STUDIES ON PIN1 AS A TARGET IN ANTICANCER THERAPY

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UNIVERSITÁ DI PISA

DIPARTIMENTO DI FARMACIA

Corso di Laurea Specialistica in Farmacia

Tesi di Laurea:

STUDIES ON PIN1 AS A TARGET IN ANTICANCER THERAPY

Relatore: Prof. Tiziano Tuccinardi

Candidato: Daniela Bartuli

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Summary

1- INTRODUCTION

1.1 PPIases

1.2 Peculiarity of Pin1

1.3 Structure of Pin1: domains 1.4 Mechanism used by Pin1 1.5 Role of Pin1 in the cell cycle 1.6 Role of Pin1 in cancer

1.6.a Factors involved in oncogenesis 1.6.b Controversy of Pin1

2 THERAPEUTIC POTENTIAL OF Pin1 INHIBITORS

2.1 Anticancer therapy

2.2 Other therapeutic potentials 2.2.a Alzheimer disease 2.2.b HIV

3 STUDIES ON Pin1 INHIBITORS

3.1 PPIase domain inhibitors

3 3.1.b NON-PEPTIDIC INHIBITORS

3.1.b.1 Pipecolate core series 3.1.b.2 Benzotiophene series 3.1.b.3 Indole series 3.1.b.4 Benzimidazole series 3.1.b.5 Naphthyl series 3.1.b.6 Pyridine-pyrazole series 3.1.b.7 TMEs

3.1.b.8 Quinazoline based and Benzophenone series 3.1.b.9 Elemonic acid derivatives

3.1.b.10 D-aspartic and D-glutamic derivatives 3.1.b.11 Phosphate-based Pin1 inhibitors

3.2 WW-domain inhibitors

3.2.a EGCG

4 TOXICITY DERIVING BY Pin1 INHIBITION

5 CONCLUSIONS

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1.INTRODUCTION

1.1 PPIases

PPIases (peptidyl-prolyl cis-trans isomerases) are ubiquitous enzymes identified in each of the three kingdoms of life1_{, with the function to catalyse the otherwise slow conversion of the cis/trans} confor-mations in the peptidyl-prolyl bond, crucial step for protein folding. There are three families of PPIases1_:

 FKBPs (FK506-binding proteins)  Cyclophilins

 Parvulins

1.2 PECULIARITY OF PIN1

One of the several regulatory mechanisms to maintain steady-state intercellular signalling is the phosphorylation of proteins on serine or threonine preceding proline residues (Ser/Thr-Pro), which is the major phosphorylation site of a superfamily of proline-directed kinases, such as CDKs, MAPKs, JNK and GSK-3β2_{. Pin1 (Protein interaction with NIMA1) belongs to the Parvulins}3 _{family and it is able to} catalytically induce transformational changes in proteins after phosphorylation. Its peculiarity is to be the only enzyme that recognizes and catalyses isomerization of phosphorylated motif of Ser/Thr-Pro in proteins1_{, which exists in the two different conformations: cis and trans, and whose conversion rate} is normally restrained by phosphorylation4 _{(Figure 1.1.a and 1.1.b).Pin1 catalyses the cis/trans}

isomerization around the peptidyl bond linking the pSer/Thr and the proline residue2_{. This reaction is} a reversible reaction accelerating both cis to trans and trans to cis isomerization, meaning that reaction products are also substrates1_{. The precise mechanism by which Pin1 isomerises} phospho-Ser/Thr-Pro bonds remains unclear.

FIGURE 1.1.A (ABOVE)ISOMERIZATION OF A PEPTIDYL -PROLYL BOND

FIGURE 1.1.B (ON THE RIGHT) SCHEMATIC DIAGRAM OF A PIN1 SUBSTRATE

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1.3 STRUCTURE OF Pin1: DOMAINS

Pin1 is located at the chromosome 19p13. It is formed by 163 amino acid residues, and consists of two domains and a linker between them (Figure 1.2).

FIGURE 1.2PIN1 STRUCTURE:DOMAINS AND CTD. FROM K.P.,ZHAO X.Z.(2007)NATURE REVIEWS MOLECULAR CELL B IOL-OGY 8,904-916

The N-terminal domain (residues 1-39) is a WW domain which is hydrophobic in nature1_{and contains} two highly conserved tryptophan residues, Trp11 and Trp34, separated by three β-sheet substructures5_. Because of their side chains, tryptophan residues are often very important for the maintenance of the structure, function and substrate affinity of a protein5_{. It has the indispensable function to recognize} phosphoserine (pSer) and phosphothreonine (pThr)-proline motifs and interact with phosphorylated Ser/Thr-Pro sequences binding the substrates and bringing them into close proximity to the PPIase catalytic site.

In this process, the phosphorylation of the Ser161 _{motif is essential for interaction of Pin1 with its} sub-strates. Phosphorylation is in fact one of the ways used by post-translational controls to regulate Pin1 function2_.

6 The C-terminal domain (residues 50-163) is a PPIase domain, so it is the one responsible for the cata-lytic function of the peptidyl-prolyl isomerization reaction. It displays unique phosphorylation-depend-ent prolyl isomerase activity that specifically catalyses the isomerization of phosphorylated Ser or Thr preceding Proline bonds.

The active site of the PPIase domain presents different portions (Figure 1.3 and 1.4):

 a hydrophobic prolyl pocket, formed by His59, His157, Met130 and Phe134 and hypothesized to anchor the prolyl residue

 a slightly shallow hydrophobic shelf that embraces His59, Ala118 and Leu122

 the cation-recognition site also called “basic cluster”, that binds the phosphate group of its substrates and it is useful to stabilize the phosphoryl moiety of the substrate peptide via elec-trostatic effect. It is formed by positively charged side chains of Lys63, Arg68 and Arg697_.  an area called "canyon", delimited by Arg69, Trp73 and Ser114

 a surface called "platform", a little bit outside the catalytic site and under an important AA Cys113, which seems to have a crucial role in the isomerization reaction, continued by Leu122.

FIGURE 1.3 AND 1.4PIN1 ACTIVE SITE

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1.4 MECHANISM USED BY PIN1

The mechanism used by Pin1 to actuate the isomerization is not yet clear. There are two main theories: a) the process may consist in a nucleophilic addition,

b) it catalyses the reaction trough a twisted-amide-intermediate

Ranganathan et al. support the nucleophilic addition theory because of the lack of activity showed by the Cys113->Ala mutants. This theory proposes Cys113 to be a nucleophile that attacks the carbonyl of the substrate peptide bond, forming a covalent tetrahedral intermediate. Otherwise, more recent data showed instead that Cys113->Asp mutation does reduce activity but it is still capable to replace the function after a Ess1 deletion in budding yeast1_.

Conversely, Daum et al. argue that the mechanism takes places through a twisted amide intermediate assuming that the aryl 1-indanyl ketones would adopt a twisted conformation around the carbonyl group1_{. Etzkorm laboratory published a study after designing a reduced-amide inhibitor to mimic the} twisted amide intermediate (compound 1)1 _{(Figure 1.5). An x-ray crystal structure of Pin1 in a complex} with a solubilized version of this compound (compound 2) showed that the prolyl ring adopted the trans-pirrolidine conformation, as expected in a twisted-amide bond1_{. Since these inhibitors do not} bind the enzyme tightly enough to be considered transition state inhibitors, the twisted amide config-uration might represent a mezzanine free-energy level in the Pin1 catalysed reaction mechanism1_.

1 R= Fmoc 2 R= Ac

FIGURE 1.5STRUCTURES OF COMPOUND 1 AND COMPOUND 2

8 In view of the fact, studies on possible substrates suggest that the mechanism may not proceed by nucleophilic addiction. The isomerization in the cis-trans direction may instead proceed by destabilizing the cis conformation of the pSer-Pro peptide bond and consequently favouring the transition state conformation.

Anyway, the enzymatic mechanism to isomerize a prolyl-amidic bond suggests that there are important H-bonds with the thiol group of Cys113 and the hydroxyl group of Ser154.

1.5 ROLE OF Pin1 IN THE CELL CYCLE

Isomerization by Pin1 is involved in a multitude of biological processes working as a conformational switcher to modulate the function of many phosphoproteins with the result of enhancing or suppress-ing cell signallsuppress-ing6_{. Pin1 acts at a post-phosphorylation step, thus the isomerization that it performs} leads to influence dynamics and outcomes within pathways regulated by mitogen-activated kinases (MAPKs), cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3β (GSK-3β) 1_{In fact, these} ones operate a regulatory mechanism about cell proliferation and transformation by phosphorylating Ser/Thr-Pro motifs. There are several proteins that have been shown to be Pin1 targets, such as cyclin D1, c-jun, c-Myc, β-catenin, p53 and tau7_{. Its action is crucial in cycle regulation, but it is also involved} in oncogenesis, Alzheimer's disease and AIDS8_{. Furthermore, it is involved in the DNA damage response,} regulating p53 function.

Pin1 is very important in eukaryotes for the cell cycle progression, transcriptional regulation and cell proliferation and differentiation. It binds several proteins in a cell cycle regulated manner and its binding activity is low during G1 and S, increases in G2/M and is the highest in M phase.

The result is a negatively regulating entry into mitosis and is also required for proper progression through mitosis9_.

1.6 ROLE OF PIN1 IN CANCER

Pin1 is normally expressed at low levels in normal human tissues and its expression is normally related with cell proliferation4_.

9 It is instead up-regulated in cancer tissues10_{, where it binds and also positively regulates a series of} protein involved in oncogenesis processes. The first evidence of Pin1 involvement in cancer develop-ment came from the observation that Pin1 was over-expressed in breast cancer, where it has been found to correlate with high cyclin D1 and beta-catenin levels (both oncogenic markers) 2_.

This correlation between Pin1 over-expression and cancer development was subsequently confirmed by a large-scale study of Pin1 expression in other kind of cancers, showing high incidence of Pin1 over-expression in common cancers such as prostate, lung and colon cancer6_.

It is of interest that its over-expression is related to tumor grades and probability of cancer recurrence after surgery11_{, while in lung and colon it has a diagnostic value of the disease.}

Depletion of Pin1 using antisense Pin1 or dominant negative Pin1 causes mitotic arrest and induction of apoptosis in cancer cell lines9_{but also growth inhibition}2_{, suppressing both cell proliferation and the} transformed phenotypes6_{induced by Neu/Ras oncogenesis. In mice, Pin1 is dispensable for viability but} seems to be required for activated Ras or ErbB2 alleles to promote breast cancer1_{because their cells} result resistant to transformation by Ras and ErbB212_{. Studies have shown that in knock-out mice,} can-cer development by ErbB2 or Ras is almost completely blocked1_.

However, Pin1 overexpression is probably not sufficient to transform a normal cell in a cancer cell since its targets must be previously phosphorylated for Pin1 to bind and operate the prolyl-bond isomeriza-tion. In fact, there is plenty of signals in cancer cells that lead to phosphorylation of serine and threo-nine residues. Therefore, we can consider Pin1 as a oncogenic catalyst because it responds and ampli-fies signals, translating them into the actual events that transform the cells and lead to cancer2_.

1.6.a Factors involved with Pin1 in oncogenesis

As mentioned before, there is a correlation between over-expression of Pin1 and high levels of cyclin D1. Pin1 acts trough different pathway signalling to increase its transcription, but it also directly inter-acts with cyclin D1 itself, stabilising and activating it1_.

Ras/AP1 signalling pathway: Ras is one of the most common oncogenes in human cancer. It is a gene involved in cell growth, differentiation and survival. It is evident that if the signalling is deregulated, it may lead to cancer. Ras activates several pathways and in particular the MAP kinases cascade. Raf ki-nases participate in activating the MAP kiki-nases cascade, and the MAP kinase, in turn, phosphorylates and inactivates Raf in a negative feedback mechanism.

10 The MAP kinase cascade leads to the synthesis of proteins such as c-jun (a positive regulator of cell proliferation), which in combination with c-Fos forms the AP-1 early response transcription factor that causes the transcription of cyclin D12_{. Pin1 acts stabilizing and so activating c-jun/Fos and inhibiting the} negative feedback mechanism on Ras.

Wnt/β-catenin pathway: this pathway is involved in control of gene expression, cell adhesion and cell polarity. Pin1 is known to up-regulate β-catenin, an important downstream effector of the WNT (wing-less-type MMTV integration site family) pathway, by inhibiting its ability to interact with APC (adeno-matous polyposis coli, a protein of the destruction complex which normally would export to the cyto-plasm for degradation). This accumulation of β-catenin induces a cellular response via gene transduc-tion alongside the TCF/LEF transcriptransduc-tional factors (where Pin1 also has binding sites) which can initiate transcriptional activation of proteins such as cyclin D1, Jun and Myc2_.

NF-kB: NF-kB is another strong inducer of cyclin D1 gene expression, having two binding sites in the human cyclin D1 promoter. Pin1 regulates the NF-kB signalling binding directly to the NF-kB protein, p65, which causes an increase in NF-KB promoter activity and consequently the transcription of cyclin D12_.

Apart from working at the transcriptional level, Pin1 has also a post-transcriptional effect because it physically interacts with cyclin D1 and this interaction results in a positive effect on cyclin D1 protein stability and nuclear localization2_.

Moreover, Pin1 is an E2F downstream target gene expression, which is a family of transcriptional fac-tors important at the G1/S checkpoint in the cell cycle. E2F is activated by various oncogenic proteins such as Cdk4 (whose gene expression is induced by cyclin D1) or Myc. E2F-1 factor binds the oncosup-pressor Rb and the result is the activation of Pin1 creating this way a positive feedback loop. Given prevalent deregulation of E2F/Rb pathways in many human cancers, it may play a critical role in the up-regulation of Pin1 in cancer cells2_.

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FIGURE 1.6SCHEME OF SOME OF THE MAINPATHWAYS INVOLVED WITH PIN1

1.6.b Controversy of Pin1

Pin1 is involved in pathways where it amplifies oncogenic signals and promotes oncogenesis but at the same time it has a role in the DNA damage response where it activates a tumour suppressing gene, protein p53.

Protein p53 has multiple roles in the cell, such as cell cycle checkpoints, genomic stability and apoptosis. When a DNA damage happens, it leads to the stabilization and accumulation of p53 and consequently to the transcriptional activation of the cell cycle inhibitor p21 and cell cycle arrest2_.

The role of Pin1 in the process is to be a positive regulator of p53 in response to DNA damage. In fact, Pin1, through its isomerization mechanism, stabilise the protein p53 preventing it from binding to its

12 ubiquitin ligase, MDM2, but also Pin1 increases the transcription of the p21 and MDM2 promoters acted by the p532_.

The fact that Pin1 participates in both stopping and promoting oncogenesis is probably due to the spe-cific cell environment, type, proliferative status and age. Thus, Pin1 controversy may be explained un-derstanding the difference between its physiological function in normal cells and its pathological role in cancer cells.

2. THERAPEUTIC POTENCIAL OF Pin1 INHIBITORS

2.1 ANTI-CANCER THERAPY

The fact that Pin1 has been shown to have a role in both cell growth and oncogenesis, led to believe that it may be applied in cancer therapy. Several studies have been done since the discovery its involvement in cancer development and the results obtained during these years of research confirm Pin1 as a promising anticancer target.

What makes of Pin1 an attractive target is:

 It presents high substrate specificity and a well-defined active site. Although it made

challenging the discovery of effective Pin1 inhibitors, the cancer therapy is relatively long and the possibility to find inhibitors that do not interfere with other functions is really attractive.  Its overexpression in cancer tissues suggests it potentiates the function of some oncogenes,

therefore to inhibit the enzyme would mean restraining the activity of those oncogenes and, consequently, the cancer progression. An example concerns the chance to indirectly interfere with the function of Ras since Pin1 is a target required for activating alleles of Ras to promote oncogenesis and that directly interfere with Ras has proven really challenging.

 Furthermore, studies on Pin1-knockout mice testify their ability to reach adulthood despite some cell proliferative abnormalities1_{, suggesting that an anti-Pin1 therapy might not have} general toxic effects.

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2.2 OTHER THERAPEUTIC POTENTIALS

Pin1 is not only involved in cancer but also in a variety of disease such as Alzheimer disease and infectious diseases8_.

2.2.a Alzheimer

Alzheimer disease cause is still unclear. It is attributed to the mutation of the amyloid precursor protein (APP) gene which increases the production of two peptides of 40-42 amino acids, called Aβ40 and Aβ42. These two proteins are the major responsible of generating the senile plaques and starting the inflam-matory process that will lead to the neuronal damage. It seems that Pin1 isomerises the pThr668-Pro motif in APP, reducing the toxic peptides formation14_{. Another feature of Alzheimer disease is the} pres-ence of aggregates of hyper-phosphorylated microtubule-associated protein tau, called neurofibrillary tangles, inside nerve cell bodies. This causes the cytoskeleton destruction and the consequently inter-ruption of the neuron's transport system. Tau phosphorylation happens in sites targeted by protein kinases such as GSK3β and Cdk5 and most of these sites are proline-directed Ser/Thr sites. Pin1 in this case, isomerizing the pThr231-Pro motif in tau, would acts enhancing tau de-phosphorylation and re-storing the correct form of the protein and its ability to bind microtubules and to promote their assem-bly13,14_.

Therefore, Pin1 also acts as a preservative of the nerve tissue.

2.2.b HIV

The human immunodeficiency virus type-1 (HIV-1) is a retrovirus that causes overtime acquired immu-nodeficiency syndrome (AIDS). Because of his very limited genome, his life activity depends on the host proteins in the infected cells. Recent studies show that Pin1 is one of this host proteins involved in the life cycle of HIV-1, including:

-the uncoating of the HIV-1 core

-the reverse transcription of the RNA genome of HIV-1

14 Furthermore, Pin1 owns the effects of activating different oncogenes and of inactivating multiple tu-mour suppressor. This may cause an extension of the HIV-infected cells life making us hypothesize at Pin1 as a possible promoter of the HIV-infection.

In conclusion, Pin1 is a promising therapeutic target for the prevention of HIV-1 but also to block the development of AIDS.

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3. STUDIES ON Pin1 INHIBITORS

3.1 PPIase DOMAIN INHIBITORS

The research of molecules able to inhibit the activity of Pin1 started with a small molecule extracted from walnut trees1_{,the naphtochinone Juglone (Figure 3.1). It showed specificity against Pin1 and a} few others Parvulines,but resulted inactive to the other PPIases1_{. It can be considered more like an} inactivator rather than an inhibitor, because it leads an irreversible reaction, rapidly forming Michael adducts with Cys1131_.

FIGURE 3.1JUGLONE

The juglone-Pin1 adducts have no stability in cells and they are quickly ubiquinated and degraded. Unluckily, it also has secondary effect, such as the action on the tubulin and interactions with other enzymes, and this lack of selectivity makes it useless as a starting point but it has been used as a tool compound to study Pin1 function in vivo1_.

We can divide the discovered inhibitors in two classes: -peptidic inhibitors

-not-peptidic inhibitors 3.1.a Peptidic inhibitors

Several peptidyl inhibitors with high selectivity have been discovered using different approaches but unfortunately these kinds of molecule are generally not-permeable to the cell membrane, which limits their application as therapeutics.

16 One of the first strategy to find peptidyl inhibitors regards a general feature of the PPIases: they can bind but not isomerize oligopeptides when the @carbon of the amino acid proximal to the peptidyl-prolyl bond presents an altered stereochemistry. Groups of researchers decided to follow the obvious strategy to find an optimal substrate and then switch the amino acid preceding proline from a L to a D isomer. After several studies the research demonstrated that cyclic peptides without the phosphate group can have high affinity to Pin1.

Recently, a study has been published which reports the discovery of a bicyclic peptidyl inhibitor of Pin116_.

The study started with Peptide 116 _{which structure presents two rings. The A ring performs a} Pin1-binding phosphopeptide motif while the B ring consist in a cell-penetrating peptide (CPP). However, this structure presents a few problems such as the presence of the phosphate that with his negative charge may interfere with the positive charge of the CPP making the molecule impermeable to the cell membrane.

Since the removal of the phosphate group causes a great decrease in potency further optimization was required. Working on the N-terminal sequence they designed a second-generation bicyclic peptide library with nonproteinogenic amino acids in order to increase both the structural diversity and the proteolytic stability. The library was screened against a S16A/Y23A mutant of Pin1, which has a defective WW domain, and the hits were then optimized.

The most potent inhibitor of this series resulted to be peptide 3716_{which after several tests resulted to} be potent and selective versus Pin1 but also cell permeable and highly resistant to proteolytic degradation (Figure 3.2).

For all these reasons peptide 37 can be considered a useful chemical probe to explore Pin1 functions in cells and also a lead compound for further optimization

17 FIGURE 3.2EVOLUTION OF BICYCLIC PEPTIDE INHIBITORS AGAINST PIN1.THE MOIETIES IN RED DERIVE FROM LIBRARY SCREENING, WHILE IN BLUE ARE SHOWN THE CHANGES MADE DURING OPTIMIZATION. FROM JIANG B., PEI D. J.MED.CHEM.,2015;58(6306-6312)

Peptide 1 structure: -A ring [D-pThr-Pip-Nal] where Pip= (R)-piperidine-2-carboxylic acid Nal= L-naphthylalanine

-B ring [Phe-Nal-Arg-Arg-Arg-Arg]

Second generation bicyclic peptide library: [Tm-(X1_X2_X3 -Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-β-Ala-β-Ala-Pra-β-Ala-Hmb-β-Ala-β-Ala-Met-resin

Where Tm= trimesic acid

Dap= 2,3-diaminopropionic acid Β-Ala= β-alanine

Pra= L-proparglycine

Hmb= 4-hydroxymethyl benzoic acid

X1 _{and X}2 _{represented any of the 27 amino acid building blocks that included 12 proteinogenic} L-amino acids [Arg, Asp, Gln, Gly, His, Ile, Lys, Pro, Ser, Thr, Trp and Tyr], 5 nonproteinogenic α-L-L-amino

18 acids [4-fluorophenylalanine (Fpa), norleucine (Nle), ornithine (Orn), phenylglycine (Phg), and L-Nal], 6 α-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, and D-Val], and 4 N^α-methylated L-amino acids [L-N^α-methyl-alanine (Mal), L-N^α-methyleucine (Mle), L-N^α-methylphenylalanine (Mpa), and sarcosine (sar)], while X3 _{was Asp, Glu, D-Asp, D-Glu, or D-Thr.}

Peptide 4: X1_{=D-Phe, X}2_{=Fpa, and X}3_=D-Thr

Peptide 37: X1_{=Fpa, X}2_{=Fpa, and X}3_=D-Thr

3.1.b. Non-peptidic inhibitors

The research of Pin1 inhibitors started with Pfizer that screened over a million compounds in HTS8_{. With} this method, they found a lot of false positives and for this reason they tried a different approach, switching to a design strategy.

As first attempt to find a starting point for design, they decided to test substrates of FKBP-12, another PPIase which has structural similarities in the catalytic site with Pin1. Both of them recognize proline residues as part of the substrate epitope even if, in comparison with FKBP-12, Pin1 also presents a rich network of H-bond potential and a larger binding site with an additional Ser-154 and Gnl-131 residues which provides two H-bond donors.

Unfortunately, the screening campaign failed and to have a starting point for inhibitor design, a genetically engineered Pin1 catalytic domain has been built and then crystallized in order to obtain an X-ray apo structure.

The apo structure does not contain the WW-domain and the sulfate ion. For this reason, the positively charged recognition pocket was occupied by water molecules or a citric acid molecule. The canyon region and the prolyl binding pocket are occupied by water molecules as well.

3.1.b.1-Inhibitors containing a Pipecolate core

Pipecolate core is a well-known scaffold for FKBP inhibitors. Basing on the knowledge of the binding mode with FKBP and trying to adapt the molecule to the Pin1 enzyme in the way to increase the interaction with it, Pfizer`s researchers added a phenyl fragment in position X to interact with the 'canyon' region1 _{(Figure 3.3).}

19 

FIGURE 3.3 JOB PROCESSING OF THE PIPECOLATE CORE

An example is compound 33 _{(Figure 3.4), which has been realized with the purpose to:} -reach the charged pocket with his phosphate group

-reach the prolyn-binding site -reach the 'canyon' region

FIGURE 3.4STRUCTURE OF COMPOUND 3

Compound 3 was determined to be a low micromolar Pin1 inhibitor (Ki = 1.7µM), so it was used for further structure-based design and optimization of the de novo lead. Unfortunately, although derived compounds showed significant selectivity over FKBP despite the pipecolate is a putative FKBP binding motif, they demonstrated shallow SAR and limited potency3_.

20 After new studies, they realized that the pipecolate core was not the optimal scaffold for the ligand efficiency, so they tried to replace it with a new better one. Retaining the phenylalaninol phosphate1_, they designed a focus combinatorial library to search for a pipecolate replacement until they arrived at compound 5b3 _{(Table 3.1).}



Library core Library focus

Compound R X Ki (μM) 4a Ph -SO2- 8.77 4b Ph -NHC(O)- 4.13 4c Ph -C(O)- 0.525 5a 2-naphthyl -C(O)- 0.1 5b 2-benzo[b]thiophenyl -C(O)- 0.179

TABLE 3.1COMPOUNDS CONTAINING A PHENYLALANINOL PHOSPHATE, DISCOVERED FROM A FOCUS COMBINATORIAL LIBRARY

Compound 5b maintains the phenylalaninol phosphate moiety but it has a benzotiophene ring instead of the pipecolate core, which has shown to improve potency and drug-like properties. They were also able to crystallize it with the PPIase K7782Q3_{and this structure was used as a guide for drug design.}

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FIGURE 3.5CRYSTALLIZED STRUCTURE COMPOUND 5B (PDBID:3IKD)

The crystal structure revealed a new binding mode, completely different from that one seen before1,3 (Figure 3.5)

-the phosphate group still interacts with the charged pocket forming H-bonds with the three residues Lys63, Arg68 and Arg69 like the previous ligands

-the phenyl group binds the prolyl-pocket

-the benzene ring forms an edge-on interaction with His157

-the thiophenyl group binds the hydrophobic area formed by Met130, Phe134 and Leu122 -the amidic nitrogen forms a weak H-bond with the sulfur of Cys113

-the amidic oxygen remains exposed to the solvent

Unfortunately, the 'canyon' region was harder to reach than expected, but a structure analysis of the co-crystalline structure showed a small hydrophobic cavity between the C3 of the phenyl ring and the Phe134 residue of Pin1. For this reason, they tried to carry on the research going to make substitutions on the C33_{. The best group was a fluoride, while other substitutions shown to reduce the binding affinity} (Table 3.2).

22 Compound R Ar 6a 3-F-phenyl 2-naphthyl 6b 3-F-phenyl 2-benzothiophenyl 7a 3-methylphenyl 2-naphthyl 7b 3-methylphenyl 2-benzothiophenyl 8a 2,3-diF-phenyl 2-naphthyl 8b 2,3-diF-phenyl 2-benzothiophenyl

TABLE 3.2COMPOUNDS RESULTED AFTER MODIFICATIONS IN C3 POSITION.

Despite the high affinity for the catalytic site, this compound series resulted not applicable to cell-based systems due to the low cell permeability arising from the presence of the phosphate group1,2,11_{. The} attempt to directly replace it with a carboxylate led to the complete loss of activity1_.

After all the previous studies, the research pointed to fix the problem of the poor cell permeability looking for a replacement of the phosphate group with better pharmaceutical properties9_.

3-Indole series

With the aim to obtain an effective way of finding starting points for drug discovery, Vernalis developed an NMR-based fragment screening platform (SeeDs) capable to identify compounds that compete with known competitor ligands. Therefore, a library of ~1200 fragments was screened and five competitively-binding compounds were identified17 _{(Table 3.3).}

23 Compound R1 R2 R3 X IC50 (μM) 9 COOH Me H C 16 10 COOH H Me C 630 11 COOH Me H N 63 12 COOH CN H N 10 13 (CH2)COOH H H N 740 14 (CH2)COOH H H C 450

TABLE 3.3COMPOUNDS IDENTIFIED FROM A LIBRARY SCREENING, CONTAINING AN INDOLE FRAGMENT

The most potent of the five resulted to be compound 9 with an IC50 of 16µM1,3 _{(Figure 3.6). It belongs} to a series that contains an indole ring and has a carboxylate group instead of the phosphate group, thus reducing the problem of the poor permeability seen in the previous series3_.

FIGURE 3.6STRUCTURE OF COMPOUND 9

The indole ring fits into a hydrophobic pocket in Pin1 active site.

The carboxylate group interacts with Lys63 in the charged pocket and a network of waters, while the indole nitrogen seems to weakly interact with the side chain of Cys113. So, this series maintains two of the binding attacks of the benzothiophene series3 _{(charged pocket and side chain of Cys113) without} having the phosphate group to reduce permeability. Anyway, this compound resulted not good even as a starting point having no portions to elaborate1_.

24 4-Benzimidazole series

The research led to benzimidazole 2-carboxilic acids (Figure 3.7) with an added side chain on the C2 of the phenyl group, in order to create an interaction with the “platform”.

FIGURE 3.7GENERAL STRUCTURE OF A BENZIMIDAZOLE

Adding an amide showed an increase in potency with this new part going to interact with the hydrophobic shelf. It is best if this side chain is rigid, because flexibility shown to confer a decrease in entropy of the binding with Pin1.

The next step was to find a substituent able to cover a bigger area of the hydrophobic shelf. It was found that a furan ring with a phenyl substituent (with substituent on his ring as well) like in compound 16e (Figure 3.8), or a pyrazole with a phenyl ring, bring to more active derivatives. (Table 3.4)

Compound R1 R2 IC50 (μM) 15a Et H 78 15b H 6.3 FIGURE 3.8STRUCTURE OF COMPOUND 16E

25 15c H 7.5 15d H 0.83 16a H 6.6 16b H 0.13 17 CH3 92 16c H 0.32 16d H 0.18 16e H 0.025 18 H 0.26

TABLE 3.4COMPOUNDS CONTAINING A BENZIMIDAZOLE WITH AN ADDED CHAIN TO INTERACT WITH THE HYDROPHO-BIC SHELF

26 Compound 16e was the best AA-based Pin1-inhibitor with IC50 of 25nM1,17 _{and SPR confirmed an} interaction17 _{with Pin1 with 1:1 stoichiometry, but resulted inactive in cell assays because of his low} permeability.

Compound 18 derives from the structure of compound 16e but instead of a furan it has a pyrazole ring, with an IC50 of 260nM1,17_{. The benzimidazole moiety maintains the binding with the hydrophobic} pocket, but the carboxylate group is slightly shifted compared to the previous ones. Furthermore, the pyrazole and the phenyl ring go on the hydrophobic shelf, but the pyrazole nitrogen does not seem to interact with protein residues and with water molecules either17 _{(Figure 3.9).}

FIGURE 3.9CRYSTAL STRUCTURE OF COMPOUND 18

Even this compound resulted inactive in cell assays, through his X-ray crystal structure they found a new series of ligands.

27 5- Naphthyl series

The previous compounds had a too high PSA (polar surface area) which conferred them a poor cell permeability1,17_{. To reduce the PSA the benzimidazole was replaced with a naphthyl group, even if this} caused a 10-20-fold loss in potency probably due to the loss of the interactions made by the benzimidazole nitrogens17_.

Compound R IC50 (μM) PC3 GI50 (μM)

19a 2.6 12.5

19b (VER1) 3.9 4.7

TABLE 3.5COMPOUNDS CONTAINING A NAPHTHYL GROUP

Compound 19b (VER1)17_{, the naphthyl analogue of compound 18, resulted to be a less efficient inhibitor} of Pin1 but its crystallized structure with Pin1R117_{shows that the replacement of the benzimidazole} with naphthyl group does not alter the binding mode (Table 3.5).

In fact, even if the carboxylate group is slightly worse positioned compared to the benzimidazole analogues, it is still able to interact with Lys63 and the substituent R still can go on the shelf-like hydrophobic surface17 _{(Figure 3.10).}

28

FIGURE 3.10CRYSTAL STRUCTURE OF COMPOUND 19B (VER1)

Encouragingly, despite the loss in potency, compounds 19a and 19b were able to inhibit the growth of PC3 prostate cancer cells1 _{under serum-free conditions}17_{, with good evidence that the mode of action} was at least in part via Pin1 inhibition.

6-Pyridine-pyrazole series

Vernalis carried on the research testing 900 fragments in a protease coupled assay at a compound concentration of 2mM. They found two active molecules, verified by two-dimensional NMR experiments12_.

One of these two molecules, compound 20 (Figure 3.11), was crystallized with Pin1R14A and showed an IC50 of 720 µM.

29 It is a pyridine-pyrazole acid and binds with Pin1 in a similar way as seen in compound 9, the indole discovered by Vernalis1,17 _{(Figures 3.12 and 3.13).}

-the carboxylate group interacts with Lys63 assuming the same position

-one of the pyrazole nitrogens overlaps with the indole nitrogen of compound 25, making the same H-bond with Cys11312_.

They decided to use compound 20 as starting point to synthetize a series of neighbours and several crystal structures of them were obtained. The most potent one resulted to be compound 21 (Figure 3.14) with an IC50 of 180 µM and LE of 0.3412_.

FIGURE 3.13CRYSTAL STRUCTURE OF COMPOUND 9

(PDBID:3KCE) FIGURE 3.12CYSTAL STRUCTURE OF COMPOUND 20

30

FIGURE 3.15CRYSTAL STRUCTURE OF COMPOUND 21(PDBID:2XP4)

This compound has an imidazole core and his imidazole nitrogens form H-bonds with Cys113 and Ser154 (Figure 3.15). At this point, they tried to investigate about possible modifications of the phenyl ring in order to reach a little hydrophobic pocket formed by side chains of Met130, Gln131 and Phe134. It has been found that the elaboration is possible in position 3 only with small groups such as: trifluoromethyl (-CF3), methyl (-CH3), nitrile (-CN) and chlorine (-Cl). Substitutions with such groups cause an improvement in potency by up to 10-fold, while bulky groups are not good instead12_.

Furthermore, adding a methyl group in position 5 of the imidazole ring would act as a vector towards the Trp73 in the canyon12_.

So, two new series of molecules were made: the first based on the addition of groups in position 3 of the phenyl ring (Table 3.6) and the addition of a methyl group in position 5 of the imidazole ring (Table 3.6), while the second shows further elaborations of the methyl group such as amides and esters12 (Table 3.7).

FIGURE 3.14STRUCTURE OF COMPOUND 21

31

Compound R IC50 (μM) LE (kcal/mol/heavy atom)

22a H 360 0.30 22b CF3 23 0.31 22c CH3 34 0.36 22d CN 19 0.36 22e Cl 20 0.38 22f OCH3 270 0.27

TABLE 3.6FIRST SERIES BASED ON THE ADDITION OF GROUPS IN POSITION 3 OF THE PHENYL RING AND THE ADDITION OF A METHYL GROUP IN POSITION 5 OF THE IMIDAZOLE RING

Modifications with amides was not encouraging at first because, except for the methyl amide 24a that maintained the high LE, the LE declines in the larger secondary ones12_.

Surprisingly, modification of the methyl group with a morpholino tertiary amide group (compound 24c showed to increase potency. This group doesn't go in the in the channel towards Trp73 but it stays stuck to the hydrophobic part of the side chain of Arg68. The carboxylate group, instead, is oriented towards Trp63. This discovery suggested to further explore tertiary amides12_.

The ester analogue 24d shows a similar way as 24c to interact with Pin1 but also an additional interaction between the carbonyl oxygen of its ester substituent and the amine nitrogen in Arg69. Unfortunately, it did not show activity on PC3 cells12_{. The attempt to increase activity on cells but also} maintain a reasonable LLE (ligand lipophilic efficiency) led to ligands with better efficiency and reasonable LLE, such as 24f which presents a N-methyl primary amide, and the most potent inhibitor of the series 24g which instead of the methyl group presents a methyl-benzothiophene moiety.

32 Although the encouraging increase of potency versus Pin1 in vitro, they showed no ability to block PC3 proliferation12_{. The solution of adding more lipophilic groups may improve the cell penetration but at} the same time it compromises the drug-likeness of the compounds.

Compound R1 R2 IC50 (μM) LE (kcal/mol/heavy atom) LLEAT (kcal/mol/heavy atom) 23 H OH 1.46 0.44 0.47 24a H NHMe 80 0.29 0.34 24b H NHBn 220 0.20 0.15 24c H 15 0.28 0.34 24d H 8.0 0.22 0.20 24e H -N(Bn)2 4.0 0.23 0.12 24f H 4.1 0.31 0.43 24g H 0.52 0.25 0.27

33 So, they went one step back, adding a chlorine atom in position 3 of the phenyl group in the previous compounds and obtaining in most cases an increase in potency, while still conserving their binding mode12 _{(Figure 3.16) (Table 3.8).}

FIGURE 3.16CRYSTAL STRUCTURES OF COMPOUND 24F AND COMPOUND 25

Compound R IC50 (μM) LE (kcal/mol/heavy

atom)

PC3 GI50 (μM)

34

26 21 0.24 >200

27 1.4 0.23 18

28 0.83 0.23 13

TABLE 3.8COMPOUNDS HAVING A CHLORINE ATOM IN C3 POSITION OF THE PHENYL RING

An example of the series with a chlorine atom is compound 28, which showed an excellent permeability in a CaCO2 assay and blocked the proliferation of serum starved PC3 cells12 _{with considerable evidence} that the effects on cells involve Pin1 inhibition.

Like the others compounds of the series it maintained the interactions with the hydrophobic pocket and the charged pocket and its nitrogens form H-bond with Cys113 and Ser154 but, unlike the previous series, this series doesn't interact with the hydrophobic shelf near Cys113 but, it instead stacks along the side chain of Arg68 and possibly Arg69 in the other side of the pocket12_.

The problem with these compounds is that they resulted to be inactive on cells and the attempt to add lipophilic groups to increase the permeability compromised the drug-likeness of the compounds. Anyway, their discovery was important because it opened the possibility to try different combinations of interactions in the research of a ligand which may be developed as a drug.

7-TMEs

In 2011 Uchida and co-workers used a new method to screen 9756 compound8_{. They used a} high-throughput screening (HTS) combined with real-time fluorescence monitoring system, followed by surface plasmon resonance (SPR) to confirm the binding specificity and get rid of the false positives8_. By adopting this procedure, SPR confirmed seven compounds8 _{which bind Pin1 in a} concentration-dependent manner, out of the 66 putative hits identified by HTS.

35 Among these, TME 001-005 have a structure formed by a phenyl ring linked to a thiazole group, while KK52 and KK54 have a Se atom instead of a S and no phenyl ring8 _{(Figure 3.17).}

TME-001 (IC50 = 6.1μM) _{TME-002 (IC50 = 4.1Μm)}

TME-003 (50%)

TME-004 (46.5%)

TME-005 (36.3%)

TME-006 (N.D.) _{KK-52 (IC50 = 61μΜ) KK-54 (IC50 = 109μΜ)}

FIGURE 3.17STRUCTURES OF THE COMPOUNDS IDENTIFIED IN THE STUDY.THE INHIBITION RATIOS AT 10ΜΜ IS SHOWN IN PARENTHESIS.N.D., NOT DETERMINED.

Among the seven compounds all resulted active except for TME-006 that resulted ineffective.

TME001 and TME002 resulted to be the most potent ones (with an IC50 of 6.1μM and 4.1μM, respectively) and both have a chlorine in position 3 of the phenyl ring.

TME001 works as a competitive inhibitor, which binds the PPIase domain but not the WW-domain, and it has IC50 of 6,1μM1_{. Studies on cell assays have shown TME001 having a cytotoxic effect when the} concentration is higher than 100μM8_{, while it has no effect at concentration lower than 30μM}1_. Moreover, at a concentration of 30μM studies on HeLa cells after release from serum starvation

36 showed they go through growth arrest, similar to what happens in Pin1 -/- cells1_{. TME001 also has an} effect on cyclophillin CypA with an IC50 value of 13.7μM, but has not effect against FKBP, thus suggesting that there could be similarities with the CypA active site8_.

Docking studies have shown that the portion 3-chloro-2-phenyl occupies the hydrophobic pocket (Phe134 and Met130), while the sulfide and the 3-ketone of the isothiazole form, H-bonds with Cys113 and the combination of Arg68/Ser134, respectively.

The docking studies8 _{have also shown that TME001 is almost completely overlapped to the} crystallographic structure of the phenyl-imidazole moiety seen previously and it shown similar binding mode, but the isothiazol-3-one is >15fold more potent, probably due to the tight interactions that it makes with Pin1.

The 3-chloro-2-fluorophenyl goes to occupy the hydrophobic pocket formed by Met130 and Phe134, while the isothiazole ring forms H-bonds with Pin1 between the sulfur and Arg68 and between the 3-ketone and Ser134.

Therefore, due to the ability of TME-001 to interfere with Pin1 functions in cells at a no cytotoxic concentration and the good binding with Pin1 which confers its potency, it is reasonable to assume that isothiazol-3-one structure might be suitable as lead compound.

8-Quinazoline-based and benzophenone series

In order to find novel Pin1 inhibitors, a library was screened and this resulted in the discovery of quinazoline-based Pin1 inhibitors and a compound containing a benzophenone motif.

Quinazoline

Through a library screening compound 29a6_{(Figure 3.18) was found and then used to synthetize other} quintazoline-based derivatives in order to investigate their SAR6_{. Compound 29a structure consists in a} quinazoline scaffold with substituents in 2,4,6-positions. The quinazoline moiety is positioned in the prolyl pocket where the nitrogen group in 6-position forms a H-bond with the –OH on the side chain of Ser154 and –NH on both side chain and amide bond on GLN131. The carboxyl group at 4-position interacts with the basic cluster, while the substituent in 2-position seems to make hydrophobic

37 interaction with the shallow surface6_{. The quinazoline scaffold was modified on the positions 2,4 and 6} (Figure 3.19) and the inhibitory activity of the compounds was investigated by a protease-coupled enzyme assay6 _{(Table 3.9 and Table 3.10).}

The results showed that:

- in 6-position (R1) the presence of a hydrogen acceptor is advantageous for the interaction with Pin1 since the nitrogen group showed the ability to form a H-bond with the –OH on the side chain of Ser154 and –NH on both side chain and amide bond on GLN131

-in 4-position (R2) it is better to have a rigid linker between the quinazoline scaffold and the group that interacts with the charged pocket.

Compound R1 R2 R3 IC50 (μM)

29a NO2 4-COOH-Ph 4-PhOPh 4.87

29b NH2 4-COOH-Ph 4-PhOPh 23.05

29c NO2 CH2COOH 4-PhOPh 16.4

29d NO2 (CH2)2COOH 4-PhOPh 31.4

29e NO2 (CH2)2COOH 3,5-CF3-PhCH2 16.4

FIGURE 3.18STRUCTURE OF COMPOUND 29A FIGURE 3.19GENERAL STRUCTURE OF

38

29f NO2 (CH2)3COOH 3,5-CF3-PhCH2 33.2

29g NO2 4-PhOPh 4-COOH-Ph 9.46

29h H 4-COOH-Ph 4-PhOPh 9.81

TABLE 3.9COMPOUNDS BEARING A QUINAZOLINE SCAFFOLD MODIFIED IN 2,4 AND 6 POSITIONS

Compound R1 IC50 (μM)

30a 4-PhOPh 9.75

30b PhCH2 46.5

30c 3-F-Ph NA

TABLE 3.10COMPOUNDS BEARING A QUINAZOLINE SCAFFOLDM, MODIFIED IN 2-POSITION

As a result of the investigation, they decided to maintain the 4-carboxyl-phenylamino and 6-nitro substituents, changing that one in 2-position6 _{(Table 3.11)}

Compound R2 R3 IC50 (μM)

39 31b 4-Cl-Ph 4-COOH-Ph 9.0 31c 3,4-Cl-Ph 4-COOH-Ph 2.90 31d 3-CF3-Ph 4-COOH-Ph 8.50 31e 4-CF3-Ph 4-COOH-Ph 5.60 31f PhCH2CH2 4-COOH-Ph 52.5 31g Cyclohexyl 4-COOH-Ph 54.2

TABLE 3.11COMPOUNDS BEARING A QUINAZOLINE SCAFFOLD, A 6-NITRO GROUP AND A 4-CARBOXYL-PHENYLAMINO AND MODIFIED IN 2-POSITION.

These compounds showed high inhibitory activity with IC50 value ranging from 2.9 to 54.2 μM.

Among the synthesized compounds those having an aromatic hydrophobic substituent resulted to be more active compared to those with alkyl hydrophobic groups6_{. In fact, compound 31c resulted to be} the most active of them with an IC50 value of 2.9 μM6.

Therefore, is preferable to use a bulky substituent to promote the interactions with the shallow hydrophobic region of Pin1.

In order to further improve the inhibitory activity, the binding mode was explored for the new compounds. The docking6 _{resulted to be similar to the binding mode of compound 29a and showed} that an additional H-bond donor on the benzene ring might helpful to the binding orientation making an H-bond with the side chains of GLN131 and Ser154.

benzophenone

From the library screening the new Compound 32a was identified10_{. It presents a benzophenone motif,} an oxalic acid instead of the phosphate group and an IC value of 10,11 µM.

The structure-activity relationship (SAR) was investigated by synthetizing a series of benzophenones with different substituents in position 4 of the A ring and in positions 3 and 4 of the B ring (Table 3.12).

40 Compound R1 R2 R3 IC50 (μM) 32a H H 10.11 32b H H 11.49 32c H H 8.93 32d H H 5.99 32e H H 6.31 32f H H 18.30 32g H H CH2CO- >100

41

32h OCH3 OCH3 54.33

32i OCH3 OCH3 53.55

32j F OCH3 10.36

32k F OCH3 14.52

TABLE 3.12BENZOPHENONES WITH DIFFERENT SUBSTITUENTS IN POSITION 4 OF THE A RING AND IN POSITIONS 3 AND

4 OF THE B RING

Substitutions on the A RING (R3)

 the best groups are: 7-nitrobenzothiophene and indole

 Little less active groups are: benzothiophene-2-carboxamido, benzothiophene-3-carboxamido and 3-chlore-benzothiophene-2-carboxamido

 An inactive group has shown to be the acetyl group, bringing to the conclusion that a bulky group is necessary for the affinity.

Substitutions on the B RING (R1 & R2 ) Compared to R1=R2=H:

 To replace with a methoxide group (-OCH3) decreases the activity

 To replace with on flourine (-F) and one methoxide group (-OCH3) brings to almost same activity

42

FIGURE3.20STRUCTUREOFTHEELEMONICACID-COMPOUND33

The binding mode was investigated using FlexX algorithm implemented in SYBYL 7.210_{. Taking} compound 32e we can exemplify the binding mode as follows:

 The 2-oxalic acid mimic the phosphate of Pin1 substrates and interacts with the basic cluster. The carboxylate group interacts with the positively charged side chains of Arg68 and Lys63. Furthermore, the 2-carbonyl oxygen of the oxalic acid makes an H-bond with the hydroxyl group of Ser114.

 The A ring is situated in a little hydrophobic area formed by His59 and Cys113, so probably the addition of a H-bonds acceptor group would lead to an increase of the binding affinity

 The B ring doesn't occupy the prolyl pocket

 The carbonyl oxygen in between the A and B rings forms a H bond with the hydroxyl group of Ser154

 The bulky substituent on the A ring is able to reach the hydrophobic shelf, included the side chains of Leu122 and Met130. Its orientation is slightly different than what observed in the naphthalene group for example. Both groups anyway make important hydrophobic interactions thanks to the large hydrophobic region.

9-Elemonic acid derivatives

During studies on elemonic acid (3-oxo-tirucallic acid) (Figure 3.20) it has been found an antitumor activity against human neuroblastoma cell lines and cytotoxic effects in human prostate cancer cell lines18_{. For this reason, the researchers tested their ability to inhibit Pin1 activity finding that}

elemonic acid inhibited Pin1 activity with an IC value of 13.16µM, similar to that of Juglone.

43 Therefore, Elemonic acid was used as lead to design novel Pin1 inhibitor, using the PDB structure of dexamethasone 21-phosphate in complex with Pin1 (PDB:3TC5)18 _{as a guide for the design. Through} computer modelling the binding sites have been explored in order to bring some structural modifications.

The tetracyclic ring structure seems to form hydrophobic interactions with the shallow surface, therefore they tried to improve the inhibitory effect by enhancing the interactions with the basic cluster, introducing carboxyl acid (or acid bioisostere) and related tetrazole moieties into elemonic acid18_.

The modifications occurred in position C24 and C2:

-in C24 they obtained compound 34, which is the benzyl ester of elemonic acid after oxidation. Thus, they synthetized its amides or esters, compounds 6,7,8,9, and its tetrazole derivative, compound 1518 (Figure 3.21).

Compound 34

44 R= Compound 36 R= Compound 38 R= Compound 37 R= Compound 39

FIGURE 3.21 MODIFICATIONS IN C24.STRUCTURES OF COMPOUNDS 34,35,43,36,37,38,39

-in C2 they introduced a carboxyl group obtaining compound 40 and its amides, compounds 41 and 42 (Figure 3.22). Compound 40 R= Compound 41 R= Compound 42

45

FIGURE 3.22MODIFICATIONS IN C2. STRUCTURES OF COMPOUNDS 40,41&42

The inhibitory activity for the resulting compounds was determined using protease-coupled assay and Juglone was taken as the positive control18 _{(Table 3.13).}

The results showed that only five ligands (compounds 34, 35, 38, 40, 43) are more potent than juglone and parent compound 33 and that heterocycle amides or ester derivatives were weaker than carboxyl acid derivatives. The most potent one is compound 40 with an IC value of 0,57 µM.

About the substitutions, compounds with carboxyl acid or small secondary amides in C24 showed a better inhibitory activity than bigger and heterocycle amide or ester derivatives. Compound 43 with a tetrazole moiety also showed a good inhibitory activity versus Pin1 but compound 40 resulted to be the most active one suggesting that an addition of a carboxyl group in C2 brings to improved results.

Compound IC50 (μM) Compound IC50 (μM)

33 13.16 ± 0.34 39 >40 34 3.68 ± 0.44 40 0.57 ± 0.19 35 1.47 ± 0.59 41 >40 36 >40 42 32.30 ± 0.40 37 31.51 ± 3.79 43 2.60 ± 0.79 38 0.83 ± 0.16 juglone 10.12 ± 1.83

TABLE 3.13INHIBITORY ACTIVITY FOR THE RESULTING COMPOUNDS, DETERMINED USING PROTEASE-COUPLED ASSAY AND JUGLONE TAKEN AS THE POSITIVE CONTROL

#i

To study their binding mode, compounds 38, 40, 41 and 43 were selected as examples for docking evaluation18_.

46 The results of the docking studies showed that the molecules fit in the hydrophobic pocket and interact with Arg68 of the basic cluster and that, as expected, compound 40 resulted the most potent one. Compound 40 is a 2-carboxylmethylene derivative. Its tetracyclic triterpenoid scaffold showed both hydrophobic and Van der Walls interactions with residues His59, Cys113, Asp121, Leu122, Phe125 and Ser154 while the carboxyl group in ring A directly forms H-bond with Arg68.

Taking stock of the situation, the results indicated that:

 The introduction of an acidic group to elemonic acid is a great way to enhance Pin1 inhibitory activity

 the presence of a 2-carboxylmethylene group at C2 resulted to increase inhibitory activity on Pin1

 the activity inhibition may be improved by addition of a carboxylic or tetrazole group at C24 on ringA, as reported in SAR (structure-activity relationships) studies.

 heterocyclic amides or esters cause a significantly decrease of activity inhibition

10-D-glutamic and D-aspartic derivatives

Focusing the research on the interactions inside the proline pocket of Pin1, they designed and synthetized a new series of ligands based on the D-glutamic and D-aspartic scaffold with aliphatic cyclic amines and cycloalkylamines as proline-pocket binding motifs.

VER17,17 _{(Figure 3.23), an old compound which previously showed cell growth-inhibitory activity, was} taken as an example to explore those interactions in a complex with Pin1.

The analysis of the complex revealed three mainly interactions7,17_: -the carboxylic group interacts with the charged pocket

-the aromatic naphthyl group makes hydrophobic interactions with the proline-binding pocket, but it doesn't completely fill it in

47

FIGURE 3.23STRUCTURE OF COMPOUND VER1

Since they noticed that the naphthyl group did not completely fill in the pocket, they tried to improve the structure replacing the naphthyl and using bulky aliphatic groups (Table 3.14).

44a-j 45a-j Compound R IC50 (μM) 44a Pyrrolidin-1-yl >100 44b Piperidin-1-yl >100 44c Homopiperidin-1-yl >100 44d 3-Methylpiperidin-1-yl 39 44e 4-Methylpiperidin-1-yl >100 44f Morpholin-1-yl 10

48 44g Cyclopentylamino >100 44h Cycloexylamino 77 44i Cycloeptylamino >100 44j Adamant-1-ylamino >100 45a Pyrrolidin-1-yl >100 45b Piperidin-1-yl >100 45c Homopiperidin-1-yl >100 45d 3-Methylpiperidin-1-yl >100 45e 4-Methylpiperidin-1-yl 81 45f Morpholin-1-yl >100 45g Cyclopentylamino >100 45h Cycloexylamino 67 45i Cycloeptylamino 18 45j Adamant-1-ylamino 91 VER1 4.2

TABLE 3.14PIN1-INHIBITORY EFFECT OF THE SYNTHESIZED COMPOUNDS 44A-J AND 45A-J

Compounds 44a-j derive from D-glutamic acid, while compounds 45a-j derive from D-aspartic acid. Ver1 and the new compounds were synthetized and tested for Pin1-inhibitory activity in the range of 1 to 200μM and IC values were calculated7_.

49 Among that ones with amine moiety, 44f and 45i showed a good inhibitory activity.

Among the Glu-derivatives, those ones with small cyclic amines or large cycloalkylamines showed no effects. As for the Asp-derivatives, the substitution with small amines brought poor activity and that one bearing a morpholino group was ineffective.

The structural analysis of the VER1/Pin1 complex7 _{showed that the distance between the proline pocket} and the cationic site is important. So probably the compounds that showed no effects were maybe too long or too short to interact with the binding site.

Furthermore, it has been revealed that a 7C cyclic amine is preferred over than one with 5 or 6, maybe because it matches better with the steric size of the proline pocket.

This last observation is supported by a new molecule, ATRA (all trans retinoic acid) (Figure 3.24), which has been recently discovered to be a Pin1 inhibitor7_{. Its good inhibitory activity is probably due to} optimal distance between the carboxylic group and the aliphatic ring.

FIGURE 3.24STRUCTURE OF COMPOUND ATRA

Derived compounds of 44f and 45j with a phenyl-thiazole moiety showed an inhibitory activity similar to VER1 making this moiety a good tool to explore the interactions within the pocket in combination with other groups that interact with the proline-pocket.

Thus, changing the aliphatic groups that bind the proline pocket but maintaining the phenyl-thiazole moiety, they arrived at compounds 46a-c and 47a-d. These new compounds have groups such as: morpholino, cycloheptylamino and azocanyl that we expect to confrere better LogP values and less planarity, becoming favourable for practical applications (Table 3.15).

50 compounds 46a-c compounds 47a-d

Compound R IC50 (μM) 46a 3-Methylpiperidin-1-yl 19 46b Azocan-1-yl >50 46c Cyclooctylamino >50 47a Cyclohexylamino >100 47b 4-Methylpiperidin-1-yl 14 47c Azocan-1-yl 3.0 47d Cyclooctylamino >100

TABLE 3.15 COMPOUNDS 46A-C AND 47A-D, BEARING A PHENYLTHIAZOLE MOIETY

That one with the best LogP value is compound 47c with a value of 1,59. This compound still need to be tested in cell assays to evaluate their effects.

11- Phosphate-based Pin1 inhibitors

The research focused then on the phosphate recognition pocket.

The presence of the phosphate group confers poor cell permeability1,11 _{and for this reason it was} necessary to be replaced with a different group. The carboxylate group successfully replaced the

51 phosphate group, even if it caused the loss of a little of binding affinity probably due to the significantly weaker carboxylate charge-charge interactions9,11_.

To do this, the researchers aimed to exploit the enzymatic mechanism that Pin1 uses to isomerize the prolyl amide bond and which seems to involve both the Cys113 thiol and the Ser154 alcohol in H-bonds formation11_.

Benzimidazole was chosen for this purpose since it has the possibility to make H-bonds and, because of its two tautomeric forms, it is also capable to satisfy the two possible directions of the H-bonding networks in Pin1 catalytic site11 _{(Figure 3.25).}

Benzimidazole analogues resulted to be potent Pin1 inhibitors (Table 3.16).

Compound X Ar 48 Cl 2-naphthyl 49 Cl 2-benzothiophenyl 50 Me 2-naphthyl 51 Me 2-benzothiophenyl 52 F 2-naphthyl 53 F 2-benzothiophenyl

TABLE 3.16BENZIMIDAZOLE ANALOGUES

COMPOUNDS 48,49,50,51,52,53

FIGURE 3.25MOST LIKELY SCENARIO OF THE TWO POSSIBLE DIRECTIONS OF THE H-BONDING NETWORKS IN THE CATALYTIC SITE OF PIN1 PROTEIN

52 The substitution in position 5 of the phenyl ring with a fluoride resulted to be optimal and the additional H-bonds formed by the two nitrogen atoms successfully enhanced the potency.

A co-crystal structure of compound 52 (Figure 3.26) with Pin1 protein confirmed what just said and also revealed an extensive H-bond network in Pin1 catalytic site and the direction of this H-bond network which consist in11 _{(Figure 3.27):}

-the benzimidazole donates an H to Ser154 that donates its –OH

-an H-bond chain starts between the other benzimidazole nitrogen and the Cys113 thiol and follows with His59, His157 and Thr152 which also forms an H-bond with the backbone amide NH of Ser154.

FIGURE 3.26CRYSTAL STRUCTURE OF COMPOUND 52

Even after making all these affinity improvements, the benzimidazole-based Pin1 inhibitors resulted inactive on cells11_{. They hypothesised that the high polarity of the benzimidazole confers them poor} permeability so they tried to substitute it to reduce molecular polarity. The indole was found to have a similar inhibitory activity but a better permeability and good aqueous solubility11_.

FIGURE 3.27H-BOND NETWORK IN THE CATALYTIC SITE OF PIN1

53 The research of substituent to improve permeability led to the dihidrothiazole-based inhibitors11 _(Table 3.17). Compound R IC50 (μM) 54 27 55 4.6 56 4.2 57 1.9

These ones demonstrated to be able to inhibit cancer cell proliferation11_.

Studies on compound 54 confirmed its ability to induce cell cycle changes by Pin1 inhibition and also histological changes such as: less cells per sample, fragmented phenotype and prematurely condensed chromatin11_.

3.2 WW-DOMAIN INHIBITORS

It has been observed that phosphorylation of Ser16 in the WW-domain shut down Pin1 by preventing the recruitment of his substrates1_{. Therefore, it is reasonable to think that the research of compounds} competing with Pin1`s substrates may be a valuable way to find effective inhibitors in cells and another potentially valid target to inhibit the enzyme.

TABLE 3.17 DIHIDROTHIAZOLE-BASED IN-HIBITORS

54 EGCG

EGCG (EpiGalloCatechin-3-Gallate) (Figure 3.28) is the major flavonoid in green tea and it has been studied as a cancer chemo-preventive compound1_.

X-ray crystal co-structures showed that EGCG may bind both the WW-domain and PPIase domain, but it looks like the major route of Pin1 inhibition is via the WW-domain18_.

A protease coupled assays give it an IC50 of 20μM. In ErbB2-transformed MEFs, 30μM of EGCG suppressed JNK signalling, cyclinD1 and Bcl-xL expression but, unfortunately, as Juglone it has additional targets that makes this compound unsuitable for us as chemical biology probes to study the action of Pin11_.

FIGURE 3.28STUCTURE OF COMPOUND EGCG

4. TOXICITY DERIVING BY PIN1 INHIBITION

After all these years of research for compounds able to inhibit Pin1, there are strong evidences that make us consider this enzyme as valid target for anticancer therapy, even though with some concerns about the toxicity deriving by this inhibition.

In fact, while up-regulation leads to pathogenesis of cancer, down-regulation may cause off target effects such as neurodegeneration similar to that one in the Alzheimer disease and hypertension19_. As mentioned earlier, Pin1 is involved in cyclinD1 synthesis and stabilization. For this reason, the inhibition of Pin1 would cause defects in the cis-trans isomerization of Tau proteins which results in neurodegeneration reminiscent of Alzheimer disease1_{, as shown by the phenotype of Pin1 deficient}

55 mice and similar to those defects observed in cyclinD1 knockout mice1_{. However, it is reasonable to} believe that these defects may be avoided, at least in part, using inhibitors that fail to cross the blood brain barrier1_.

Pin1 may also affects endothelial function and blood pressure regulation. The eNOs activity is regulated by its phosphorylation status. It contains the sequence p-Ser116-pro117, which is a potential Pin1 substrate in endothelial cells when Ser116 is phosphorylated. Depletion or inhibition of Pin1 increases Ser116 phosphorylation and this lead to a decrease of NO production and relaxation response thus hypertension19_.

Furthermore, studies on Pin1 knockout mice revealed that they develop normally in young age but in adult age they develop several abnormalities in specific tissues1 _{such as smaller body size, testicular} atrophy, defects in breast development during pregnancy, neurodegeneration, and retinal degradation. Interestingly, cyclin D1 levels are lower especially in those tissues that display a severe phenotype2_.

However, it is reasonable to believe that a therapy with pin1-specific inhibitors is possible and might not have general toxic effects, even if it is not possible to demonstrate until the development of them.

5. CONCLUSIONS

Several lines of evidence suggest that inhibiting Pin1 would result in the suppression of tumorigenesis, providing a unique way to disturb oncogenic pathway signals. Cyclin D1 showed to be the key target downstream of Pin11_{and there is a correlation between Pin1 overexpression and high levels of cyclin} D1 in cancer tissues. Importantly, in Pin1-knockout mice the levels of cyclin D1 are significantly decreased and the Pin1-null phenotype resembles the cyclinD1-null phenotype1_{, suggesting that Pin1} is important in the regulation of cyclin D1 in vivo. MEFs (mouse embryonic fibroblasts) derived from Pin1-/- mice showed to have a lower proliferation rate, probably caused by the downregulation of cyclin D1 and pRb. Studies on mice have also shown that Pin1 certainly has a role in signal amplification and translation but its role in cancer initiation it does not seems to be essential since it has been demonstrated it can be bypassed. As an example, cancer initiation after MMTV-driven expression of ErbB2 or Ras results being almost completely blocked but, in contrast, the overexpression of c-Myc

56 readily leads to breast cancer1_{. Furthermore, Pin1 deficient mice are viable, despite some deficiency} in cell proliferation1_{, rising the chances that an anti-cancer therapy based on Pin1 inhibition may be} possible.

Unfortunately, even though Pin1 has given reasons to be an attractive target, the research of effective Pin1 inhibitors is being challenging. The goal is to create highly potent and non-phosphate based molecules. Until now, several series of compound have been discovered and even if none of them can be used as a therapeutic, they have been useful because they showed different possibilities of interaction with the enzyme.

More studies on Pin1 must be done. It would be interesting to probe Pin1 functions in different cell environments and maybe to generate human cancer cells with knockout mutations of Pin1 by using biological tools. It is also important to further investigate the binding mode since a compound combining different interactions with the enzyme could have impressive activity.

6. BIBLIOGRAPHY

1. Moore J.D., Potter A. Pin1 inhibitors: Pitfalls, progresses and cellular pharmacology. Bioorg.Med.Chem.Lett. , 2013; 23 (4283-4291)

2. Ryo A., Liou Y. Prolyl isomerase Pin1: a catalyst for oncogenesis and a potential therapeutic target in cancer. J.Cell.Sci. , 2003; 116 (773-783)

3. Guo C., Hou X. Structure based design of novel human Pin1 inhibitors (I). Bioorg.Med.Chem.Lett. , 2009; 19 (5613-5616)

4. Bao L., Kimzey A. Prevalent overexpression of prolyl isomerase Pin1 in human cancers. Am.J.Pathol. , 2004; 164 (1727-1737)

5. Wang J.Z., Xi L. The structural and functional role of the three tryptophan residues in Pin1. J.Photoch.Photobio.B , 2015; 146 (58-67)

6. Zhu L., Jin J. Synthesis and biological evaluation of novel quinazoline-derived Human Pin1 inhibitors. Bioorg.Med.Chem. , 2011; 19 (2797-2807)