CHAPTER 2
p53-MDM2 INTERACTION
INHIBITORS
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2.1 INTRODUCTION
Since its discovery 30 years ago, p53 (Figure 22) has been a subject of intense studies: this protein was first characterized as an oncogene, a confounding issue derived from the study of the mutant p53 variants expressed in tumors. Years later, the wild type version of the p53 gene was unequivocally identified as a tumor suppressor. Eventually, the p53 protein was characterized as a DNA-binding transcription factor.79
Figure 22. Tumor suppressor p53.80
The high interest for this molecule is largely due to the fact that p53 is one of the most frequently altered protein in human cancer. Approximately 50% of all human malignancies harbour mutations or deletions in the p53 gene that disable the tumor suppressor function of the encoded protein. This high rate of genetic alterations underscores the important cellular function of p53. The tumor suppressor controls a signal transduction pathway evolved to protect multicellular organisms from cancer development that could be initiated by diverse stresses including DNA damage.81
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Genotoxic stress that damages DNA induces cell cycle arrest, activation of DNA repair, and in the event of irreparable damage, induction of apoptosis. The decision by cells either to repair DNA lesions and continue through the cell cycle or to undergo apoptosis is relevant to the incidence of mutagenesis and, subsequently, carcinogenesis. In this context, incomplete repair of DNA damage prior to replication or mitosis can result in the accumulation of heritable genetic changes. However, the nature of the cellular signaling response that determines cell survival or death is far from being understood.82
2.1.1 p53 : structure and function
The human p53 protein contains 393 amino acids and has been divided structurally and functionally into four domains: an acidic amino-terminus domain (aa 1-43) which is required for transcriptional activation; a central core sequence-specific DNA-binding domain (aa 100-300); a tetramerization domain (aa 324-355) and a C-terminus regulatory domain (aa 363-393), rich in basic amino acids and believed to regulate the core DNA-binding domain (Figure 23).
Figure 23. Structure of p53 protein showing the different domains, and hot spots for mutations in human cancer. The 393 aminoacid p53 protein is diagrammed from the amino-terminus (1) to the carboxy-terminus (393) with boundaries for various domains shaded with different colors. The list of different cellular and viral proteins reported to bind to different regions of p53 is given below.83
95 The N-terminus activation domain interacts with the transcription factors TFIID (TBP, TAFs), and TFIIH which form part of the basal transcriptional machinery and positively regulate gene expression. Amino acids residues Phe19, Leu22, and Trp23 in p53 have been shown to be required for transcriptional activation in vivo and are also involved in the interaction with the TATA-associated factors.
Human ubiquitin E3 ligase Murine Double Minute 2 protein, MDM2, and adenovirus E1B- 55 kDa protein negatively regulate the p53 transcriptional activity through binding to the N-terminus activation domain. The importance of the negative regulation of p53 by MDM2 is highlighted by the fact that homozygous deletion of MDM2 in mice results in embryonic lethality, which is rescued by simultaneous homozygous deletion of p53. The central core domain of p53 contains the sequence-specific DNA-binding region. There are four highly conserved regions within the central core domain, where most of the p53 missense mutations were found. The tetrameric p53 protein, which is a dimer of a dimer, binds to four repeats of a consensus DNA sequence 5'-PuPuPuC(A/T)-3' (where Pu is a purine) and this sequence is repeated in two pairs, each arranged as inverted repeats. The purified core DNA-binding domain can bind co-operatively to DNA (Figure 24).
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The C-terminus domain regulates the ability of p53 to bind to specific DNA sequences at its central core domain. This extreme C-terminus domain, including amino acids 363-393, is rich in basic residues. This domain can bind non-specifically to different forms of DNA such as DNA breaks or internal mismatches. Evidence suggests that a structural change at the C-terminal domain is required to activate p53 for sequence-specific DNA-binding. It was found that deletion of this domain, its phosphorylation by protein kinase C (PKC) or casein kinase II at specific residues or binding of antibody PAB421 can activate sequence-specific DNA-binding by the central core domain.83
The protein p53 is a potent transcription factor capable of activating multiple target genes, leading to cell cycle arrest, apoptosis, or senescence (Figure 25). The biological role of p53 is to ensure genome integrity of cells. p53 can stimulate the repair processes and protective mechanisms, or the cessation of cell division and the induction of acquired cell death. To achieve its aims, p53 may use a wide spectrum of activities, such as its ability to function as transcription factor, by inducing or repressing different genes or as a regulatory protein, within numerous signaling pathways. The activity of p53 is stimulated in response to DNA damage and to various genotoxic insults that eventually compromise genome integrity. Following genotoxic stress, p53 primarily determines the cell fate to induce growth arrest, DNA repair, or apoptosis. In particular, upon exposure to severe DNA damage, p53 conducts to elicit cell death by apoptosis .
The loss of p53 functions and the deregulated activities of p53 are involved not only in the development of malignant diseases, but also are in relation to cardiovascular, neurodegenerative, infectious and metabolic diseases, as well as participate in the process of organism aging. p53 has the ability to bind specific responsive DNA elements and the specificity of the transcriptional activation depends on the ability of p53 DNA-binding domain to interact with the regulatory regions of certain genes. The transcriptional activation is determined by the N-terminus of p53, which contains several regions interacting with the transcription
97 machinery and recruiting factors that modify local chromatin structure.82
Figure 25. p53 regulates cellular response to stress.84
2.1.2 Stabilization of p53 in response to DNA damage
p53 is regulated primarily through post-translational modifications, especially phosphorylation, and the accumulation of p53 is the first step in response to cellular stress (Figure 26). The N-terminus is heavily phosphorylated, while the C-terminus contains phosphorylated, acetylated, methylated and sumoylated residues. Post-translational modifications on the N-terminus are important for stabilizing p53 while those on the C-terminus inhibit the ability for regulation of sequence-specific DNA binding, the oligomerization state, the nuclear import/export process, and the ubiquitination.82 The MDM2 gene is a transcriptional target of p53, and once synthesized, the MDM2 protein can bind to p53 at the N-terminus leading to its rapid degradation through the ubiquitin-proteasome machinery. In response to DNA damage, the ATM kinase rapidly phosphorylates p53 at Ser15. The serine/threonine kinase Chk2 acts downstream of ATM phosphorylating p53 at Ser20. These phopshorylated sites in the N-terminus of p53 are close to the MDM2-binding region of the protein, thereby they block the interaction p53-MDM2, leading to stabilization of p53, that escapes
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from proteasomal degradation. Recent findings suggested that constitutive phosphorylation of p53 by PKC at the C-terminus domain contributes to its degradation via the ubiquitin-proteasome pathway.82
Figure 26. Functional domain and modification residues in p53. TAD indicates transactivation domain. NLS and NES mean nuclear localization and export signals, respectively. Modifications related to apoptosis are highlighted in red.82
2.1.3 p53 pathway
As above stated, p53 is a transcription factor that controls cellular response to genotoxic, oncogenic and hypoxic stress through the induction of cell cycle arrest and apoptosis (Figure 27). This protein acts in different ways:
- by inducing cell cycle arrest through the stimulation of transcription of the genes encoding p21 and 14-3-3σ protein;
- by inducing apoptosis in two different manners: the extrinsic and the intrinsic pathway (through activation of target genes’ transcription, such as PUMA and NOXA, or interacting with anti-apoptotic proteins, such as Bcl-2 and Bcl-X, or by inducing activation of caspases).
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Figure 27. The tumor suppressor p53 is a powerful anti-proliferative and pro-apoptotic molecule.85
2.1.4 The p21 circuit
A direct transcriptional target of p53 is p21, a potent cycline-dependent kinase (CDK) inhibitor that effectively blocks cell cycle progression in G1 phase. p21 is exquisitely regulated at various stages, from transcription to protein degradation, and these regulatory events tune p53-dependent cell cycle arrest.79
p53 is one of many well documented transcriptional regulators of the p21 locus. Positive and negative regulation by other factors dictate the extent to which p53 transactivates p21. For example, MIZ1 (MYC interacting zinc-finger protein-1) and SP1 (specificity protein 1) have been shown to regulate p21 in p53-dependent and/or -independent manners. Recently, ZAC1 (zinc-finger protein which regulates apoptosis and cell cycle arrest) was shown to synergistically transactivate p21 through a direct interaction with SP1 and through a functional interaction with p53.86
In contrast, p53-dependent p21 induction is subject to a myriad of antagonist factors. For example, the transcription factor MYC represses p21 activation in response to multiple forms of DNA damage via its interaction with MIZ1. ZBTB4 (zinc-finger and BTB domain containing 4) is a member of the POZ domain-containing family of transcriptional repressors that can also inhibit p21
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transcription through interaction with MIZ1. Other POZ repressors also regulate p21 transactivation through direct competition with p53 and/or SP1. These examples demonstrate the potential for regulatory complexity that exists at just one step of gene regulation.87
After transcription, splicing and 3′ end formation, mature mRNAs are regulated by numerous factors that influence their stability, localization and access to ribosomes for active translation. Increased RNA stability plays a major role in facilitating p21 protein upregulation in response to DNA damage via the binding of HuR (ELAVL1) to an AU-rich element (ARE) in the p21 3′ untranslated region (UTR). RNPC1 (RBM38, RNA binding motif 38) is a direct transcriptional target of p53 which also binds to an ARE in the p21 3′ UTR to stabilize it under DNA damaging conditions, thus creating a link between two adjacent levels of the circuit. In fact, HuR and RNPC1 act cooperatively to bind the 3′ UTR and increase the stability of the p21 mRNA. In contrast, AUF1 (AU-rich binding protein 1, hnRNP D) competes with HuR to regulate p21 mRNA stability antagonistically. Intriguing set of experiments demonstrated that while AUF1 and HuR can bind to adjacent sites in the p21 3′ UTR to promote nuclear export, in the absence of HuR, AUF1 shifts its binding position to promote destabilization of the mRNA. Elements in the 5′ coding region of the p21 mRNA are also subject to regulation, as CUGBP1 and calreticulin (CALR) compete for binding to CG-rich elements to increase or decrease translation, respectively, in an interplay shown to affect differentiation and senescence. Thus, post-transcriptional regulation by competing RNA binding factors significantly influences p21 protein levels. Interestingly, p53 activates the expression of p21 both directly, at the transcriptional level, and indirectly, by regulating factors that increase mRNA stability.88
The ultimate signal integrator of post-transcriptional events is the ribosome, which synthesizes protein that can in turn be post-translationally regulated. p21 is subject to a variety of post-translational modifications that alter its activity, subcellular localization and/or stability. p21 can be phosphorylated on at least seven Ser/Thr residues with variable effects on localization and stability. For example, AKT/PKB can phosphorylate p21 on Thr145 or Ser146. This is an
101 intriguing case because AKT phosphorylation at Thr145 was shown to increase p21 stability and block its association with PCNA, promoting cell cycle arrest, whereas the Ser146 modification activates a Cyclin D1-CDK4 complex to drive proliferation.89 Additionally, phosphorylation of Thr145 by the oncogenic PIM1 kinase was shown to alter p21 localization, sequestering it in the cytoplasm where it is not competent to block cell cycle progression. Furthermore, p21 protein stability can be altered both positively and negatively by p38α/JNK1 phosphorylation of Ser130 and GSK3β phosphorylation of Ser114, respectively. These examples represent only a small subset of the kinases and phosphorylation sites that regulate p21, but they surely serve to demonstrate how a single type of post-translational modification can modulate p21 activity well after p53-dependent transactivation. The final layer of regulation in this circuit comes at the level of proteasomal degradation. Ubiquitin-mediated degradation of p21 occurs at different phases of the cell cycle, directed by different E3 ligases including SCF-Skp2, APC-Cdc20 and CRL4Cdt2. It has also been reported that p21 can be degraded by the proteasome independent from its ubiquitination status. p21 can be stabilized without interceding post-translational modifications through the formation of complexes with Cyclin D1, mediated by RAS overexpression, or by binding to WISp39/Hsp90, both of which prevent the association of p21 with the proteasome. This last layer of regulation can amplify or dampen the signal initiated by active p53 to determine the level and activity of p21 in the cell, as well as its final effect on the cell cycle.79
2.1.5 Target protein 14-3-3σ
Cell cycle arrest in response to p53 activation is mediated by two main proteins: p21, which arrests cells mainly in the G1 phase, and 14-3-3σ, which arrests cells at the G2/M transition. 14-3-3σ is a direct transcriptional target of p53 that binds to the mitotic phosphatase Cdc25 and sequesters it in the cytoplasm, thus preventing it from activating CDK1-cyclin B complexes. p21 and 14-3-3σ work coordinately to enforce cell cycle arrest in response to various DNA damaging agents and protect from apoptosis. Relatively little is known about additional transcriptional coregulators of 14-3-3σ; however, BRCA1 has been shown to
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increase 14-3-3σ mRNA levels in a p53-dependent fashion. In mouse embryonic steam cells, BRCA1 is required for full expression of 14-3-3σ, a finding which translated to several p53 wild-type human cancer cell lines where overexpression of BRCA1 resulted in upregulation of 14-3-3σ. Negative regulation of 14-3-3σ transcription by promoter DNA hypermethylation occurs in many normal tissues and is widespread in human cancers, but the mechanisms by which it is established remain largely elusive. Of note, the IKKα kinase shields 14-3-3σ from silencing in keratinocytes by binding to the promoter and blocking the action of the histone- and DNA-methyl-transferases SUV39H1 and DNMT3A, respectively.79
2.1.6 The extrinsic apoptotic pathway
Activation of death receptors by their specific ligands is responsible for the induction of the extrinsic apoptotic pathway. Briefly, signals from outside the cell, which determine that a certain cell should undergo apoptosis, are mediated via specific receptors, e.g. the Fas receptor. Binding of the Fas-ligand to the Fas receptor (Cd95/Apo1), a member of the TNF-R family of receptors and key component of the extrinsic pathway, results in a clustering of receptors that lead to the recruitment of FADD and pro-caspase-8 to the complex. Caspase-8 auto-activates itself, upon which it auto-activates pro-caspase-3, a principal effector caspase of apoptosis. p53 can promote apoptosis through transcriptional activation of genes encoding death receptors located at the plasma membrane, including Fas, DR4, DR5 and PERP.90
Additionally to enhancing Fas transcription, overexpressed p53 may increase the levels of the Fas receptor at the cell surface by promoting trafficking of the Fas receptor from the Golgi.
Two additional members of the death receptor family that are transcriptional induced by p53 include DR4 and DR5/KILLER, which are death-domain-containing receptors for TNF-related apoptosis-inducing ligand (TRAIL). Both DR4 and DR5 are induced upon DNA-damage and can trigger or enhance apoptosis induced by TRAIL and chemotherapeutic agents.91
103 Another pro-apoptotic gene, PERP, is activated in response to DNA damage in cells transduced either with E2F1 or with the adenoviral E1A protein. In PERP knockout mice it was demonstrated that deficiency of PERP leads to a compromised p53-dependent apoptosis response in thymocytes and neurons. Although PERP expression was significantly elevated in all cell types tested, the functional requirement for PERP in apoptosis induction was different in each cell type. The kinetics of PERP induction upon DNA damage and the presence of p53 responsive element support the idea that PERP may be a direct transcriptional target of p53. The exact mechanism by which PERP mediates apoptosis still remains an unanswered question.
Another way by which p53 inhibits the pro-survival signaling is via the up-regulation of the PTEN tumor suppressor. Activation of surface receptors by binding of their ligands (mitogens, cytokines) or the activation of oncogenes such as Ras activates the Akt/PKB signaling pathway. The signals are transduced by the phosphatidylinositol 3-kinase (PI3K), which thereby promotes cell viability and proliferation. The PI3-kinase phosphorylates phosphatidyl-inositol 2-phosphate (PIP2) to PIP3. PIP3 is needed for certain kinases, like PDK1, that phosphorylate and activate Akt/PKB. However, upon activation of the lipid phosphatise PTEN, which converts PIP3 to PIP2, PDK1 is not activated anymore, thereby impairing the Akt/PKB signaling pathway. The activation of PTEN halts cell division and leads to the induction of apoptosis by inhibition of the Akt/PKB signaling. Interestingly, activation of transcription of the PTEN gene by p53 is strongly stimulated by Ser46-phosphorylation.91
2.1.7 The intrinsic apoptotic pathway
The intrinsic apoptotic pathway is dominated by members of the Bcl2 family that directly impact on mitochondrial outer membrane permeabilization (MOMP) (Figure 28). MOMP occurs through the actions of the pro-apoptotic Bcl2 family members such as Bax (Bcl-2-associated X) and Bak (Bcl-2 antagonist/killer). After MOMP, cytochrome c is released from the mitochondrial intermembrane space, which is followed by cytochrome c binding to the cytosolic Apaf-1 protein and a large multi-protein structure known as the apoptosome is formed. Once
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formed, the apoptosome recruits the inactive initiator caspase-9 and activates it by removing the pro-domain. Active caspase-9 subsequently triggers a cascade by activation of the executioner caspases pro-caspase-3 and -7, which can cleave several cellular targets and ultimately leads to cell death, with the characteristic apoptosis hallmarks such as chromatin condensation and cellular blebbing.92
Figure 28. Apoptosis mitochondrial pathway.93
The Bcl2 family consists of several anti-apoptotic (prosurvival) and pro-apoptotic members. The classification of the different members is based on structural similarity of the Bcl2 homology (BH) domain named BH1, BH2, BH3 and BH4 and the presence of a transmembrane domain. The BH3 domain is the minimum requirements for the pro-apoptotic function and was shown to be essential for heterodimerization between members of the Bcl2 family. The Bcl2 family can be divided into three classes: antiapoptotic or pro-survival, whose members are structurally related the most to Bcl2 (e.g. Bcl-XL). Pro-apoptotic proteins such as Bax and Bak, that are structurally related to Bcl2, but antagonize its pro-survival function. The third group of proteins that only carry the BH3 domain are pro-apoptotic and termed “BH3-only” proteins.
105 Intriguingly, several Bcl2 family members have been implicated in p53-dependent apoptosis (Figure 29). The first member to be identified as a p53-inducible target was the pro-apoptotic Bax protein, while the precise p53 responsive elements within the Bax gene were identified later. In a mouse model system in which p53 dependent apoptosis attenuates tumor growth of the brain, it was observed that absence of Bax increased tumor growth, while apoptosis levels decreased. Later it was shown that Bax interacts with another p53-induced protein, the apoptosis-associated speckle-like protein (ASC), which is suggested to translocate Bax to the mitochondria.91
Figure 29. p53 mediated apoptosis.95
A very important p53 regulated target gene is encoding the “BH3 only” protein PUMA (p53 upregulated modulator of apoptosis), that regulates apoptosis upstream from Bak and Bax. In humans the PUMA gene, which contains a high affinity p53-responsive element in the first intron, encodes for two
BH3-106
containing isoforms, PUMA-α and PUMA-β, which are both upregulated in response to p53 activation. PUMA is a potent activator of the apoptosis cascade by modulating Bax activity to facilitate cytochrome c release from the mitochondria94. The physiological relevance validating the role of PUMA in p53-mediated apoptosis came from PUMA knockout mice. The PUMA knockout mice remarkably recapitulate several key apoptotic deficiencies observed in p53 knockout mice. It was demonstrated that PUMA is required for the p53 dependent apoptotic response to the c-Myc and E1A oncogenes in MEFs.
While also several p53 independent effects were observed. More recently it has been shown that in hematopoietic cells the PUMA expression is inhibited by Slug, another p53 induced gene upon γ-irradiation. Here, the authors observed that Slug expression prevents hematopoietic progenitors to undergo DNA damage induced apoptosis, and identify PUMA as the key target.91
Yet an additional member of the pro-apoptotic “BH3 only” family of proteins that is regulated by p53 is Noxa. Noxa knockout mice showed notable resistance to the adenoviral protein E1A mediated- and DNA damage induced apoptosis. Similarly to the mechanism by which PUMA activates cytochrome c release from the mitochondria, Noxa is also believed to act as a sensitizer by interacting and interfering with the pro-apoptotic members of the Bcl2 family. In contrast to PUMA that binds promiscuously with pro-survival members of the Bcl2 family, Noxa selectively interacts with Mcl-1 and A1 of this family.96
p53 is also shown to activate Bid, a particular “BH3 only” domain protein, which has the ability to couple the extrinsic death receptor pathway to MOMP, a feature of the intrinsic apoptotic pathways. Activation of cytoplasmic Bid involves cleavage at residue 60 by death receptor activated caspase-8, thereby exposing a glycine at the N-terminus, and generating the tBid (truncated Bid). The exposed glycine residue in tBid is subsequently myristoylated, which targets it to the mitochondrial membrane. At the membrane it is thought to bind and inhibit the pro-apoptotic member Bcl-xL, resulting in the release of cytochrome c and apoptosome formation.
Besides the activation of genes involved in apoptosis, it has also been reported that p53 represses the expression of several genes. Although the exact mechanism
107 by which p53 represses transcription is unknown, it seems not to involve recognizable p53 binding sites, but may rather involve a large repressor complex.91
2.1.8 Positive and negative p53 feedback loops
A variety of studies in the literature have identified ten positive or negative feedback loops in the p53 pathway. Each of these loops creates a circuit composed of proteins whose activities or rates of synthesis are influenced by the activation of p53, and this in turn results in the alteration of p53 activity in a cell. Of these, seven are negative feedback loops that modulate down p53 activity (MDM2, Cop-1, Pirh-2, p73 delta N, cyclin G, Wip-1 and Siah-1) and three are positive feedback loops (PTEN-AKT, p14/19 ARF and Rb) that modulate up p53 activity. All of these networks or circuits are autoregulatory in that they are either induced by p53 activity at the transcriptional level, transcriptionally repressed by p53 (p14/19 ARF) or regulated by p53-induced proteins. Six of these feedback loops act through MDM2 (MDM2, cyclin G, Siah-1, p14/19 ARF, AKT and Rb) to modulate p53 activity.97
An exciting finding is that the p53 pathway is intimately linked to other signal transduction pathways that play a significant role in the origins of cancer. One of the first connection studied involves p14/p19 ARF and MDM2. The p14/19 ARF protein binds to the MDM2 protein and modulates down its ubiquitin ligase activity, increasing the levels of the p53 protein. The transcription of the p14/19 ARF gene is positively regulated by E2F-1 and beta-catenin and negatively regulated by p53 itself. In addition, the levels of p14/19 ARF protein are increased by Ras and Myc activities in a cell (Figure 30).98
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109 The p14/19 ARF-MDM2 complexes are often localized in the nucleolus of the cell due to the nucleolar localization of the signals present within p14/p19 ARF. The nucleolus is the site of ribosomal biogenesis and p14/19 ARF activity itself can alter the rate of RNA processing of the ribosomal RNA precursor into mature ribosomal subunits. Thus, p14/19 ARF plays an important role in cell cycle regulation by controlling MDM2 and p53 levels and coordinating this with ribosomal biogenesis.97
The MDM2 in the nucleolus is not, however, a passive entity. The MDM2 protein has been shown to bind specifically to three large ribosomal subunit proteins Leu5, Leu11 and Leu23, and the binding of Leu5 or Leu11 to MDM2 lowers its ubiquitin ligase activity.
In addition, the ringfinger domain of MDM2 binds specifically to an RNA sequence found in the large ribosomal RNA subunit. The Rb protein can be found in cells in a complex with MDM2 and p53, resulting in high p53 activity and enhanced apoptotic activity. High levels of active E2F-1 not bound to Rb switches the p53 response from G-1 arrest to apoptosis. Both Rb and MDM2 are phosphorylated and inhibited by cyclin E-cdk2. When p53 is activated, it stimulates the synthesis of the p21 protein, which inhibits cyclin Ecdk2 activity, and this in turn acts upon the Rb-MDM2 complex that promotes p53 activity and apoptosis. After DNA damage, both the MDM2 protein and the p53 protein are modified by the ATM protein kinase. This enhances p53 activity in the same way that the p53-MDM2-Rb complex increases p53 function and is proapoptotic (Figure 31). 99
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Figure 31. Cyclin/Cdk/Rb/MDM2 loop.97
Part of the activation of the p53 protein involves the phosphorylation of the p53 protein at serines located at residues 33 and 46 by the p38 MAP kinase (Figure
32).
Figure 32. Wip-1/p38 MAPK loop.97
This p38 MAP kinase is itself activated by phosphorylation (regulated by the Ras-Raf- Mek-Erk pathway) that can be reversed or inactivated by the Wip-1
111 phosphatase. Wip-1 is a p53-responsive or p53-regulated gene forming a negative autoregulatory loop and connecting the p53 and Ras pathways.100
An activated p53 protein positively regulates the transcription of the ubiquitin ligase Siah- 1, which in turn acts to degrade the beta-catenin protein. Beta-catenin levels can regulate the p14/19 ARF gene, which in turn negatively regulates MDM2 and results in higher p53 levels. Siah-1 thus connects the Wnt-beta-catenin- APC pathway to the p53 pathway (Figure 33). 101
Figure 33. Siah-1/beta-catenin/p14/19 ARF loop.97
In some cell types, the p53 protein induces the transcription of the PTEN gene. The PTEN protein is a PIP-3 phosphatase that activates the AKT kinase, which has a number of antiapoptotic protein substrates including the MDM2 protein. Phosphorylation results in the translocation of MDM2 into the nucleus where it inactivates p53 (Figure 34).97
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These positive and negative feedback loops accomplish two things: they modulate p53 activity in the cell and they coordinate p53 activity with other signal transduction pathways that regulate entry of the cell into the cell cycle.97
There are two additional p53 autoregulatory circuits that negatively feedback upon p53 function. One of the most active of the p53-responsive genes is the cyclin G gene. It is rapidly transcribed to high levels after p53 activation in a wide variety of cell types. The cyclin G protein makes a complex with the PP2A phosphatase, which removes a phosphate residue from MDM2, which is added to the MDM2 protein by a cdk kinase. Phosphorylation of MDM2 by cyclin A/cdk2 inhibits its activity, thus the cyclin G-PP2A phosphatise enhances MDM2 activity and inhibits p53 (Figure 35). 102
Figure 35. Cyclin G/ MDM2 loop.19
The second negative feedback loop involves a member of the family of p53 transcription factors, which include p53, p63 and p73 that are related by structure and function and have evolved from a common precursor. After a stress response, the p53 gene is activated, which in turn stimulates the transcription of a particular spliced m-RNA from the p73 gene, called p73 delta N. This translates a p73 protein without its amino-terminal domain (Figure 36).97
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Figure 36. p73 delta N loop.97
All three proteins of the p53 family have similar domain structures composed of an N-terminal transcriptional activation domain linked to a central core domain that binds to a specific DNA sequence discussed above. All three of the p53 family transcription factors recognize the same DNA sequence, even though p53, p63 and p73 are capable of initiating distinct transcriptional programs. Thus, when p53 activates the transcription of p73 delta N, the p73 delta N protein can bind to many of the p53-regulated genes, but the absence of a transactivation domain makes it act as a repressor or competitor of p53 transcriptional activation.97
2.1.9 p53-MDM2 interaction
The MDM2 gene was originally identified on double-minute chromosomes of spontaneously transformed mouse 3T3 fibroblasts, and the MDM2 protein was later found to be physically associated with p53. Since then, compelling evidence has emerged for MDM2 to have a physiologically critical role in controlling p53. MDM2-null mouse embryos die early after implantation, but are fully rescued if they are co-deficient for p53. This provides compelling genetic evidence that the most important role of MDM2 is the physiological regulation of p53 function, at least in early development. Importantly, MDM2 itself is the product of a p53-inducible gene. Thus, the two molecules are linked to each other through an autoregulatory negative feedback loop aimed at maintaining low cellular p53 levels in the absence of stress.
Principally, MDM2 is an E3 ligase and promotes p53 degradation through a ubiquitin-dependent pathway on nuclear and cytoplasmic 26S proteasomes. Protein modification by ubiquitin conjugation is a general intracellular targeting
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mechanism and covalently attached polyubiquitin chains on lysine residues target proteins to proteasomes for degradation (Figure 37).
Recent studies, using various MDM2 and p53 mutants, elucidated structural requirements for the MDM2-mediated destruction of p53. Combined, these results revealed the importance of several regions on both proteins and showed that in order for p53 to accumulate in response to stress, the regulatory mechanism has to be interrupted so that p53 can escape the degradation-promoting effects of MDM2.
MDM2 harbors a self- and p53-specific E3 ubiquitin ligase activity within its evolutionarily conserved COOH terminal RING finger domain (Zinc-binding), and its RING finger is critical for the E3 ligase activity. MDM2 transfers monoubiquitin tags onto lysine residues mainly in the COOH terminus of p53. MDM2-mediated p53 ubiquitination takes place in the nucleus in a complex with the p300/CREB-binding protein (CBP) transcriptional coactivator proteins, serving as a scaffolding. The majority of endogenous MDM2 is bound to p300/CBP in the nucleus. However, whereas MDM2 catalyzes p53 monoubiquitination, which in itself is not a substrate for proteasome degradation, p300/MDM2 complexes are mediating the final p53 polyubiquitination.
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2.1.10 Structural requirements for p53-MDM2 interaction
Human MDM2 is a 491-amino acid (aa)-long phosphoprotein that interacts through its NH₂ terminal domain with an α-helix present in the NH₂ terminal transactivation domain of p53. This entails several negative effects on p53. MDM2 directly blocks p53 transcriptional activity by binding to its NH₂ terminal transcription domain. More importantly, MDM2 functions as E3 ligase that ubiquitinates p53 for proteasome degradation. The crystal structure of the p53-MDM2 complex has been solved (Figure 38).104
The biochemical basis of MDM2-mediated inhibition of p53 function was further elucidated by crystallographic data that showed that the NH₂ terminal domain of MDM2 presents a deep hydrophobic cleft into which the transactivation domain of p53 binds, thereby concealing itself from interaction with the transcriptional machinery. This has been confirmed by biochemical analysis. The direct interaction between the two proteins has been localized to a relatively small (aa 25–109) hydrophobic pocket domain at the NH₂ terminus of MDM2 and a 15-aa amphipathic peptide at the NH₂ terminus of p53. The minimal MDM2-binding site on the p53 protein was subsequently mapped within residues 18–26. Site-directed mutagenesis has shown the importance of p53 residues Leu14, Phe19, Leu22, Trp23, and Leu26, of which Phe19, Trp23, and Leu26 are the most critical. The bond for the interaction between p53-MDM2 consist of only three hydrogen bonds and the most buried one is contributed by Trp23 on p5326. Accordingly, the MDM2-binding site p53 mutants are resistant to degradation by MDM2. Similarly, mutations of MDM2 at residues Gly58, Glu68, Val75, or Cys77 result in lack of p53 binding. The interacting domains show a tight keylock configuration of the p53-MDM2 interface. The hydrophobic side of the amphipathic p53 α-helix, which is formed by aa 19–26 (with Phe19, Trp23, and Leu26 making contact), fits deeply into the hydrophobic cleft of MDM2. Thr18 is very important for the stability of the p53 α-helix. The MDM2 cleft is formed by the aa 26–108 and consists of two structurally similar portions that fold up into a deep groove lined by 14 hydrophobic and aromatic residues. Experimental measurements of the strength of the p53-MDM2 bond range from a Kd of 60 to
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700 nM, depending on the length of the p53 peptide. Phosphorylation of Ser15 and Ser20 does not affect binding, but Thr18 phosphorylation weakens binding 10-fold, indicating that phosphorylation of Thr18 only is responsible for abrogating p53-MDM2 binding. DNA damage mediated disruption of this complex in vivo also requires only Thr18 phosphorylation. However, stabilization of p53 after ionizing radiation results from an inhibition of MDM2 binding through a phosphorylation cascade that first requires phosphorylation of p53 Ser15, which is required for subsequent phosphorylation of Thr18.105
In conclusion, conformation and hydrophobicity appear to be two critical requirements for the interaction between MDM2 and p53. A basic but insufficient requirement for p53 degradation is the direct interaction between p53 and MDM2 through their NH2 termini.
Additional requirements on p53 include its homo-tetramerization region, which enhances degradability possibly via improved MDM2 binding, because a monomeric p53 mutant has lost sensitivity to degradation by MDM2. As stated above, lysine residues, mainly in the COOH terminus of p53, are required anchors for monoubiquitination. Also, the extreme COOH terminus of p53 (aa 363–393) is required, presumably again due to its lysine residue.103
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Figure 38. Crystal structure of the p53-MDM2 protein-protein interaction.104
2.1.11 p53 as a therapeutic target
The p53 tumour suppressor protein is a transcription factor that is activated in response to cellular stress. Depending on the severity of the threat to genome integrity, p53 then imposes cell cycle arrest or apoptosis. Because p53 has strong growth-suppressive activity, it must be tightly regulated to allow normal cells to function. This is achieved to a large extent by MDM2 protein.
The functions of p53 are ablated in all cancers as a means of evading apoptosis, either by disabling p53 directly through mutation or deletion, or indirectly by alterations of various components of the pathways that regulate p53. About half of all cancers retain wild-type p53 and in these the normal regulation of p53 is sometimes disrupted through direct overexpression of MDM2 (in ca. 7% of cancers). MDM2 overexpression due to gene amplification is especially frequent (ca. 30%) in human osteogenic sarcomas and soft tissue sarcomas.106
Because of the central role of p53 in tumour suppression, nongenotoxic therapeutic strategies that activate p53 in one way or another are highly desirable.
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There are many potential therapeutic targets within the p53 pathway, downstream of the stress response, which offer the possibilities of nongenotoxic p53 activation and bypassing the particular defects that could render an upstream target ineffective. The p53-MDM2 regulatory system is one such target. Modulation of this system with small molecules is a very active area of research.106
The key question for any therapeutic strategy that aims to activate the p53 response is whether of not this will result in a selective effect on tumour cells as opposed to the cells of healthy tissues. Such specificity of p53 to kill tumour cells, but not normal cells, appears to underlie the safety of p53 gene therapy, which has gained approval in China and is now being developed elsewhere. It has been shown that mice with a hypomorphic MDM2 allele produce only about 30% of the normal level of MDM2 and exhibit increased transcriptional and functional activation of p53. The effects of p53 under these circumstances are not lethal as one might expect, although the animals are small and show p53-dependent apoptosis of lymphoid cells. Nevertheless they are viable, do not age prematurely, and are resistant to tumour formation.107
Similarly, in vivo suppression of MDM2 using antisense oligonucleotides has been demonstrated to result in therapeutic antitumour effects without overt toxicity. From these and other results it is clear that the p53 pathway differs significantly in normal and p53 wild-type cancer cells and that the latter are selectively sensitive to increases in p53 effector functions.
Different approaches have been exploited to release p53 from MDM2 control including inhibition of MDM2 expression, MDM2 ubiquitin ligase activity and p53-MDM2 binding.106
2.1.12 Blocking MDM2 expression
Several studies using antisense oligonucleotides to inhibit MDM2 expression have established the proof-of-concept of this approach in cells and mouse models of human cancer. MDM2 downregulation by antisense oligonucleotides has resulted in p53 stabilization and activation of the p53 pathway in cancer cells that grow in tissue culture in vitro or as tumor xenografts in nude mice. Interestingly, not only wild-type p53 cells but also cells that express mutant p53 have responded equally
119 well to MDM2 inhibition. This unexpected observation has been difficult to explain with the selective inhibition of MDM2 expression. A recent study has suggested that p53-independent stabilization of the cyclin-dependent kinase inhibitor p21 as a result of MDM2 downregulation might contribute to the antitumoral activity of MDM2 antisense oligonucleotide. However, this mechanism has not been independently confirmed.85
2.1.13 Inhibiting MDM2 ubiquitin ligase activity
Because p53 ubiquitylation and degradation is the major way of negative control by MDM2, targeting the ubiquitin ligase activity of MDM2 has been pursued as a p53-activating strategy.108
Recently, small-molecule inhibitors have been identified that specifically target the E3 ligase activity of MDM2. Several compounds from this class, referred to as HL198C (Figure 39), inhibited in vitro p53 ubiquitylation with IC₅₀ in the 20–50
M range. In cancer cells, they activated p53 signaling and induced apoptosis in a p53-dependent manner. However, these compounds have low potency and selectivity. Further optimization of these inhibitors to increase their potency, E3 ligase specificity and to eliminate p53-independent off-target activities is necessary to assess the potential of this p53-activating strategy.85
2.1.14 Inhibiting MDM2–p53 binding
MDM2 is unable to downregulate p53 if prevented from interacting with it. Therefore, inhibition of MDM2–p53 binding is a desirable strategy for p53 stabilization and activation. However, targeting of protein–protein interactions by small molecules is challenging.
Protein–protein interactions usually involve large and flat surfaces that are difficult to break by low molecular weight compounds. In the case of the p53-MDM2 interaction, however, it has been demonstrated that a limited number of amino acid residues are crucial for the binding of these two proteins. In fact, just three p53 amino acids –Phe19, Trp23 and Leu26 – have been found essential for the binding between the two proteins and they are inserted into a deep
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hydrophobic pocket on the surface of the MDM2 molecule. Thus, one can envision designing small molecules that might successfully mimic this interaction.85
2.1.15 Small-molecule inhibitors of the p53-MDM2 interaction
An increasing number of small-molecule p53-MDM2 binding inhibitors have been discovered and published in the past three years. Here, only recent compounds are examined that have acceptable cellular potency and selectivity for their molecular target and might represent viable leads for development of therapeutic agents.
The first potent and selective small-molecule MDM2 antagonists, the nutlins (Figure 40), were identified from a class of cis-imidazoline compounds.109 These inhibitors could displace p53 from MDM2 in vitro with nanomolar potency (IC₅₀ = 90 nM for nutlin-3a, the active enantiomer of nutlin-3). Crystal-structure studies demonstrated that nutlins bind to the p53 pocket of MDM2 in a way that remarkably mimics the molecular interactions of the crucial amino acid residues from p53 (Figure 39. Nutlins have been shown to enter multiple types of cultured cells and inhibit the p53-MDM2 interaction in the cellular context with a high degree of specificity, leading to stabilization of p53 and activation of the p53 pathway. Proliferating cancer cells were effectively blocked in G1 and G2 phases, and underwent apoptosis when exposed to low micromolar concentrations of nutlins.109 Gene array analysis also confirmed that nutlins are highly selective for their molecular target in the cellular context. Administration of nutlin-3a to nude mice that bear established human cancer xenografts for 21 days showed effective tumor-growth inhibition and tumor shrinkage at non-toxic doses. These studies provided the first in vivo proof-of-concept that activation of wild-type p53 by pharmacological inhibitors of the p53-MDM2 interaction is feasible and might exploited for cancer therapy.85
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Figure 39. Structural aspects of the p53–MDM2 interaction and nutlin binding. (a) MDM2 and p53 interact with each other with their N-terminal domains through a well defined p53 binding pocket. The crystal structure of the binding revealed that three amino acid residues of the p53 peptide (green) are essential for the binding with MDM2, and they are inserted into a fairly deep cavity on the MDM2 surface (yellow). The figure depicts the most important functional domains of p53 and MDM2 proteins. (b) Nutlin-2 (red) binds to the p53 pocket of MDM2 by mimicking the interaction of the three crucial amino acid residues from the p53 peptide (green).85
Four other classes of p53 activators have been reported since the discovery of the nutlins but their activity has not been independently confirmed yet. A class of MDM2 antagonists with a benzodiazepine core (Figure 40) has been reported that can disrupt p53-MDM2 binding in vitro with IC₅₀ in the 0.5–2 M range. The first published compounds had low cellular penetrability but improved compounds could suppress the growth of wild-type p53 cells with IC₅₀ in the 10– 30 M range and 3–9-fold selectivity for cells with functional p53. Low cellular potency and selectivity of these agents makes it difficult to evaluate their antitumoral potential85. A small-molecule compound, termed RITA (reactivation of p53 and induction of tumor cell apoptosis) (Figure 40), has been identified recently by a cell-based screen. RITA can activate the p53 pathway through a nongenotoxic mechanism and induce p53- dependent growth arrest and apoptosis.
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Treatment of severe combined immune deficiency (SCID) mice that bear HCT116 human tumor xenografts inhibited their growth in a p53-dependent manner. It has been proposed that RITA activates p53 by preventing its interaction with MDM2. In contrast to other reported inhibitors, RITA accomplishes this by binding to p53 but not to MDM2. However, other studies with RITA have not confirmed the ability of the compound to block the p53–MDM2 interaction in vitro. Therefore, the molecular mechanism of p53 activation by RITA awaits further clarification. Using structure-based design, Ding et al. identified several compounds with a spiro-oxindole core structure (Figure 40) that can inhibit p53-MDM2 binding in vitro with potency in the 20–2000 nM IC50 range. A good correlation between in vitro and cellular activity has also been demonstrated. The most potent compounds in this class have shown anti-proliferative activity in the low micromolar range with nearly 30-fold selectivity for cells with wildtype p53 over cells with mutant p53, but their biological activity has not been well characterized. These authors also recently reported another class of quilinol MDM2 antagonists (Figure 40) with a good cellular activity and selectivity for wild-type p53. However, no in vivo activity has been reported yet.85
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2.2 TSPO/MDM2 dual –target ligands
Combination therapy has been the standard of care in several diseases, such as diabetes and immunoinflammatory disorders110, since it is a rationale strategy to decrease drug resistance and to increase response and tolerability.
The benefits of multi-target action are well established in cancer, because the process of oncogenesis is known to be multigenic, multifactorial and characterized by the misregulation of more than one protein.111
The multi-target strategy may be of great importance against glioblastoma multiforme (GBM), a particularly aggressive form of brain cancer. Despite massive research efforts, the surgical resection is the only cure for GBM, and the currently approved anti-GBM agents, such as Temozolamide, Bevacizumab, or Cilengitide offer a limited improvement in progression free survival.112-114 Evasion of cell death is a hallmark of cancers and a major cause of treatment failure; in this respect, apoptosis inducers that act through the mitochondria death pathway have been emerging as promising drugs in a large number of tumors115, particularly in GBM.116 Activation of the cancer cell death machinery through the mitochondrial membrane permeabilization has been obtained so far also by the use of drugs targeting the mitochondrial translocator protein (TSPO). Ligands such as PK11195, Ro5-4864 and diazepam have demonstrated antitumor effects in
vitro and in vivo, both as single agents or combined with the chemotherapeutic
agents etoposide or ifosfamide.117 Importantly, in a variety of systems, PK11195 can reduce or abrogate the antiapoptotic effect of Bcl-2-family proteins, suggesting that TSPO could be exploited to bypass Bcl-2-imposed chemoresistance. 117 In this respect, we have recently demonstrated that newly synthesized selective TSPO ligands are able to trigger apoptosis also in human GBM cell lines and in rat C6 glioma cells, modulating the opening of the mitochondrial permeability transition pore (MPTP), of which TSPO is an important constitutive protein.118,119
Another important role in mitochondria-mediated cell apoptosis involves the tumor suppression protein p53. Indeed, in addition to target gene regulation, p53 can directly induce permeabilization of the outer mitochondrial membrane, by forming complexes with the protective Bcl2-family proteins, resulting in
125 cytochrome c release.120 The deregulation of this pro-apoptotic protein is widely described in literature, and the reactivation of its endogenous function represents an important anti-cancer therapeutic strategy121, at least for those tumors with a no mutant p53. P53 is negatively regulated by the murine double minute 2 (MDM2), and thus inhibitors of p53-MDM2 interaction currently represent another viable approach in GBM therapy.121-123
Thus, while agents targeting either the TSPO or p53-MDM2 interaction have been already investigated and provided to have some survival benefit in cancers, molecules targeting both proteins are not known so far. With the perspective that co-targeting TSPO and p53-MDM2 with one molecule could maximize the anti-tumor efficacy, dual binders were rationally designed, synthesized and biologically characterized in human GBM cells.
2.2.1 Design and synthesis of novel TSPO/MDM2 dual –target ligands
With the aim to target TSPO and p53-MDM2 at the same time, molecular modeling studies were conducted to identify, among our in-house database of TSPO ligands118,119,51,52,66, those that would disrupt the p53-MDM2 interaction if adequately decorated. Among a number of chemically diverse structures, we focused on indole-containing structures, as indole is considered a “privileged scaffold” for drug discovery.124
Specifically, we focused on phenylindolylglyoxylamides (PIGA), which are prone to be easily substituted (eg. on the amide nitrogen) and for which an exhaustive SARs study has already been performed by our group providing a clear picture of PIGA-TSPO interaction.51,52,125 PIGA SARs clearly suggested the essential requirements for binding to TSPO: (i) the presence of one H-bond acceptor (the oxygen atom of the second carbonyl group of the oxalyl bridge); (ii) an aromatic moiety at the 2-position of the indole nucleus; (iii) N,N-disubstitution at the amide nitrogen. Herein, bearing in mind all this information, we decided to substitute the alkyl groups with chemical features mimicking the p53 trans-activation domain helix. Thus, for the design of the novel MDM2 inhibitors, both crystallographic data, revealing the mode of binding of p53-MDM2, and the MDM2 ligands reported so far126 were taken into account. The three critical residues of p53 transactivation
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domain are bound to MDM2 N-terminal domain in a well-defined surface pocket characterized by three hydrophobic clefts hereon named: Phe19, Trp23 and Leu26 pockets.
Very recently, new MDM2 “hot spots” for the development of potent inhibitors have been available and suggested that the MDM2 N-terminus region, which is a disordered loop in the apo-structure, can adopt an ordered folding and form additional van der Waals and hydrogen bond contacts upon the binding of an inhibitor, considerably augmenting the activity of the last.127 Considering all this information, we attempted, as first step, the docking calculation of unsubstituted 2-phenylindolglyoxylylamide in the MDM2 pocket, through the Autodock 4 program. In the lowest energy binding pose, the phenyl ring was buried in the Trp23 pocket, the indole ring occupied the Phe19 region, while the glyoxylylamide moiety was oriented toward the Leu26 pocket offering chances for a further decoration. Thus, similarly to p53 transactivation domain helix, we functionalized the glyoxylylamide group with a Leu- residue. Subsequently, we decided to further elongate our compound in the attempt to reach the region nearby the MDM2 N-terminus. The design strategy resulted in compounds 81 and
82 (Figure 41). H3C H3C NH NH O N H O O 82 HO O H3C H3C NH NH O N H O O 81 O O
Figure 41.Structure of novel indolylglyoxyldipeptides 81 and 82.
As proof of concept of the design hypothesis, docking calculation of 81 and 82 in the MDM2 binding site was attempted and two main binding poses were found in the lowest energy clusters.
127 In particular, in the first one (hereon referred as binding mode A, Figure 42 a) the ligand was inserted in the MDM2 binding site so that the phenyl substituent bound to the indole ring was buried in the Trp23 pocket making favorable van der Waals contacts with L57, I61, F86, F91, I99 and I103 side-chains. The MDM2 Phe19 subpocket was occupied by the indole core that was able to make hydrophobic contacts with I61, M62, V75 and Y67 residues. On the other side of
81, Phe residue was accommodated within the MDM2 N-terminus region where it
formed hydrophobic contacts with I19, Y100, L54 and M50 and engaged an amide-π interaction with the Q24 side chain, while the ethyl ester moiety was found sandwiched between F55 and the methylene groups of the K51 side chain. Oddly, Leu26 cleft was not filled by the Leu side chain of 81 that, instead, establishes hydrophobic interactions with F55 residue. Accordingly, in a very recent work by Olson’s group the benzyl moiety of their morpholinones ligands engaged an edge-face π−π interaction with the same F55 leaving Leu26 subpocket unoccupied just as in our case.128 However, the occupancy of Leu26 pocket by the Leu residues of 81 cannot be strictly excluded.
Figure 42. Docking poses of Compound 81 in the MDM2 binding site.The ligand is represented as golden stick, the protein residues and surface as cyan sticks and transparent blue respectively. MDM2 binding pockets are labeled according to p53 side chains and are highlight in red dots.
In fact, in the energetically equivalent binding mode B (Figure 42 b), the inhibitor was positioned so that the isobutyl side chain was well inserted into Leu26 subpocket establishing lipophilic contacts with H96, I99 and Y100 residues. Notably, in the case of 82 (where the binding mode B is preferred), docking results showed that the carboxylic group flipped toward the Leu26 pocket H-bonding the Nδ of the H96 imidazole ring, thus compensating the loss of the hydrophobic interactions established by the ethyl ester 81. In both cases, the found
128
binding modes definitely suggested that the design hypothesis was sound and that the synthesis of 81 and 82 was worthwhile.
The synthesis of the target indole derivative 81 was achieved by the convergent procedure outlined in Scheme 8.
Scheme 8 ClCOCOCl anh. Et2O O°C N H NH Cl O O + 79 CH3 H3C N H COOH O O H3C H3C NH O N H O 76 77 H3C H3C NH2 NH O 80 TFA, CH2Cl2, rt anh. toluene NEt3, 0°C H3C H3C NH NH O N H O O 81 78 O O O O O O O COOC2H5 H2N CDI, anh. DMF,rt H3C H3C NH NH O N H O O 82 HO O LiOH MeOH:H2O / 3:1
129 The first dipeptide intermediate 80 was obtained in two steps starting from the Boc-L-leucine 76 that was condensed with L-phenylalanine ethyl ester, in the presence of N,N'-carbonyldiimidazole (CDI) in anhydrous dimethylformamide to obtain compound 77, and then deprotected by treatment with trifluoroacetic acid in dichloromethane. The commercially available 2-phenylindole 78 was acylated with oxalyl chloride, in anhydrous ethyl ether at 0 °C to obtain the corresponding 2-phenylindolglyoxylyl chloride 79.51,52 Derivative 79 was allowed to react with compound 80, in the presence of triethylamine in dry toluene solution at 0 °C, to give compound 81, which was finally purified by flash chromatography. The ester moiety of 81 was finally hydrolyzed with lithium hydroxide monohydrate in a MeOH/H2O (3:1) solution under reflux overnight to yield the corresponding carboxylic acid 82.
2.2.2 TSPO activity in “cell-free” models
At first, the ability of the novel compounds 81 and 82 to bind TSPO was tested. For this purpose, radioligand binding assays with the TSPO-selective radioligand [3H]PK11195 were performed. Compounds 81 and 82 were able to displace specific [3H]PK11195 binding, in a concentration-dependent manner, with Ki values of 438 ± 35 nM and 759 ± 56 nM (Figure 43), respectively.
Figure 43. New synthesized compounds bind to TSPO and induce Δψm collapse.
[3H]PK11195 radioligand binding: membranes homogenates obtained from kidney (20 µg of
proteins) were incubated with 0.6 nM [3H]PK11195 and different compounds concentrations.
-10 -8 -6 -4 0 50 100 Compound 1 Compound 7 Log [compound] [ 3H ]P K 1 1 1 9 5 sp eci fi c b in d in g ( % ) 81 82
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Reaching equilibrium, samples were filtered and bound radioactivity was counted. Data are expressed as percentage of specific binding versus basal value (set to 100%) and represent the mean ± SEM of three different experiments.
Several evidences have shown that TSPO is a constituent of the MPTP, taking therefore part in dissipation of Δψm that occurs after MPTP opening, as well as in the release of proapoptotic inter-membrane proteins and in the induction of apoptosis2. To assess whether compounds 81 and 82 could modulate MPTP opening through their selective binding to TSPO, Δψm was measured in mitochondria isolated from the human GBM cell line U87MG. The treatment of the isolated mitochondria with compound 81 or 82 (5 M) for 15 min lead to a significant reduction in tetrachloro-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) accumulation compared to control untreated mitochondria, demonstrating the collapse of Δψm (Figure 44 and 45).
Figure 44. Evaluation of Δψm in isolated mitochondria: Mitochondria (5µg of proteins) were treated with compound 81 (5 µM), or compound 82 (5 µM), or DMSO (control) for 15 min. The Δψm was evaluated using JC-1 protocol. The data are expressed as variation of JC1 uptake into mitochondria, calculated as the difference between RFU read after 10 minutes and RFU read at the beginning. Data represented the mean ± SEM of three different experiments. Each experiment was performed in duplicate. Statistical significance was determined with a one-way ANOVA with Bonferroni post-test:**P<0.01, ***P<0.001 vs control.
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Figure 45. Representative graph of mitochondria potential evaluation using JC-1 protocol. The results were expressed as RFU units in time.
2.2.3 Human p53-MDM2 complex dissociation in “cell-free” models
The ability of the new compounds to bind MDM2, thus dissociating the p53-MDM2 complex was investigated by NMR and ELISA-based in vitro assays on recombinant and native human p53-MDM2 complex, respectively.
Holak et al. have recently described a two-dimensional 15N-HSQC based NMR assay to determine the effect of antagonists on protein-protein interactions named AIDA (for antagonist induced dissociation assay). 129,130 AIDA requires the use of a large protein fragment (larger than 30 kDa) to bind to a small reporter protein (less than 20 kDa). However, in appropriate conditions (flexible residues), just 1D proton NMR spectra may suffice for monitoring the states of proteins in complexes upon treatment with ligands. Because of the highly flexible nature of the N-terminal domain of p53, p53-MDM2complex is suitable for 1D proton NMR application.129,131 In particular, the NH side chains of W23 and W53 produce sharp lines in the free p53 1D proton spectrum. On forming the complex with MDM2, W23 signal disappears, as W23, together with the p53 residues 17-26, comprises the primary binding site for MDM2. Upon binding, these residues participate in well-defined structures of large MDM2/p53 complexes, whereas W53 is still not structured when p53 is bound to MDM2.129,132 The observed 1/T2 transverse relaxation rate of the bound W23 in the complexes increases significantly, and the broadening of NMR resonances results in the disappearance
0 100 200 300 400 500 600 0 250 500 750 1000 1250 DMSO Compound 1 Compound 7 Time (sec) R F U 81 82
132
of this signal from the spectra. Figure 25 shows the 1H NMR signals of the tryptophan residues of p53 and of p53-MDM2 complex (0.1 mM, only W53 NH side chains signal can be detected in the last). After the addition of compound 81 or 82 (0.2 mM formal final concentration) to the p53-MDM2 complex, the W23 peak appears (Figure 25C and Figure 25D, respectively) indicating a complete p53 release.129
To confirm the qualitative results obtained in the NMR experiments, a quantitative sandwich immune-enzymatic assay technique, on crude cell lysates obtained from U87MG cells was developed. As a first step, protein dependence of the assay was revealed by adding increased aliquots of U87MG cell lysate. As shown in Figure 47A, specific absorbance values at 450 nm proportionally increased with protein concentration of U87MG cell lysates, with a trend toward hyperbole saturation starting from 40 µg of proteins. The absorbance at 450 nm of blank wells, obtained in the absence of p53 antibody, remained always under the 20% of total values. The assay was also validated using different concentration of human recombinant p53-MDM2 complex (Figure 47B). As reference compounds, the two characterized MDM2 inhibitors, Nutlin-3 and ISA27 were used.123,132 As shown in Figure 47C, both Nutlin-3 and ISA27 were able to inhibit the formation of p53-MDM2 complex, with IC50 values of 108.0 ± 4.5 nM and 121.7 ± 14.5 nM, respectively. These values are comparable to those reported using Biacore’s surface plasmon resonance technology133
, or a similar ELISA assay on recombinant p53-MDM2 proteins.134 Compounds 81 and 82 were thus tested with the validated ELISA assay, showing to be able to efficaciously dissociate p53-MDM2 complex, with IC50 values in the nanomolar range (Figure 47D). Noticeably, compound 81 appeared to be approximately 10 times more
potent than the reference MDM2 inhibitors Nutlin-3 or ISA27, with an IC50 value of 11.65 ± 0.49 nM, whereas compound 82 showed similar potency to the reference MDM2 inhibitors (IC50 value of 202.0 ± 21.2 nM).
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Figure 46. Dissociation of recombinant p53-MDM2 complex by NMR studies: One-dimensional proton spectrum of the side chains of tryptophans (W) of p53 alone (a), p53-MDM2 complex (b), p53-MDM2 complex after addition of compound 81 (c), or compound 82 (d). Figure shows the 1H NMR signals of the tryptophan residues of p53 and of p53-MDM2 complex (0.1 mM, only W53 NH side chains signal can be detected in the last). After the addition of compound 81 or 82 (0.2 mM formal final concentration) to the p53-MDM2 complex, the W23 peak appears (c and d, respectively) indicating a complete p53 release.
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Figure 47. ELISA-based in vitro MDM2/p53 protein-protein interaction assay (A, B) Different amount of cell lysates (panel A) or human recombinant p53-MDM2 protein (panel B) were captured on wells pre-coated with MDM2 antibody. After extensive washes, levels of the p53-MDM2 complex were quantified using an antibody specific for p53, and subsequently an HRP-conjugated antibody and a TMB substrate kit. (C, D) U87MG cell lysates, containing the native p53-MDM2 complex, were pre-incubated with DMSO (control) or different concentrations of the indicated compounds. Then, lysates were captured on wells pre-coated with MDM2 antibody. After extensive washes, levels of the p53-MDM2 complex were quantified using an antibody specific for p53, and subsequently an HRP-conjugated antibody and a TMB substrate kit. Blank wells were obtained in the absence of p53 antibody. Data are expressed as absorbance at 450 nm minus blank values (A, B), or as % of control set to 100 % (C, D), and represent the mean±SEM of at least three independent experiments.
2.2.4 p53-MDM2 complex dissociation and reactivation of p53 function in
U87MG cells
The cellular parameters commonly studied to support the reactivation of p53 pathway, following cell exposure with a MDM2 inhibitor,122,123,133 were then evaluated. First, we examined the effect of compounds 81 and 82 on p53 protein
0 50 100 150 200 0.00 0.25 0.50 0.75 510 25 A
cell lysate (µg proteins)
A b s 4 5 0 nm 0 0.00 0.25 0.50 0.75 5/1.6 12.5/4 25/8 50/16 B recombinant p53/MDM2 complex (ng/ml) A b s 4 50 nm -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 20 40 60 80 100 Compound 1 Compound 7 D Log [compound] p5 3 /M D M 2 c o m p le x (% v s c o nt ro l) -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 20 40 60 80 100 ISA27 Nutlin-3 C Log [compound] p 5 3/ M D M 2 c o m p le x (% vs c on tr ol ) 81 82
