.; Bond, G. L. The Genetics of the P53 Pathway, Apoptosis and Cancer Therapy.

Nat. Rev. Drug Discov. 2008, 7 (12), 979–

987.

(41) Fridman, J. S.; Lowe, S. W. Control of Apoptosis by P53. Oncogene 2003, 22 (56), 9030–9040.

(42) Sengupta, S.; Harris, C. C. P53: Traffic Cop at the Crossroads of DNA Repair and Recombination. Nat. Rev. Mol. Cell Biol. 2005, 6 (1), 44–55.

(43) Giono, L. E.; Manfredi, J. J. The P53 Tumor Suppressor Participates in Multiple Cell Cycle Checkpoints. J. Cell. Physiol. 2006,

209 (1), 13–20.

(44) Hainaut, P.; Hollstein, M. P53 and Human Cancer: The First Ten Thousand Mutations. Adv. Cancer Res. 2000, 77, 81– 137.

(45) Vogelstein, B.; Lane, D.; Levine, A. J. Surfing the P53 Network. Nature 2000,

408 (6810), 307–310.

(46) Khoury, K.; Holak, T. A.; Dömling, A. P53/MDM2 Antagonists: Towards Nongenotoxic Anticancer Treatments. In

Protein-Protein Interactions in Drug Discovery; Dömling, A., Ed.; Wiley-VCH

Verlag GmbH & Co. KGaA, 2013; pp 129– 163.

(47) Khoury, K.; Domling, A. P53 Mdm2 Inhibitors. Curr. Pharm. Des. 2012, 18 (30), 4668–4678.

(48) Popowicz, G. M.; Dömling, A.; Holak, T. A. The Structure-Based Design of

Mdm2/Mdmx–p53 Inhibitors Gets Serious.

Angew. Chem. Int. Ed. 2011, 50 (12),

2680–2688.

(49) Khoo, K. H.; Verma, C. S.; Lane, D. P. Drugging the P53 Pathway: Understanding the Route to Clinical Efficacy. Nat. Rev.

Drug Discov. 2014, 13 (3), 217–236.

(50) Dömling, A. P53-Mdm2 Antagonists. Patent Application WO 2012/033525 A3, 2012.

(51) Dömling, A.; Holak, T. Novel P53-Mdm2/P53-Mdm4 Antagonists to Treat Proliferative Disease. WO2011106650 A3, January 19, 2012.

(52) Liu, Q.; Chi, X.; Leber, B.; Andrews, D. W. Bcl-2 Family and Their Therapeutic Potential. In Cell Death; Wu, H., Ed.; Springer New York, 2014; pp 61–96. (53) O’Neill, J.; Manion, M.; Schwartz, P.;

Hockenbery, D. M. Promises and Challenges of Targeting Bcl-2 Anti-Apoptotic Proteins for Cancer Therapy.

Biochim. Biophys. Acta 2004, 1705 (1), 43–

51.

(54) Stauffer, S. R. Small Molecule Inhibition of the Bcl-XL-BH3 Protein-Protein

Interaction: Proof-of-Concept of an In Vivo Chemopotentiator ABT-737. Curr. Top.

Med. Chem. 2007, 7 (10), 961–965.

(55) Gavathiotis, E. Structural Perspectives on BCL-2 Family of Proteins. In Cell Death; Wu, H., Ed.; Springer New York, 2014; pp 229–251.

(56) Schenk, R. L.; Strasser, A.; Dewson, G. BCL-2: Long and Winding Path from Discovery to Therapeutic Target. Biochem. Biophys.

Res. Commun. 2017, 482 (3), 459–469.

(57) Huang, Y.; Park, Y. C.; Rich, R. L.; Segal, D.; Myszka, D. G.; Wu, H. Structural Basis of Caspase Inhibition by XIAP: Differential Roles of the Linker versus the BIR Domain.

Cell 2001, 104 (5), 781–790.

(58) Deveraux, Q. L.; Takahashi, R.; Salvesen, G. S.; Reed, J. C. X-Linked IAP Is a Direct Inhibitor of Cell-Death Proteases. Nature 1997, 388 (6639), 300–304.

(59) Listen, P.; Roy, N.; Tamai, K.; Lefebvre, C.; Baird, S.; Cherton-Horvat, G.; Farahani, R.; McLean, M.; Lkeda, J.-E.; Mackenzie, A.; Korneluk, R. G. Suppression of Apoptosis

A Small Molecule Smac Mimic Potentiates TRAIL- and TNFɲ-Mediated Cell Death.

Science 2004, 305 (5689), 1471–1474.

(61) Schimmer, A. D.; Welsh, K.; Pinilla, C.; Wang, Z.; Krajewska, M.; Bonneau, M.-J.; Pedersen, I. M.; Kitada, S.; Scott, F. L.; Bailly-Maitre, B.; Glinsky, G.; Scudiero, D.; Sausville, E.; Salvesen, G.; Nefzi, A.; Ostresh, J. M.; Houghten, R. A.; Reed, J. C. Small-Molecule Antagonists of Apoptosis Suppressor XIAP Exhibit Broad Antitumor Activity. Cancer Cell 2004, 5 (1), 25–35. (62) Vellanki, S. H. K.; Grabrucker, A.; Liebau,

S.; Proepper, C.; Eramo, A.; Braun, V.; Boeckers, T.; Debatin, K.-M.; Fulda, S. Small-Molecule XIAP Inhibitors Enhance ɶ-Irradiation-Induced Apoptosis in Glioblastoma. Neoplasia N. Y. N 2009, 11 (8), 743–752.

(63) Carter, B. Z.; Mak, D. H.; Morris, S. J.; Borthakur, G.; Estey, E.; Byrd, A. L.; Konopleva, M.; Kantarjian, H.; Andreeff, M. XIAP Antisense Oligonucleotide (AEG35156) Achieves Target Knockdown and Induces Apoptosis Preferentially in CD34+38- Cells in a Phase 1/2 Study of Patients with Relapsed/Refractory AML.

Apoptosis Int. J. Program. Cell Death 2011, 16 (1), 67–74.

(64) West, A. C.; Martin, B. P.; Andrews, D. A.; Hogg, S. J.; Banerjee, A.; Grigoriadis, G.; Johnstone, R. W.; Shortt, J. The SMAC Mimetic, LCL-161, Reduces Survival in Aggressive MYC-Driven Lymphoma While Promoting Susceptibility to Endotoxic Shock. Oncogenesis 2016, 5 (4), e216. (65) Orvig, C.; Abrams, M. J. Medicinal

/ŶŽƌŐĂŶŝĐŚĞŵŝƐƚƌLJ͗ര/ŶƚƌŽĚƵĐƚŝŽŶ͘Chem.

Rev. 1999, 99 (9), 2201–2204.

(66) Dollwet, H. H. A.; Sorenson, J. R. J. Historic Uses of Copper Compounds in Medicine.

Trace Elem. Med. 1985, 2 (2), 80–87.

(67) Mjos, K. D.; Orvig, C. Metallodrugs in Medicinal Inorganic Chemistry. Chem. Rev. 2014, 114 (8), 4540–4563.

(68) Huaizhi, Z.; Yuantao, N. China’s Ancient Gold Drugs. Gold Bull. 2001, 34 (1), 24–29. (69) Sadler, P. J. Inorganic Chemistry and Drug

Design. Adv. Inorg. Chem. 1991, 36, 1–48. (70) Bindoli, A.; Rigobello, M. P.; Scutari, G.;

Gabbiani, C.; Casini, A.; Messori, L. Thioredoxin Reductase: A Target for Gold

1

2

3

4

5

6

7

8

9

Pommier, Y.; Lippard, S. J.; Hemann, M. T. A Subset of Platinum-Containing Chemotherapeutic Agents Kills Cells by Inducing Ribosome Biogenesis Stress. Nat.

Med. 2017, 23 (4), 461–471.

(72) Zhang, C. X.; Lippard, S. J. New Metal Complexes as Potential Therapeutics. Curr.

Opin. Chem. Biol. 2003, 7 (4), 481–489.

(73) Bruijnincx, P. C. A.; Sadler, P. J. New Trends for Metal Complexes with Anticancer Activity. Curr. Opin. Chem. Biol. 2008, 12 (2), 197–206.

(74) de Almeida, A.; Oliveira, B. L.; Correia, J. D. G.; Soveral, G.; Casini, A. Emerging Protein Targets for Metal-Based Pharmaceutical Agents: An Update. Coord. Chem. Rev. 2013, 257 (19–20), 2689–2704.

(75) Rosenberg, B. Platinum Complexes for the Treatment of Cancer: Why the Search Goes On. In Cisplatin; Lippert, B., Ed.; Verlag Helvetica Chimica Acta, 1999; pp 1– 27.

(76) Alderden, R. A.; Hall, M. D.; Hambley, T. W. The Discovery and Development of Cisplatin. J. Chem. Educ. 2006, 83 (5), 728. (77) Rosenberg, B.; Van Camp, L.; Krigas, T.

Inhibition of Cell Division in Escherichia Coli by Electrolysis Products from a Platinum Electrode. Nature 1965, 205 (4972), 698–699.

(78) Rosenberg, B.; Vancamp, L.; Trosko, J. E.; Mansour, V. H. Platinum Compounds: A New Class of Potent Antitumour Agents.

Nature 1969, 222 (5191), 385–386.

(79) Siddik, Z. H. Cisplatin: Mode of Cytotoxic Action and Molecular Basis of Resistance.

Oncogene 2003, 22 (47), 7265–7279.

(80) Dasari, S.; Tchounwou, P. B. Cisplatin in Cancer Therapy: Molecular Mechanisms of Action. Eur. J. Pharmacol. 2014, 0, 364– 378.

(81) Hardie, M. E.; Kava, H. W.; Murray, V. Cisplatin Analogues with an Increased Interaction with DNA: Prospects for Therapy. Curr. Pharm. Des. 2016, 22 (44), 6645–6664.

(82) Canetta, R.; Rozencweig, M.; Carter, S. K. Carboplatin: The Clinical Spectrum to Date. Cancer Treat. Rev. 1985, 12, 125– 136.

(83) Mathé, G.; Kidani, Y.; Segiguchi, M.; Eriguchi, M.; Fredj, G.; Peytavin, G.; Misset, J. L.; Brienza, S.; de Vassals, F.; Chenu, E. Oxalato-Platinum or 1-OHP, a Third-Generation Platinum Complex: An Experimental and Clinical Appraisal and Preliminary Comparison with Cis-Platinum

and Carboplatinum. Biomed.

Pharmacother. Biomedecine Pharmacother. 1989, 43 (4), 237–250.

(84) Meggers, E. Targeting Proteins with Metal Complexes. Chem. Commun. 2009, No. 9, 1001–1010.

(85) Griffith, D.; Parker, J. P.; Marmion, C. J. Enzyme Inhibition as a Key Target for the Development of Novel Metal-Based Anti-Cancer Therapeutics. Anticancer Agents

Med. Chem. 2010, 10 (5), 354–370.

(86) Casini, A.; Reedijk, J. Interactions of Anticancer Pt Compounds with Proteins: An Overlooked Topic in Medicinal Inorganic Chemistry? Chem. Sci. 2012, 3 (11), 3135–3144.

(87) Che, C.-M.; Siu, F.-M. Metal Complexes in Medicine with a Focus on Enzyme Inhibition. Curr. Opin. Chem. Biol. 2010, 14 (2), 255–261.

(88) Fricker, S. P. Cysteine Proteases as Targets for Metal-Based Drugs. Met. Integr.

Biometal Sci. 2010, 2 (6), 366–377.

(89) Casini, A.; Gabbiani, C.; Sorrentino, F.; Rigobello, M. P.; Bindoli, A.; Geldbach, T. J.; Marrone, A.; Re, N.; Hartinger, C. G.; Dyson, P. J.; Messori, L. Emerging Protein Targets for Anticancer Metallodrugs: Inhibition of Thioredoxin Reductase and Cathepsin B by Antitumor Ruthenium(II)-Arene Compounds. J. Med. Chem. 2008,

51 (21), 6773–6781.

(90) Failes, T. W.; Cullinane, C.; Diakos, C. I.; Yamamoto, N.; Lyons, J. G.; Hambley, T. W. Studies of a Cobalt(III) Complex of the MMP Inhibitor Marimastat: A Potential Hypoxia-Activated Prodrug. Chem. Weinh.

Bergstr. Ger. 2007, 13 (10), 2974–2982.

(91) Dalla Via, L.; Nardon, C.; Fregona, D. Targeting the Ubiquitin-Proteasome Pathway with Inorganic Compounds to Fight Cancer: A Challenge for the Future.

Future Med. Chem. 2012, 4 (4), 525–543.

(92) Zhang, X.; Frezza, M.; Milacic, V.; Ronconi, L.; Fan, Y.; Bi, C.; Fregona, D.; Dou, Q. P. Inhibition of Tumor Proteasome Activity by Gold–dithiocarbamato Complexes via Both Redox-Dependent and -Independent Processes. J. Cell. Biochem. 2010, 109 (1), 162–172.

(93) Verani, C. N. Metal Complexes as Inhibitors of the 26S Proteasome in Tumor Cells. J. Inorg. Biochem. 2012, 106 (1), 59– 67.

(94) Thompson, K. H.; Lichter, J.; LeBel, C.; Scaife, M. C.; McNeill, J. H.; Orvig, C. Vanadium Treatment of Type 2 Diabetes:

A View to the Future. J. Inorg. Biochem. 2009, 103 (4), 554–558.

(95) Sava, G.; Zorzet, S.; Turrin, C.; Vita, F.; Soranzo, M.; Zabucchi, G.; Cocchietto, M.; Bergamo, A.; DiGiovine, S.; Pezzoni, G.; Sartor, L.; Garbisa, S. Dual Action of NAMI-A in Inhibition of Solid Tumor Metastasis.

Clin. Cancer Res. 2003, 9 (5), 1898–1905.

(96) Bergamo, A.; Gagliardi, R.; Scarcia, V.; Furlani, A.; Alessio, E.; Mestroni, G.; Sava, G. In Vitro Cell Cycle Arrest, in Vivo Action on Solid Metastasizing Tumors, and Host Toxicity of the Antimetastatic Drug NAMI-A and Cisplatin. J. Pharmacol. Exp. Ther. 1999, 289 (1), 559–564.

(97) Gaynor, D.; Griffith, D. M. The Prevalence of Metal-Based Drugs as Therapeutic or Diagnostic Agents: Beyond Platinum.

Dalton Trans. 2012, 41 (43), 13239–13257.

(98) Bertrand, B.; Casini, A. A Golden Future in Medicinal Inorganic Chemistry: The Promise of Anticancer Gold

Organometallic Compounds. Dalton Trans. 2014, 43 (11), 4209–4219.

(99) Nardon, C.; Boscutti, G.; Fregona, D. Beyond Platinums: Gold Complexes as Anticancer Agents. Anticancer Res. 2014,

34 (1), 487–492.

(100) Nobili, S.; Mini, E.; Landini, I.; Gabbiani, C.; Casini, A.; Messori, L. Gold Compounds as Anticancer Agents: Chemistry, Cellular Pharmacology, and Preclinical Studies.

Med. Res. Rev. 2010, 30 (3), 550–580.

(101) Clinicaltrials.Gov Identifier for Auranofin: NCT01747798, NCT01419691,

NCT02063698, NCT02736968, NCT01737502, NCT02961829, NCT02968927.

(102) Arnér, E. S. J.; Holmgren, A. The Thioredoxin System in Cancer. Semin.

Cancer Biol. 2006, 16 (6), 420–426.

(103) Tonissen, K. F.; Di Trapani, G. Thioredoxin System Inhibitors as Mediators of Apoptosis for Cancer Therapy. Mol. Nutr.

Food Res. 2009, 53 (1), 87–103.

(104) Ott, I. On the Medicinal Chemistry of Gold Complexes as Anticancer Drugs. Coord.

Chem. Rev. 2009, 253 (11–12), 1670–

1681.

(105) Berners-Price, S. J.; Filipovska, A. Gold Compounds as Therapeutic Agents for Human Diseases. Met. Integr. Biometal Sci.

(107) Messori, L.; Marchetti, L.; Massai, L.; Scaletti, F.; Guerri, A.; Landini, I.; Nobili, S.; Perrone, G.; Mini, E.; Leoni, P.; Pasquali, M.; Gabbiani, C. Chemistry and Biology of Two Novel Gold(I) Carbene Complexes as Prospective Anticancer Agents. Inorg.

Chem. 2014, 53 (5), 2396–2403.

(108) Liu, W.; Gust, R. Update on Metal N-Heterocyclic Carbene Complexes as Potential Anti-Tumor Metallodrugs. Coord.

Chem. Rev. 2016, 329, 191–213.

(109) Cinellu, M. A.; Ott, I.; Casini, A. Gold Organometallics with Biological Properties. In Bioorganometallic

Chemistry; Jaouen, G., Salmain, M., Eds.;

Wiley-VCH Verlag GmbH & Co. KGaA, 2014; pp 117–140.

(110) Rubbiani, R.; Kitanovic, I.; Alborzinia, H.; Can, S.; Kitanovic, A.; Onambele, L. A.; Stefanopoulou, M.; Geldmacher, Y.; Sheldrick, W. S.; Wolber, G.; Prokop, A.; Wölfl, S.; Ott, I. Benzimidazol-2-Ylidene Gold(I) Complexes Are Thioredoxin Reductase Inhibitors with Multiple Antitumor Properties. J. Med. Chem. 2010,

53 (24), 8608–8618.

(111) Citta, A.; Schuh, E.; Mohr, F.; Folda, A.; Massimino, M. L.; Bindoli, A.; Casini, A.; Rigobello, M. P. Fluorescent Silver(I) and Gold(I)-N-Heterocyclic Carbene Complexes with Cytotoxic Properties: Mechanistic Insights. Metallomics 2013, 5 (8), 1006– 1015.

(112) Barnard, P. J.; Baker, M. V.; Berners-Price, S. J.; Day, D. A. Mitochondrial Permeability Transition Induced by Dinuclear Gold(I)– carbene Complexes: Potential New Antimitochondrial Antitumour Agents. J.

Inorg. Biochem. 2004, 98 (10), 1642–1647.

(113) Barnard, P. J.; Berners-Price, S. J. Targeting the Mitochondrial Cell Death Pathway with Gold Compounds. Coord. Chem. Rev. 2007, 251 (13–14), 1889–1902. (114) Hickey, J. L.; Ruhayel, R. A.; Barnard, P. J.;

Baker, M. V.; Berners-Price, S. J.; Filipovska, A. Mitochondria-Targeted Chemotherapeutics: The Rational Design of Gold(I) N-Heterocyclic Carbene Complexes That Are Selectively Toxic to Cancer Cells and Target Protein Selenols in Preference to Thiols. J. Am. Chem. Soc. 2008, 130 (38), 12570–12571.

1

2

3

4

5

6

7

8

9

Detailed in Vitro and Cellular Study. J.

Med. Chem. 2008, 51 (15), 4790–4795.

(116) Bertrand, B.; Stefan, L.; Pirrotta, M.; Monchaud, D.; Bodio, E.; Richard, P.; Le Gendre, P.; Warmerdam, E.; de Jager, M. H.; Groothuis, G. M. M.; Picquet, M.; Casini, A. Caffeine-Based Gold(I) N-Heterocyclic Carbenes as Possible Anticancer Agents: Synthesis and Biological Properties. Inorg. Chem. 2014,

53 (4), 2296–2303.

(117) Bazzicalupi, C.; Ferraroni, M.; Papi, F.; Massai, L.; Bertrand, B.; Messori, L.; Gratteri, P.; Casini, A. Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G-Quadruplex: A Joint ESI MS and XRD Investigation. Angew. Chem. Int. Ed

CHAPTER

2

This thesis is divided in two sections, exploring two different approaches to develop and validate potential anticancer agents, organic compounds and experimental metal complexes, to target different proteins and unravel or gain insights on the mechanism of action.

A

IMS

:

P

ART

A:

I

NHIBITORS OF P

53/MDM2

INTERACTION

:

x To design and synthetize potent inhibitors of the p53/MDM2 interaction.

x To establish a structure activity relationship and validate their affinity towards the receptor, comparing the new series of compounds with previously described inhibitors.

P

ART

B:

B

IOLOGICAL ACTIVITY OF VARIOUS FAMILIES OF METAL COMPLEXES

x To evaluate in vitro the toxicity of the different families of metal complexes in human cancer cell lines to determine their potential as anticancer agents

x To evaluate the toxicity of potential anticancer metallodrugs in healthy tissue using rat precision cut liver and kidney slices.

x To study the mechanisms of transport of well-known and new metal based drugs using rat precision cut kidney slices.

OUTLINE

In chapter 1, an introduction about the main topics disclosed in this thesis is given, including basic background about cancer, currently used chemotherapeutic strategies and different kinds of targets, like protein families and DNA. A main focus is directed to small molecule inhibitors of protein-protein interactions with oncological importance, such as p53/MDM2, BCL-XL/BH3, XIAP/SMAC interactions. Additionally, an overview of the use of metals and metal complexes through history, and metallodrugs used currently in the clinic, their mechanism of action, with emphasis on cisplatin and experimental gold complexes.

Chapter 3 is a systematic review of p53/MDM2 interaction inhibitors, with emphasis on crystallographic structures and observed binding modes for different types of scaffold, their properties as well as preclinical and clinical studies of these small molecules and peptides. General guidelines for design of inhibitors based of the structural data, including conserved water molecules are given. Chapters 4 and 5, describe the chemical synthesis of a new class of p53/MDM2 inhibitors, designed based on previously described compounds. For this we used our pharmacophore based virtual screening platform ANCHOR.QUERY as a tool to obtain multicomponent reaction scaffolds to

2

3

4

5

6

7

8

9

software and an aliphatic handle to cover a large hydrophobic surface area formed by MDM2_Tyr67,

MDM2

Gln72, MDM2His73 MDM2Val93 and MDM2Lys94, increasing the affinity to the receptor. Chapter 5

presents the synthesis and biochemical evaluation of the affinity of a new class of 1,2,3-trisubstituted bis(indoles) heterocycles derivatives designed to mimic the three key p53’s aminoacids for the binding

with MDM2, and an extra interaction over MDM2_Val93.

Chapter 6 examines the toxicity in cancer cells and in healthy tissue of a series of gold compounds synthetized as bifunctional compounds to act as chimeric compounds combining the cytotoxicity of gold and the drug-resistance reduction of a lansoprazole moiety through the decrease of the acidic microenvironment in cancer cells. The toxicity assessment was performed in an ex-vivo model, using rat precision cut kidney and liver slices, to determine the toxicity profile and was compared with the formerly reported toxicity in cancer cells. Classical pathways involved in cellular stress were studied to get insight in the mechanism of action.

Chapter 7 studies the mechanisms of uptake as well as toxicity of cisplatin in comparison to a cytotoxic cyclometallated gold(III) compound in precision cut kidney slices. The involvement of the Organic Cation Transporters (OCTs) in the uptake mechanism of cisplatin was studied using specific OCT inhibitors.

In chapter 8, a series of organometallic N-heterocyclic carbene (NHC) complexes was synthesized and characterized. The cytotoxic activities of the compounds were tested in 4 human cancer cell lines and their toxicity in healthy tissue was determined using rat precision cut kidney slices as a tool to determine the potential selectivity towards cancer cells.

Finally, the outcome of the work presented in this thesis is summarised and discussed in chapter 9. Additionally, future perspectives are included for the development and study of potential anticancer compounds, including the use of Precision Cut Tissue Slices to assess the possible undesirable side effects and toxicity in healthy tissue.

PART

A

CHAPTER

3

H

OW TO DESIGN A SUCCESSFUL P

53-

MDM

2/

X

INHIBITOR

:

A THOROUGH OVERVIEW BASED ON

CRYSTAL STRUCTURES

Natalia Estrada-Ortiz, Constantinos G. Neochoritis and Alexander Dömling*

Department of Drug Design, University of Groningen Antonius Deusinglaan 1, 9700 AD Groningen, The Netherlands.

Abstract: A recent therapeutic strategy in oncology is based on blocking the protein-protein interaction MDM2/X-p53. Inhibiting the binding between wild-type p53 and its negative regulators MDM2 and/or MDMX has become an important target in oncology to restore the anti-tumor activity of p53, the so-called guardian of our genome. Interestingly, based on the multiple disclosed compound classes and structural analysis of small molecule-MDM2 adducts, the p53-MDM2 complex is perhaps the best studied and most targeted protein-protein interaction. Several classes of small molecules have been identified as potent, selective and efficient inhibitors and many co-crystal structures with the protein are available. In this report we are describing properties, preclinical and clinical studies of the small molecules and peptides classified in categories of scaffolds. A special focus is on crystal structures and the binding mode of the compounds including conserved waters.

3

4

5

6

7

8

9

1. INTRODUCTION

Protein-protein interactions are of outmost importance and implicated in almost all biological processes. Proteins should not be considered to function as single, isolated entities but display their roles by interacting with other cellular components and the different interaction patterns of a protein is at least as important as the intrinsic biochemical activity of the protein itself. Therefore, the biological role of a protein is heavily based on the protein-protein interactions. Especially for diseases, the majority of cases ultimately relies on regulating the PPIs. Identifying and successfully targeting the

PPIs by finding inhibitors or activators is the basis of the drug discovery.1

P53 is the principal regulator of the cell division and growth,2,3 _{being able to control genes implicated}

in cell cycle control, apoptosis, angiogenesis metabolism, senescence and autophagia.4 Mutations in

this protein are present in about 50% of human cancers; altering the DNA binding domain ceasing

p53’s activity as a transcription factor.5 _{The remaining tumors, p53 pathway is inactivated by}

up-regulation of p53 inhibitors, such as the mouse double minute proteins (MDM2 and MDMX, -also known as MDM4- HDM2 and HDMX in humans), or by down-regulation of p53 cooperators, such as

ARF.6,7

The MDM2 gene was found to be upregulated in approximately 7% of tumors, with increased transcript levels and enhanced translation. Mutation of p53 and upregulation of MDM2 do not usually occur within the same tumor, indicating that MDM2 over-expression is an effective path for

inactivation of p53 function in tumorgenesis.8 MDM2 functions as an inhibitor of the N-terminal

trans-activation domain (TAD) of p53, and promotes p53 degradation through the ubiquitin-proteasome

system (E3 ligase activity).9,10 _{On the other hand, MDMX can downregulate p53 via inhibition of the}

TAD domain, and it can upregulate MDM2.11

Moreover, it does not have E3 ligase activity, but its binding with MDM2 increases the rates of

ubiquitinylation of p53 by MDM2.11,12 _{The C-terminal RING domains of both MDM2 and MDMX, is}

involved in dimerization; MDM RING domains can form homodimers, the heterodimers can be form by

a reduced autoubiquitylation of MDM2 and increased p53 ubiquitylation.13 _{Consequently, MDM2 is}

stabilized by MDMX, keeping the levels of p53 low in healthy cells.13–15 _{The use of dual action}

MDM2/MDMX antagonists in cancer cells expressing wild type p53 should activate p53 more

significantly than agents that only inhibit MDM2, resulting in more effective anti-tumor activity.16–18

The p53-MDM2 interaction is well druggable by small molecules based on a buried surface area of

~700 Å2, a well-structured deep and hydrophobic binding site of similar dimension than small

molecules. For comparison the immune-oncology target protein-protein interaction PD1-PD1L has a

buried surface area of ~2000 Å2, and is flat and featureless (figure 1). 19,20

The binding site of p53 in MDM2 is formed by 14 residues: Leu57, Met62, Tyr67, Gln72, Val75, Phe86, Phe91, Val93, His96, Ile99 and Tyr100. The cleft at the surface of MDMX is highly similar, 4 out of 14

residues are however different: MDM2_Leu54>MDMX_Met53, MDM2_Phe86>MDMX_Leu85, MDM2_His96> MDMX_Pro95

and MDM2Ile99>MDMXLeu98.18 MDM2 and MDMX have a deep hydrophobic pocket on which the p53 protein binds as an alpha helix. Three p53 amino acids are deeply buried in the MDM2 and MDMX clefts: Phe19, Trp23, and Leu26 and the interaction is mostly governed by hydrophobicity (PDB: 1YCR,

Figure 1. Two important protein-protein interaction targets: left p53 (green surface) with MDM2 (grey surface), PDB: 1YCR; below: footprint of p53 on MDM2 shown as blue surface. Right: PD1 (green surface) with PD1L (grey surface, PDB: 4ZQK); below: footprint of PD1 on PD1L shown as blue surface

Figure 2. p53 key amino acids Phe19, Trp23 and Leu26 bound to MDM2 (PDB: 1YCR). Red dotted lines indicate the polar contacts

Additionally, the Trp23 indole-NH forms a hydrogen bond with the backbone carbonyl of MDM2_{Leu54. In}

MDMX, Met53 and Tyr99 are bulged into the hydrophobic surface groove making it smaller and

slightly different in shape (PDBs: 3DAB, 3DAC).21 The known three finger pharmacophore model for

p53-MDM2 is now widely accepted to be responsible for the binding of small molecules and peptides

to the MDM2.23–25 Recently an extended four finger model was proposed taking into account the

intrinsically disordered MDM2 N-terminus which can be ordered by certain small molecules as shown

by co-crystallization.26,27

Due to the importance of p53-MDM2/X protein-protein interaction, several reviews have been

published in the last years.28–31 This review is giving a broad recent overview of inhibitors of the

p53-MDM2/MDMX protein-protein interaction, mostly focusing on the available structural data. Additionally, some insights into the rational design and optimization of MDM2/X binder, their activities

in vitro, in vivo and clinical trials are given. Moreover, a preliminary water analysis of the current

3

4

5

6

7

8

9

2. I

NHIBITORS OF THE P

53-MDM2/MDMX

INTERACTIONS

2.1. N

UTLIN

-

TYPE COMPOUNDS

The first class of highly potent, specific and orally active MDM2 inhibitors, was disclosed in 2004 by

scientists from Hoffmann-La Roche,32 _{cis-diphenyl substituted imidazolines, known as Nutlins (1).}

Modifications and optimizations resulted in derivatives named Nutlin 1 (1a), Nutilin 2 (1b) and Nutlin

3a (1c) (figure 3). Nutlin compounds show inhibitory concentration values (IC50) of 260, 140 and 90

nM, respectively. These compounds displaced recombinant p53 fragment (corresponding to residues 1-312 of human p53) from its complex with MDM2 using surface plasmon resonance in a competition

assay (SPR).32 Nutlin 2 complexed with MDM2 co-crystal structure (PDB: 1RV1, figure 4) led to the

elucidation, for the first time of a non-peptide, small molecule structural information of the

interaction.32 _{Superimposition of the co-crystal structures MDM2-Nutlin 2 and MDM2-p53 (PDB:}

1YCR), showed that the two bromo-substituted phenyl rings and the ethoxyl group of Nutlin 2 mimic the Trp23, Leu26 and Phe19, respectively, the key hydrophobic binding residues of the p53 peptide

(figure 1).32 _{Nutlin 3a bound to MDM2 (PDB: 4J3E)}33 _{shows that both 4-chlorophenyl groups perfectly}

fill the Leu26 and Trp23 pockets, while the isopropoxy group reaches deep into the Phe19 pocket. It

activates wild-type p53 and selectively kills cancer cells, with IC50 of 1-2 μM in the SJSA-1, HCT116 and

RKO cell lines (osteosarcoma, colorectal and colon carcinoma, respectively) and showed 10-fold selectivity compared with p53 mutated cell lines MDA-MB-435 and SW480 (melanoma and colorectal

adenocarcinoma, respectively).32 Furthermore, in vivo studies demonstrate the capability of Nutlin 3a

to reduce tumor growth by 90% in SJSA-1 in mice xenograft model.32

In 2008, compound 2 (RG7112) based on Nutlins (PDB: 4IPF),34 completed phase I clinical trials against

advanced solid and soft tissue tumors and hematological malignancies.35,36 Four key modifications

were made to enhance the binding activity to MDM2, cellular growth inhibition and improve pharmacokinetic properties: Two methyl groups were introduced in the imidazole ring to protect from metabolism, the isopropyl ether was replaced to ethyl ether to reduce the molecular weight maintaining the hydrophobic interactions, the methoxy group was changed by a tert-butyl moiety to decrease the metabolic liability and lastly, methyl sulphonyl group was added onto the piperidine ring to increase the binding affinity and improve the pharmacokinetics via reduced logD. After these

modifications, compound 2, displayed enhanced binding activity towards MDM2 with a Kd 10.7 nM

and cellular growth inhibition 3-fold more potent compared with Nutlin 3a.33 _{The main drawback}

shown during the clinical trials in patients with liposarcoma, was thrombocytopenia, but the data obtained from biopsies in this preliminary trial suggested that the p53 pathway can be reactivated despite the presence of excess of MDM2, resulting in cytostatic and possibly apoptotic effects in

O N N S O O N N Cl Cl O O _N NH N N O O Cl Cl O 1c (Nutlin-3a) 2 (RG7112) Cl N N CO2H NH Cl Cl N N NH Cl N O N 3 (WK-23) 4 (WK-298) Cl N N NH Cl NH O N O 5 N _N F Cl O N N NH2 F Cl HN O 7 N _N Cl OH O Cl 6 N O NH O S N N Cl Cl S N N Cl Cl O N Cl O S N N Cl N NH O 8 9 10 N N N O O N N OH N N O O Br Br O N N O N N O O Cl Cl 1b (Nutlin-2) 1a (Nutlin-1)