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HAL Id: tel-01647215

https://tel.archives-ouvertes.fr/tel-01647215

Submitted on 24 Nov 2017

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Thi Phuong Anh Tran

To cite this version:

Thi Phuong Anh Tran. Design, synthesis and biological evaluation of new RNA ligands as in-hibitors of oncogenic microRNAs production. Other. Université Côte d’Azur, 2016. English. �NNT : 2016AZUR4076�. �tel-01647215�

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THESE

Pour obtenir le titre de

Docteur en Sciences

de l'UNIVERSITE de Nice Sophia-Antipolis

Discipline : Chimie

Présentée et soutenue par

Thi Phuong Anh TRAN

Design, synthesis and biological evaluation of new RNA ligands as inhibitors of

oncogenic microRNAs production

Conception, synthèse et évaluation biologique de nouvea

ux ligands d’ARN en

tant qu’inhibiteurs de la production de microARNs oncogènes

Thèse dirigée par Dr. Maria DUCA

Soutenue le 14 Octobre 2016

Jury:

Dr. Florence

MAHUTEAU-BETZER

Chargé de Recherche CNRS

Institut Curie

Rapporteur

Pr. Philippe URIAC

Professeur

Université de Rennes

Rapporteur

Pr. Alain BURGER

Professeur

Université Nice Sophia Antipolis

Examinateur

Dr. Maria DUCA

Chargé de Recherche CNRS

Université Nice Sophia Antipolis

Directrice de

thèse

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Acknowledgements

As Ale Hope put it, The o l a to fi d out ho to do a PhD is to do o e . No I k o hat it is by doing a PhD studies. It is full of peaks and valleys. And this thesis is the final peak in the process.

This occasion I wish to acknowledge the financial support from the Vietnamese Ministry of Education and Training and the Campus France, without it there is no chance for me to spend my most memorable time studying and researching in the beautiful city of Nice.

I am also indebted to Dr. Maria Duca, my supervisor, the one who was willing to host and has always been behind my valleys and peaks. Her direction, supports and encouragements are the only reason of my timely and fruitful research. I am particularly grateful for her closely support during the time I drafted my thesis and also for the care that she paid to every aspect of my daily life.

Dr. Audrey Digiorgio is also credited for her detailed instructions and explanations. Her straight and insightful comments influentially contributed to my work. Every discussion with her was a great source of pleasure as well as scientifically productive.

My work is also not completed without the contribution by Dr. Cathy Staedel and Dr. Fabien Darfeuille from ARNA lab, University of Bordeaux 2. I really owe them for taking the intracellular tests, thank you.

With the ICN lab, big thank goes to the head of Molécules Bioactives Group, Dr. Rachid Benhida for his warm welcome. Special thanks to Prof. Nadia Patino for her lovely care; I will miss your kisses that always cheered my days. I would also like to appreciate Dr. Stéphane Azoulay for his kindness during my 3-years stay, Dr. Marc Gaysinski for the NMR spectroscopy, Dr. Lionel Massi for the mass spectrometry and Dr. Nadine Martinet for her kindness care.

For my fellow lab-mates, I thank you for the time we share working and grilling together. You are all the reason to get me out of the blue by the time I spent it in the valleys of research. To name all of you is impossible and someone who is not presented is surely because my lack of time and you all are equally important to me. Thank you to Najiba and Pauline – my first teachers of French; to Sofia – the young Spanish girl, who always starts the conve sio ith pou uoi a d despite distance, has updated me everything in life. To Janah – the aziest a I e e e et ho ade e laugh a lot and gave me helpful advices in chemistry. To Nicolas, Adeline, Marie and Soukaina - I had joyful time

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worked together for just a short time but keep in touch until now. To Cécile and Gilles - I will never forget the night of 14th Jul , the da e ade ‘ouleu de p i te ps a d ou ki d ess

encouragements during my redaction. And all the others, Coralie, Hela, Alexandra, Nelli, Claudio, Mauro, Bibi, Hamza, Dominique you will be truly missed.

Out of the lab back home is my dear beloved Mother. I know that I am the little stubborn to you, but you know deep in my heart, I love you the most and I know you do, too. Without you, definitely I could not achieve any of this. This thesis is the one I dedicate to you. Father, I thank you for giving me this life. I will always remember you. I also want to thank my classmate and my dear husband. You are all the things I have got, thank you for being in my life.

To Duy, many thanks for sharing, encouraging and advising about chemistry knowledge. To Khanh, Hà for personal advices and sharing of all the ups and downs. To Hường and Yến, my roommates for almost two years, who cooked for me Vietnamese food and eased my homesickness. Big hugs to you. And to the group of intercontinental chatting, you are my friends forever.

This thesis, it is just the conclusion of one chapter of my life. From the height it brings, a brand new horizon opens before me in the quest to find solutions to fight cancer. It is hard, full of unexpected turns and dead-ends. It is like finding a needle in a haystack, but as long as we fight for it, we have hope for it and who knows, maybe one day, mi-RNA-based therapeutic is the most efficient approach for the treatment of cancer and this thesis is one contribution to it.

Nice,

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Table of Contents

ACKNOWLEDGEMENTS ...5 LIST OF ABBREVIATIONS ... 13 GENERAL INTRODUCTION ... 19 CHAPTER I ... 23 BIBLIOGRAPHICAL REVIEW ... 23 1 MIRNAS BIOGENESIS ... 23 2 MIRNAS FUNCTIONS ... 25

3 MIRNAS AND HUMAN CANCERS ... 28

4 MIRNAS AS THERAPEUTIC TARGETS IN CANCER ... 33

4.1 RESTORATION OF TUMOR SUPPRESSOR MIRNAS ACTIVITY ... 34

4.1.1 miRNAs mimics ... 34

4.1.2 miRNA expression vectors ... 35

4.1.3 Small molecules ... 36

4.2 INHIBITION OF ONCOGENIC MIRNAS ... 36

4.2.1 Oligonucleotides-based approaches ... 37

(a) Antisense oligonucleotides ... 38

(b) miRNA sponges ... 39

(c) miRNA masks ... 39

4.2.2 Small molecules-based approaches ... 40

(a) High throughput screening approach ... 42

(i) Discovery of small molecules inhibiting miRNAs transcription ... 42

(ii) Discovery of small molecules interfering with Drosha or Dicer cleavage step... 43

(iii) Discovery of small molecules interfering with RISC loading ... 46

(b) Focused screening approach ... 46

(c) Design of miRNA inhibitors ... 50

4.2.3 Contributions of our research group to design and synthesis of new inhibitors of oncogenic miRNAs production ... 53

5 PROJECT OBJECTIVES ... 56

CHAPTER II ... 59

RIBOSOME-TARGETING ANTIBIOTICS AS INHIBITORS OF ONCOGENIC MICRORNAS BIOGENESIS: OLD SCAFFOLDS FOR NEW PERSPECTIVES IN RNA TARGETING ... 59

1 INTRODUCTION ... 59

2 RESULTS AND DISCUSSION ... 60

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2.3 MEASUREMENT OF BINDING AFFINITY (DISSOCIATION CONSTANTS,KD) ... 68

2.4 ENZYMATIC FOOTPRINTING AND IDENTIFICATION OF THE INTERACTION SITES ... 73

2.5 DICER CLEAVAGE INHIBITION OF PRE-MIRNA-372 ... 76

2.6 THERMODYNAMIC SIGNATURE OF ANTIBIOTICS ... 78

3 CONCLUSION ... 79

CHAPTER III ... 81

ONCOGENIC MICRORNAS BIOGENESIS AS A DRUG TARGET: ... 81

STRUCTURE-ACTIVITY RELATIONSHIP STUDIES ON NEW AMINOGLYCOSIDE CONJUGATES ... 81

1 INTRODUCTION ... 81

2 RESULTS AND DISCUSSION ... 84

2.1 DESIGN AND SYNTHESIS OF AMINOGLYCOSIDE CONJUGATES ... 84

2.1.1 Modification of the artificial nucleobase S ... 84

2.1.2 Modification of the linker ... 88

2.1.3 Modifications of the aminoglycoside moiety ... 90

(a) Preparation of apramycin analog of Neo-S ... 91

(b) Preparation of neamine and 2-deoxystreptamine analogs of Neo-S ... 92

2.2 BIOCHEMICAL AND BIOLOGICAL EVALUATION OF THE SYNTHESIZED COMPOUNDS ... 97

2.2.1 Screening of the synthesized conjugates ... 97

2.2.2 Binding studies ... 100

2.2.3 Biological activity in gastric adenocarcinoma cells ... 107

2.2.4 Study of intracellular uptake of compounds 3.14a, 3.14d and Neo-S ... 113

3 CONCLUSION ... 115

CHAPTER IV ... 117

NOVEL RNA LIGANDS CONTAINING 2-DEOXYSTREPTAMINE: ... 117

SIMPLE APPROACHES TOWARD POTENTIAL INHIBITORS OF ONCOGENIC MIRNAS BIOGENESIS ... 117

1 INTRODUCTION ... 117

1.1 SYNTHESIS OF A 2-DEOXYSTREPTAMINE SCAFFOLD ... 118

1.2 2-DOS CHEMICAL MODIFICATION FOR THE PREPARATION OF NEW RNA LIGANDS ... 120

1.3 AIMS OF THE WORK ... 124

2 RESULTS AND DISCUSSION ... 126

2.1 PREPARATION OF 2-DOS CONJUGATES ... 126

2.1.1 Preparation of 2-DOS derivatives containing carbamate linkers ... 126

2.1.2 Preparation of 2-DOS derivatives containing 1,2,3-triazole linkers... 133

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(c) Preparation of alkyne-modified artificial nucleobases ... 136

(d) Preparation of 2-DOS conjugates containing triazole linker ... 136

2.1.3 Preparation of 2-DOS derivatives containing an ether linker ... 138

2.2 BIOLOGICAL STUDY ... 140

2.2.1 Screening of the newly synthesized conjugates for their ability to inhibit the cleavage of Dicer enzyme on pre-miRNA-372 ... 140

2.2.2 Screening of the newly synthesized conjugates for their ability to bind to pre-miRNA-372 . 143 2.3 PREPARATION OF NEW ANALOGS OF 4.57E ... 146

3 CONCLUSION ... 152

CONCLUSIONS AND PERSPECTIVES ... 153

CHAPTER V ... 157

EXPERIMENTAL PART ... 157

1 CHEMISTRY ... 157

1.1 LIST OF SYNTHESIZED COMPOUNDS ... 157

1.1.1 Chapter III... 157

1.1.2 Chapter IV ... 167

1.2 MATERIALS & SYNTHETIC PROTOCOLS ... 171

1.2.1 Chapter III... 173 1.2.2 Chapter IV ... 182 2 BIOLOGICAL EVALUATION ... 203 2.1 MATERIALS ... 203 2.2 BIOLOGICAL PROCEDURES ... 205 REFERENCES ... 209 PUBLICATIONS ... 229

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List of abbreviations

2-DCS 2-dimensional combinatorial screening

2-DOS 2-dexoxystreptamine

4-DMAP 4-dimethylaminopyridine

A adenine

Ago2 argonaute 2 protein

AGS human gastric adenocarcinoma cell line

AGL1/AGL2 asparagine-linked glycosylation 1/ asparagine-linked glycosylation 2

AML acute myeloid leukemia

ASO antisense oligonucleotide

AAV adeno-associated vectors

BCL2 B-cell CLL/lymphoma 2

BIM B-cell CLL/lymphoma 2-like protein 11  Boc2O di-tert-butyl dicarbonate

BTBC 1,1-bis[6-(triluoromethylbenzotriazoyl]carbonate

C cytosine

CDK4 cyclin dependent kinase 4

CDK6 cyclin dependent kinase 6

CEBPB CCAAT/enhancer-binding protein beta

CHX cyclohexane

CLL chronic lymphocytic leukemia

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Cx43 connexin 43

DABCYL 4-((4-(dimethylamino)phenyl)azo)benzoic acid  DGCR8 DiGeorge syndrome chromosomal region 8  DIPEA N,N-diisopropylethylamine

DIS dimerization initiation site

DMA dimethylacetamide

DMF dimethylformamide

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

DPC di-(2-pyridyl) carbonate

DSC N,N’-disuccinimidyl carbonate

dsDNA double-stranded deoxyribonucleic acid  dsRNA double-stranded ribonucleic acid

DTT 1,4-dithiothreitol

EC50 effective concentration

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

eq. equivalent

ESI electrospray ionisation

FAM 6-carboxyfluorescein

FID fluorescent intercalator displacement  FRET fluorescence resonance energy transfer

FP fluorescence polarization

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15

HCC hepatocellular carcinoma

HCV hepatitis C virus

HIV human immunodeficiency virus

HOBt hydroxybenzotriazole

HPLC high performance liquid chromatography  HRMS high resolution mass spectrometry  HSQC heteronuclear single-quantum correlation  IC50 inhibitory concentration

Kb kilobase

KD dissociation constant

KIT tyrosine-protein kinase

LATS2 large tumor suppressor kinase 2

LC/MS liquid chromatography/mass spectrometry

MCL-1 induced myeloid leukemia cell differentiation protein  Mef2 myocyte enhancer factor-2 protein

miRNA micro RNA

mRNA messenger RNA

ncRNA non-coding ribonucleic acid

NMR nuclear magnetic resoncance

NOESY nuclear overhauser spectroscopy

nt(s) nucleotide(s)

ODN oligonucleotide

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P53 tumor protein p53

Pd/C palladium on carbon

piRNA P-element induced wimpy testis (PIWI)-interaction ribonucleic acid

PLL polylysine

pre-miRNA precursor miRNA  pri-miRNA primary miRNA

PTEN phosphatase and tensin homolog  pTsOH p-toluenesulfonic acid

qRT-PCR quantitative polymerase chain reaction

r.t. room temperature

Rf retention factor

RISC RNAs induced silencing complex

RNA ribonucleic acid

RNAi RNA interfering

RNase ribonuclease

rRNA ribosome RNA

RT-PCR reverse transcription polymerase chain reaction  SAR structure-activity relationship

SD standard deviation

SHIP1 phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1  siRNA small interfering RNA

StARTS structure-activity relationships through sequencing

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17  TAR trans-activation response element

TBAF tetrabutylammoniumfluoride

TBAI tetrabutylammonium iodide

TBDMSCl tert-butyldimethylsilyl chloride

TBTA tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine

TFA trifluroacetic acid

THF tetrahydrofuran

TIPS-Cl triisopropylsulfonyl chloride

TIS triisopropylsilane

TLC thin-layer chromatography

TPF trypaflavine

TRBP the human immunodeficiency virus transactivating response RNA-binding protein

tRNA transfer RNA

U uracil

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19

General introduction

In the last decades, despite critical efforts of scientists, cancer remains a global health threat. Current developments of therapeutics against cancer have been focused on discovering small-molecule drugs that act on new molecular targets through specific interactions, thus leading to the desired clinical effects.

The discovery of miRNAs and of their functions in the regulation of gene expression is one of the most important discoveries in genetics of the last thirty years. Especially, the dysregulation in miRNA expression is associated to the initiation and the development of a wide number of human diseases. miRNA has thus become a research subject capturing major interest of scientists worldwide, particularly in the field of miRNA-based oncology therapeutics. In addition to provide viable markers for the detection and characterization of cancers, miRNAs represent a new class of potential drug targets for the development of anticancer therapy.

Our group is interested in discovering new RNA ligands targeting oncogenic miRNAs production. We are focussing our attention on small-molecule RNA ligands, capable to bind selectively to stem-loop structured precursors of targeted miRNAs. Indeed, the interaction between these molecules and precursors of oncogenic miRNAs is responsible for the inhibition of their processing toward the mature miRNAs.

During the last years, our research group has developed various series of RNA ligands able to bind selectively to pre-miRNA-372 and pre-miRNA-373, precursors of two oncogenic miRNAs responsible for the development of various cancers, such as gastric cancer. We also have developed a screening assay allowing the rapid identification of the most promising inhibitors that have been then tested for their antiproliferative activity in gastric cancer cells thanks to a collaboration with Dr. Fabien DARFEUILLE and Dr. Cathy STAEDEL in ARNA laboratory at the University of Bordeaux. So, a lead compound (Neo-S in Figure 1) has been identified as a very promising inhibitor of the targeted miRNAs.

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20 Figure 1. Chemical structure of lead compound Neo-S previously identified in our group

This molecule is composed of the aminoglycoside neomycin, an aminoglycoside commonly employed in therapy as an antibiotic because of its ability to bind to prokaryotic ribosomal RNA, and of an artificial nucleobase known to interact selectively with DNA and RNA base pairs

via specific hydrogen bonds. These two parts have been linked together by a triazole linker

arm. The most active compound Neo-S bears an antiproliferative effect which is highly specific for gastric cancer cells overexpressing targeted miRNAs. The reported experiments have demonstrated that the biological activity is directly correlated to the inhibition of the production of oncogenic miRNA-372 and miRNA-373, thus leading to the restoration of normal mRNA translation.

In this context, my PhD project has thus been focused on the discovery of new and better inhibitors of oncogenic miRNAs production. My work has consisted of the screening of known RNA ligands as well as the synthesis of new compounds inspired by our previously identified lead Neo-S. All compounds have been studied for their ability to inhibit the production of various oncogenic miRNAs and in particular of our privileged targets, i.e. miRNA-372 and miRNA-373. The biochemical study of the ability to bind the targeted RNAs and to inhibit the processing of oncogenic miRNAs has then been performed in order to establish structure-activity relationships. We have also identified the exact interaction of the best inhibitors in order to understand their molecular mechanisms of action. Finally, intracellular studies have been carried out in collaboration with biologists.

This manuscript is organized in five parts:

1. The first part (Chapter I) consists of a general introduction on miRNAs biogenesis, functions and relationships with human pathologies, especially with human cancers. Furthermore, a

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21 detailed, as published in a recent review by our group (Di Giorgio, A., Tran, T.P.A. and Duca, M., 2016. Small-molecule approaches toward the targeting of oncogenic miRNAs: roadmap for the discovery of RNA modulators. Future medicinal chemistry, 8(7), pp.803-816). This part includes describing previous works of our research group about oncogenic miRNAs targeting and with the main objectives of my PhD work.

2. The second part (Chapter II) reports the screening and the biochemical evaluation of currently employed antibiotics known as binders of prokaryotic ribosomal RNA. Eighteen compounds were studied as inhibitors of oncogenic miRNAs production on four different miRNA precursors and as selective binders of these RNA sequences. This part illustrates the results we obtained as well as a detailed study of the biological activity of the most active antibiotics in terms of inhibition, binding affinity, selectivity and thermodynamics of the interaction and finally the study of the site of the interaction on the targeted RNAs. The results described in this section have already been published (Tran, T.P.A., Vo, D.D., Di Giorgio, A. and Duca, M., 2015. Ribosome-targeting antibiotics as inhibitors of oncogenic microRNAs biogenesis: old scaffolds for new perspectives in RNA targeting. Bioorganic & medicinal

chemistry, 23(17), pp.5334-5344).

3. The third part (Chapter III) describes the medicinal chemistry optimization of the previously identified lead Neo-S where we chemically modified the three parts constituting this compound in order to improve the biological activity. The synthesis of thirteen new analogs as well as their biological evaluation in vitro and in cancer cells is described. The results described in this section have been recently published (Vo, D.D., Tran, T.P.A., Staedel, C., Benhida, R., Darfeuille, F., Di Giorgio, A. and Duca, M., 2016. Oncogenic MicroRNAs Biogenesis as a Drug Target: Structure–Activity Relationship Studies on New Aminoglycoside Conjugates.Chemistry–

A European Journal, 22(15), pp.5350-5362).

4. The fourth part (Chapter IV), focuses on a new series of RNA ligands that could act as inhbitors of miRNAs production. They are constituted of conjugates of 2-deoxystreptamine, which is part of neomycin aminoglycoside, and various (hetero)aromatic moieties. This section describes the synthesis of 13 new compounds and the preliminary results about their biological activity in vitro.

5. The last part of this thesis (Chapter V) reports the experimental details both for the chemical procedures employed for the synthesis of new compounds and for the protocols used for the biochemical studies.

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Chapter I

Bibliographical review

1

miRNAs biogenesis

Cells survival relies on their ability to respond to changes in their environment. Regulation of gene expression is critical to this adaptability by allowing the cell to express proteins when needed. The expression of a gene involves two main steps including transcription and translation. Transcription is the production of messenger RNA (mRNA) from the DNA sequence containing genomic information. Translation is the use of mRNA to direct protein synthesis (Figure 1.1).

Figure 1.1. Simplified representation of gene expression. (from Nature Education 1).

It is estimated that about 75% of the human genome is transcribed into RNAs, whereas only 3% is transcribed into protein-coding mRNAs.2,3 Functional RNA molecules that are transcribed

from DNA but not translated into proteins are known as non-coding RNAs (ncRNAs). They function as regulators of gene expression at the transcriptional and post-transcriptional level.4,5 These ncRNAs can be divided into two main groups: the short ncRNAs (<30 nucleotides

(nts)) and the long ncRNAs (>200 nts). The three major classes of short ncRNAs are microRNAs (miRNAs), short interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs). All theses ncRNAs play a crucial role in the regulation of gene expression and represent goldmines for

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24 basic research, biomarkers discovery and therapeutic applications.6,7 To date, the most studied

ncRNAs are miRNAs which represent the main subject of this thesis.

miRNAs are a class of single-stranded non-coding RNAs of 18-25 nucleotides that play an important role in the regulation of gene expression.8 These powerful molecules are able to

turn on or off genes by selectively interacting with messenger RNAs (mRNAs) that are necessary for translating the genetic information into proteins. The first miRNA, called lin-4, was discovered by Victor Ambros et al. in 1993 in worm.9 Since then, approximately 20,000

miRNAs have been identified in animals, plants and viruses including over 2,000 human miRNAs which can target more than 60% of the entire genome (www.mirbase.org). In fact, a single miRNA can target a large number of genes (about 500 for each miRNA family).10–13 In

Figure 1.2, historical discoveries have been collected to give a quick overview about the achievements in miRNAs research, starting with the discovery of the first miRNA twenty-two years ago, passing through the elucidation of miRNAs basic biology, miRNAs involvement in cancer, miRNAs transition for therapy to recent clinical trials involving the use of miRNAs-based therapeutics.14–16

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25 As illustrated in Figure 1.3, biogenesis of miRNAs starts within the nucleus: the genes coding for miRNAs are transcribed by RNA polymerase II into primary transcripts of variable length (usually between 1kb and 3 kb), called pri-miRNAs that contain a typical stem-loop structure (Figure 1.3-1).17 The double-stranded region of pri-miRNAs is then recognized by protein

DGCR8 which associates with a ribonuclease called Drosha to form a microprocessor complex. This complex is able to cut the pri-miRNAs into smaller hairpin precursor miRNAs (pre-miRNAs) of about 70-100 nucleotides 18 (Figure 1.3-2). In the following step, pre-miRNAs are exported

into the cytoplasm through a nuclear pore by the transport molecule Exportin 5 (Figure 1.3-3).19 Inside the cytoplasm, pre-miRNAs are cleaved by another ribonuclease, called Dicer, to

yield mature double-stranded miRNAs of variable length (18-25 nucleotides, miRNA: miRNA* duplexes) (Figure 1.3-4).20 After unwinding, the active guide strand is recognized by Argonaute

2 protein (Ago2), whereas the passenger strand is degraded (Figure 1.3-5). Ago2 is an endogenous polymerase H and it is the core component of RISC (i.e. RNAs induced silencing complex) (Figure 1.3-6).21 The o ple i‘NA/‘I“C i ds to the -untranslated

single-stranded mRNAs leading directly to either a repression of the corresponding mRNA translation or induction of its degradation, thus providing post-transcriptional control of gene expression.22

Figure 1.3. Simplified overview of the miRNAs biogenesis pathway where each step of miRNAs biogenesis and action is indicated.

2

miRNAs functions

As described in the previous section, biogenesis of miRNA is a complex multi-step process starting from the nucleus, passing over some post-transcriptional modifications and ending in the cytoplasm. The final step of this process is the interaction between the guide-stranded

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26 miRNA and the target mRNA leading to the regulation of mRNA translation and modulation of the expression of target proteins.22 This step makes miRNAs acting as regulators of gene

expression and participating in numerous physiological and pathological processes, including cell cycle progression and proliferation, differentiation, cell survival and development.23 Due to

this pivotal role, miRNAs raised as interesting targets for new therapeutic approaches and a vast number of studies related to miRNAs has been reported.14

Among small regulatory molecules which modulate gene expression, the small interfering RNAs (siRNAs) seem to be the most similar to miRNAs by either their chemical composition or mechanism of action.24 These two classes of ncRNAs represent two pathways of the process of

RNA interference (RNAi) inside multicellular organisms and participate in the process of RNA-mediated gene silencing.18 miRNAs and siRNAs show many similarities in their structure,

biogenesis and functions (Figure 1.4). Indeed, they share part of the biogenesis process: being processed by Dicer from double-stranded RNAs into a small double-stranded RNA segment (18-25 nts for miRNA, 21-23 nts for siRNA) contai i g -monophosphates and a two-nucleotide overhang at its -end in the cytoplasm. That small RNA segment is then incorporated into RISC due to the recognition of Argonaute protein and it drives the silencing of a target mRNA via degradation and/or transcriptional repression.18 However, siRNAs differ

from miRNAs in the molecular origin and the mode of mRNA target recognition (Figure 1.4). First, contrary to miRNAs, siRNAs are primarily exogenous in origin, derived directly from a virus, a transposon or a transgene trigger and depend on Dicer only, not on Drosha.25 Second,

double-stranded siRNAs are normally perfectly self-complementary, whereas the miRNAs hairpin typically exhibit one or more bulges or loops, leading to only partial complementarity of the strands.26 Third, siRNAs show a perfect complementarity to the targeted mRNAs leading

to the degradation of the said mRNAs, whereas miRNAs are not perfectly complementary to the corresponding mRNAs. Due to the regions where miRNA/RISC binds to their target mRNA p i a il ith the -UTR), miRNA cannot have a perfect matching to the corresponding mRNA.18,27 This mismatch allows each miRNA to target hundreds of mRNAs and results in the

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27 Figure 1.4. Scheme illustrating the RNA interference process and comparing siRNAs with miRNAs within

their biogenesis.

Despite all these similarities, siRNAs seem to be presented only in plants and lower animals, while miRNAs are the main regulators of gene expression in mammals. For this reason, we will focus the rest of the introduction on miRNAs regulation and involvement in human pathologies. In fact, because of the essential regulatory role of miRNAs, their overexpression or underexpression have been linked to the pathophysiology of various diseases.29,30 As a

single miRNA has the capability of regulating hundreds of mRNAs, it is clear that the deregulation of miRNAs levels can destabilize the entire cellular machinery.28 Many efforts

have been focused toward the development of miRNAs-based therapies targeting genetic and viral diseases.18,24

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28 Table 1.1 List of some major examples of miRNAs involved in physiological and pathophysiological

processes.

miRNA Species Target Function Ref.

Lin-4 C.elegans Lin-14 Lin-14 is negatively regulated by Lin-4. 9

miRNA-1 D.melanogaster Twist, Mef2 miRNA-1 knockout fruit flies lead to death in 3-instar larval stage.

31

miRNA-206 Pig Cx43 Highly expressed at one or more stages of

muscle development.

32

miRNA-15a/16-1

Mice BCL2 Control B cell proliferation and its deletion leads to chronic lymphocytic leukemia.

33

miRNA-155 Mice Unknown miRNA-155 promotes autoimmune inflammation

by enhancing inflammatory T cell development.

34

miRNA-34a Human p53 p53-independent up-regulation of miR-34a

during oncogene-induced senescence represses MYC.

35

miRNA-31 Human, mice Unknown miRNA-31 overexpression causes the regression of metastases with an effect on the primary tumor.

36

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miRNAs and human cancers

Cancer begins when genes in a cell become abnormal and the cell starts to grow and divide out of control. Figure 1.5 shows the nature of genetic changes that can induce the conversion of a normal cell into a cancer cell. There are two main types of genes that play a role in cancer: proto-oncogenes and tumor suppressor genes. In normal cells, proto-oncogenes are genes that normally help cells grow and tumor suppressor genes slow down cell division, repair DNA mistakes or tell cells when to die. Thus, in cancer cells, the activation of oncogenes or the inactivation of tumor suppressor genes induce cells to modify their rate of growth and proliferation thus becoming uncontrolled and pathogenic.37

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29 Figure 1.5. Transformation of normal cells into cancer cells under the action of oncogenes and tumor

suppressor genes.

A number of studies have proved that the aberrant expression of miRNAs is linked to various types of cancers.38 According to recent estimations, cancer represents 34% of all human

diseases-associated with deregulated miRNAs, thus being the major pathology where miRNAs are implicated, followed by immunological and infectious (15 %), cardiovascular (7 %), and others.28

The fi st epo t de o st ati g that i‘NAs a e elated to a e s a e f o C o e s g oup.39

In their study, they found that the miRNA-15a-16-1 cluster was frequently deleted or down-regulated in the majority (~68%) of B cells of patients with chronic lymphocytic leukemia (CLL) (Table 1.2). Three years later, the miRNA1792 cluster (consisting of six miRNAs: miRNA17, -18a, -19a, -19b-1, -20a and -92-1), which is involved in lymphoma, was found to be up-regulated in many different tumors (Table 1.2).40

Normal cell Cancer cells

oncogenes

tumor supressor genes

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30 Table 1.2. Examples of miRNAs involvement in cancer and their targets

Along with many findings concerning miRNAs implicated in cancer, whose some examples are summarized in Table 1.2, miRNA profiling studies revealed that there are significant differences in expression levels of miRNAs between normal and tumor tissue types that allowed also for an accurate discrimination between different types of cancer.40,51 Noteworthy,

deregulation of miRNAs in cancers was classified into two different categories: up-regulated miRNAs and down-regulated miRNAs. If a miRNA is up-regulated and inhibits the synthesis of one or more tumor suppressor proteins, it can be termed as an oncogenic miRNA (or oncomiR). Conversely, if a miRNA is down-regulated and inhibits the synthesis of one or more oncogenic proteins, it is called a tumor suppressor miRNA (Figure 1.6).28

miRNA Targets Function Cancer types Ref

miRNA-15a-16-1

BCL-2, MCL1 Tumor suppressor

CLL, Prostate cancer, pituitary adenomas.

39

Let-7 NRAS, KRAS, MYC

Tumor suppressor

Lung, colon, breast, ovarian, stomach.

41,42

miRNA-372 miRNA-373

LATS2 Oncogenic Testicular germ tumors, gastric, breast. 43,44 miRNA-34a, miRNA-34b, miRNA-34c BCL-2, CDK4, CDK6, MYC Tumor suppressor

Colon, lung, breast, kidney, bladder cancer.

35,45–47

miRNA-155 SHIP1, CEBPB

Oncogene CLL, AML, lung, colon, breast 48

miR-17-92 BIM, PTEN, Oncogene Lung, breast, colon and stomach.

40

miRNA-221 miRNA-222

PTEN Oncogene CLL, lung, breast, colon. 49

miRNA-221 miRNA-222

KIT Tumor suppressor

(32)

31 Figure 1.6. miRNAs as tumor suppressors and oncogenes in cancer.

It should be noted that the definition of miRNAs as oncogenes or as tumor suppressors requires an indication of the type of cells in which they act.52 Some miRNAs can behave like

oncogenes in one cell type and as tumor suppressors in others.7 For example, when

miRNA-221 is up-regulated in liver cancer, it exerts an oncogenic function by down-regulating the expression of the tumor suppressor phosphatase and tensin homolog (PTEN),49 but in

erythroblastic leukemia it acts as a tumor suppressor by reducing the expression of the KIT (tyrosine-protein kinase KIT or CD117) oncogene (Table 1.2).50 One possible explanation is that

the same miRNAs can participate in distinct pathways, having different effects on cell survival, growth and proliferation that are dependent on the cell type.6 This function is a result of

miRNA/mRNA interactions in which a unique miRNA could have different targets and a unique mRNA could be targeted by different miRNAs in different cells.6

Beside their involvement in cell proliferation, miRNAs have recently also been implicated in cell migration and metastasis as observed for instance with miRNA-10a highly expressed in metastatic breast cancer, which positively regulates cell migration and invasion.53

Due to the involvement of miRNAs in cancer, many recent researches have focused on the utility of miRNAs as effective tools in the fight against cancer. miRNAs can be used as biomarkers for cancer diagnosis and prognosis due to their specific expression in cancer.51

Indeed, each tumor type has a distinct miRNA signature that distinguishes it from normal tissues and other cancer types. Most cancers can be further sub-classified into different prognosis groups based on these signatures.40 Furthermore, a recent work has revealed that it

is possible to successfully classify poorly differentiated tumors using miRNAs expression profiles, whereas mRNAs profiles were highly inaccurate when applied to the same samples.51

(33)

32 Moreover, due to their small size, miRNAs are relatively resistant to RNase degradation suggesting that they would be promising candidates for detection in biological fluids.54 These

findings highlight the potential of miRNAs profiling in cancer diagnosis. Finally, miRNAs not only represent viable markers for the detection and characterization of cancers, but also provide potential tools for the development of new anticancer therapeutics (Figure 1.7).

miRNA

Biomarkers miRNA-targeted therapeutics

tissuses biological fluids

miRNA profiles

oncomiRNAs tumor- suppressor miRNAs

miRNA inhibitors or miRNA mimics

Figure 1.7. Application of miRNAs in cancer diagnosis and therapeutics.

Both oncogenic and tumor suppressor miRNAs play a central role in cancer development and progression thus being excellent candidates as biological targets for anticancer treatments. Two therapeutic approaches for miRNAs targeting have been reported to date: the inhibition of the expression of an oncogenic miRNA or the substitution for the loss of expression of a tumor suppressor miRNA.28 As cancer is a multigenic disease, the advantage of these

miRNA-targeted approaches is that miRNAs can target several genes involved in a specific pathway in cancer development. For this reason, compared to siRNA-targeted approaches which are designed to target individual genes, miRNA-targeted approaches are a potential rising star in the fight against cancer diseases since miRNAs can target multiple genes.7 However, there are

also substantial challenges associated with miRNAs targeted approaches. As mentioned above, the function of miRNAs depends on the expression of their crucial target. A miRNA can have different protein targets in different cell types of a same organism, thus consequently leading to opposite effects. Therefore, the specific modulation of miRNA expression using miRNA-targeted approaches might have beneficial effects in one cell type but harmful effects in another.7

(34)

33

4

miRNAs as therapeutic targets in cancer

It was only few years ago that the first human miRNA was discovered, and yet, a miRNA-targted therapeutic has recently entered a Phase II human clinical trial for HCV-infected patients.55 This rapid progress reflects the importance of deregulated miRNAs in human

pathologies and shows great promises for the development of new therapies, especially in the field of cancer treatment, in the foreseeable future.

This section aims to provide an overview of the field of miRNA-targeted therapeutics by giving a brief introduction about the strategies and the current approaches that have been experimentally validated (Figure 1.8). As mentioned above, there are two major strategies for developing miRNA-targeted therapeutics: (1) restoration of tumor suppressor miRNAs activity and (2) inhibition of oncogenic miRNAs. To reach this goal, various approaches have been proposed. Among them, oligonucleotides-based approaches and small molecules-based approaches have been employed to either directly inhibit or activate miRNAs or indirectly interfere with miRNAs expression upon targeting specific genes or transcription factors. The advantages and limitations of each individual approach will be discussed below.

(35)

34

4.1 Restoration of tumor suppressor miRNAs activity

A few successful approaches have been employed to restore levels of tumor suppressor miRNAs, including (i) miRNAs mimics, (ii) miRNAs expression vectors and (iii) small-molecule activators.

4.1.1 miRNAs mimics

miRNAs mimics are non-natural double-stranded miRNA-like RNA fragments. Such an RNA fragment is an oligonucleotide designed to have its e d pa tiall o ple e ta to the sele ted se ue e i the -UTR of the target mRNA.56 Once introduced into cells, this RNA

fragment, mimicking an endogenous miRNA, can bind specifically to its target mRNA thus regulating protein expression (a, Figure 1.9). In 2010, the Wiggins group has shown successful restoration of tumor suppressors miRNA-34a, leading to reduced cellular proliferation in the xenograft model of colon cancer.57 Following this study, other in vivo studies have been

focused on the improvement of delivering systems into cells of miRNA mimics, such as a viral vehicle transporting miRNA-34a to the xenograft model mentioned above,57 a neutral lipid

emulsion in a murine model of non-small cell lung cancer (NSCLC) for the same miRNA,58 a

lentivirus to deliver miRNA-34a in an aggressive mouse model of NSCLC in lung cancer.59 In

2013, the first clinical trial using miRNA-34a mimic (MRX34) was performed in patients with liver cancer,60 showing promising results with a safe profile.61 The application of this strategy

presents some advantages since (i) miRNA mimics can be designed to either direct translational process repression or to induce translational process repression and mRNA degradation depending upon the degree of complementarity between the sequence of miRNA mimic and the targeted mRNA;62 (ii) miRNA mimics act only on their specific targeted mRNA

and influence selectively the corresponding protein, while native miRNAs act on various mRNAs carrying the complementary binding sequence thus being less specific.63 However, the

lack of very efficient delivery systems may become a barrier for the advancement into the clinic of many miRNA mimics candidates.56

(36)

35

4.1.2 miRNA expression vectors

Another approach to increase the amounts of tumor suppressor miRNAs is to use miRNAs expression vectors. miRNA expression vectors are engineered with promoters that enable the expression of the miRNA of interest in a tissue- or tumor-specific fashion (b, Figure 1.9).14 As

an example, Kota et al. demonstrated that miRNA-26a, that is strongly down-regulated in hepatocellular carcinoma cells (HCC), could be systemically administrated in a mouse model of HCC using an adeno-associated virus (AAV), thus leading to the inhibition of cancer cells proliferation and the activation of tumor-specific apoptosis, resulting in dramatic suppression of tumor progression without toxicity.64 Although no study so far has reported the clinical

usage of such an approach for miRNAs restoration, AAV-based gene therapy holds great promise for highly-specific miRNAs-based therapeutics.64

Pre-miRNA miRNA duplex mature miRNA Nucleus Cytoplasm miRNA mimics miRNA vectors RISC ORF 3'UTR target mRNA

miRNA level increases to normal

Diminution of oncogene expression

miRNA as tumor suppressor genes

a. b.

Figure 1.9. Approaches for the overcoming of tumor suppressor miRNAs underexpression. (a) Use of oligonucleotides designed to mimic endogenous miRNAs. (b) Introduction of genes coding for miRNAs

(37)

36

4.1.3 Small molecules

While restoration of miRNA levels using synthetic oligonucleotides, such as miRNAs mimics or miRNA expression vectors, are straightforward, the use of small molecules for the same purpose, i.e. the increase of miRNAs amounts by activating their expression, holds challenges. The few studies which have identified small-molecule candidates increasing the intracellular amounts of miRNAs have been the result of high throughput screenings and two examples are reported in Figure 1.10.65,66 Both these molecules are able to activate transcription of miRNAs

genes in a non-specific manner leading to a global upregulation of miRNAs. The rational design of small molecules to up-regulate specific miRNAs has never been reported to date.

H N Cl Cl O 1.1 N N O OH O F N HN 1.2

Figure 1.10. Chemical structure of compounds 1.1 and 1.2 acting as activators of miRNA production, discovered after the screening of chemical libraries.

As a conclusion, the restoration of tumor suppressor miRNAs has emerged as a highly promising therapeutic strategy. However, this approach is very recent and only few achievements have been obtained so far, most notably with the clinical trials of miRNA-34a in patients with liver cancer.60,61 Improvement of in vivo delivery systems remains the main

challenge for successful translation into the clinic of tumor suppressors miRNAs as therapeutics.

4.2 Inhibition of oncogenic miRNAs

Compared to the restoration of tumor suppressor miRNAs, efforts towards the inhibition of overexpressed oncogenic miRNAs have been a major research topic over the past years. The current strategies have been divided into two major categories: oligonucleotides-based approaches (antisense oligonucleotides, miRNA sponges, miRNA masks) and small molecules-based approaches (Figure 1.8).

(38)

37

4.2.1 Oligonucleotides-based approaches

Oligonucleotides-based approaches rely on direct inhibition of miRNA function. The design of oligonucleotides complementary to either mature miRNA strand (antisense oligonucleotides, miRNAs sponges) or targeted mRNA (miRNAs masks) leads to the inhibition of miRNAs functions (Figure 1.11).67

Pre-miRNA miRNA duplex mature miRNA Nucleus Cytoplasm ORF 3'UTR target mRNA miRNA as oncogene

a. Antisense oligonucleotides (ASOs)

RISC/miRNA GFP Sponge constructs sponge construct b. miRNA sponges ORF 3'UTR target mRNA c. miRNA masks ORF 3'UTR target mRNA

miRNA level decreases to normal

Increase of tumor suppressor gene expression

miRNA/ASO duplex

RISC/miRNA

Figure 1.11. Oligonucleotides-based approaches for the inhibition of oncogenic miRNAs (a) Synthetic antisense oligonucleotides can target mature miRNAs and induce either degradation or duplex formation. (b) miRNA sponges transfected into cells containing multiple and tandem binding sites for the

targeted miRNAs, lead to a decrease in the expression levels of oncogenic miRNAs. (c) miRNA-masks designed to have the same seed se ue e tha iRNAs of i te est i o de to i d to ’-UTR of its target

(39)

38

(a)

Antisense oligonucleotides

Antisense oligonucleotides (ASOs, also known as anti-miRNAs) work as competitive inhibitors of miRNAs.52 The very first illustration for the functional inhibition of miRNAs was successfully

achieved by using ASOs against miRNA-2 and miRNA-13 in Drosophila in the study of Alexandre

et al.68 Further experiments using these simple ASOs were ineffective in other organisms such

as Caenorhabditis elegans due to their poor stability.69 Moreover, unmodified ASOs can cause

off-target effects upon binding to miRNAs bearing very similar sequences (miRNAs belonging to the same family).28 Thus, researchers introduced modifications in the chemical structure of

oligonucleotides to increase stability, binding affinity and specificity. This has been achieved through a variety of modifications such as (1) -positio odifi atio s of the suga -OMe and LNA) and (2) internucleotide linkage modifications such as phosphorothioates (Figure 1.12).

Figure 1.12. Most common structural modifications employed in ASOs (B: nucleobase)

A o g these odifi atio s, -O-methyl-modified oligonucleotides led to effective inhibitors with improved binding affinities for mature miRNAs in the study of association of ALG-1/ALG-2 (two C.elegans Argonaute proteins) with worm miRNA let-7, and better nuclease resistance than that of unmodified ASOs.69 Following these findings, Krutzfeldt et al. de eloped

-O-methyl-modified cholesterol-conjugated single-stranded RNA analogues, with phosphorothiorate linkage (also known as antagomirs), complementary to miRNA-122, which is abundant in the liver and implicated in hepatitis C virus proliferation.70 The silencing of

endogenous miR-122 by this method was specific, efficient and long lasting in in vivo experiments in liver cancer in mouse.70

(40)

39 The modified structure that displayed the highest affinity for miRNA targets, hence reducing the off-target effects,71 are the locked nucleic acids (LNA), a class of nucleic acid analogues in

which the sugar ring is locked by a methyle e idge o e ti g the -O ato a d the -C atom, thus locking the sugar in a C -endo conformation.72 LNA-anti-miRNAs with

phosphorothioate linkages were more efficiently delivered in cells and were able to effectively silence miRNA-122 at much lower doses than cholesterol conjugated analogues with mouse liver cancer model.70,73 At last, Miravisen, a 15-mer LNA-DNA hybrid (mixture of LNA and DNA

nucleoside units connected by phosphorothioate linkages) has recently entered a phase IIa clinical trials for treatment of hepatitis C virus (HCV) infection as an anti-miRNA-122 drug.55

This is the first miRNA-targeted drug to enter human clinical trials opening the door to a potential new treatment for patients with chronic hepatitis C infection.

(b)

miRNA sponges

Many miRNAs are members of families that share the same seed sequence (2-8nt motif at -end) but may have one or more nucleotide changes in the remaining sequence.74 Similar to

ASOs, miRNA sponges are all ASOs acting on a particular miRNA by base-pairing mechanisms and producing miRNAs inhibition effects. Compared to ASOs, which are designed to block a single miRNA, miRNA sponges contain multiple, tandem binding sites for a miRNA seed sequence thus being able to target all members of a miRNAs family, and representing a major improvement.75 In recent years, miRNA sponges have been proved effective in several cell

lines, including non-small cancer cell,76 embryonic neural stem cell77 and B cell lymphoma.78

(c)

miRNA masks

The fundamental mechanism of miRNA function is the base-paring interaction between a miRNA and the targeted mRNAs (miRNA/mRNA interaction).79 Thus, either miRNAs or mRNA

may serve as targets to alter miRNA function.

miRNA mask approach is based on the use of antisense oligonucleotides (ASOs). In contrast to antimiRs, miRNA masks consist of single-st a ded -O-methyl-modified oligonucleotides that do not directly interact with targeted miRNA, but that are complementary to the binding site of i‘NA i the -UTR of the target mRNA.80 In this way, a miRNA mask blocks the access of

the binding site of miRNA of interest on a specific target, thus leading to a miRNA mask/mRNA interaction instead of miRNA/mRNA interaction. In other words, miRNA masks act like

(41)

40 protectors of the gene from being inhibited by miRNA and their effect is sequence-specific. This approach has been applied successfully in a zebrafish model to prevent the repressive actions of miRNA-430 in transforming growth factor-β sig ali g path a s.81

In conclusion to this section, the application of the oligonucleotide strategy proved that a specific targeting of miRNAs leads to the desired biological effect. According to publicly available information, several miRNA candidates have already progressed into product and clinical development.82 However, it is known that there are challenges involved in the delivery

of these non-small-molecules, and their pharmacodynamic and pharmacokinetic properties are not ideal for therapeutic applications.83 These tasks require improvements in

oligonucleotides chemical design and in efficient delivery systems, which hopefully will continue to shed light on ways to increase therapeutic efficacy and specificity.

4.2.2 Small molecules-based approaches

In order to circumvent the implicit challenges of the use of oligonucleotides in therapy, indirect strategies, i.e. the use of drugs to modulate miRNA expression by targeting their transcription and processing, have also been developed. The discovery of small-molecule drugs targeting specific miRNAs and modulating their activities could be very useful as these drugs could be further employed for the development of targeted cancer therapies as well as therapies for other miRNA-related pathologies.

As explained earlier, biogenesis of miRNA is a complex multi-step process starting from the nucleus and going toward a mature miRNA duplex in the cytoplasm. Thus, manipulations of each step involved in this process may alter the biogenesis and/or the function of miRNAs. Moreover, during the course of their biogenesis, miRNA molecules are maintained in structural forms that are rich of secondary structures such as stem-loop structures and internal loops and bulges (Figure 1.13) for proper recognition by microprocessor and Dicer enzyme.84,85 These

structures offer the possibility for selective/specific binding interactions with small-molecule ligands.86,87

(42)

41 Figure 1.13. Four general classes of RNA secondary structure

An overview of the potential targets for small molecules intervention to activate or inhibit miRNAs inside cells is illustrated in Figure 1.14 (A-E) along with the multi-step miRNA biogenesis pathway.

Figure 1.14. Simplified overview over the miRNAs biogenesis pathway where each step of miRNAs biogenesis and action is indicated. Steps that have been inhibited or activated by the use of small

molecules are indicated by letters A-E.

A: transcription inhibition. B: Drosha-cleavage inhibition.

C: Dicer cleavage inhibition. D: RISC complex formation inhibition. E: miRNA/mRNA recognition inhibition.

(43)

42 Various reviews relating the targeting of miRNAs using small molecules28,88–91 have been

recently published. To date, a large number of examples of small molecules targeting transcription (A in Figure 1.14), Drosha or Dicer cleavage (B and C in Figure 1.14) and the formation of RISC complex with mRNA recognition by miRNAs (D and E in Figure 1.14) have been reported. Despite this, the main limitation of the use of small molecules for the inhibition of miRNAs pathway is the difficulty to rationally design compounds that would be specific for a particular RNA sequence. For this reason, the major part of the examples reported to date have been discovered after the random screening of large chemical libraries or based on known RNA ligands such as aminoglycoside antibiotics. Thus, in this section, we will describe most of the reported examples focusing on the medicinal chemistry approach that has been employed in order to discover small-molecule drugs able to specifically or selectively interfere with oncogenic miRNAs. These include (a) the high-throughput screening (HTS) of large libraries of compounds (> 1000 compounds) using cell-free as well as cell-based assays; (b) the focused screening of small libraries (< 100 compounds) of molecules that are known to be able to interact with RNA thus being supposed modulators of miRNAs pathway and (c) the design of small molecules based on the secondary structure of targeted RNA and/or three-dimensional structure of enzymes involved in miRNAs pathway.

(a)

High throughput screening approach

The first studies concerning the use of small molecules to interfere with oncogenic miRNAs were based on high throughput screenings (HTS). Some of these screenings are cell-free and are based on the use of fluorescence to detect small-molecule binding to the RNA target but cell-based assays were also largely applied to the screening of chemical libraries toward the discovery of miRNAs modulators. Most of them are based on luciferase reporter systems and have been recently reviewed.92 Other innovative screening methodologies have also been

recently validated but they could not be applied to actual libraries so far.93–95 In this first

section, we will describe HTS that led to the discovery of small molecules able (i) to inhibit miRNA transcription (ii) to inhibit Drosha or Dicer cleavage steps and (iii) to impede RISC loading.

(i) Discovery of small molecules inhibiting miRNAs transcription

One of the first studies describing the inhibition of an oncogenic miRNA by small molecules by HTS was reported by Deiters and co-workers.96 The luciferase-based screening of more than

(44)

43 1000 compounds led to the discovery of compound 1.3 (Figure 1.15C), a diazobenzene derivative, as an efficient inhibitor of miRNA-21.96 The reporter construct contained the

complementary sequences to mature miRNA-21, to miRNA-30 as control for specificity or to a negative-control linker sequence downstream of a luciferase reporter gene (Figure 1.15A). In the presence of miRNA-21, the luciferase signal is inhibited, while in the presence of efficient inhibitors of this miRNA, the luciferase expression is restored (Figure 1.15B). Several rounds of structural modification/screening led to the establishment of structure-activity relationships and to the identification of compound 1.4 (Figure 1.15C) twice more active than 1.3 and bearing an EC50of M. A detailed stud o the ode of a tio of 1.3 and 1.4 suggested that

both compounds target the transcription of miRNA-21 gene into pri-miRNA-21.

N N

O

NH

1.4

luciferase miR target sequence

miR inhibitors

luciferase miR target sequence

miR H2N N N NO2 1.3 A B C

Figure 1.15. Intracellular assay for the evaluation of miRNA inhibition by small molecules. In the absence of inhibitors, the presence of miRNA blocks luciferase expression (A) while in the presence of efficient inhibitors, the biogenesis of miRNA is inhibited and luciferase expression is restored (B). (C) miRNA-21

inhibitors 1.3 and 1.4 discovered after the screening of small molecule libraries.

(ii) Discovery of small molecules interfering with Drosha or Dicer

cleavage step

Various examples of small molecules able to bind to miRNAs precursors (pri-miRNAs or pre-miRNAs) have also been reported in the literature (Figure 1.16).

(45)

44 H2N N N H N NH N O OH O O O NH H2N NH2 O NH O NH2 O 1.5 NH O NH2 n 1.6 N NH2 H2N 1.7 N H NH NH2 H2N C N 1.9 S O NH2 O H2N O 1.8

Figure 1.16. Chemical structures of small molecules identified after high-throughput screening and that are able to interfere with Drosha or Dicer cleavage step.

Compound 1.5 was identified during a screening of peptoid microarrays containing 7680 compounds for the targeting of miRNA-21 precursors (pri-miRNA-21 and pre-miRNA-21).97 This

compound showed a good affinity for the target with a KD of . M a d a -fold

discrimination against a closely related RNA hairpin. However, it was not possible to define if these peptoids act at the level of Drosha or Dicer cleavage and no intracellular studies were performed.

Compounds 1.6 (polylysine, PLL) and 1.7 (trypaflavine, TPF) were discovered by Jeang et al. after the screening of 530 compounds in a luciferase reporter-based intracellular assay.98 They

demonstrated that these two compounds were able to inhibit miRNA-mediated silencing functions with different mechanisms. While compound 1.6 (PLL) impaired Dicer and Drosha activity, 1.7 (TPF) was an inhibitor of miRNA-loading into the Ago2 complex. The authors demonstrated that both compounds could reverse the tumorigenicity of miRNA-93 and miRNA-130b-overexpressing cells. However, this biological effect was not specific for particular miRNAs since both compounds affect general miRNA activities. On the other hand, Nakatani and co-workers discovered inhibitors of the Dicer cleavage step via a fluorescent indicator displacement (FID) assay using thioxanthone X2SS (1.8 in Figure 1.16) as a fluorescent probe since its fluorescence is efficiently quenched when bound to a pre-miRNA structure (Figure 1.17).99 FID assay is based on the fact that a stronger binder would cause its release from RNA,

(46)

45 of 9600 compounds over 10 different pre-miRNAs in order to investigate its potential. Various hits were identified: some of them were specific for a particular pre-miRNA, while others were able to bind to two or more pre-miRNAs. The best results were obtained with four compounds that showed specificity for pre-miRNA-29 demonstrating the efficacy of this screening, but no further studies were performed on these molecules.

Figure 1.17. FID assay using 2,7-bis (2-aminoethoxy)-9H-thioxanthen-9-one (X2SS). (adatepd from Ref. 95).

Another very efficient screening was employed by Kang and co-workers in 2013.101 In this

work, the screening was performed in silico based on the three-dimensional structure of pre-miR-21 generated from the miRbase database followed by MC-Fold/MC-Sym pipeline.102 The

high-throughput molecular docking for pre-miRNA-21 against 1990 compounds was performed using Autodock and led to the identification of 5 hits that were further studied for miRNA-21 inhibition in 4 human epithelial cancer cells (U87 and LN229 glioblastoma cells, MCF-7 breast cancer cells and SGC7901 gastric cancer cells). Compound AC1MMYR2 1.9 (Figure 1.16) showed the best results inhibiting specifically miRNA-21 in all cancer cells (about 50% i hi itio at M . However, an indirect inhibition effect was also observed for other intracellular miRNAs, as determined by RT-PCR quantification of miRNA-21 and of eleven other intracellular miRNAs. Indeed, the authors demonstrated that AC1MMYR2 is an inhibitor of the Dicer processing step of pre-miRNA-21 and in vivo preclinical trials showed a remarkable antitumor effect in both glioblastoma and breast cancer orthotopic models. This remarkable study proved that compounds able to inhibit specifically the biogenesis of oncogenic miRNAs can be efficient antitumor molecules even if other miRNAs are also influenced by their presence.

(47)

46

(iii) Discovery of small molecules interfering with RISC loading

miRNAs loading in RISC complex is another step that has been studied as a potential target for small molecules. H N N H O O S NH O O OH CH3 O 1.11 OH OH O O HO O OH OH O 1.10

Figure 1.18. Small molecules interfering with RISC loading identified after HTS.

Compound 1.10 (Figure 1.18) was identified when Kiriakidou and co-workers performed the screening of 2320 compounds using fluorescence polarization (FP) assay against miRNA-21.103

This cell-free assay is based on the combination of TAMRA-labeled miRNA-21 and recombinant Ago2 enzyme. A high polarization is observed with miRNA-21/Ago2 complex, whereas polarization value is lower in the presence of an inhibitor since the complex miRNA-21/Ago2 is blocked. Compound 1.10 was the most efficient inhibitor impeding the loading of miRNA-21 on Ago2 at a concentration of M (IC50 = 0.47 M) in human cells containing a luciferase

reporter system.

Finally, compound 1.11 (Figure 1.18) was identified by Del Rio et al,104 as a lead inhibitor by

high-throughput docking screening of around 260.000 compounds that were designed based on the crystallographic structure of the human Ago2 in complexation with miRNA-20a. This compound showed efficient binding to Ago2 in surface plasmon resonance (SPR) studies (KD =

126 µM) and inhibited miRNA-20a/Ago2 interaction in NB4 cells.

(b)

Focused screening approach

The second approach that has been envisaged in the field of miRNAs targeting is based on focused screenings. Most of the reported examples were applied to the research of binders of miRNAs or their precursors. In this context, smaller libraries containing compounds that are known to bind to RNAs and inhibit their interaction with intracellular cofactors or proteins are studied as binders of one of the intermediates of miRNA biogenesis as well as of mature

(48)

47 miRNAs. This approach is based on the hypothesis that a compound that would be able to efficiently and specifically bind to a miRNA precursor (pri-miRNA or pre-miRNA) would inhibit its processing toward the final miRNA, while a compound that would be able to interact with mature miRNA would inhibit its interaction with the target mRNA and eventually inhibit its function.

In 2006, Davies and Arenz have developed an in vitro assay based on the Fluoresence Resonance Energy Transfer (FRET) technique to identify small RNA binders targeting miRNAs precusors.105,106 The pre-miRNA is double-la eled ith a fluo opho e a d a ue he at the

a d e d, espe ti el . I the p ese e of e o i a t Di e e z e, the p e-miRNA is cleaved and fluorescence is observed. If a ligand is able to strongly bind to the structured pre-miRNA and inhibit the cleavage by Dicer, no fluorescence is detected (Figure 1.19).

Figure 1.19. A homogenous assay for miRNA maturation. The doubly labeled RNA beacon is hydrolyzed by Dicer leading to a fluorescence increase (left) and no fluorescence increase is seen in the presence of a

pre-miRNA ligand (right). (after Ref 101)

As a proof of principle, they showed that Dicer-mediated increase in fluorescence was inhibited by kanamycin A, an aminoglycoside antibiotic, with an IC50 of approximately 70 µM.

Figure

Figure 1.3. Simplified overview of the miRNAs biogenesis pathway where each step of miRNAs biogenesis  and action is indicated
Figure 1.7. Application of miRNAs in cancer diagnosis and therapeutics.
Figure 1.8. Strategies for miRNAs-based therapeutics.
Figure 1.9. Approaches for the overcoming of tumor suppressor miRNAs underexpression. (a) Use of  oligonucleotides designed to mimic endogenous miRNAs
+7

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