[Cell Cycle 5:22, 2575-2578, 15 November 2006]; ©2006 Landes Bioscience
Perspective
Modulating Molecular Functions of p53 with Small Molecules Abstract Association of the human tumor suppressor p53 with many human cancers makes it a valuable therapeutic target. Stress‑induced molecular interactions of p53 with other effector proteins are immensely intertwined with regulation of its functions in orchestrating a wide array of cellular responses, thereby defying analysis of the underlying molecular mechanisms with conventional molecular and cellular biology methods. Recent discoveries of small molecules that can selectively modulate the molecular interactions of p53 offer promising opportunities to address the challenge of dissecting these complex mechanisms and increase the hope for pharmacological control of p53 for clinical benefits of cancer patients.
RIB
*Correspondence to: Ming-Ming Zhou; Department of Molecular Physiology and Biophysics; Mount Sinai School of Medicine; New York University; 1425 Madison Avenue; New York, New York 10029 USA; Tel.: 1.212.659.8652; Fax: 1.212.849.2456; Email:
[email protected]
IST
of Molecular Physiology and Biophysics; Mount Sinai School of Medicine; New York University; New York, New York USA
Original manuscript submitted: 09/29/06 Manuscript accepted: 10/02/06 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3464
introduction
Acknowledgements
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This work was supported by grants from the National Institutes of Health to M.-M.Z. and S.M.
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tumor suppressor p53, CREB-binding protein (CBP), bromodomain, lysine acetylation, small molecules
The human tumor suppressor protein p53 is known to control a wide array of cellular events in response to stress signals that risk integrity of the human genome.1‑4 During stress, p53 induces expression or silencing of a series of target genes that in turn control cellular responses such as cell cycle arrest, senescence, DNA repair or apoptosis.5,6 p53 function is controlled by multiple post‑translational modifications that are clustered in short stretches of the protein sequence.7 These modifications not only modulate the cellular protein level of p53 through control of its stability, but also regulate a network of molecular interactions between p53 and effector proteins that exert p53 functions in transcriptional regulation of its target genes. Because of such a tight balance between protein stability and function, conventional methods such as site‑directed mutagenesis accompanied by transient cell transfection of a mutated protein expression vector often lead to inconclusive results due to marked perturbation of the protein expression level, or possible masking of mutation effects by different neighboring modifications. Similar problems exist even with the RNAi gene knock‑down approach that affects an entire protein of interest rather than certain aspects of the protein’s functions. However, small‑molecule chemical ligands that are capable of selectively targeting specific molecular interactions and regulation of a target protein have shown promises in overcoming these challenging problems in studying p53 function in the cell in a temporal manner. Here, we review the new findings of p53 functions from these studies.
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Shiraz Mujtaba1 Lei Zeng1 Ming-Ming Zhou1,*
www.landesbioscience.com
Regulation of p53 Function with Post‑Translational Modifications The structure of p53 is consistent with its role as a transcription factor with identified domains responsible for transcriptional activation, sequence‑specific DNA binding, and oligomerization as a tetramer. The biological activity of p53 is tightly regulated by post‑translational modifications in its N‑ and C‑terminal regions3,8 (Fig. 1B). Upon DNA damage, p53 is extensively phosphorylated within the N‑terminal activation domain that relieves its association with the negative regulator MDM2 and results in p53 stabilization and activation.9,10 In addition, phosphorylation occurring in the C‑terminus of p53 was suggested to enhance its DNA binding in vitro, as studies have implicated the C‑terminal 30 residues of p53 in exerting a negative regulatory effect on the DNA binding activity of the protein.11,12 Deletion of these C‑terminal 30 amino acids containing phosphorylation within this region by casein kinase II and protein kinase C as well as the binding site of bacterial dnaK activates the DNA binding activity of p53.13,14 Consistent with this view, a monoclonal antibody which recognizes an epitope in this region activates the ability of p53 to bind to DNA15 as does a peptide derived from the carboxyl end of p53.16 Cell Cycle
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Figure 1. (A) A schematic diagram highlighting molecular interactions and regulation of p53 as a transcriptional factor; (B) an array of post‑translational modifications in N‑ and C‑terminal segments of p53. Flags colored in green, yellow and blue represent acetylation, phosphorylation, and ubiquitination, respectively.
In response to DNA damage, p53 also becomes acetylated on multiple lysine residues in its C‑terminal sequence.17,18 The transcriptional coactivator histone acetyltransferases (HATs) p300/CBP (CREB‑binding protein) and p300/CBP‑associated factor (PCAF) have been shown to acetylate K373 and K382 (to a lesser extent K372 and K381), and K320 respectively (Fig. 1B). Lysine acetylation or de‑acetylation of p53 has been directly linked to its ability to regulate p53 stabilization and activation as a transcription factor in cell cycle arrest and apoptosis19‑21 or senescence.22 Growing evidence from recent studies in cells show that lysine acetylation of p53 may not result in enhancement of its DNA binding ability as hypothesized previously based on in vitro studies,17,18 but rather promotes its recruitment of coactivators that leads to histone acetylation and transcriptional activation of target genes.19 In addition to acetylation, the same C‑terminal lysine residues of p53 are also subject to ubiquitination in a mutually exclusive manner, thereby leading to proteasomal degradation23,24 (Fig. 1B).
Molecular Interactions of p53 with Chromosomal Proteins Post‑translational modifications of amino acids in proteins can positively or negatively modulate protein‑protein interactions as illustrated in receptor‑mediated signal transduction and chromatin‑ mediated gene transcription. N‑terminal serine phosphorylation in p53 relieves its repressive association with MDM2, whereas C‑terminal lysine acetylation promotes p53 recruitment of transcriptional coactivators that possess HAT activity.25 The latter transcriptional coactivators are responsible for chromatin modifications and remodeling that are required for transcriptional activation of p53 target genes. As shown in recent studies, p53/coactivator recruitment is mediated by acetyl‑lysine binding of the bromodomains of the coactivators.25,26 The evolutionarily conserved bromodomain, originally reported in the Drosophila protein brahma (hence the 2576
name), is present in a large number of chromatin‑associated proteins and nuclear HATs,27,28 and has been shown to function as an acetyl‑lysine binding domain.29,30 The bromodomains adopt a conserved structural fold of a left‑handed four‑helix bundle (aZ, aA, aB and aC), as first shown in the bromodomain of PCAF.29 The ZA and BC loops at one end of the helical bundle form a hydrophobic pocket for acetyl‑lysine binding.29 Recent structural analyses of the PCAF bromodomain recognition of histone H4 acetylated at lysine 8 (H4‑K8ac)29 and of HIV‑1 trans‑activator Tat acetylated at lysine 50 (Tat‑K50ac)26 reinforce the notion that bromodomain/acetyl‑lysine recognition serves as a pivotal mechanism in regulating protein‑ protein interactions in chromatin‑mediated gene transcription.30 The dynamic and reversible lysine acetylation of p53 has been linked to its protein stability and biological activity, as acetylation of a lysine precludes its ubiquitination that would lead to protein degradation. However, our knowledge about the overall sequential and/or combinatorial effects of acetylation of multiple C‑terminal lysines in p53 is still very limited. Recently, acetylation on lysine 382 (K382ac) has been shown to be required for its recruitment of CBP through bromodomain/p53‑K382ac binding—a molecular interaction that is essential for activation of p21 that leads to cell cycle arrest in response to DNA damage.25 This molecular mechanism of acetyl‑lysine‑mediated coactivator recruitment may be utilized in p53 interactions with the subunits of the TFIID complex, TAFII40 and TAFII6031 as well as TRRAP.19
Relieving p53 Negative Regulation with Small Molecules Small‑molecule ligands blocking p53/MDM2 interaction can be used not only to study the negative regulation of p53, but also for the development of therapeutic agents.32 It is also notable that over‑expression of MDM2 is observed in several forms of human cancers. Using screening and design strategies, three peptidomimetics of
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p53/MDM2 as a new target for anti‑cancer therapy, and highlight the exciting therapeutic potential of these small molecule inhibitors.
Blocking p53 Activation with Small Molecules
Figure 2. Three‑dimensional structures of p53 effector proteins in complex with their ligands. (A) MDM2 in complex with an N‑terminal p53 peptide (aa 17‑27) (PDB 1YCQ); (B) Nutlin‑2 (imidazoline 2) (1RV1). The bromodomain of the transcriptional coactivator CBP in complex with (C) a C‑terminal p53 K382ac‑containing peptide (aa 367‑386) (1JSP); and (D) MS7972 (2D82). The peptide and chemical ligands are color‑coded by atom type. The protein structures of MDM2 and the CBP bromodomain in their peptide and chemical ligand bound forms are correspondingly present in a similar orientation.
peptoids, b‑amino acids and terphenyl helix mimics have been developed that target this molecular interaction. For example, Kodadek and colleagues33 identified an amphipathic hexamer peptoid from a library screening that binds to MDM2 with KD of 37 mM. Robinson and coworkers34 developed an b‑turn mimic that projects the key hydrophobic amino acid side‑chains similarly as those in an a‑helix of p53 activation domain when bound to MDM2, but with an eight‑fold affinity increase (IC50 of 140 nM). Similarly, guided by the crystal structure of p53/MDM2 complex, Schepartz et al.35,36 designed a 14‑mer helix of b‑amino acids targeting MDM2 with a KD of ~368 nM. Several such terphenyl compounds inhibit the p53/MDM2 interaction in human cell culture, but p53‑dependent activation was lost at higher concentration, possibly due to nonspecific toxicity. Novel small‑molecule chemicals inhibiting p53/MDM2 binding have also been reported in the literature. From an NCI chemical library screening, Galatin and Abraham37 identified a sulfonamide compound that matches the proposed pharmacophore of the p53/ MDM2 interaction that inhibits p53/MDM2 binding with IC50 of 32 mM and causes a 20% increase of p53 transcriptional activity in an MDM2‑overexpressing cell line. Grasberger et al38,39 discovered a benzodiazepine for MDM2 (with KD of 80 nM) from a combinatorial library screening. This p53/MDM2 inhibitor was shown to induce expected anti‑proliferative effects in cancer cell lines at micromolar concentrations. Moreover, researchers at Hoffmann‑La Roche40 developed small‑molecule chemicals, termed nutlins, that show improved drug‑like chemical properties and inhibitory activity for p53/MDM2 (with IC50 of 0.09–0.26 mM) as compared to other small‑molecule inhibitors (Figs. 2A and B). Although target specificity was not directly addressed, their study reported that only one enantiomer of nutlin‑3 exerts the inhibitory activity. The nutlins are functional in an in vivo assay, with 90% inhibition of tumor growth observed upon treatment of osteosarcoma xenografts with nutlin‑3. In a more recent study, small molecule HLI98, which inhibits MDM2 E3 activity as identified in a high‑throughput assay was shown to enhance p53 stability and activity.41 Collectively, these results confirm the negative regulation of p53 by MDM2, verify www.landesbioscience.com
Bromodomain binding to a biological ligand can be highly specific through recognition by specific amino acid residues anchoring around an acetyl‑lysine, but the interaction is typically not very tight with affinity (KD) ranging from 10–100 mM.25,42‑44 The ligand binding site in a bromodomain that recognizes the acetyl‑lysine and the flanking residues contains predefined structural cavities, thus making it suitable for structure-based design of small molecules capable of blocking acetyl‑lysine ligand binding. Using a combined strategy of a target structural knowledge‑based construction of a “focused” library and nuclear magnetic resonance (NMR) spectroscopy guided screening, a series of small‑molecule chemicals were identified in a recent study that block the coactivator CBP bromdomain binding to p53 at the acetylated lysine 382.45 The detailed NMR structural analysis of the lead chemical MS7972 (9‑acetyl‑2,3,4,9‑tetrahydro‑carbazol‑1‑one) bound to the CBP bromodomain (with KD of 19.6 mM) revealed that the ligand is situated at the entrance of the acetyl‑lysine binding pocket by association with residues in the ZA loop (Fig. 2C and D), thereby blocking CBP interaction with p53‑K382ac. These chemical ligands were used to assess p53 function as a transcription activator in response to DNA damage in a cell‑based assay.45 As reported previously in the literature, p53 expression in U2OS cells is low in a resting state, due to its negative regulation by MDM2. Upon DNA damage stimulation by doxorubicin treatment, p53 becomes serine‑phosphorylated in the N‑terminal activation domain. Additionally, p53 becomes acetylated on its C‑terminal lysines, promoting CBP recruitment via bromodomain/p53‑K382ac binding, leading to histone acetylation and transcriptional activation of target genes, such as the cyclin‑dependent kinase inhibitor p21 in cell cycle arrest. As expected, treatment of U2OS cells with the CBP bromodomain ligand MS7972 at 200 mM, prior to the doxorubicin stimulation, results in a dramatic decrease of the doxorubicin‑ induced increase in p53. This instability of the lysine‑acetylated cellular p53 in a free state is likely due to rapid deacetylation by histone deacetylases and subsequent ubiquitination and degradation by MDM2. This effect is consistent with the decrease in p53‑ mediated p21 activation. Collectively, these results strongly suggest that inhibition of CBP bromodomain association with p53‑K382ac by small molecules can cause a dramatic inactivation of p53 transcriptional activity through promoting protein instability by changes of its post‑translational modification states.
Conclusions Our understanding of molecular mechanisms underlying eukaryotic gene transcriptional regulation is considerably enhanced over the past years by employing strategies such as molecular genetics, RNAi, and small‑molecule based approaches. Individually, each of these approaches has certain advantages as well as limitations. For example, molecular genetics has tremendous capability to target a specific protein, but it takes a relatively long time, minutes if not hours, to see phenotypic changes, which is typically dependent upon the developmental process. Moreover, genetic manipulations such as point or deletion mutations are impossible to reverse. Similarly, effectiveness of the RNAi method is dependent upon the half‑life of a target protein, and it affects all functions of the target protein. On the
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contrary, the small‑molecule based approach is capable of providing rapid and reversible effects on a protein in its endogenous form under physiological conditions. However, discovery of small‑molecule modulators with high specificity for a protein of interest can be challenging. An ideal strategy for complete interpretation of the in‑depth molecular mechanisms would be to combine the specificity of genetics and the reversibility by small molecules that may be developed using target structure-based design, a concept that has been illustrated in the recent study of small molecules for p53 function.45 Recent advances in the study of p53 biology highlight the central role of this versatile biological molecule in orchestrating a wide array of cellular processes in response to stress signals, which is accomplished with a highly coordinated and complex network of molecular interactions and regulation. The recently discovered small molecules of the p53 pathway have demonstrated the power of using such novel research tools in probing the functional complexity of transcriptional activity of the endogenous p53 in physiological conditions. These encouraging results emerging from the cell‑based study of p53 protein level and functional activation using these small molecules have increased the hope that pharmacological modulation of p53 function may provide clinical benefits for novel anti‑cancer treatment. The small‑molecule‑based approaches described in the p53 studies are applicable to rational design and development of chemical ligands to study other important cellular proteins that may lead to new disease therapeutics. References 1. el‑Deiry WS. Regulation of p53 downstream genes. Semin Cancer Biol 1998; 8:345‑57. 2. Oren M. Decision making by p53: Life, death and cancer. Cell Death Differ 2003; 10:431‑42. 3. Prives C, Hall PA. The p53 pathway. J Pathol 1999; 187:112‑26. 4. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408:307‑10. 5. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997; 88:323‑31. 6. Vousden KH. Apoptosis. p53 and PUMA: A deadly duo. Science 2005; 309:1685‑6. 7. Giaccia AJ, Kastan MB. The complexity of p53 modulation: Emerging patterns from divergent signals. Genes Dev 1998; 12:2973‑83. 8. Vousden KH. Activation of the p53 tumor suppressor protein. Biochim Biophys Acta 2002; 1602:47‑59. 9. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature 1997; 387:296‑9. 10. Kubbutat MH, Jones SN, Vousden KH. Regulation of p53 stability by Mdm2. Nature 1997; 387:299‑303. 11. Ahn J, Prives C. The C‑terminus of p53: The more you learn the less you know. Nat Struct Biol 2001; 8:730‑2. 12. Jayaraman J, Prives C. Activation of p53 sequence‑specific DNA binding by short single strands of DNA requires the p53 C‑terminus. Cell 1995; 81:1021‑9. 13. Hupp TR, Lane DP. Allosteric activation of latent p53 tetramers. Curr Biol 1994; 4:865‑75. 14. Takenaka I, Morin F, Seizinger BR, Kley N. Regulation of the sequence‑specific DNA binding function of p53 by protein kinase C and protein phosphatases. J Biol Chem 1995; 270:5405‑11. 15. Hupp TR, Meek DW, Midgley CA, Lane DP. Regulation of the specific DNA binding function of p53. Cell 1992; 71:875‑86. 16. Hupp TR, Sparks A, Lane DP. Small peptides activate the latent sequence‑specific DNA binding function of p53. Cell 1995; 83:237‑45. 17. Gu W, Roeder RG. Activation of p53 sequence‑specific DNA binding by acetylation of the p53 C‑terminal domain. Cell 1997; 90:595‑606. 18. Liu L, Scolnick DM, Trievel RC, Zhang HB, Marmorstein R, Halazonetis TD, Berger SL. p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage. Mol Cell Biol 1999; 19:1202‑9. 19. Barlev NA, Liu L, Chehab NH, Mansfield K, Harris KG, Halazonetis TD, Berger SL. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. Mol Cell 2001; 8:1243‑54. 20. Ito A, Kawaguchi Y, Lai CH, Kovacs JJ, Higashimoto Y, Appella E, Yao TP. MDM2‑HDAC1‑mediated deacetylation of p53 is required for its degradation. Embo J 2002; 21:6236‑45. 21. Luo J, Su F, Chen D, Shiloh A, Gu W. Deacetylation of p53 modulates its effect on cell growth and apoptosis. Nature 2000; 408:377‑81.
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