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Small molecular weight protein–protein interaction antagonists—an insurmountable challenge?
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Small molecular weight protein–protein interaction antagonists—an insurmountable challenge? Alexander Do¨mling Several years ago small molecular weight protein–protein interaction (PPI) antagonists were considered as the Mount Everest in drug discovery and generally regarded as too difficult to be targeted. However, recent industrial and academic research has produced a great number of new antagonists of diverse PPIs. This review structurally analyses small molecular weight PPI antagonists and their particular targets as well as tools to discover such compounds. Besides general discussions there will be a focus on the PPI p53/mdm2. Address Departments of Pharmaceutical Sciences and Chemistry, University of Pittsburgh, United States Corresponding author: Do¨mling, Alexander ([email protected])
However, more and more scientists recognize the eigenvalue of protein interaction antagonization. The growing importance of PPIs as oncology targets was underlined by many talks and posters at the recent AACR-EORTC meeting in San Francisco. Covered examples included p53/mdm2/mdm4, Bcl family interactions, IAPs, c-Myc, SPRY2-Cb, ERCC1/XPF, FAK–protein interactions, orphan nuclear receptor COUP-TFI, Smad4-SKI, cSrc-SH3, Smad2/3/4, Rb/Raf-1, SDF-1/CXCR4, tissue factor/FVIIa, HOX/PBX and tubulin. In the following the small molecule antagonists of the p53/mdm2 interaction are discussed, as an example of a successfully targeted PPI.
The p53/mdm2 case Structure
Current Opinion in Chemical Biology 2008, 12:281–291 This review comes from a themed issue on Molecular diversity Edited by Gerry Maggiora and Christopher Hulme Available online 21st May 2008 1367-5931/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2008.04.603
PPI signatures Systematic structural analyses of PPI interfaces yield a highly diverse picture from large and flat to narrow and structured interactions [1,2]. However, the majority of PPIs involve large binding sites where affinity is obtained by a multitude of often weak interactions. As a consequence these widely spaced interactions are difficult to mimic with small molecules, and thus targeting PPIs is a challenging area of drug discovery. Classical concepts established in drug discovery, for example, transition state mimics cannot be applied here. On the contrary, allosteric small molecular weight PPI antagonists and agonists are very common as modulators of the dynamic (de)polymerization of the tubulin cytoskeleton, and they are generally used to treat cancers in a clinical setting. It is therefore difficult to make predictions on the druggability of specific protein–protein interaction surfaces. Figure 1 shows different potentially druggable PPIs with high-resolution X-ray structure information. In all these cases the PPI exists as a rather confined, narrow and highly structured ‘hot spot’. www.sciencedirect.com
P53 is a transcription factor that regulates the cell cycle in response to cellular stress leading to DNA damage, for example, oxygen, radiation and xenobiotics. P53 holds the cell cycle and activates DNA repair proteins; if DNA damage proves to be beyond repair, p53 initiates apoptosis. In normal, noncancerous cells p53 is usually inactive, bound to the protein mdm2. This complex prevents p53’s action and promotes its degradation (by ubiquitination and proteasomal degradation). In cancer, tumour suppression by p53 is severely reduced. Thus, antagonizing the p53/mdm2 complex is a promising new strategy in cancer therapy [3]. In 1998 Pavletich described the crystal structure of the interaction of p53 and mdm2 (Figure 1) [4]. The negative regulator of p53 mdm2 has a very defined and deep binding pocket for p53. It became clear that the p53–mdm2 interface is an atypical protein– protein interaction. It is small and highly structured, that is, the calculated buried accessible surface area of mdm2 in the interface is only 660 A˚2. The diameter of the mdm2-binding groove with 18 A˚ (from Phe100CH2 to Phe67CH2) is comparable to a small molecule. It comprises a ‘hot spot’ where most of the binding affinity is concentrated [5]. The apparent dissociation constant of natural p53 peptides ranges from 60 to 700 nM depending on the peptide length. Mdm4 is also a negative regulator of p53 in a cellular context. Mdm4, a homologous protein of mdm2 has been described to bind the same p53 mini helix motif [6]. Mdm4 was described for the first time in 1996 as a novel p53-binding protein that shared in common some functional properties with mdm2 a high sequence homology to mdm2 [7]. Upon binding to p53 via the C-terminal ring domain, mdm4 stimulates p53 degradation through mdm2 ubiquitination. The mechanism of the mdm2/mdm4 regulation of p53, however, is not clearly understood; in particular, the contribution of Current Opinion in Chemical Biology 2008, 12:281–291
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Figure 2
P53/mdm2/mdm4 interactions. Residues shown, M50, P92 and Y96 for mdm4, and L54, H96 and Y100 for mdm2, are important for binding to p53. The structure reveals that although the principal features of the mdm2–p53 interaction are preserved in the mdm4–p53 complex, the mdm4 hydrophobic cleft on which the p53 peptide binds is significantly altered: a part of the cleft is blocked by side chains of M54 and Y100 of the p53-binding pocket of mdm4. (Right) Close up of the comparison of the high-resolution X-ray structure of p53 model peptides bound to mdm4 (above, PDB ID: 2Z5S) and mdm2 (below, PDB ID: 1YCR). Both complexes are oriented the same way. The green surfaces are the respective mdm2 and mdm4 proteins. Highlighted in blue are the crucial rim amino acids. The blue sticks represent the side chain of the p53 binding hot spot. The mdm4-binding cleft is significantly altered as compared with the mdm2 cleft: The western part of the binding cleft is blocked by the amino acids M54 and Phe100. Moreover the mdm4 pocket is flatter than mdm2.
mdm2 versus mdm4 to the regulation of p53 stability and activity is unclear. Several recent research lines of study underscore the idea that both mdm2 and mdm4 must be inhibited for effective restoration of p53 activity (when p53 exists as wild type in cancer) [8]. Mdm4 and mdm2 have been reported to cooperatively inhibit p53 activity in proliferating and quiescent cells in vivo [9]. The importance of independent p53 regulation of mdm4 over mdm2 was also demonstrated in vivo [10]. Owing to elevated high levels of mdm4 in several tumour types including head and neck squamous carcinoma, pre-B acute lymphoblastic leukaemia, childhood cancer retinoblastoma,
brain and breast, mdm4 is a promising target in its own right besides mdm2 [7]. Recently the three-dimensional structure of a p53 peptide binding into a humanized Xenopus mdm4 protein has been published with a 2.1 A˚ resolution by the Holak group and validated as an excellent human mdm4 model (Figure 2) [5]. As opposed to the plethora of reported small molecule antagonists of mdm2, currently none have been described for mdm4. Nutlin3 that is a potent mdm2 antagonist binds only very weakly to mdm4 and is incapable to disrupt p53/mdm4 in vivo at relevant concen-
(Figure 1 Legend ) Examples of different high-resolution structures of ‘hot spots’ PPIs. (a) Interaction of SMAD with SKI (yellow sticks) with implications in cancer apoptosis highlights the crucial string T271C272H273W274G275 (PDB-ID 1MR1); (b) Bir2-caspase 7 interaction with implications in cancer apoptosis; grey surface caspase-7, yellow sticks and cartoon XIAP Bir2 domain; highlighted in green the very hydrophobic V146 cage made up from the three aromatic amino acids Y230W232F282 (PDB-ID: 1I4O); (c) homodimer DPPIV with implications in diabetes shown as golden surface DPPIV monomer A, highlighting S720 forming a hydrogen bond with the indol of W734; shown as blue sticks hot spot triad F731W734Y735; W734 is deeply buried (71%) in the corresponding monomer with 160 A˚2 (PDB–ID: 1N1M); (d) interaction of XPF with ERCC1 with potential implications in cis platin resistance; the two XPF F840 and F888 (black sticks) form strong hydrophobic contacts with and are deeply buried in ERCC1 (PBD-ID: 2A1J). The pictures were generated using PyMol (www.pymol.com). www.sciencedirect.com
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trations. By comparison of the high-resolution X-ray structures of mdm4 and mdm2-binding p53 peptides, the selectivity of nutlin3 to mdm2 can be smoothly explained. Antagonistic compounds
Interestingly, no other protein–protein interaction attracted as much interest in the medicinal chemistry
community as p53/mdm2. More than 20 different chemotypes have been described to date that antagonize p53/ mdm2 (Figure 3). Here we concentrate on the discussion on small molecules with preferred properties that enhance oral bioavailability and generally have higher metabolic stability than peptides or proteins. Therefore, peptides, peptoids and high molecular weight natural products (chlorofusin, Mw = 1363 Da) are out of the scope
Figure 3
Currently described p53/mdm2 small molecular weight antagonists. Current Opinion in Chemical Biology 2008, 12:281–291
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of this review. The reader is referred to reference [11] for further information.
recently separation of the enantiomers by supercritical chiral HPLC has been described [18,19].
Amongst the first inhibitors of p53/mdm2 described were chalcone derivatives, 1 [12]. The molecules bind to a subsite of the p53-binding cleft of human mdm2 as shown by multidimensional NMR spectroscopy. According to a model based on the NMR data the p-chloro phenyl moiety of the chalcone binds deeply in the Trp23 pocket. Later boronic acid chalcones were reported with improved activity against cancer cell, also exhibiting selectivity against cancer cells [13]. However, titration of the boronic acid chalcones to 15N-labeled mdm2 revealed that these compounds are not able to dissociate the p53/mdm2 complex, even at very high concentration of 2 mM [14]. Novel chalcones and bis-chalcones based on the above studies have been recently described exhibiting selectivity in apoptosis induction with Vogelstein’s HCT-116 (p53+/+ and p53 / ) cell lines [15].
The benzodiazepine scaffold (BDA) was discovered to potently antagonize the p53/mdm2 interaction by a high throughput ThermoFluor screening technology [20]. Similar to nultin-3, a high-resolution X-ray structure of derivative 3 exists, which gives valuable structural insights into the binding interactions of the BDA to the p53-binding site in mdm2 (Figure 4). Although the BDA and imidazoline scaffolds chemically are very different, their substituents not surprisingly show a very strong overlap in the corresponding X-ray structures. The two chlorophenyl substituents mimic the Leu26 and Trp23 and the iodophenyl substituent sits in the Phe19-binding pocket. The benzodiazepine scaffold was convergently synthesized by a two-step one-pot multicomponent reaction (Ugi reaction). Therefore, many derivatives are easily accessible, and an in-depth structure activity relationship has been published, which is very valuable in understanding the nature of the hydrophobic binding groove in mdm2 [21]. For example introduction of a pentenylicacid substituent at the N1 of the benzodiazepine ring improved potency, solubility and cell-based activities and enabled access to modified ADMET properties. Several biological studies have been reported on the BDA mdm2 scaffold including xenograft studies indicating potent anticancer activity in synergy with doxorubicin [22]. Interestingly the BDA scaffold was until this point typically thought of as a b-turn mimetic. These studies imply that the privileged BDA backbone can also be developed as a potent a-helix mimic [23]. The same group later described the 1,4-diazepine-2,5-dione scaffold [24]. Crystallographic analysis of ligands bound to mdm2 suggested that 7-substituted 1,4-diazepine-2,5-diones could mimic the a-helix of p53 peptide and may represent a promising scaffold to develop mdm2/p53 antagonists of potentially reduced molecular weight.
By far the best characterized p53/mdm2 antagonist to date is without doubt nutlin-3, 2, an imidazoline derivative [16], discovered by high throughput screening. The high binding enantiomer displaces p53 from the mdm2 protein with an IC50 of 90 nM determined by surface plasmon resonance experiments. The binding mode of nutlin-3 has been elucidated by high-resolution X-ray structure analysis (Figure 4). Analysis reveals that the chlorophenyl moieties in positions 4 and 5 bind deeply into the Leu26 and Trp23 pockets, respectively. The Phe19 of p53 is mimicked by the isopropyloxy phenyl moiety of nutlin-3. In addition, the 4-methoxy functionality mimics the p53 Leu22 to a certain degree. The cell and xenograft biology of nutlin-3 is impressively described in more than 100 publications [17]. Nutlin-3 is made available to the scientific community as a racemic mixture serving as a valuable tool for cancer biology. It is prepared in this form via a sequential 8-step route, and
Figure 4
Comparison of the binding sites of p53 (PDB-ID: 1YCR), nutlin3 (PDB-ID: 1RV1) and a benzodiazepine (PDB-ID: 1T4E) into the mdm2-binding site (all poses show the same approximate orientation). Left, the p53 mdm2 ‘hot spot’. Only the hydrophobic amino acid side chains LWY of the p53 amphiphatic a-helix are shown as yellow sticks. Middle, left: the binding of the J&J benzodiazepine into the mdm2-binding groove; right: the binding of a Roche nultin derivative into the mdm2-binding groove. www.sciencedirect.com
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Thus, compound 17 was reported to have an mdm2 affinity of 3 mM. In addition, the introduction of a pentyl carboxylic chain rendered the compound water soluble and cell membrane permeable (CACO-2). No cellular anticancer cellular activity was reported. The norbornane scaffold has been described to antagonize p53/mdm2 [25]. Compound 4 was shown in cell-based studies to induce apoptosis in cancer cells. No affinities to mdm2 or any molecular mode of action is reported. Compound 5 has been reported to inhibit the p53/mdm2 interaction on the basis of virtual high throughput docking exercises of the NCI database [26]. In a p53 peptide displacement assay it was shown to have a Ki of 31.8 mM. Subsequently, however, NMR-based studies unequivocally showed that 5 precipitated the mdm2 protein at relevant concentrations in the NMR tube (promiscuous inhibitor?) [14]. The isoindolone scaffold was reported to yield p53/mdm2 antagonists [27]. Compound 6, the most potent antagonist, showed a Ki of 5 mM in an ELISA assay. Initial compounds of this series were tested in the NCI-60 cell line panel, and the programme COMPARE revealed a new class of anticancer agents so far not present in the NIH collection [28]. Compound 6 induced p53-dependent gene transcription in a dose-dependent manner, in the SJSA human sarcoma cell line. Docking studies were also reported and according to these the following 3D picture was drawn: The p-chlorophenyl substituent is mimicking Trp23 and pointing deeply into the central binding pocket; the propyl residue is occupying the Phe19-binding site and the 3,5-dimethoxy-4-hydroxybenzyl moiety is sitting over the Leu26 pocket, with one methoxy group sneaking in the hydrophobic pocket. Interestingly, a new H bond is postulated between the hydroxyl group of the benzyl substituent and Tyr100 of mdm2. Compound 7 is based on a spiroindolone scaffold and is currently the most potent p53/mdm2 antagonist reported with a fluorescence polarization (FP) derived Ki of 3 nM [29]. In addition it showed activation of p53 function and inhibition of cell growth in cancer cells with wild-type p53 status. Moreover, the compound shows excellent specificity over cancer cells with deleted p53 and shows a minimal toxicity to normal cells. According to modeling studies the oxindole moiety occupies the p53 Trp23binding site and also forms a hydrogen bond to Leu54 backbone carbonyl of mdm2. The tert-butyl side chain seemingly mimics the Leu26 of p53 and the m-chlorophenyl substituent mimics the p53 Phe19 side chain. In addition this molecule features a morpholino-2-ethylamide moiety mimicking the p53 Leu22 that is not deeply buried in the published p53/mdm2 X-ray structure, nevertheless undergoing extensive hydrophobic contacts. Current Opinion in Chemical Biology 2008, 12:281–291
In addition the morpholino-2-ethyl amide side chain presumably contributes to the water solubility of the compound. Although, the affinity of compound 7 is excellent, it seems to have reduced cell permeability. A similar compound 21 with a non-basic, however, solubilizing side chain has been recently reported including very promising cell based and xenograft data [30]. Compound 21 disrupts the mdm2–p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumour cells. MI-219 stimulates rapid but transient p53 activation in established tumour xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis and complete tumour growth inhibition. This promising compound is currently undergoing preclinical development. Compound 8 is an interesting aniline derivative with an 8hydroxyquinoline moiety and was discovered by a virtual database screening exercise based on a simple p53 peptide derived pharmacophore [31]. This quinolinol is reported to bind to mdm2 with a Ki of 120 nM (FP-based binding assay) and activates p53 in cancer cells with a mechanism of action consistent with targeting the mdm2– p53 interaction using Vogelstein’s HCT-116 (p53+/+ and p53 / ) cell lines. The authors postulate that 8 mimics the three p53 residues crucial in the binding to mdm2. Molecule 9 was discovered by screening of a designed library of 173 compounds by an FP-based binding assay [32]. The computational structure-based design was guided by Hamilton’s concept of small molecular weight a-helix mimetic. The most potent compounds have been further characterized by NMR shift perturbation assays. These assays provide a good, although not high-resolution indication of the binding site of the molecule on the surface of mdm2. According to these experiments with 9 and 14N-labeled mdm2, the most significantly perturbed mdm2 residues are Glu25, Phe55, His73, and Val93. This is in good agreement with a model where 9 bind to the p53binding pocket in mdm2. The general synthesis of the scaffold comprises a 10-step process. The natural fermentation product hexylitaconic acid 10 has been reported to inhibit p53/mdm2 by an ELISA assay [33]. The structure comprises a reactive Michael acceptor—an a,b-unsaturated carboxylic acid. The apparent IC50 is rather high at 230 mM. The absolute stereochemistry of the active compound is unknown. No cellbased activities were reported. Compounds 11–15 were discovered by a computational high throughput docking approach of a 35 000 compound library using an ensemble-based receptor model [34]. These compounds were identified using a multiple protein structure (MPS) method that incorporates protein flexibility into a receptor-based pharmacophore model. www.sciencedirect.com
Small molecular weight protein[19_TD$IF]–protein interaction antagonists[20_TD$IF]—an insurmountable challenge? Do¨mling 287
Impressive potent compounds were found that include compound 11, Ki of 110 nM in a FP assay. This potent binding is explained by an improved shape complementarity of the compound and the receptor, a more complete occupancy of the Phe19 pocket and the formation of two hydrogen bonds, between the catechol moiety and the Gln72 backbone carbonyl and the phenol moiety and His96 backbone carbonyl, respectively. For compound 14, the authors propose an interesting binding mode based on docking, in which the (2-ethylthio) ethylester portion of the molecule in a helical way sneaks down the bottom of the Trp23 pocket. On the contrary the Leu26 pocket seems to be unoccupied by 14. Surprisingly, the affinity for 14 is considerable with 21 mM. Another compound found in this virtual screening exercise is sulfonamide 15 (Ki = 37.2 mM). The binding of compound 16 to mdm2 has been investigated by NMR spectroscopy [6]. 16 was found to have an affinity (Ki) for mdm2 of 4 mM. In addition it has been shown that 16 can dissociate preformed p53/mdm2 com-
plex with a Kd of 3 mM. The Kd of nutlin-3 in the same experiment was 0.7 mM. No relative or absolute stereochemistry of 16 is given, nor is there any cell-based activity reported. Compound 18, a 2,5-dithiophenofuran with the trivial name RITA, was reported to bind to p53 and thus antagonize the p53/mdm2 interaction on the basis of ELISA and Fluorescence Correlation Spectroscopy [35], Multiple biological activities were reported in accordance with the proposed biochemical mode of action. However, scrutinizing the in vitro binding of compound 18 to p53 and mdm2 did not show any affinity nor was the compound able to dissociate the preformed complex [36], Therefore the observed biological activities seem to be derived from another unknown biochemical mechanism. Compound 19 (Ki = 20 mM, ELISA), interestingly was rationally designed by a general a-helix mimetic approach [37]. Herein, Hamilton and co-workers use
Figure 5
The three finger, thumb–index–middle finger pharmacophore model of p53/mdm2 antagonists. Above: Stereoview of six different p53/mdm2 antagonists aligned to fit the three-finger model (clockwise, 11a.m.: oxindol, benzodiazepinone, spirooxindol, imidazoline, bicycle and terphenyl). www.sciencedirect.com
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the terphenyl scaffold to place substituents in the right position in space that mimic the hydrophobic residues i, i + 4 and i + 7 of an amphiphatic a-helix. This concept seems to be general and several successful examples of antagonized a-helix interactions have been reported, including gp41 helix bundle interactions (HIV viral fusion) and Bcl2 family interactions (cancer apoptosis). A potential issue of this class of compounds is their very high lipophilicity and their lengthy synthetic access. Additionally, 19 only activate transcription in cells at a concentration of 30 mM. Finally, compound 20 (JNJ-26854165) an unusual tryptamine-derived (4-pyridino)-1,4-diaminophenyl was recently reported to be advanced into phase I human clinical trials [38]. No affinity data of 20 to mdm2 were reported. The compound seems to induce conformational changes in mdm2 as indicated by the abrogation of tryptic digestion of mdm2 in its presence. The compound induces p53 and activates p53 transcriptional activity in different cell lines. Mdm2 driven p53 degradation is suppressed. In glioblastoma and non-small-cell-lung-cancer xenografts 20 showed a broad spectrum tumour activity equipotent to taxol and outperforming Tarceva. From the above discussion of recently described p53/ mdm2 antagonists it becomes clear that only the full arsenal of biophysical, biochemical and cell biological methods will give a comprehensive and meaningful picture of the value of a compound [20]. Moreover, by
applying several complementary screening and characterization methods reported inaccurate inhibitor profiles may be minimized [14].
A compound-based pharmacophore model for p53/mdm2 antagonists On the basis of the published high-resolution information and the overlap of other described p53/mdm2 inhibitors a simple pharmacophore model is presented here. Owing to the 3D nature of the pharmacophore it is here called ‘The three finger model pharmacophore model’ (Figure 5). In this crude model several additional pharmacophoric features are not represented, for example, the potential to gain additional H bonding to Gln72. This protein–protein interaction is largely governed by highly hydrophobic interactions, and owing to the very deep and structured mdm2-binding site, a high degree of shape complementarity is demanded by the protein for an efficient antagonist. Even small chemical additions to a backbone, for example, addition of a methyl group or a halogen at a wrong position can largely destroy the activity of antagonists. Selectivity
One of the claims often made is that an antagonist of a PPI owing to its unique interaction motif should be intrinsically highly selective, as opposed to, for example, a kinase inhibitor with a more generic binding site. Besides the question of the therapeutic value of a drug with high selectivity in a multi factorial disease such as
Figure 6
Selectivity of a drug is driven by its structural biology. The p53-binding site of mdm2 and probably also mdm4 can be occupied by several other transcription factors as well. Therefore, a potential p53/mdm2 antagonist will probably also interrupt other protein interactions involving mdm2 and mdm4. The biological effects of these interactions have to be delineated in the future to yield potent novel agents to fight cancer. Current Opinion in Chemical Biology 2008, 12:281–291
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cancer has to be scrutinized. Thus, continuing with the theme of this article, mdm2 is a negative regulator of the transcription factor p53. The p53 tumour suppressor is vital in cell cycle regulation, DNA repair and apoptosis. Once DNA damage is beyond repair capabilities p53 induces apoptosis. Since mdm2 is overexpressed in many cancers and/or there are multiple gene copies in cancer cells with wild-type p53, antagonizing this PPI seemed a logical approach to restore p53 activity. In fact, three independent groups simultaneously published convincing data suggesting that p53 restoration should be pursued as a drug target approach: Ventura et al. found that restoration of p53 function leads to tumour regression in vivo [39]. Xue et al. demonstrated that p53 restoration induced senescence and tumour clearance in a murine liver carcinoma model [40]. Martins et al. constructed a reversible, switchable p53 knockin (KI) mouse model and observed after ‘switching’ on p53 expression, rapid apoptosis, and significantly increased survival was observed in an aggressive lymphoma model [41]. The picture, however, is much more complicated. There exist homologous proteins to p53, p63 and p73 that have very similar mdm2-binding motifs. Moreover a second remote p53 mdm2-binding motif has been recently described [42]. A genome-wide sequence query for mdm2-binding motifs also revealed a plethora of potential interaction partners (Figure 6) [39]. Several other transcription factors and proteins have been discovered to bind to mdm2 via the p53-binding site. These include the well-known E2F1 and HIF1-a [43,44]. These findings are very exciting, since E2F1 and HIF1-a play an important role in cancer development and progression. E2F1 transcription factor is best known for its ability to regulate the expression of genes required for DNA replication and cell cycle progression. Thus, the potent p53/mdm2 antagonist nutlin-3 induced apoptosis even in p53-mutant malignant peripheral nerve sheath and p53-null HCT116 cell lines. In fact, a clear dosedependent transcriptional activation of free E2F1 was observed [44]. Antagonizing HIF1-a/mdm2 with Nutlin3 showed a dose-dependent reduction of the HIF1-a transcriptional target VEGF under conditions of normoxia or hypoxia [43]. Thus, in the case of E2F1 and HIF1-a there seems to be a potential therapeutic synergy to p53. By sequence alignment to the hydrophobic p53-binding motif, other proteins have been predicted as binding partners of mdm2 in the p53-binding site, including VP16, TREB5, C-Jun, NFkB p65 and ALL1 [42]. However, for many of the other interactions involving the p53-binding site of mdm2 and mdm4 the biological role is unknown. Thus, the interactions and regulation of p53 and other proteins by mdm2-type binding sites is a highly complex matrix that is not understood in detail (Figure 6). It can be assumed that many such interactions are biologically relevant and can occur in a time and space-dependent manner, whereas others are biologically impossible. www.sciencedirect.com
Conclusion The recent propagation of several compounds into human clinical trial including Genentech’s XIAP inhibitor, Abbott’s Bcl2 antagonists and J&J-26854165 p53/mdm2 antagonist is a major advancement for the development of PPI antagonists. However, despite these success stories, antagonizing PPIs by small molecules is still a major challenge. There is no established structure based and general approach for the discovery of PPI antagonists. Importantly, PPIs structural appearance is highly diverse and they generally do not show obvious signatures, for example, an ATP-binding site as in kinases or an active site triad as in serine proteases. Clearly, there are PPIs that are more accessible to small molecule medicinal chemistry and other more difficult PPIs still await the discovery of their antagonists. PPIs, however, comprise a growingly important class of targets of high relevance for the development of new drugs in therapeutic areas of unmet medical needs. Moreover, from an intellectual standpoint of view targeting PPIs is an outstanding challenge. Efforts to target PPIs with small molecules are worthy undertakings and will most probably yield high return.
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10. Xiong S, Van Pelt CS, Elizondo-Fraire AC, Liu G, Lozano G: Synergistic roles of mdm2 and mdm4 for p53 inhibition in central nervous system development. PNAS 2006, 103:32263231. 11. Murray JK, Gellman SH: Targeting protein-protein interactions: lessons from p53/mdm2. Pept. Sci. 2007, 88:657. 12. Stoll R, Renner C, Hansen S, Palme S, Klein C, Belling A, Zeslawski W, Kamionka M, Rehm T, Muhlhahn P et al.: Chalcone derivatives antagonize interactions between the human oncoprotein mdm2 and p53. Biochemistry 2001, 40:336-344. 13. Kumar SK, Hager E, Petit C, Gurulingappa H, Davidson NE, Khan SR: Design, Synthesis, and Evaluation of Novel BoronicChalcone Derivatives as Antitumor Agents. J Med Chem 2003, 46:2813. 14. D’Silva L, Ozdowy P, Krajewski M, Rothweiler U, Singh M, Holak TA: Monitoring the effects of antagonists on protein– protein interactions with NMR spectroscopy. J Am Chem Soc 2005, 127:13220-13226. Description of a medium throughput, however, high content screening method for antagonist of p53 and mdm2. The experiments described herein can be used to obtain multiple information of putative protein interaction antagonists. Questions that can be answered are What is the affinity of the molecule for the protein? Which protein does the molecule bind to? Is the molecule able to disrupt the protein interaction? Are the disrupted proteins still structurally integer, or are they denaturated or did major structural rearrangements in a protein happen? Is the molecule a promiscuous inhibitor? Is the compound precipitating a protein? 15. Modzelewska A, Pettit C, Achanta G, Davidson NE, Huang P, Khan SR: Anticancer activities of novel chalcone and bischalcone derivatives. Bioorg Med Chem 2006, 14:3491.
2,5-diones as a-helix mimetic antagonists of the hdm2–p53 protein–protein interaction. Chem Biol Drug Des 2006, 67:201. 24. Raboisson P et al.: Structure-based design, synthesis, and biological evaluation of novel 1,4-diazepines as hdm2 antagonists. Bioorg Med Chem Lett 2005, 15:1857. 25. Zhao J, Wang M, Chen J, Luo A, Wang X, Wu M, Yin D, Liu Z: The initial evaluation of non-peptidic small-molecule hdm2 inhibitors based on p53–hdm2 complex structure. Cancer Lett 2002, 183:69. 26. Galatin PS, Abraham DJ: A nonpeptidic sulfonamide inhibits the p53–mdm2 interaction and activates p53-dependent transcription in mdm2-overexpressing cells. J Med Chem 2004, 47:4163-4165. 27. Hardcastle IR et al.: Isoinolinone-based inihibitors of the mdm2–p53 protein–protein interaction. Bioorg Med Chem Lett 2005, 15:1515. 28. Paul KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L, Plowman J: Boyd MR: Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. J.N.C.I. 1989, 81:1088-1092. 29. Ding K, Lu Y, Nikolovska-Coleska Z, Wang G, Qui S, Shangary S, Gao W, Qin D, Stuckey J, Krajewski K, Roller PP, Wang S: Structure-based design of spiro-oxindoles as potent, specific small-molecule inhibitors of the mdm2–p53 interaction. J Med Chem 2006, 49:3432-3435. Design and synthesis of the currently most potent p53 mdm2 antagonist. Binding mode according to docking studies as well as cellular activities is discussed. 30. Shangary S et al.: Temporal activation of p53 by a specific mdm2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. PNAS 2008, 105:3933. 31. Lu Y, Nikolovska-Coleska Z, Fang X, Gao W, Shangary S, Qiu S, Qin D, Wang S: Discovery of a nanomolar inhibitor of the human murine double minute 2 (mdm2)–p53 interaction through an integrated. Virtual database screening strategy. J Med Chem 2006, 49:3759.
16. Vassilev LT et al.: In vivo activation of the p53 pathway by small molecule antagonists of mdm2. Science 2004, 303:844. First high-resolution X-ray structure analysis of a small molecule binding into mdm2. Cellular and xenograft studies are discussed therein.
32. Lu F, Chi S-W, Kim D-H, Han K-H, Kuntz ID, Guy RK: Proteomimetic libraries: design, synthesis, and evaluation of p53–mdm2 interaction inhibitors. J Chem Chem 2006, 8: 315.
17. Vassilev LT: Mdm2 inhibitors for cancer therapy. TRENDS Mol Med 2006, 13:23.
33. Tsukamoto S, Yoshida T, Hosono H, Ohta T, Yokosawa H: Hexylitaconic acid: a new inhibitor of p53–hdm2 interaction isolated from a marine-derived fungus, Arthrinium sp.. Bioorg Med Chem Lett 2006, 16:69.
18. Fry DC, Emerson SD, Palme S, Vu BT, Liu C-M, Podalski F: NMR structure of a complex between mdm2 and a small molecule inhibitor. J Biomol NMR 2004, 30:163. 19. Wang Z, Jonca M, Lamnros T, Ferguson S, Goodnw R: Exploration of liquid and supercritical fluid chromatographic chiral separation and purification of Nutlin-3—a small molecule antagonist of mdm2. J Pharm Biomed Anal 2007, 45:720. 20. Grassberger BL et al.: Discovery and cocrystal structure of benzodiazepinedione hdm2 antagonists that activate p53 in cells. J Med Chem 2005, 48:909. One of the two published high-resolution X-ray structures of a small molecule binding mdm2, providing a lot of structural insight into the binding interactions and the signature of the binding site. Also the synthetic approach, which uses multicomponent reaction chemistry (MCR) is noteworthy. This allowed for the rapid and efficient optimization of the compound series by preparing 20 000 analogues. 21. Parks DJ et al.: Enhanced pharmacokinetic properties of 1,4benzodiazepine-2,5-dione antagonists of the hdm2–p53 protein–protein interaction through structure-based drug design. Bioorg Med Chem Lett 2006, 16:3310-3314. 22. Koblish HK et al.: Benzodiazepinedione inhibitors of the hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo. Mol Cancer Ther 2006, 5:160. 23. Cummings MD, Schubert C, Parks DJ, Calvo RR, LaFrance LV, Lattanze J, Miliewicz KL, Lu T: Substituted 1,4-benzodiazepineCurrent Opinion in Chemical Biology 2008, 12:281–291
34. Bowman AL, Nikolovska-Coleska Z, Zhong H, eWang S, Carlson HA: Small molecule inhibitors of the mdm2–p53 interaction discovered by ensemble-based receptor models. J Am Chem Soc 2007, 129:12809. 35. Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, Masucci M, Pramanik A, Selivanova G: Small molecule RITA binds to p53, blocks p53–mdm2 interaction and activates p53 function in tumors. Nat Med 2004, 10:1321. 36. Krajeski M, Ozdowy P, D’Silva L, Rothweiler U, Holak TA: NMR indicates that the small molecule RITA does not block p53– mdm2 binding in vitro. Nat Med 2005, 11:1135. 37. Yin H, Lee G, Park HS, Payne GA, Rodriguez JA, Sebti SM, Hamilton AD: Terphenyl-based helical mimetics that disrupt the p53/hdm2 interaction. Angew Chem Intl Ed Engl 2005, 117:2764. 38. Arts J et al.: Preclinical anti-tumor studies with JNJ-26854165— a novel class of hdm2 antagonists in clinical development [abstract]. AACR-NCIEORTC International Conference on Molecular Targets and Cancer Therapeutics, October 22–26; San Francisco, CA, Philadelphia (PA): AACR: 2007. . Abstract No A264. 39. Ventura A, Kirsch DG, McLaughlin ME, Tuveson DA, Grimm J, Lintault L, Newman J, Reczek EE, Weissleder R, Jacks T: Restoration of p53 function leads to tumour regression in vivo. Nature 2007, 445:661-665. www.sciencedirect.com
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40. Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V, Cordon-Cardo C, Lowe SW: Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 2007, 445:656-660. 41. Martins CP, Brown-Swigart L, Evan GI: Modeling the therapeutic efficacy of p53 restoration in tumors. Cell 2006, 127:1323-1334. 42. Chi S-W, Lee SH, Kim DH, Ahn MJ, Kim JS, Woo JY, Torizawa T, Kainosho M, Han KH: Structural details on mdm2–p53 interaction. J Biol Chem 2005, 280:38795.
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43. Ambrosini G, Sambol EB, Carvajal D, Vassilev LT, Singer S, Schwartz GK: Mouse double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1. Oncogene 2007, 26:3473. 44. LaRusch GA, Jackson MW, Dunbar JD, Warren RS, Donner DB, Mayo LD: Nutlin3 blocks vascular endothelial growth factor induction by preventing the interaction between hypoxia inducible factor 1A and hdm2. Canc Res 2007, 67:450.
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Small molecular weight protein–protein interaction antagonists—an insurmountable challenge? Alexander Do¨mling Several years ago small molecular weight protein–protein interaction (PPI) antagonists were considered as the Mount Everest in drug discovery and generally regarded as too difficult to be targeted. However, recent industrial and academic research has produced a great number of new antagonists of diverse PPIs. This review structurally analyses small molecular weight PPI antagonists and their particular targets as well as tools to discover such compounds. Besides general discussions there will be a focus on the PPI p53/mdm2. Address Departments of Pharmaceutical Sciences and Chemistry, University of Pittsburgh, United States Corresponding author: Do¨mling, Alexander ([email protected])
However, more and more scientists recognize the eigenvalue of protein interaction antagonization. The growing importance of PPIs as oncology targets was underlined by many talks and posters at the recent AACR-EORTC meeting in San Francisco. Covered examples included p53/mdm2/mdm4, Bcl family interactions, IAPs, c-Myc, SPRY2-Cb, ERCC1/XPF, FAK–protein interactions, orphan nuclear receptor COUP-TFI, Smad4-SKI, cSrc-SH3, Smad2/3/4, Rb/Raf-1, SDF-1/CXCR4, tissue factor/FVIIa, HOX/PBX and tubulin. In the following the small molecule antagonists of the p53/mdm2 interaction are discussed, as an example of a successfully targeted PPI.
The p53/mdm2 case Structure
Current Opinion in Chemical Biology 2008, 12:281–291 This review comes from a themed issue on Molecular diversity Edited by Gerry Maggiora and Christopher Hulme Available online 21st May 2008 1367-5931/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2008.04.603
PPI signatures Systematic structural analyses of PPI interfaces yield a highly diverse picture from large and flat to narrow and structured interactions [1,2]. However, the majority of PPIs involve large binding sites where affinity is obtained by a multitude of often weak interactions. As a consequence these widely spaced interactions are difficult to mimic with small molecules, and thus targeting PPIs is a challenging area of drug discovery. Classical concepts established in drug discovery, for example, transition state mimics cannot be applied here. On the contrary, allosteric small molecular weight PPI antagonists and agonists are very common as modulators of the dynamic (de)polymerization of the tubulin cytoskeleton, and they are generally used to treat cancers in a clinical setting. It is therefore difficult to make predictions on the druggability of specific protein–protein interaction surfaces. Figure 1 shows different potentially druggable PPIs with high-resolution X-ray structure information. In all these cases the PPI exists as a rather confined, narrow and highly structured ‘hot spot’. www.sciencedirect.com
P53 is a transcription factor that regulates the cell cycle in response to cellular stress leading to DNA damage, for example, oxygen, radiation and xenobiotics. P53 holds the cell cycle and activates DNA repair proteins; if DNA damage proves to be beyond repair, p53 initiates apoptosis. In normal, noncancerous cells p53 is usually inactive, bound to the protein mdm2. This complex prevents p53’s action and promotes its degradation (by ubiquitination and proteasomal degradation). In cancer, tumour suppression by p53 is severely reduced. Thus, antagonizing the p53/mdm2 complex is a promising new strategy in cancer therapy [3]. In 1998 Pavletich described the crystal structure of the interaction of p53 and mdm2 (Figure 1) [4]. The negative regulator of p53 mdm2 has a very defined and deep binding pocket for p53. It became clear that the p53–mdm2 interface is an atypical protein– protein interaction. It is small and highly structured, that is, the calculated buried accessible surface area of mdm2 in the interface is only 660 A˚2. The diameter of the mdm2-binding groove with 18 A˚ (from Phe100CH2 to Phe67CH2) is comparable to a small molecule. It comprises a ‘hot spot’ where most of the binding affinity is concentrated [5]. The apparent dissociation constant of natural p53 peptides ranges from 60 to 700 nM depending on the peptide length. Mdm4 is also a negative regulator of p53 in a cellular context. Mdm4, a homologous protein of mdm2 has been described to bind the same p53 mini helix motif [6]. Mdm4 was described for the first time in 1996 as a novel p53-binding protein that shared in common some functional properties with mdm2 a high sequence homology to mdm2 [7]. Upon binding to p53 via the C-terminal ring domain, mdm4 stimulates p53 degradation through mdm2 ubiquitination. The mechanism of the mdm2/mdm4 regulation of p53, however, is not clearly understood; in particular, the contribution of Current Opinion in Chemical Biology 2008, 12:281–291
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Figure 2
P53/mdm2/mdm4 interactions. Residues shown, M50, P92 and Y96 for mdm4, and L54, H96 and Y100 for mdm2, are important for binding to p53. The structure reveals that although the principal features of the mdm2–p53 interaction are preserved in the mdm4–p53 complex, the mdm4 hydrophobic cleft on which the p53 peptide binds is significantly altered: a part of the cleft is blocked by side chains of M54 and Y100 of the p53-binding pocket of mdm4. (Right) Close up of the comparison of the high-resolution X-ray structure of p53 model peptides bound to mdm4 (above, PDB ID: 2Z5S) and mdm2 (below, PDB ID: 1YCR). Both complexes are oriented the same way. The green surfaces are the respective mdm2 and mdm4 proteins. Highlighted in blue are the crucial rim amino acids. The blue sticks represent the side chain of the p53 binding hot spot. The mdm4-binding cleft is significantly altered as compared with the mdm2 cleft: The western part of the binding cleft is blocked by the amino acids M54 and Phe100. Moreover the mdm4 pocket is flatter than mdm2.
mdm2 versus mdm4 to the regulation of p53 stability and activity is unclear. Several recent research lines of study underscore the idea that both mdm2 and mdm4 must be inhibited for effective restoration of p53 activity (when p53 exists as wild type in cancer) [8]. Mdm4 and mdm2 have been reported to cooperatively inhibit p53 activity in proliferating and quiescent cells in vivo [9]. The importance of independent p53 regulation of mdm4 over mdm2 was also demonstrated in vivo [10]. Owing to elevated high levels of mdm4 in several tumour types including head and neck squamous carcinoma, pre-B acute lymphoblastic leukaemia, childhood cancer retinoblastoma,
brain and breast, mdm4 is a promising target in its own right besides mdm2 [7]. Recently the three-dimensional structure of a p53 peptide binding into a humanized Xenopus mdm4 protein has been published with a 2.1 A˚ resolution by the Holak group and validated as an excellent human mdm4 model (Figure 2) [5]. As opposed to the plethora of reported small molecule antagonists of mdm2, currently none have been described for mdm4. Nutlin3 that is a potent mdm2 antagonist binds only very weakly to mdm4 and is incapable to disrupt p53/mdm4 in vivo at relevant concen-
(Figure 1 Legend ) Examples of different high-resolution structures of ‘hot spots’ PPIs. (a) Interaction of SMAD with SKI (yellow sticks) with implications in cancer apoptosis highlights the crucial string T271C272H273W274G275 (PDB-ID 1MR1); (b) Bir2-caspase 7 interaction with implications in cancer apoptosis; grey surface caspase-7, yellow sticks and cartoon XIAP Bir2 domain; highlighted in green the very hydrophobic V146 cage made up from the three aromatic amino acids Y230W232F282 (PDB-ID: 1I4O); (c) homodimer DPPIV with implications in diabetes shown as golden surface DPPIV monomer A, highlighting S720 forming a hydrogen bond with the indol of W734; shown as blue sticks hot spot triad F731W734Y735; W734 is deeply buried (71%) in the corresponding monomer with 160 A˚2 (PDB–ID: 1N1M); (d) interaction of XPF with ERCC1 with potential implications in cis platin resistance; the two XPF F840 and F888 (black sticks) form strong hydrophobic contacts with and are deeply buried in ERCC1 (PBD-ID: 2A1J). The pictures were generated using PyMol (www.pymol.com). www.sciencedirect.com
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trations. By comparison of the high-resolution X-ray structures of mdm4 and mdm2-binding p53 peptides, the selectivity of nutlin3 to mdm2 can be smoothly explained. Antagonistic compounds
Interestingly, no other protein–protein interaction attracted as much interest in the medicinal chemistry
community as p53/mdm2. More than 20 different chemotypes have been described to date that antagonize p53/ mdm2 (Figure 3). Here we concentrate on the discussion on small molecules with preferred properties that enhance oral bioavailability and generally have higher metabolic stability than peptides or proteins. Therefore, peptides, peptoids and high molecular weight natural products (chlorofusin, Mw = 1363 Da) are out of the scope
Figure 3
Currently described p53/mdm2 small molecular weight antagonists. Current Opinion in Chemical Biology 2008, 12:281–291
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of this review. The reader is referred to reference [11] for further information.
recently separation of the enantiomers by supercritical chiral HPLC has been described [18,19].
Amongst the first inhibitors of p53/mdm2 described were chalcone derivatives, 1 [12]. The molecules bind to a subsite of the p53-binding cleft of human mdm2 as shown by multidimensional NMR spectroscopy. According to a model based on the NMR data the p-chloro phenyl moiety of the chalcone binds deeply in the Trp23 pocket. Later boronic acid chalcones were reported with improved activity against cancer cell, also exhibiting selectivity against cancer cells [13]. However, titration of the boronic acid chalcones to 15N-labeled mdm2 revealed that these compounds are not able to dissociate the p53/mdm2 complex, even at very high concentration of 2 mM [14]. Novel chalcones and bis-chalcones based on the above studies have been recently described exhibiting selectivity in apoptosis induction with Vogelstein’s HCT-116 (p53+/+ and p53 / ) cell lines [15].
The benzodiazepine scaffold (BDA) was discovered to potently antagonize the p53/mdm2 interaction by a high throughput ThermoFluor screening technology [20]. Similar to nultin-3, a high-resolution X-ray structure of derivative 3 exists, which gives valuable structural insights into the binding interactions of the BDA to the p53-binding site in mdm2 (Figure 4). Although the BDA and imidazoline scaffolds chemically are very different, their substituents not surprisingly show a very strong overlap in the corresponding X-ray structures. The two chlorophenyl substituents mimic the Leu26 and Trp23 and the iodophenyl substituent sits in the Phe19-binding pocket. The benzodiazepine scaffold was convergently synthesized by a two-step one-pot multicomponent reaction (Ugi reaction). Therefore, many derivatives are easily accessible, and an in-depth structure activity relationship has been published, which is very valuable in understanding the nature of the hydrophobic binding groove in mdm2 [21]. For example introduction of a pentenylicacid substituent at the N1 of the benzodiazepine ring improved potency, solubility and cell-based activities and enabled access to modified ADMET properties. Several biological studies have been reported on the BDA mdm2 scaffold including xenograft studies indicating potent anticancer activity in synergy with doxorubicin [22]. Interestingly the BDA scaffold was until this point typically thought of as a b-turn mimetic. These studies imply that the privileged BDA backbone can also be developed as a potent a-helix mimic [23]. The same group later described the 1,4-diazepine-2,5-dione scaffold [24]. Crystallographic analysis of ligands bound to mdm2 suggested that 7-substituted 1,4-diazepine-2,5-diones could mimic the a-helix of p53 peptide and may represent a promising scaffold to develop mdm2/p53 antagonists of potentially reduced molecular weight.
By far the best characterized p53/mdm2 antagonist to date is without doubt nutlin-3, 2, an imidazoline derivative [16], discovered by high throughput screening. The high binding enantiomer displaces p53 from the mdm2 protein with an IC50 of 90 nM determined by surface plasmon resonance experiments. The binding mode of nutlin-3 has been elucidated by high-resolution X-ray structure analysis (Figure 4). Analysis reveals that the chlorophenyl moieties in positions 4 and 5 bind deeply into the Leu26 and Trp23 pockets, respectively. The Phe19 of p53 is mimicked by the isopropyloxy phenyl moiety of nutlin-3. In addition, the 4-methoxy functionality mimics the p53 Leu22 to a certain degree. The cell and xenograft biology of nutlin-3 is impressively described in more than 100 publications [17]. Nutlin-3 is made available to the scientific community as a racemic mixture serving as a valuable tool for cancer biology. It is prepared in this form via a sequential 8-step route, and
Figure 4
Comparison of the binding sites of p53 (PDB-ID: 1YCR), nutlin3 (PDB-ID: 1RV1) and a benzodiazepine (PDB-ID: 1T4E) into the mdm2-binding site (all poses show the same approximate orientation). Left, the p53 mdm2 ‘hot spot’. Only the hydrophobic amino acid side chains LWY of the p53 amphiphatic a-helix are shown as yellow sticks. Middle, left: the binding of the J&J benzodiazepine into the mdm2-binding groove; right: the binding of a Roche nultin derivative into the mdm2-binding groove. www.sciencedirect.com
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Thus, compound 17 was reported to have an mdm2 affinity of 3 mM. In addition, the introduction of a pentyl carboxylic chain rendered the compound water soluble and cell membrane permeable (CACO-2). No cellular anticancer cellular activity was reported. The norbornane scaffold has been described to antagonize p53/mdm2 [25]. Compound 4 was shown in cell-based studies to induce apoptosis in cancer cells. No affinities to mdm2 or any molecular mode of action is reported. Compound 5 has been reported to inhibit the p53/mdm2 interaction on the basis of virtual high throughput docking exercises of the NCI database [26]. In a p53 peptide displacement assay it was shown to have a Ki of 31.8 mM. Subsequently, however, NMR-based studies unequivocally showed that 5 precipitated the mdm2 protein at relevant concentrations in the NMR tube (promiscuous inhibitor?) [14]. The isoindolone scaffold was reported to yield p53/mdm2 antagonists [27]. Compound 6, the most potent antagonist, showed a Ki of 5 mM in an ELISA assay. Initial compounds of this series were tested in the NCI-60 cell line panel, and the programme COMPARE revealed a new class of anticancer agents so far not present in the NIH collection [28]. Compound 6 induced p53-dependent gene transcription in a dose-dependent manner, in the SJSA human sarcoma cell line. Docking studies were also reported and according to these the following 3D picture was drawn: The p-chlorophenyl substituent is mimicking Trp23 and pointing deeply into the central binding pocket; the propyl residue is occupying the Phe19-binding site and the 3,5-dimethoxy-4-hydroxybenzyl moiety is sitting over the Leu26 pocket, with one methoxy group sneaking in the hydrophobic pocket. Interestingly, a new H bond is postulated between the hydroxyl group of the benzyl substituent and Tyr100 of mdm2. Compound 7 is based on a spiroindolone scaffold and is currently the most potent p53/mdm2 antagonist reported with a fluorescence polarization (FP) derived Ki of 3 nM [29]. In addition it showed activation of p53 function and inhibition of cell growth in cancer cells with wild-type p53 status. Moreover, the compound shows excellent specificity over cancer cells with deleted p53 and shows a minimal toxicity to normal cells. According to modeling studies the oxindole moiety occupies the p53 Trp23binding site and also forms a hydrogen bond to Leu54 backbone carbonyl of mdm2. The tert-butyl side chain seemingly mimics the Leu26 of p53 and the m-chlorophenyl substituent mimics the p53 Phe19 side chain. In addition this molecule features a morpholino-2-ethylamide moiety mimicking the p53 Leu22 that is not deeply buried in the published p53/mdm2 X-ray structure, nevertheless undergoing extensive hydrophobic contacts. Current Opinion in Chemical Biology 2008, 12:281–291
In addition the morpholino-2-ethyl amide side chain presumably contributes to the water solubility of the compound. Although, the affinity of compound 7 is excellent, it seems to have reduced cell permeability. A similar compound 21 with a non-basic, however, solubilizing side chain has been recently reported including very promising cell based and xenograft data [30]. Compound 21 disrupts the mdm2–p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumour cells. MI-219 stimulates rapid but transient p53 activation in established tumour xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis and complete tumour growth inhibition. This promising compound is currently undergoing preclinical development. Compound 8 is an interesting aniline derivative with an 8hydroxyquinoline moiety and was discovered by a virtual database screening exercise based on a simple p53 peptide derived pharmacophore [31]. This quinolinol is reported to bind to mdm2 with a Ki of 120 nM (FP-based binding assay) and activates p53 in cancer cells with a mechanism of action consistent with targeting the mdm2– p53 interaction using Vogelstein’s HCT-116 (p53+/+ and p53 / ) cell lines. The authors postulate that 8 mimics the three p53 residues crucial in the binding to mdm2. Molecule 9 was discovered by screening of a designed library of 173 compounds by an FP-based binding assay [32]. The computational structure-based design was guided by Hamilton’s concept of small molecular weight a-helix mimetic. The most potent compounds have been further characterized by NMR shift perturbation assays. These assays provide a good, although not high-resolution indication of the binding site of the molecule on the surface of mdm2. According to these experiments with 9 and 14N-labeled mdm2, the most significantly perturbed mdm2 residues are Glu25, Phe55, His73, and Val93. This is in good agreement with a model where 9 bind to the p53binding pocket in mdm2. The general synthesis of the scaffold comprises a 10-step process. The natural fermentation product hexylitaconic acid 10 has been reported to inhibit p53/mdm2 by an ELISA assay [33]. The structure comprises a reactive Michael acceptor—an a,b-unsaturated carboxylic acid. The apparent IC50 is rather high at 230 mM. The absolute stereochemistry of the active compound is unknown. No cellbased activities were reported. Compounds 11–15 were discovered by a computational high throughput docking approach of a 35 000 compound library using an ensemble-based receptor model [34]. These compounds were identified using a multiple protein structure (MPS) method that incorporates protein flexibility into a receptor-based pharmacophore model. www.sciencedirect.com
Small molecular weight protein[19_TD$IF]–protein interaction antagonists[20_TD$IF]—an insurmountable challenge? Do¨mling 287
Impressive potent compounds were found that include compound 11, Ki of 110 nM in a FP assay. This potent binding is explained by an improved shape complementarity of the compound and the receptor, a more complete occupancy of the Phe19 pocket and the formation of two hydrogen bonds, between the catechol moiety and the Gln72 backbone carbonyl and the phenol moiety and His96 backbone carbonyl, respectively. For compound 14, the authors propose an interesting binding mode based on docking, in which the (2-ethylthio) ethylester portion of the molecule in a helical way sneaks down the bottom of the Trp23 pocket. On the contrary the Leu26 pocket seems to be unoccupied by 14. Surprisingly, the affinity for 14 is considerable with 21 mM. Another compound found in this virtual screening exercise is sulfonamide 15 (Ki = 37.2 mM). The binding of compound 16 to mdm2 has been investigated by NMR spectroscopy [6]. 16 was found to have an affinity (Ki) for mdm2 of 4 mM. In addition it has been shown that 16 can dissociate preformed p53/mdm2 com-
plex with a Kd of 3 mM. The Kd of nutlin-3 in the same experiment was 0.7 mM. No relative or absolute stereochemistry of 16 is given, nor is there any cell-based activity reported. Compound 18, a 2,5-dithiophenofuran with the trivial name RITA, was reported to bind to p53 and thus antagonize the p53/mdm2 interaction on the basis of ELISA and Fluorescence Correlation Spectroscopy [35], Multiple biological activities were reported in accordance with the proposed biochemical mode of action. However, scrutinizing the in vitro binding of compound 18 to p53 and mdm2 did not show any affinity nor was the compound able to dissociate the preformed complex [36], Therefore the observed biological activities seem to be derived from another unknown biochemical mechanism. Compound 19 (Ki = 20 mM, ELISA), interestingly was rationally designed by a general a-helix mimetic approach [37]. Herein, Hamilton and co-workers use
Figure 5
The three finger, thumb–index–middle finger pharmacophore model of p53/mdm2 antagonists. Above: Stereoview of six different p53/mdm2 antagonists aligned to fit the three-finger model (clockwise, 11a.m.: oxindol, benzodiazepinone, spirooxindol, imidazoline, bicycle and terphenyl). www.sciencedirect.com
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the terphenyl scaffold to place substituents in the right position in space that mimic the hydrophobic residues i, i + 4 and i + 7 of an amphiphatic a-helix. This concept seems to be general and several successful examples of antagonized a-helix interactions have been reported, including gp41 helix bundle interactions (HIV viral fusion) and Bcl2 family interactions (cancer apoptosis). A potential issue of this class of compounds is their very high lipophilicity and their lengthy synthetic access. Additionally, 19 only activate transcription in cells at a concentration of 30 mM. Finally, compound 20 (JNJ-26854165) an unusual tryptamine-derived (4-pyridino)-1,4-diaminophenyl was recently reported to be advanced into phase I human clinical trials [38]. No affinity data of 20 to mdm2 were reported. The compound seems to induce conformational changes in mdm2 as indicated by the abrogation of tryptic digestion of mdm2 in its presence. The compound induces p53 and activates p53 transcriptional activity in different cell lines. Mdm2 driven p53 degradation is suppressed. In glioblastoma and non-small-cell-lung-cancer xenografts 20 showed a broad spectrum tumour activity equipotent to taxol and outperforming Tarceva. From the above discussion of recently described p53/ mdm2 antagonists it becomes clear that only the full arsenal of biophysical, biochemical and cell biological methods will give a comprehensive and meaningful picture of the value of a compound [20]. Moreover, by
applying several complementary screening and characterization methods reported inaccurate inhibitor profiles may be minimized [14].
A compound-based pharmacophore model for p53/mdm2 antagonists On the basis of the published high-resolution information and the overlap of other described p53/mdm2 inhibitors a simple pharmacophore model is presented here. Owing to the 3D nature of the pharmacophore it is here called ‘The three finger model pharmacophore model’ (Figure 5). In this crude model several additional pharmacophoric features are not represented, for example, the potential to gain additional H bonding to Gln72. This protein–protein interaction is largely governed by highly hydrophobic interactions, and owing to the very deep and structured mdm2-binding site, a high degree of shape complementarity is demanded by the protein for an efficient antagonist. Even small chemical additions to a backbone, for example, addition of a methyl group or a halogen at a wrong position can largely destroy the activity of antagonists. Selectivity
One of the claims often made is that an antagonist of a PPI owing to its unique interaction motif should be intrinsically highly selective, as opposed to, for example, a kinase inhibitor with a more generic binding site. Besides the question of the therapeutic value of a drug with high selectivity in a multi factorial disease such as
Figure 6
Selectivity of a drug is driven by its structural biology. The p53-binding site of mdm2 and probably also mdm4 can be occupied by several other transcription factors as well. Therefore, a potential p53/mdm2 antagonist will probably also interrupt other protein interactions involving mdm2 and mdm4. The biological effects of these interactions have to be delineated in the future to yield potent novel agents to fight cancer. Current Opinion in Chemical Biology 2008, 12:281–291
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cancer has to be scrutinized. Thus, continuing with the theme of this article, mdm2 is a negative regulator of the transcription factor p53. The p53 tumour suppressor is vital in cell cycle regulation, DNA repair and apoptosis. Once DNA damage is beyond repair capabilities p53 induces apoptosis. Since mdm2 is overexpressed in many cancers and/or there are multiple gene copies in cancer cells with wild-type p53, antagonizing this PPI seemed a logical approach to restore p53 activity. In fact, three independent groups simultaneously published convincing data suggesting that p53 restoration should be pursued as a drug target approach: Ventura et al. found that restoration of p53 function leads to tumour regression in vivo [39]. Xue et al. demonstrated that p53 restoration induced senescence and tumour clearance in a murine liver carcinoma model [40]. Martins et al. constructed a reversible, switchable p53 knockin (KI) mouse model and observed after ‘switching’ on p53 expression, rapid apoptosis, and significantly increased survival was observed in an aggressive lymphoma model [41]. The picture, however, is much more complicated. There exist homologous proteins to p53, p63 and p73 that have very similar mdm2-binding motifs. Moreover a second remote p53 mdm2-binding motif has been recently described [42]. A genome-wide sequence query for mdm2-binding motifs also revealed a plethora of potential interaction partners (Figure 6) [39]. Several other transcription factors and proteins have been discovered to bind to mdm2 via the p53-binding site. These include the well-known E2F1 and HIF1-a [43,44]. These findings are very exciting, since E2F1 and HIF1-a play an important role in cancer development and progression. E2F1 transcription factor is best known for its ability to regulate the expression of genes required for DNA replication and cell cycle progression. Thus, the potent p53/mdm2 antagonist nutlin-3 induced apoptosis even in p53-mutant malignant peripheral nerve sheath and p53-null HCT116 cell lines. In fact, a clear dosedependent transcriptional activation of free E2F1 was observed [44]. Antagonizing HIF1-a/mdm2 with Nutlin3 showed a dose-dependent reduction of the HIF1-a transcriptional target VEGF under conditions of normoxia or hypoxia [43]. Thus, in the case of E2F1 and HIF1-a there seems to be a potential therapeutic synergy to p53. By sequence alignment to the hydrophobic p53-binding motif, other proteins have been predicted as binding partners of mdm2 in the p53-binding site, including VP16, TREB5, C-Jun, NFkB p65 and ALL1 [42]. However, for many of the other interactions involving the p53-binding site of mdm2 and mdm4 the biological role is unknown. Thus, the interactions and regulation of p53 and other proteins by mdm2-type binding sites is a highly complex matrix that is not understood in detail (Figure 6). It can be assumed that many such interactions are biologically relevant and can occur in a time and space-dependent manner, whereas others are biologically impossible. www.sciencedirect.com
Conclusion The recent propagation of several compounds into human clinical trial including Genentech’s XIAP inhibitor, Abbott’s Bcl2 antagonists and J&J-26854165 p53/mdm2 antagonist is a major advancement for the development of PPI antagonists. However, despite these success stories, antagonizing PPIs by small molecules is still a major challenge. There is no established structure based and general approach for the discovery of PPI antagonists. Importantly, PPIs structural appearance is highly diverse and they generally do not show obvious signatures, for example, an ATP-binding site as in kinases or an active site triad as in serine proteases. Clearly, there are PPIs that are more accessible to small molecule medicinal chemistry and other more difficult PPIs still await the discovery of their antagonists. PPIs, however, comprise a growingly important class of targets of high relevance for the development of new drugs in therapeutic areas of unmet medical needs. Moreover, from an intellectual standpoint of view targeting PPIs is an outstanding challenge. Efforts to target PPIs with small molecules are worthy undertakings and will most probably yield high return.
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