Xiaohong Lu

Isoindolinone Inhibitors of the Murine Double Minute 2 (MDM2)-p53 Protein-Protein Interaction: Structure-Activity Studies Leading to Improved Potency

Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 μM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 μM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.

The MYCN oncoprotein as a drug development target

The transcription factor and proto-oncogene MYCN is reviewed as a potential specific target for cancer therapy. Amplification of MYCN is frequently found in a number of advanced-stage tumours, including neuroblastoma (25%), small cell lung cancers (7%), alveolar rhabdomyosarcoma and retinoblastoma. It is associated with rapid tumour progression and poor outcome in human neuroblastoma. MYCN is a member of the myc family of proto-oncogenes which encode nuclear proteins that form heterodimers with MAX protein through their conserved HLHZip domains. The MYC/MAX complexes transactivate a number of MYC-target genes in a sequence-specific manner. MYC-MAX interaction is essential for MYC-induced cell cycle progression, cellular transformation, and transcriptional activation. A causal link between the transformed phenotype and MYCN has been established by a range of in vitro and in vivo studies, including a transgenic model of neuroblastoma in which MYCN overexpression is targeted to neuronal tissue by the use of a tyrosine hydroxylase promoter. Downregulation of MYCN expression either by antisense treatment targeted against MYCN mRNA or by retinoids has been shown to decrease proliferation and/or induce neuronal differentiation of neuroblastoma cells. Inhibition of MYC-MAX dimerisation by small-molecule antagonists has recently been shown to interfere with MYC-induced transformation of chick embryo fibroblasts, indicating that functional inhibitors of the MYC family of oncoproteins have potential as therapeutic agents. Finally, we describe the development and validation of a functional MYCN reporter gene assay using neuroblastoma cells (NGP) which have been stably transfected with a luciferase gene construct under control of the ornithine decarboxylase gene promoter. This assay has been used for a pilot screen of 2800 compounds from the Cancer Research-UK collection, identifying five compounds showing a consistent significant reduction of MYCN-dependent luciferase activity (>50%) in repeated screens. This cell-based, MYCN reporter gene assay will be scaled up for high throughput screens of compound libraries and will aid in the future development of specific therapeutic strategies in neuroblastoma and other tumours in which MYCN amplification has been implicated.

The role of MYCN in the failure of MYCN amplified neuroblastoma cell lines to G1 arrest after DNA damage.

We previously reported that 3 p53 wild type (wt) MYCN amplified (MNA) neuroblastoma cell lines failed to G1 arrest after DNA damage despite induction of p53, p21WAF1 and MDM2. We hypothesised that this was due to high MYCN expression. p53 responses to DNA damage were examined in an additional 13 p53 wt neuroblastoma cell lines. MNA was significantly associated with a failure to G1 arrest after DNA damage (p

MDM2-p53 protein-protein interaction inhibitors: a-ring substituted isoindolinones.

Structure-activity relationships for the MDM2-p53 inhibitory activity of a series of A-ring substituted 2-N-benzyl-3-(4-chlorophenyl)-3-(1-(hydroxymethyl)cyclopropyl)methoxy)isoindolinones have been investigated, giving rise to compounds with improved potency over their unsubstituted counterparts. Isoindolinone A-ring substitution with a 4-chloro group for the 4-nitrobenzyl, 4-bromobenzyl and 4-cyanobenzyl derivatives (10a-c) and substitution with a 6-tert-butyl group for the 4-nitrobenzyl derivative (10j) were found to confer additional potency. Resolution of the enantiomers of 10a showed that potent MDM2-p53 activity resided in the (-)-enantiomer ((-)-10a; IC(50)=44 ± 6 nM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compounds 10a and (-)-10a increase p53 protein levels, activate p53-dependent MDM2 and p21 transcription in MDM2 amplified cells, and show improved selectivity for growth inhibition in wild type p53 cell lines over the parent compound.

Heat shock factor-1 modulates p53 activity in the transcriptional response to DNA damage

Here we define an important role for heat shock factor 1 (HSF1) in the cellular response to genotoxic agents. We demonstrate for the first time that HSF1 can complex with nuclear p53 and that both proteins are co-operatively recruited to p53-responsive genes such as p21. Analysis of natural and synthetic cis elements demonstrates that HSF1 can enhance p53-mediated transcription, whilst depletion of HSF1 reduces the expression of p53-responsive transcripts. We find that HSF1 is required for optimal p21 expression and p53-mediated cell-cycle arrest in response to genotoxins while loss of HSF1 attenuates apoptosis in response to these agents. To explain these novel properties of HSF1 we show that HSF1 can complex with DNA damage kinases ATR and Chk1 to effect p53 phosphorylation in response to DNA damage. Our data reveal HSF1 as a key transcriptional regulator in response to genotoxic compounds widely used in the clinical setting, and suggest that HSF1 will contribute to the efficacy of these agents.

Diaryl- and triaryl-pyrrole derivatives: inhibitors of the MDM2–p53 and MDMX–p53 protein–protein interactions

Triarylpyrroles e.g. 4c and 4s inhibit the MDM2–p53 and MDMX–p53 protein–protein interactions.

Searching for Dual Inhibitors of the MDM2-p53 and MDMX-p53 Protein–Protein Interaction by a Scaffold-Hopping Approach

Two libraries of substituted benzimidazoles were designed using a ‘scaffold‐hopping’ approach based on reported MDM2‐p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X‐ray structure. Benzimidazole libraries were prepared using an efficient solution‐phase approach and screened for inhibition of the MDM2‐p53 and MDMX‐p53 protein–protein interactions. Key examples showed inhibitory activity against both targets.

TP53 mutant MDM2 -amplified cell lines selected for resistance to MDM2-p53 binding antagonists retain sensitivity to ionizing radiation

Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR.

Abstract A140: Identification of substituted isoindolinones as potent inhibitors of the MDM2‐p53 protein‐protein interaction

The p53 tumor suppressor plays a pivotal role in the cell by reacting to stress, which may be caused by hypoxia, DNA damage, or oncogenic signalling. Activation of p53 protein results in the transcription of a number of genes that govern progression through the cell cycle, the initiation of DNA repair, and apoptosis. The activity of p53 is tightly regulated by the MDM2 protein, which is transcribed in response to p53 activation. MDM2 binds to and inactivates p53 and also ubiquitylates the MDM2‐p53 complex to target it for proteosomal degradation. In normal cells the balance between active p53 and inactive MDM2‐bound p53 is maintained in a negative feedback loop. Inhibition of the MDM2‐p53 protein‐protein complex by small molecule inhibitors is expected to reactivate normal p53 pathways in cells overexpressing MDM2, consequently exerting an anti‐cancer effect. Potent small molecule inhibitors of the MDM2‐p53 interaction have been identified e.g. the Nutlins [Science2004, 303, 844], the benzodiazepinediones and indolinones [J. Med. Chem.2006, 49, 3759–3762]. The antitumor activities of these compounds in cellular and in vivo models are promising. We have reported previously inhibitors of the MDM2‐p53 interaction, based on an isoindolinone scaffold [J. Med. Chem.2006, 49, 6209–6221], and SAR studies leading to compounds with improved potency e.g. NU8345A; IC50 = 171 ± 15 nM. NMR structural studies suggested that substitution of the isoindolinone A‐ring may provide additional favourable interactions with the protein. A small series of examples was prepared, and 4‐substitution with small hydrophobic groups was found to improve potency e.g. rac‐4‐methyl‐3‐(4‐chlorophenyl)‐3‐((1‐(hydroxymethyl) cyclopropyl)methoxy)‐2‐(4‐nitrobenzyl)isoindolin‐1‐one (NU8405; IC50 = 274 ± 35 nM) and rac‐4‐chloro‐3‐(4‐chlorophenyl)‐3‐((1‐(hydroxymethyl)cyclopropyl)methoxy)‐2‐(4‐nitrobenzyl)isoindolin‐1‐one (NU8406; IC50 = 143 ± 26 nM). Introduction of a 6‐tert‐butyl group was also found to be favourable i.e. rac‐6‐tert‐butyl‐3‐(4‐chlorophenyl)‐3‐((1‐(hydroxymethyl)cyclopropyl)methoxy)‐2‐(4‐nitrobenzyl)isoindolin‐1‐one (NU8399; IC50 = 152 ± 27 nM). Resolution of the enantiomers of NU8406, using chiral HPLC, gave NU8406A and NU8406B (IC50s of 43.8 ± 6.2 nM and 1.27 ± 0.08 M, respectively). The cellular activities of key compounds have been determined and show dose dependent induction of p53 regulated genes by Western blotting. NU8406 shows selective cytotoxicity in p53wt HCT116 cells compared with the HCT116−/− line (GI50s = 9.8 ± 1.1 and 21.3 ± 3.1 M, respectively) which is comparable to Nutlin‐3 (ELISA IC50 = 66.2 ± 5.7 nM; GI50s = 8.9 ± 0.5 and 38.8 ± 3.4 M, respectively). Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A140.

Abstract A154: Mechanisms of cellular resistance to the growth inhibitory and cytotoxic effects of MDM2‐p53 binding antagonists

Background: About 50% of solid tumors and more than 80% of haematological malignancies express wild‐type p53 at diagnosis. Therefore, activation of the p53 pathway by antagonizing its negative regulator Murine Double Minute 2 (MDM2) may offer a new therapeutic strategy for cancer treatment. Recently, several potent and selective small molecule antagonists of the p53‐MDM2 protein‐protein interaction have been developed. Studies with these compounds have strengthened the concept that selective, non‐genotoxic p53 activation may in some circumstances represent an alternative to current cytotoxic chemotherapy. Aims: To investigate the potential for drug resistance against p53‐MDM2 interaction antagonists and to establish therapeutic strategies to circumvent this. Methods: NGP neuroblastoma and SJSA‐1 osteosarcoma cell lines have been used to select a series of clonal variants resistant to two potent small molecule inhibitors of the MDM2‐p53 interaction Nutlin‐3 [ Science 2004, 303, 844] and MI63 [ J.Med.Chem. 2005, 48, 909]. The drug resistance phenotype of these clones was evaluated by SRB growth inhibition assay, western blotting for activation of p53 and downstream mediators of p53 dependent growth inhibition and cytotoxicity, and caspase‐3/7 activation analysis, in response to drug treatment. DNA sequence analysis for p53 mutation detection was also conducted to explore possible resistance mechanisms. Results: Following 60‐day exposure to 1‐5µM plus 60‐day exposure to 5‐40µM of MI63 or Nutlin‐3 respectively, several clones were selected showing 11.5–43.2 fold increases in GI50 values. Clones selected for resistance to one of the agents showed cross‐resistance to the other. This increased resistance was reflected in reduced activation of growth inhibition and apoptosis pathways downstream of p53, although induction of p53 protein itself was little changed compared to parental cell lines. All clones exhibited undetectable accumulation of MDM2 and p21 proteins compared with the marked changes seen with parental cells after treatment with 20 µM of Nutlin‐3 or 5 µM MI63 for 4–6 hours, even though the p53 protein accumulated to the same extent as parental cells. Decreased cleavage of caspase‐3 and PARP proteins were also observed, consistent with the reduced apoptosis. Caspase‐3/7 enzymatic activity in the resistant clones in response to exposure to MI63 or Nutlin‐3 for 48 hours was also significantly reduced compared to the response in parental cell lines. Point missense mutations of the p53 gene were observed within the resistant cell clones of both SJSA‐1 and NGP cell lines. Conclusions: As with other therapeutic agents, treatment with MDM2‐p53 binding antagonists is likely to be subject to the development of drug resistance mechanisms. A panel of clones selected for resistance to Nutlin‐3 and MI63 showed impaired activation of growth inhibitory and apoptotic pathways downstream of p53 in response to treatment with these agents compared to parental cells. DNA sequencing and p53 pathway analysis were consistent with a p53 mutation resistance mechanism. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A154.