Review

Oncogene (2010) 29, 4245–4252; doi:10.1038/onc.2010.188; published online 24 May 2010

Pharmacological reactivation of mutant p53: from protein structure to the cancer patient

K G Wiman1

1Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden

Correspondence: Professor KG Wiman, Department of Oncology-Pathology, Karolinska Institute, Cancer Center Karolinska, Stockholm SE-171 76, Sweden. E-mail: klas.wiman@ki.se

Received 8 January 2010; Revised 18 April 2010; Accepted 21 April 2010; Published online 24 May 2010.

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Abstract

The p53 tumor suppressor pathway blocks tumor development by triggering apoptosis or cellular senescence in response to oncogenic stress. A large fraction of human tumors carry p53 mutations that disrupt DNA binding of p53 and transcriptional regulation of target genes. Reconstitution of wild-type p53 in vivo triggers rapid elimination of tumors. Therefore, pharmacological reactivation of mutant p53 is a promising strategy for novel cancer therapy. Several approaches for identification of small molecules that target mutant p53 have been applied, including rational design and screening of chemical libraries. The compound PhiKan083 binds with high affinity to a crevice created by the Y220C mutation in p53 and stabilizes the mutant protein. The compound PRIMA-1 (p53 reactivation and induction of massive apoptosis) restores wild-type conformation to mutant p53 by binding to the core and induces apoptosis in human tumor cells. The PRIMA-1 analog APR-246 is currently tested in a clinical trial. Successful development of mutant p53-reactivating anticancer drugs should have a major impact on the treatment of cancer.

Keywords:

mutant p53; drug discovery; reactivation; apoptosis; cancer therapy

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Introduction

p53 is a key tumor suppressor that is implicated in wide range of cellular processes, such as cell-cycle arrest, senescence, apoptosis, DNA repair, autophagy and metabolism (for references, see Hainaut and Wiman, 2005; Green and Kroemer, 2009; Vousden and Prives, 2009). p53 binds specifically to DNA and regulates transcription of target genes, such as p21 that blocks cell-cycle progression and Bax, Puma and Noxa that trigger cell death by apoptosis (Vousden and Lu, 2002; Hainaut and Wiman, 2005). In addition, p53 inhibits transcription of specific genes, including the anti-apoptotic Bcl-2 and hTERT (Xu et al., 2000; Mirza et al., 2003). p53 can also promote apoptosis by transcription-independent mechanisms upon translocation to mitochondria (Mihara et al., 2003).

The p53 protein has a short half-life under normal conditions. The p53 target MDM2 is an E3 ligase that, together with the structurally related partner protein MDMX (HDM4), ubiquitinates p53 and thus targets it for proteasome-mediated degradation. MDM2 also blocks p53's transactivation domain. Thus, p53 and MDM2 form a negative feedback loop that maintains low levels of p53 in the absence of cellular stress (Vousden and Lu, 2002). Cellular stress such as oncogene activation and DNA damage stabilizes p53 through post-translational modifications, including phosphorylation that prevents MDM2 binding, and in part also through increased mRNA levels and enhanced translation. As a result, p53 accumulates and triggers a biological response (Hainaut and Wiman, 2005).

p53-dependent senescence and apoptosis is a critical barrier against tumor development. Oncogenic stress as result of oncogene activation and disruption of cell-cycle control leads to aberrant DNA replication with stalled replication forks, which triggers a DNA damage response involving activation of the ATM-Chk2 or ATR-Chk1 kinase pathways and accumulation of p53 (Bartkova et al., 2005; Gorgoulis et al., 2005; Bartek et al., 2007). Furthermore, oncogene activation induces expression of the p14ARF protein (p19ARF in the mouse) that inhibits MDM2, leading to p53 accumulation and a p53-dependent biological response (Sherr and Weber, 2000). Thus, activation of p53 on oncogenic stress serves to eliminate incipient tumor cells by apoptosis and/or senescence.

Mutation of the p53 gene occurs in a significant fraction of human tumors, although at variable frequency in different tumor types. Tumors such as small cell lung cancer and ovarian carcinoma have the highest incidence of p53 mutations whereas, for example, testis cancer only rarely carries mutant p53 (Olivier et al., 2002; www-p53.free.frwww-p53.iarc.fr). Most p53 mutations are missense mutations in the DNA-binding core domain that result in deficient DNA binding, and as a consequence, failure to transactivate target genes (Soussi and Wiman, 2007). Inactivation of p53 by mutation will disrupt the DNA damage response pathway and thus permit survival upon oncogenic stress. Ample evidence indicates that the p53 pathway is dysfunctional also in tumors carrying wild-type p53; for example, by overexpression of MDM2, loss of p14ARF or mutation of ATM. Therefore, inactivation of the p53 response is a critical step during tumor evolution, allowing evasion of apoptosis and sustained tumor growth. In addition, many tumor-derived mutant p53 proteins have acquired tumor growth-stimulating activities, such as illegitimate transactivation of the c-Myc oncogene or the MDR1 (multidrug resistance) gene (Sigal and Rotter, 2000), and hetero-oligomerization with p53 family proteins p63 and p73, resulting in attenuated apoptosis (Gaiddon et al., 2001). Recent work shows that mutant p53 can promote tumor invasion by affecting integrin and epidermal growth factor receptor trafficking (Muller et al., 2009).

Given the key role of the p53 pathway in tumor suppression, its presumably universal inactivation in human tumors, and numerous studies indicating that p53 mutation is associated with poor prognosis (see for example Olivier et al., 2006), novel strategies for treatment of cancer based on restoration of this pathway are very attractive. This notion is strongly supported by in vivo studies showing that restoration of wild-type p53 function by various means in mice causes rapid tumor regression in vivo (Martins et al., 2006; Ventura et al., 2007; Xue et al., 2007). These results suggest that restoration of wild-type p53 expression is sufficient for elimination of tumors even in the presence of multiple tumor-associated genetic alterations.

Various strategies for reconstitution of wild-type p53 function in tumors have been developed, and some have even reached the clinic. Gene therapy, that is, introduction of an intact copy of the p53 gene in tumors that carry mutant p53 using a tailor-made virus (in most cases adenovirus), has been tested in clinical trials in patients with lung cancer, head and neck cancer and other types of cancer. Only relatively mild side effects have been observed, and a fraction of the patients have shown at least partial clinical responses. Combination with conventional chemotherapeutic drugs such as cisplatin has enhanced the clinical efficacy (reviewed by Wiman, 2006). However, gene therapy has some limitations. It is not possible to introduce wild-type p53 in all tumor cells; therefore, the clinical effect of the treatment depends on the so-called bystander effect, that is, the ability of reconstituted tumor cells to inhibit growth of neighboring non-reconstituted cells. Moreover, the p53 virus has to be injected locally in the tumor. Treatment of disseminated disease will require improved techniques for systemic delivery of wild-type p53-carrying viruses and tumor cell targeting in vivo.

Over the past decade, screening approaches have led to the identification of small molecules that restore p53 function in tumor cells. The current list of p53-reactivating compounds includes those that activate a p53 response in wild-type p53-carrying tumors. In the case of the compound Nutlin, this is achieved by binding to MDM2 and blocking the interaction between p53 and MDM2, resulting in accumulation of wild-type p53 (Vassilev et al., 2004). MI-219 is another small molecule inhibitor of the p53–MDM2 interaction (Shangary et al., 2008). Other compounds such as RITA and Tenovin-6 act on wild-type p53 through different mechanisms (Issaeva et al., 2004; Lain et al., 2008). These approaches have recently been reviewed by Brown et al. (2009).

As shown in Figure 1, a basic idea behind mutant p53 reactivation is restoration of wild-type conformation and DNA binding, followed by transcriptional transactivation of target genes and induction of apoptosis and/or senescence. Targeting mutant p53 by small molecules appears as an even greater challenge than activating wild-type p53 in a tumor. A wide range of different mutant p53 proteins are expressed in tumors, and although reduced thermostability is a common feature of many p53 mutants, mutations may give rise to different and unique structural alterations in the protein. The design of drugs that are capable of restoring wild-type function to such a heterogenous target is no doubt a formidable undertaking. Nonetheless, small molecules that bind and/or restore wild-type conformation and function to mutant p53 have been identified using various approaches. This review will focus on mutant p53-reactivating molecules and highlight two main approaches, that is, rational drug design and random screening of chemical libraries.

Figure 1.
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General strategy for mutant p53 reactivation by small molecules. Restoration of wild-type conformation and specific DNA binding to abundantly expressed mutant p53 will result in transactivation of p53 target gene transcription, followed by induction of apoptosis and/or cellular senescence, leading to efficient tumor elimination.

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Mutant p53 as a therapeutic target

The p53 core domain (residues 100–300) binds specifically to DNA. Its structure when bound to DNA was determined by X-ray crystallography (Cho et al., 1994). The p53 core domain is intrinsically unstable; it is correctly folded at 37°C but has a melting temperature of 44°C, and a short half-life at physiological temperature (Joerger and Fersht, 2008). p53 mutations in human tumors are scattered over the core domain. The most frequently mutated residues include Arg175, Ser245, Arg248 and Arg273. Tumor-associated p53 mutations can be divided in two main categories. Mutations in the first category affect residues that make direct contact with DNA, for instance Arg273. Substitution of such residues will disrupt specific DNA binding, but as in the case of the His273 mutant, have little or no effect on the overall structure of the core domain. In contrast, mutations in the second category affect residues that are important for the structural integrity of the core, such as Arg175. These mutations, for example, His175 and Ser249, destabilize the core domain and therefore abrogate binding to DNA. For both types of mutants, p53's ability to transactivate target genes is ablated. Mutations in the core domain may also disrupt the proapoptotic activity of cytoplasmic and mitochondrial p53, including the interaction with Bcl-2/Bcl-XL. The observation that many p53 mutants share a common property, that is, reduced thermostability and complete or partial unfolding, raises the possibility of designing drugs that stabilize the wild-type conformation and thus restore wild-type function. However, mutant p53 rescue by small molecules must also take into account local structural disturbances of the DNA-binding surface.

Another relevant feature of mutant p53 as a therapeutic target is its high level of expression in many tumor cells. Immunohistochemistry frequently reveals intense p53 staining in tumors tissues, and this is often, but not always, associated with p53 mutation. The enhanced levels of mutant p53 in tumor cells may be due to failure to transactivate the MDM2 gene whose protein product targets p53 for degradation, and constitutive stress signaling as result of oncogene activation and dysregulation of cell growth. Restoration of wild-type function to mutant p53 expressed at elevated levels should trigger a robust biological response and efficient elimination of tumor cells through apoptosis or senescence.

As we shall see, both rational design and random screening approaches have led to the identification of mutant p53-targeting compounds with different structures and properties. Interestingly, several of the compounds identified by random screening share a common chemical activity that is likely to have a key role in mutant p53 reactivation.

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Mutant p53-reactivating drugs by rational design

Structural studies of mutant p53 core domain proteins by X-ray crystallography have provided detailed information about diverse structural effects of various tumor-derived mutations (Joerger et al., 2006). This information can be used as a basis for rational design of compounds that will bind and stabilize the wild-type conformation of the core domain. For some β-sandwich mutants, for example, Ala143 and Cys220, the binding of a small-molecule drug is expected to stabilize the wild-type conformation and thus rescue wild-type p53 function. In the case of other mutants, such as Ser249, the situation is more complex as local structural changes in the DNA-binding surface will prevent efficient DNA binding.

Fersht and co-workers have focused on the Cys220 p53 mutant as a potential therapeutic target. This is the most common mutation outside the DNA-binding surface of the p53 core domain, occurring in some 75000 tumors annually worldwide. It is located at the end of the β-sandwich that serves as a basic scaffold for the DNA-binding surface. The mutation results in a crevice that destabilizes the protein by 4kcal/mol and could potentially be targeted by a small molecule. Because it is located at some distance from the DNA-binding surface, small molecule binding is unlikely to interfere with DNA binding that is crucial for p53 activity.

Based on structural information from X-ray crystallography, Fersht and colleagues carried out an in silico screening to identify molecules that were able to bind the mutation-induced cleft in the Cys220 p53 protein. More than 2.5 million structures were analyzed in silico and small group of 80 compounds was finally tested in vitro by NMR spectroscopy for binding to the Cys220 protein. One compound, PhiKan059, was shown to bind to the cleft created by Cys220, and further screening of analogs led to the identification of PhiKan083 with improved binding affinity. This compound stabilizes the Cys220 mutant in a concentration-dependent manner, raising the melting temperature by almost 2°C and increasing the half-life from 3.8 to 15.7min (Boeckler et al., 2008). Analysis of the crystal structure of the protein–PhiKan083 complex provided detailed information about interactions between the compound and the protein, and indicated specific conformational changes upon binding. PhiKan083 is an interesting compound but its true potential can only be assessed when biological data on its effect on tumor cells in vitro and in vivo will be available. Although PhiKan083 targets the Cys220 mutant only, it could nevertheless be important for cancer therapy because the total number of cancer patients worldwide carrying this particular mutation is substantial.

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Screening for mutant p53-reactivating compounds

A different approach for identification of mutant p53-reactivating small molecules is screening chemical libraries for compounds that restore folding and DNA binding to mutant p53. The compound CP-31398 was shown to prevent unfolding of wild-type or mutant p53 in vitro, and inhibit tumor growth in vivo (Foster et al., 1999). CP-31398 stabilizes p53 through reduced ubiquitination (Wang et al., 2003) and induces expression of classical p53 target genes, such as p21, but also induces p53-independent cell death (Wischhusen et al., 2003). NMR analysis did not detect binding to the p53 core domain (Rippin et al., 2002). It is noteworthy, however, that CP-31398 potentially shares a chemical activity with several other mutant p53-targeting compounds (see below). Another mutant p53-targeting compound, P53R3, was identified by screening of a small chemical library using an in vitro DNA binding assay (Weinmann et al., 2008). This compound restores sequence-specific DNA binding of both His273 and His175 mutant p53, enhances the recruitment of p53 to target promoters and induces p53-dependent expression of p53 target genes. Its potential antitumor activity in vivo has yet to be determined.

An alternative strategy is cellular screening to identify compounds that selectively target mutant p53-expressing tumor cells. One advantage with this approach is that the readout is cell death or growth arrest, and therefore the identified hit compounds are able to enter cancer cells and induce a desired biological effect. Also, this approach is unbiased with regard to the molecular mechanism of action, which may be direct or indirect. Conversely, elucidation of the exact mechanism may be a demanding and tedious job.

Ellipticine was initially identified by analysis of drug sensitivity profiles of the National Cancer Institute tumor cell line panel as a drug that selectively affects cell lines with mutant p53 (Shi et al., 1998). Further studies showed that ellipticine can enhance sequence-specific DNA binding and transcription transactivation of mutant p53, and can induce mutant p53-dependent cell death (Peng et al., 2003). More recent work has shown that the combination of ellipticine with 5-fluorouracil allows depletion of putative cancer stem cell populations (Huang et al., 2009).

We have used a simple cell-based assay for screening of chemical libraries, plant extracts and compounds identified by molecular modeling (Figure 2). Our screening of the Diversity set from the National Cancer Institute led to the discovery of two small molecules with mutant p53-reactivating capacity; that is, PRIMA-1 (p53 reactivation and induction of massive apoptosis) and MIRA-1 (mutant p53-dependent induction of rapid apoptosis). In a separate screen of a small compound library, we also identified STIMA-1 (SH group-targeting compound that induces massive apoptosis). MIRA-1 is a maleimide with a reactive double bond that can participate in reactions of Michael addition. Therefore, MIRA-1 is a so-called Michael acceptor with potential ability to modify cysteines in proteins (Bykov et al., 2005a). Consistent with this notion, several MIRA-1 analogs that contain the reactive double bond show biological activity against mutant p53-expressing cells, whereas analogs lacking the double bond are inactive. Similarly, STIMA-1 is a Michael acceptor that potentially targets SH groups (Zache et al., 2008a). In vivo studies with MIRA-1 and STIMA-1 indicated that both compounds are unsuitable for further drug development due to poor solubility and/or toxicity. Still, they have been useful in that they have indicated a common mechanism for mutant p53 reactivation, as will be further discussed below.

Figure 2.
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Cellular screening for identification of mutant p53-targeting compounds. Human osteosarcoma (Saos-2) or lung adenocarcinoma (H1299) cells that lack p53 and their sublines carrying exogenous His273 or His175 mutant p53, respectively, are used for screening of chemical libraries, compounds identified by molecular modeling or collections of plant extracts. Compounds or extracts that show strong selectivity for the mutant p53-expressing cells are further characterized with regard to their effect on mutant p53 conformation and DNA binding, upregulation of p53 target genes, induction of apoptosis and antitumor effect in vivo in mice.

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PRIMA-1 (2,2-bis(hydroxymethyl)-1-azabicyclooctan-3-one) shows selective growth-inhibitory and apoptosis-inducing effect on mutant p53-expressing Saos-2 cells (Figure 3) (Bykov et al., 2002). Our further studies of PRIMA-1 and its structural analog PRIMA-1MET (APR-246) showed that these compounds inhibit human xenograft tumor growth in SCID mice (Bykov et al., 2002, 2005b) and growth of mouse tumors in a syngeneic host (Zache et al., 2008b). They also show potent effect on primary human acute myeloid leukemia and chronic lymphoid leukemia cells (Nahi et al., 2004, 2006). Furthermore, we showed that PRIMA-1MET (APR-246) is able to synergize with certain chemotherapeutic drugs, including adriamycin and cisplatin (Bykov et al., 2005b). In theory, such synergy could arise from the restoration of wild-type p53 function, as wild-type p53-carrying tumors often show better response to chemotherapy. However, we have also suggested the possibility that induction of mutant p53 protein levels on treatment with DNA-damaging chemotherapeutic drugs might enhance the potency of PRIMA-1 and APR-246, because higher levels of mutant p53 increase sensitivity to these compounds.

Figure 3.
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The low-molecular-weight compound PRIMA-1 induces mutant p53-dependent apoptosis through the mitochondrial pathway. Reactivation of mutant p53 leads to upregulation of the pro-apoptotic p53 target genes, Bax, Puma and Noxa, and ER stress. PRIMA-1 also activates caspase-2, followed by activation of caspase-3 and caspase-9. This triggers cell death by apoptosis (Shen et al., 2008; Lambert et al., 2010).

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Molecular mechanism of PRIMA-1 and APR-246

The exact molecular mechanism of mutant p53 reactivation by PRIMA-1 and APR-246 is clearly a critical issue that is highly relevant for further drug optimization and the design of novel compounds with improved potency and target selectivity. Our data have indicated that treatment with PRIMA-1 or APR-246 leads to upregulation of at least some of p53's target genes; for example, Bax, Puma and Noxa (Figure 3). Others have shown that PRIMA-1 is capable of inducing apoptosis in a transcription-independent manner (Chipuk et al., 2003). We found that PRIMA-1 and APR-246 induce activation of caspase-2, caspase-3 and caspase-9, consistent with induction of apoptosis via the mitochondrial pathway (Shen et al., 2008). Moreover, microarray analysis revealed that APR-246 induces a limited set of genes in a mutant p53-dependent manner, followed by ER stress (Lambert et al., 2010; Figure 3). Yet the question remained as to how these compounds affect mutant p53. Do they bind directly to mutant p53 or do they act through indirect mechanisms?

We have examined the stability of PRIMA-1 and APR-246 and found that both compounds are relatively rapidly converted to other compounds under physiological conditions. At least one of these compounds, MQ, has a reactive double bond that is likely to participate in Michael addition reactions, making it a Michael acceptor. We showed that it binds covalently to the p53 core domain, and that such modification per se is sufficient to endow mutant p53 with pro-apoptotic properties (Lambert et al., 2009). Transfer of recombinant PRIMA-1/APR-246/MQ-modified mutant p53 protein into p53 null human tumor cells resulted in upregulation of pro-apoptotic genes, such as Bax, Puma and Noxa, and induction of apoptosis as shown by loss of cell viability and caspase activation. Importantly, a PRIMA-1 analog that cannot be converted to MQ is biologically inactive. Thus, similar to MIRA-1 and STIMA-1, PRIMA-1 and APR-246 rely on a Michael acceptor activity for mutant p53 reactivation, although the reactive compound is a metabolite of the original compounds. PRIMA-1 and APR-246 can therefore be viewed as prodrugs that are converted to the active substance in vivo.

WR1065, a metabolite of amifostine, restores wild-type conformation and transcription transactivation function to Met272 mutant p53 (North et al., 2002). This compound was shown to bind to p53 (Shen et al., 2001) and has a thiol group, suggesting that it binds covalently to cysteines in mutant p53 by forming a disulfide bond.

The observation that several mutant p53-targeting compounds are able to modify cysteines in p53 is particularly interesting in view of previous studies showing that p53 is regulated in a redox-dependent manner. A reducing environment promotes correct p53 folding and DNA binding, whereas the formation of disulfide bonds in p53 can inactivate DNA binding and may lead to p53 aggregation. Zinc binding to reduced p53 is critical for proper conformation. The redox state of p53 is controlled by several cellular factors, including thioredoxin-thioredoxin reductase and the enzyme APE1/Ref1 that acts as a p53 reductase (reviewed by Bykov et al., 2009). The fact that several identified mutant p53-targeting molecules possess Michael acceptor activity suggests that mutant p53 reactivation by these compounds involves redox regulatory pathways that normally control wild-type p53 activity. Thiol modification could block the formation of intramolecular disulfide bonds that might lock the core domain in an inactive conformation, and could also prevent intermolecular disulfide bonds that cause mutant p53 aggregation (Figure 4). Modification of cysteines could also directly restore wild-type conformation and function to mutant p53. Conceivably, adducts in the core domain might form protein–DNA bridges that stabilize DNA binding or promote correct folding due to their hydrophobicity, as shown in Figure 1. A final answer to the question of the mechanism should come from structural studies of PRIMA-1/APR-246/MQ-modified mutant p53 by X-ray crystallography and/or NMR.

Figure 4.
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Proposed effect of thiol modification on mutant p53 aggregation. Unfolding of mutant p53 may expose cysteines that are normally hidden in the core domain. This might lead to the formation of intra- and intermolecular disulfide bonds that lock mutant p53 in an inactive conformation and cause protein aggregation. Thiol modification by compounds such as MQ derived from PRIMA-1/APR-246 could prevent formation of such disulfide bonds and thus promote correct folding and restoration of wild-type function.

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These results also raise the question as to how a reactive compound such as MQ could achieve target specificity, as it might potentially react with multiple proteins and other molecules inside cells. There are several possible reasons for selectivity toward mutant p53-expressing cells. First, the structural context of a specific cysteine might dictate selectivity so that only a limited number of proteins are modified. Interestingly, there are other examples of target-selective thiol-modifying compounds, such as leptomycin B that targets the nuclear export protein CRM1 by covalent modification of Cys528 (Kudo et al., 1999), and HKI-272, a follow-up compound to Iressa that targets the epidermal growth factor receptor (Kwak et al., 2005). Second, thiol modification would not necessarily trigger apoptosis in all cases; for many proteins, such modification might have little or no impact on cell survival. Third, because the active compound MQ is generated from a prodrug, that is, PRIMA-1 or APR-246 (Lambert et al., 2009), cellular uptake may occur before conversion. If so, MQ would be, to a large extent, produced inside tumor cells in proximity to its target mutant p53, rather than bind to proteins in the extracellular environment. Because extensive modification of extracellular proteins might lead to toxicity, MQ itself is unlikely to be suitable as a therapeutic agent.

Apart from restoration of mutant p53 conformation and DNA binding, PRIMA-1 and APR-246 could conceivably have effects on the complexing between mutant p53 and the p53 family proteins p63 and p73. Mutant p53 binding is thought to inhibit p63/p73-dependent apoptosis (Gaiddon et al., 2001) and, therefore, disruption of such complexes might unleash a p63 and/or p73 response that could contribute to PRIMA-1-induced tumor cell death.

Does PRIMA-1/APR-246 target a wide range of mutant p53 proteins or only a subset of mutants? Clearly, both DNA contact mutants, such as His273, and structural mutants, such as His175, confer sensitivity to PRIMA-1/APR-246 when expressed in p53 null tumor cells. Gel shift assays revealed enhanced DNA binding of a range of p53 mutants, with the interesting exception of the Phe176 mutant (Bykov et al., 2002). Normally, Cys176 holds a zinc atom in p53. Substitution of this residue will probably result in severe unfolding that cannot be rescued by PRIMA-1 and APR-246. The ability of PRIMA-1 and APR-246 to target several different mutant p53 isoforms may be due to stabilization of the wild-type conformation; as discussed above, reduced thermostability and complete or partial unfolding is a common feature of mutant p53 proteins. Further studies should address the effect of PRIMA-1 on a larger number of p53 mutants in a quantitative manner.

In principle, a reactive compound like MQ might modify cysteines in wild-type p53 as well, particularly if cysteines are exposed upon protein unfolding. We confirmed binding to wild-type p53 and found that the degree of binding is correlated with the extent of unfolding (Lambert et al., 2009). Thus, PRIMA-1/APR-246 could presumably also reactivate unfolded wild-type p53 in tumors, triggering apoptosis and/or senescence.

Importantly, normal cells such as human diploid fibroblasts are significantly more resistant to PRIMA-1 in vitro (Bykov et al., 2002). This is consistent with the lack of any obvious toxicity at therapeutic doses in mice (Bykov et al., 2002; Zache et al., 2008b) and indicates a sufficiently large therapeutic window. Nonetheless, it is likely that PRIMA-1 and APR-246 also target other proteins in cells, at least at higher concentrations.

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Summary and future perspectives

Mutant p53 is an attractive target for novel cancer therapy, given the high frequency of p53 mutation and high levels of expression of mutant p53 in many human tumors. However, reactivation of mutant p53 requires different strategies than inhibition of protein kinases such as the Bcr–Abl fusion protein or the epidermal growth factor receptor that are targets of the clinically used drugs imatinib (Gleevec) and gefitinib (Iressa), respectively. These kinases have active sites that can be blocked by small molecules. Such targets are considered more readily ‘druggable’ than mutant p53, which lacks a well-defined common site for binding of a reactivating small molecule, and shows considerable heterogeneity as to the structural changes induced by specific mutations. Also, conformational and functional rescue of a misfolded protein is likely to pose greater difficulties for drug design than inhibition of a dysregulated kinase. Small molecules that target mutant p53 and trigger apoptosis in tumor cells have been identified by either rational design or random screening approaches. APR-246, a structural analog of the compound PRIMA-1 that was identified by screening of chemical library, is currently being tested in the clinic in a phase I trial. The progress thus far should encourage further studies to identify more potent and selective mutant p53-targeting substances.

Although identified mutant p53-targeting compounds show potent antitumor effects in vitro and in vivo by themselves, they should also be tested in combination with currently used chemotherapeutic drugs, for example cisplatin, that in general show better efficacy on tumors that carry wild-type p53. Synergy between PRIMA-1/APR-246 and cisplatin has been shown both in vitro and in vivo (Bykov et al., 2005b). Combination therapy is a key strategy against the development of drug resistance. The identification of novel targeted anticancer drugs opens possibilities for testing synergy between substances that affect different growth-regulating cellular pathways or different components of the same pathway. For instance, the combination of PRIMA-1 and Nutlin could enhance PRIMA-1-induced apoptosis by preventing MDM2-mediated inhibition of reactivated mutant p53. Moreover, the availability of more advanced mouse models, that is, mutant p53 knock-in mice (reviewed by Donehower and Lozano (2009)), opens possibilities for improved studies of antitumor efficacy in vivo, and assessment of the effect on mutant p53 gain-of-function activities.

We also need to elucidate exactly how compounds like PRIMA-1 and APR-246 interact with the mutant p53 core domain and promote its correct folding. A better understanding of the precise mechanism at the molecular level will be the basis for improved drug design and eventually more potent and selective targeting of mutant p53 in cancer. Our hope is that further progress in this direction will lead to significantly improved cancer therapy.

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Conflict of interest

Professor Wiman is cofounder and shareholder of Aprea AB, a start-up company that develops p53-based cancer therapy. He is also a member of its board.

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Acknowledgements

I thank the Swedish Cancer Society (Cancerfonden), the Swedish Medical Research Council (VR), the Cancer Society of Stockholm (Cancerföreningen) and the EU 6th Framework Program for generous support. I am cofounder and shareholder of Aprea AB, a company that develops p53-based cancer therapy, and member of its board.