Current Pharmaceutical Design, 2011, 17, 000-000 1 1381-6128/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd. Small-Molecule Inhibitors of p53-MDM2 Interaction: the 2006-2010 Update Melissa Millard, Divya Pathania, Fedora Grande, Shili Xu, and Nouri Neamati* Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121 Abstract: Increasing knowledge of the relationship between p53 and MDM2 has led to development of potential small molecule inhibi- tors useful for clinical studies. Herein, we discuss the patented (2006-2010) inhibitors of p53-MDM2 interaction. The anticancer agents discussed in this review belong to several different chemical classes including benzodiazepinediones, cis-imidazolines, oxindoles, spiro- oxindoles, and numerous miscellaneous groups. This review also provides comprehensive information on inhibitors of p53-MDM2 inter- action that are currently being tested in clinical trials. It is important to note that many of the disclosed inhibitors need further validation to be considered as bona fide inhibitors of p53-MDM2 interaction and some will not be further considered for future studies. On the other hand, JNJ-26854165, a novel tryptamine derivative and RG7112, a cis-imidazoline representative have shown promising results in early phases of trials in cancer patients. AT-219, a spiroindolinone in late stage preclinical studies is a likely candidate to proceed into clinical trials. It remains to be seen how these inhibitors will perform in future clinical studies as single agents and in combination with the cur- rently approved chemotherapeutic agents. Keywords: p53, MDM2, protein-protein interaction, nutlin, benzodiazepinediones, cis-imidazolines, oxindoles, spiro-oxindoles, small- molecule anticancer agents, MDM2 antagonists, p53 wild type, spiroindolinone, JNJ- 26854165, RG7112, AT-219. 1. INTRODUCTION A hallmark of cancer is its ability to continuously proliferate and escape apoptosis. Among the molecules that regulate cell cycle progression and apoptosis, p53 has been extensively studied mainly due to its role in carcinogenesis. The p53 protein was originally discovered in 1979 as a 53 kD protein bound by the large T antigen of the sarcoma-associated virus SV40, and was described as a cell- cycle accelerator [1, 2]. It was ten years after its discovery that peo- ple started to unravel its tumor-suppressing function through ge- netic and functional studies [3]. Ever since then, p53 has drawn increasing research attention, which expanded our knowledge on the role of p53 in cancer progression. The tumor suppressor protein p53 can be stabilized and acti- vated by (1) aberrant growth signals (p14 ARF and oncogenes Ras, Myc or Fas involved), (2) DNA damage (ATM and Chk2 in- volved), and (3) a range of chemotherapeutic drugs, ultraviolet light, and protein-kinase inhibitors (ATR and CKII involved) (Fig. 1A). These three pathways maintain a high concentration of intra- cellular p53, which binds to a vast number of DNA promoter re- gions [4] and activates the expression of genes in control of cell growth arrest (p21, 14-3-3 ), DNA repair (GADD45), apoptosis (Scotin, PERP, NOXA, Bax, Fas, reactive oxygen species, KIL- LER/DR5, P53/AIP1, PIDD, PUMA), and the prevention of new blood vessel formation (TSP1, GD-AIF, Maspin BAL1) [5, 6]. In addition to its transcriptional activities in regulating cellular behav- iors, p53 has also been reported to induce apoptosis in a transcrip- tion-independent manner through interaction with Bcl-2 family members at the mitochondrial membrane [7]. In normal cells, and under physiological conditions, p53 is short-lived and the activation of p53 network is tightly under con- trol. One of the most important negative regulators of p53 is MDM2 (murine double minutes-2) [8], a 491 amino-acid protein originally cloned from the transformed mouse cell line 3T3-DM in 1987 [9]. The homolog of MDM2 in human is sometimes referred to as HDM2. MDM2 has been reported to inhibit p53 functions in at least three different ways. First, as an E3 ubiquitin ligase, MDM2 *Address correspondence to this author at the Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, 1985 Zonal Avenue, PSC 304, Los Angeles, CA 90089-9121, USA; Tel: 323-442-2341; Fax: 323-442-1390; E-mail: neamati@usc.edu binds to p53 and attaches ubiquitin groups to the carboxy terminus of p53, promoting ubiquitin-dependent degradation of p53 through the 26S proteasome [10-12]. Secondly, MDM2 can also directly interrupt p53 transcriptional activity via interaction with the N- terminal transcription activation domain of p53 [13, 14]. The X-ray crystal structure of the MDM2-p53 complex (PDB entry: 1YCR) was published in 1996 (Fig. 1B). Thirdly, MDM2 carries p53 away from its target genes through nuclear export [15]. On the other hand, the expression as well as the cellular concentration of MDM2 is regulated by p53, which means MDM2 acts as an internal feed- back regulator in the p53 network. The decreased intracellular amount of p53 due to the negative regulation of MDM2 in turn reduces MDM2 expression, hence shutting down this feedback loop and allowing p53 protein to be again activated [6]. In vivo studies on the relationship between MDM2 and p53 have shown that ho- mozygous deletion of MDM2 in the mouse germline causes lethal- ity at the blastocyst stage with inappropriate apoptosis, which can be rescued by p53 deletion [16, 17]. The inhibitory effects of MDM2 on p53 are also positively or negatively regulated by several cellular mechanisms. The most important positive regulation depends on the functions of MDMX, also known as MDM4, a non-redundant homolog of MDM2. MDMX not only enhances MDM2-mediated p53 ubiquitination via the formation of the MDMX-MDM2 heterodimer through their RING finger domains [18-20], but also directly blocks p53 tran- scriptional activity via a protein-protein interaction with p53 [21]. The negatively regulation on MDM2 is switched on under patho- logical conditions, stabilizing p53 and thus increasing its cellular concentration. These negative regulatory mechanisms include MDM2 association with ARF under oncogenic stress [22] or with ribosomal proteins (L5, L11, L23) under ribosomal stress [23-25], and post-translational modifications of MDM2 and p53 mainly induced by DNA damage-induced kinases [8, 26-28]. These nega- tive regulations result in the dissociation of the p53-MDM2 interac- tion and restore p53-dependent gene expression. Ever since the first p53 mutation reported in 1989 in a cancer patient [29], loss of p53 activity has been found in ~50% of human cancers, and is often associated with resistance in conventional cancer therapies. The currently known p53 inactivation mechanisms include (1) gene mutation; (2) p53 interaction with viral proteins; (3) p53 protein interaction with cellular oncoproteins, especially