Revisión de metodologías utilizadas en la investigación primaria

2.1 INTRODUCTION

The tumor suppressor p53 protein, “the guardian of the genome”, has an overarching role in protecting organisms from cancer.65 As a transcription factor, human p53 is 393 amino acids long (43.7 kDa) and includes an N-terminus transcription-activation domain (TAD), which is important for apoptotic activity (Figure 3).66 p53 is present at a very low cellular concentration in normal cells, while p53 accumulates in cells and becomes activated under certain conditions, such as hypoxia, DNA damage, or oncogene activation. p53 then binds to DNA and induces the transcription of downsteam genes (i.e., p21), which initiate cell cycle arrest, DNA repair, and apopotosis to prevent the proliferation of the damaged cells.66 Loss of the p53 tumor suppressor pathway contributes to the development of most human cancers.67 Restoration of the function of tumor suppressor p53 (such as Gendicine®) results in considerable therapeutic responses of tumors in cancer patients.68

Mdm2 (murine double minute 2) protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal TAD of the p53 tumor suppressor,69 and an inhibitor of p53 transcriptional activation (Figure 3).70 Mdm2 acts as an oncogene in tissue culture systems, and the oncogenic potential of Mdm2 has been demonstrated.71 Mdm2 (the human homolog is also called Hdm2) has been found amplified in more than 10% of 8000 human cancers from various

sites, including lung or stomach.72 Mdm4 (the human homolog is also known as Mdmx, Hdm4 or Hdmx) was later identified as a p53-binding protein sharing structural homology with Mdm2.73 Mdm4 has been found amplified or overexpressed in 10-20% of over 800 diverse tumors including lung, colon, stomach, and breast cancers, and 65% of retinoblastomas.72

The human p53 protein consists of five regions: the N-terminal intrinsically unstructured transactivation domain (TAD), which interacts with Mdm2 or Mdm4, the regulatory proline-rich domain (PRD), a well- structured DNA binding core domain (DBD), the tetramerization domain (4D) and the C- terminal region.

A model depicting some of the mechanisms that may regulate p53 subcellular localization, stability and transcriptional activity.

(Adapted with permission from Elsevier:2770980154366) Figure 3. Structure and function of p5367

Overall, Mdm2 (491 amino acids) and Mdm4 (490 amino acids) exhibit 32.6% amino acid sequence identity.74 The N-terminal domain of Mdm4 shows the highest sequence similarity with the related domain of Mdm2 (53.6% identity). The BoxI BD is the most conserved domain, and a sequence comparison of amino acids most important for interaction with p53 are shown (Figure 4), with residues in bold that constitute the p53-binding hydrophobic pocket.75 As shown in the co-crystal structures, the interactions between p53 and both its transcriptional inhibitors Mdm2 and Mdm4 are very conserved (Figure 4). On the other hand, the differential elucidation of the biological functions of both proteins are awaited with great interest.76

In view of the strong growth suppressive and pro-apopototic function of p53, the oncoprotein Mdm2 (and/or Mdm4) inhibits the function of wild type p53 in about 50% of human cancers.77 The overexpression of the oncogene product Mdm2 (or Mdm4) is often observed in cancer cells, which negatively regulates the activity of wild-type p53. The relevance of Mdm2 (or Mdm4) on the regulation of p53 levels and activity has fostered the development of strategies aimed at restoring p53 functions.78 A number of different small molecules and peptidomimetics disclosed in the last decade have been shown to block the physical interaction between p53 and Mdm2.79 On the other hand, small molecule inhibitors of Mdm2 ubiquitin ligase activity have also shown the ability to stabilize and activate p53 in tumors that retain wild-type p53.80

Comparison of Mdm2 and Mdm4 primary structures. The p53-BoxI binding domain (BoxI BD; amino acids ca. 25- 110), the Zinc finger domain (ZD; aa ca. 290-330) and the RING domain (RING; aa ca. 435-482) are conserved. The rest sequence is not conserved.75 (Adapted with permission

from Elsevier: 2770990646612)

Strucutres of p53-Mdm2 complex (PDB: 1YCR; p53- yellow, Mdm2-green) and p53-Mdm4 complex

(PDB: 3DAB; p53-purple, Mdm2-blue). Figure 4. The interaction between p53 and Mdm2 (Mdm4)

Targeted manipulation of apoptosis in tumor cells provides a strategy for the rational design and discovery of novel anticancer agents. The PPI site between the transcription factor p53 and its negative regulator Mdm2 is a major target in current cancer drug discovery.81 Disrupting the interaction between p53 and Mdm2 was shown to restore the wild type p53 activity and drive cancer cells selectively into apoptosis.81 Many investigations of small

molecule p53-Mdm2 inhibitors in different cancer cell lines and animal models support their usefulness as potential anticancer agents with a novel mode of action.82 In fact, recent interim results from the first-in-class clinical p53-Mdm2 inhibitor RG7112 (a nutlin-3 derivative) in patients with relapsed/refractory acute myeloid and lymphoid leukemia and refractory chronic lymphocytic leukemia/small cell lymphocytic lymphomas are encouraging.83 While several classes of small molecule p53-Mdm2 inhibitors have been described in the past, only some are of sufficient potency and few have been characterized by co-crystal structure analysis.28, 84-88 The interface between p53 and Mdm2 is, however, accessible to small molecule drug discovery due to its dimension, concavity and hydrophobicity of the binding site.89 Current p53-Mdm2 inhibitors have been discovered by different techniques, including high throughput screening (HTS), computational HTS and structure-based design (Table 3).27, 28, 86, 87

Table 3. Inhibitors of p53-Mdm2 interaction

Ligand Structure Ki (µM) MW (Da) Ref.

p53 residues 15-29 (PDB: 1YCR) 0.6 1808 90 Stapled peptide (SAH-p53-8) 0.055 2180 91 Nutlin-2 (PDB: 1RV1) O N N HO N N O O Br Br 0.09 581 28 TDP222669 (PDB: 1T4E) CO2H H N N O O I Cl Cl 0.067 566 27

MI-63 analog (PDB: 3LBL) H N N H O H N O N O F Cl Cl 0.036 577 92 Amgen 1a (PDB: 3JZK) N N N H N O Br Br 11 536 93

The visualization tool of the ANCHOR Database shows the interaction between Mdm2 (surface representation) and three anchor residues from p53 (stick representation) in Figure 5.62 These three residues are among those with the largest ΔSASA and lowest (i.e. favorable) predicted binding interaction energy. Note that the table of interacting residues also shows the C- terminal Asn29 as having a large value of ΔSASA and unfavorable predicted energy (+3.7 kcal/mol) due to the extra carboxylic acid group. Thus, as is, Asn29 is predicted to be not a good group to target for drug design. On the other hand, the three selected residues (Phe19, Trp23 and Leu26) are indeed hotspot anchors that have been exploited on the design of compounds that inhibit the interaction between p53 and Mdm2.94

Figure 5. Visualization tool showing anchor residues Phe19, Trp23 and Leu26 of p53 (PDB: 1YCR)62 (Adapted with permission from Oxford University Press: 2770971385014)

2.2 STRUCTURE-BASED APPROACH FOR THE RATIONAL DESIGN OF P53-