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Activation of the P53 Pathway by Small-Molecule- Induced MDM2 and MDMX Dimerization

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Activation of the P53 Pathway by Small-Molecule- Induced MDM2 and MDMX Dimerization Activation of the p53 pathway by small-molecule- induced MDM2 and MDMX dimerization Bradford Graves1, Thelma Thompson, Mingxuan Xia, Cheryl Janson, Christine Lukacs, Dayanand Deo, Paola Di Lello, David Fry, Colin Garvie, Kuo-Sen Huang, Lin Gao, Christian Tovar, Allen Lovey, Jutta Wanner, and Lyubomir T. Vassilev1 Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110 Edited by Alan R. Fersht, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved May 30, 2012 (received for review March 3, 2012) Activation of p53 tumor suppressor by antagonizing its negative marginal in many tumor cell lines expressing normal levels of regulator murine double minute (MDM)2 has been considered an MDM2, suggesting that cancer uses other mechanisms to at- attractive strategy for cancer therapy and several classes of p53- tenuate or disable p53 signaling (20), such as the overexpression MDM2 binding inhibitors have been developed. However, these of the other negative p53 regulator, MDMX. High levels of compounds do not inhibit the p53-MDMX interaction, and their MDMX protein can make MDM2 antagonists, which have effectiveness can be compromised in tumors overexpressing shown very low activity against p53-MDMX binding, ineffective – MDMX. Here, we identify small molecules that potently block in killing cancer cells (21 23). Thus, simultaneous inhibition of p53 binding with both MDM2 and MDMX by inhibitor-driven MDM2 and MDMX is needed to release the full activity of stabilized p53 (15, 17). Therefore, recent efforts have been fo- homo- and/or heterodimerization of MDM2 and MDMX proteins. fi Structural studies revealed that the inhibitors bind into and oc- cused on identi cation of dual MDM2/MDMX antagonists. Because of distinct structural differences between MDM2 and clude the p53 pockets of MDM2 and MDMX by inducing the for- – mation of dimeric protein complexes kept together by a dimeric MDMX in their p53-binding pockets (24 26), small molecules optimized for MDM2 have shown very low affinity for MDMX small-molecule core. This mode of action effectively stabilized p53 (27). For example, the first potent and selective small-molecule and activated p53 signaling in cancer cells, leading to cell cycle MDM2 antagonist, nutlin-3a, has ∼400-fold lower potency arrest and apoptosis. Dual MDM2/MDMX antagonists restored against MDMX than MDM2 (28). This trend has been followed p53 apoptotic activity in the presence of high levels of MDMX by other MDM2 inhibitors (19). Efforts to identify MDMX- MEDICAL SCIENCES and may offer a more effective therapeutic modality for MDMX- specific inhibitors have recently yielded a class of small molecules overexpressing cancers. with in vitro binding activity in the high nanomolar range but relatively poor cellular potency and uncertain mechanism of he tumor suppressor p53 is a powerful growth-suppressive cellular activity (29). Nearly equipotent MDM2/MDMX peptide Tand proapoptotic protein tightly controlled by its negative inhibitors have been identified and characterized structurally but regulators: murine double minute (MDM)2 and MDMX (1, 2). their activity has been detected only in cell-free systems (30). These proteins bind p53 with their structurally similar N-termi- Recently, a cell-penetrating “stapled” peptide with good MDMX nal domains and effectively inhibit p53 transcriptional activity (1, binding affinity has been identified and evaluated in cancer cells 3). They both possess a RING (really interesting new gene) (31). Although cellular potency against p53-MDMX interaction domain in their C termini, but it is only functional in MDM2, has been found adequate, this peptide was unable to disrupt which serves as a specific E3 ligase and main regulator of p53 effectively p53-MDM2 binding, and it has been combined with stability (4, 5). Despite its RING domain, MDMX does not have the MDM2 antagonist, nutlin-3, to assess the antitumor potential an intrinsic ligase activity and does not affect directly p53 sta- of this emerging therapeutic modality. bility (6). However, MDMX can enhance ligase activity of Here, we identify a class of small molecules that can potently MDM2 toward p53 by forming MDM2/MDMX heterodimers (7, inhibit p53 interactions with both MDM2 and MDMX by in- 8). It has been reported that the MDM2/MDMX complex is duced protein dimerization and effectively restore p53 activity in responsible for polyubiquitination of p53, whereas MDM2 alone MDMX-overexpressing cancer cells. We show that antagonizing primarily induces monoubiquitination (9). Targeted disruption both negative p53 regulators significantly improves the apoptotic of MDM2/MDMX heterocomplexes is embryonic-lethal in mice, outcome in cancer cells overproducing MDMX. suggesting that complex formation is essential for p53 regulation in vivo (10). On the other hand, MDM2 can also ubiquitinate Results MDMX and is, therefore, responsible for its stability as well (11, Identification of Indolyl Hydantoins as MDM2/MDMX Antagonists. A 12). MDM2 is a transcriptional target of p53, and both proteins diverse library of small molecules was screened for suppression form an autoregulatory feedback loop by which they mutually of p53-MDMX binding (Table S1). The hits were then tested for control their cellular levels (13). activity against the p53-MDM2 interaction. One series of indolyl The functional relationship between MDM2 and MDMX is hydantoin compounds emerged as potent, dual MDM2/MDMX still being refined at the molecular level, but it is well established that these two negative regulators play a critical role in con- trolling p53 tumor-suppressor function in normal cells (2, 14). Author contributions: B.G., T.T., P.D.L., D.F., C.G., K.-S.H., L.G., C.T., J.W., and L.T.V. de- This is why they are frequently overproduced through gene signed research; B.G., T.T., M.X., C.J., C.L., D.D., P.D.L., C.G., L.G., C.T., and J.W. performed amplification and/or overexpression in tumors that retain wild- research; A.L. and J.W. contributed new reagents/analytic tools; B.G., T.T., M.X., C.L., D.D., type p53 (14). Therefore, antagonizing the binding of MDM2 P.D.L., D.F., C.G., K.-S.H., L.G., C.T., and L.T.V. analyzed data; and B.G. and L.T.V. wrote and MDMX to p53 is expected to restore p53 function and may the paper. offer a strategy for cancer therapy (15). Recently identified Conflict of interest statement: The authors are employees of Hoffmann-La Roche Inc. small-molecule inhibitors of the p53-MDM2 interaction have This article is a PNAS Direct Submission. fi validated this approach, and the rst pharmacological MDM2 Data deposition: The atomic coordinates and structure factors have been deposited in the antagonists are now undergoing clinical evaluation (16, 17). Protein Data Bank, www.pdb.org (PDB ID codes 3U15 and 3VBG). MDM2 inhibitors have shown effective p53 activation followed 1To whom correspondence may be addressed. E-mail: [email protected] or by cell cycle arrest, induction of apoptosis, and tumor regression [email protected]. in cancer cells with mdm2 gene amplification (18, 19). However, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. their apoptotic activity has been found to be moderate to 1073/pnas.1203789109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1203789109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 antagonists. For example, RO-2443 (Fig. 1A) showed a re- MDMX N-terminal fragment (Fig. 1B) in the presence and ab- markably similar inhibitory activity against both MDM2 (IC50 = sence of the small molecule indicated that: (i) the compound was 33 nM) and MDMX (IC50 = 41 nM) binding to p53. For its size, binding to the p53 pocket; and (ii) there was a substantial up- RO-2443 appeared highly potent (ligand efficiency, defined as field shift for Y63, which is consistent with shielding by an aro- binding energy per heavy atom, is 0.36) and likely to bind into, at matic group. A similar shift had been observed upon binding of most, two of the three subpockets on the surface of MDMX or a p53 peptide to MDMX attributable to shielding by Phe19.In MDM2. These pockets were defined by the original structure of addition, there was an overall resonance broadening in the a p53 peptide bound to MDM2 (32) which showed that there presence of the compound, which was manifested in the HSQC 19 23 were three key residues from the p53 peptide, Phe , Trp , and spectrum as a decrease in cross-peak intensities, suggesting for- 26 Leu . Throughout this report, these binding pockets on the mation of a higher-molecular-mass species. To determine the surface of MDM2 and MDMX are referred to as the Phe, Trp, effect of RO-2443 on the state of MDMX in solution, size-ex- and Leu pockets. clusion chromatography with static light scattering (SEC-SLS) To investigate the mechanism of action of RO-2443, we first 1 15 was performed (Fig. 1C). In the absence of RO-2443, the protein used NMR spectroscopy. The [ H- N]HSQC spectrum of the gave a SLS-calculated mass of 12.8 kDa, which agrees with the theoretical mass of a monomer (12.3 kDa). Addition of RO-2443 resulted in the protein eluting at an earlier elution volume, in- dicating that the shape and/or mass of the protein had changed. A B The SLS-calculated mass of the complex was 24.1 kDa, which F suggests that MDMX forms a dimer when bound to RO-2443. RO-2443 O F G54 100 N Kinetic analysis of the binding of RO-2443 to MDMX (Fig. 1D) G54 N O showed that the binding was in line with a two-molecule binding 80 H model.
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