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The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.

Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers., Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 μM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 μM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 μM (Nek2); GI50 (SKBR3) 2.2 μM] which exhibited >5–10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 μM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 μM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.

Inhibition of CDKs is an attractive approach to cancer therapy due to their vital role in cell growth and transcription. Pan-CDK inhibitors have shown some clinical benefit, and trials are ongoing. Selective CDK4 and CDK6 inhibitors have been licensed for the treatment of hormone responsive, RB-positive breast cancer in combination with antihormonal agents. Selective inhibitors of CDKs 5, 7, 8, 9 and 12 have been identified across a range of chemotypes.

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 μM for ERK5; IC50 > 120 μM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.

Purines and related heterocycles substituted at C-2 with 4’-sulfamoylanilino and at C-6 with a variety of groups have been synthesized with the aim of achieving selectivity of binding to CDK2 over CDK1. 6-Substituents that favour competitive inhibition at the ATP binding site of CDK2 were identified and typically exhibited 10-80-fold greater inhibition of CDK2 compared to CDK1. Most impressive was 4-((6-([1,1'-biphenyl]-3-yl)-9H-purin-2-yl)amino) benzenesulfonamide (73) that exhibited high potency towards CDK2 (IC50 0.044 μM), but was ~ 2000-fold less active towards CDK1 (IC50 86 μM). This compound is therefore a useful tool for studies of cell cycle regulation. Crystal structures of inhibitor-kinase complexes showed that the inhibitor stabilizes a glycine-rich loop conformation that shapes the ATP ribose binding pocket, and that is preferred in CDK2, but has not been observed in CDK1. This aspect of the active site may be exploited for the design of inhibitors that distinguish between CDK1 and CDK2.

Potent and selective small-molecule inhibitors of the p53-MDM2 interaction intended for the treatment of p53 wild-type tumors have been designed and optimized in a number of chemical series. This review details recent disclosures of compounds in advanced optimization and features key series that have given rise to clinical trial candidates. The structure–activity relationships for inhibitor classes are discussed with reference to x-ray structures, and common structural features are identified.

Structure-guided expansion of a fragment hit for the ATAD2 bromodomain enabled improvement in ATAD2 inhibition and selectivity over BRD4., ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure–activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.

BACKGROUND The emergence of novel psychoactive substances (NPS), particularly synthetic cannabinoid receptor agonists (SCRA), has involved hundreds of potentially harmful chemicals in a highly dynamic international market challenging users', clinicians', and regulators' understanding of what circulating substances are causing harm. We describe a toxicovigilance system for NPS that predicted the UK emergence and identified the clinical toxicity caused by novel indole and indazole carboxylate SCRA. METHODS To assist early accurate identification, we synthesized 5 examples of commercially unavailable indole and indazole carboxylate SCRA (FUB-NPB-22, 5F-NPB-22, 5F-SDB-005, FUB-PB-22, NM-2201). We analyzed plasma and urine samples from 160 patients presenting to emergency departments with severe toxicity after suspected NPS use during 2015 to 2016 for these and other NPS using data-independent LC-MS/MS. RESULTS We successfully synthesized 5 carboxylate SCRAs using established synthetic and analytical chemistry methodologies. We identified at least 1 SCRA in samples from 49 patients, including an indole or indazole carboxylate SCRA in 17 (35%), specifically 5F-PB-22 (14%), FUB PB-22 (6%), BB-22 (2%), 5F NPB-22 (20%), FUB NPB-22 (2%), and 5F-SDB-005 (4%). In these 17 patients, there was analytical evidence of other substances in 16. Clinical features included agitation and aggression (82%), reduced consciousness (76%), acidosis (47%), hallucinations and paranoid features (41%), tachycardia (35%), hypertension (29%), raised creatine kinase (24%), and seizures (12%). CONCLUSIONS This toxicovigilance system predicted the emergence of misuse of indole and indazole carboxylate SCRA, documented associated clinical harms, and notified relevant agencies. Toxicity appears consistent with other SCRA, including mental state disturbances and reduced consciousness.

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.

The study of cellular mechanisms of cancer growth and survival has revealed a complex web of signalling pathways that produce the ‘hallmarks of cancer’. A number of critical cellular signalling nodes have been characterised from which many new drug targets have been proposed. A number of these nodes are regulated by protein–protein interactions, and so present attractive, if challenging, targets for drug discovery. Drug discovery efforts towards three PPI families have resulted in clinically investigated drugs: i.e. Bcl-2 family inhibitors, MDM2-p53 inhibitors and IAP inhibitors. The discovery and optimisation of these drug classes are detailed in this chapter.

Background: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. Methods: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. Results: 2,6-Diaminopyrimidines with an O4-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50

International audience; The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57 617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure−activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

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.

The interaction between murine double minute (MDM2) and p53 is a major target in anticancer drug design. Several potent compound series, including the nutlins and spirooxindoles, have previously been established as high-affinity antagonists of MDM2. In this paper, we describe the interaction of isoindolinone inhibitors with MDM2, as characterized by nuclear magnetic resonance spectroscopy. Isoindolinone inhibitors bind specifically to the MDM2 p53 binding site and exploit all sub-pockets used by p53, nutlins and spirooxindoles. Furthermore, isoindolinones bind with low micromolar to high nanomolar affinities, with the best compound approaching the potency of nutlin-3.

In response to cellular stress, the p53 tumor suppressor is activated to modulate cell cycle progression, DNA repair, and cell death. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 can therefore reactivate p53 and elicit an anti-cancer effect. Small molecule inhibitors of the MDM2-p53 interaction remains a promising strategy for cancer therapy and a number of these compounds are in clinical development. An isoindolinone series, identified by the Northern Institute for Cancer Research (NICR), has been used as a starting point for the development of potent MDM2-p53 inhibitors. Structure based drug design was applied during lead optimisation to gain potency whilst also focusing on stabilizing the main metabolically labile position and reducing lipophilicity. This approach led to potent compounds with EC50 Citation Format: Lynsey Fazal, Maria Ahn, Luke Bevan, Ildiko Buck, Juan Castro, Gianni Chessari, Ben Cons, Keisha Hearn, Steven Howard, Chris Johnson, Judith Reeks, Emiliano Tamanini, Neil Thompson, Hugh Walton, Pamela Williams, Ruth H. Bawn, Tim J. Blackburn, Celine Cano, Sarah J. Cully, Bernard Golding, Roger Griffin, Karen Haggerty, Ian Hardcastle, Herbie Newell, Martin Noble, Huw Thomas, Elaine Willmore, Yan Zhao, Steve Wedge. Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1652.

We investigated the cellular response to two MDM2-p53 antagonists (RG7388 and Cpd 1) in a panel of 19 tumor cell lines and in patient-derived chronic lymphocytic leukemia (CLL) cells. RG7388 (Idasanutlin) is an MDM2-p53 antagonist developed by Roche and Cpd1 a novel potent and selective MDM2-p53 antagonist developed as part of an ongoing Alliance between Newcastle University, Cancer Research UK and Astex Pharmaceuticals. RG7388 and Cpd1 potently inhibited the proliferation of many cell lines, with MDM2-amplified SJSA-1 osteosarcoma cells and cells from haematological malignancies being the most sensitive (e.g. GI50 values in the acute myeloid leukemia cell line MOLM13 were 33 + 16 nM for RG7388 and 8 + 1 nM for Cpd 1). We also examined induction of apoptosis by measuring caspase 3/7 activation (24 h treatment). SJSA-1 cells showed a 20-fold increase in caspase activation and Molm13 cells a 6-fold increase (measured at 300nM with RG7388). However, cell lines from solid tumours such as the colorectal carcinoma HCT116 or the hepatocellular carcinoma HepG2 did not show any induction of apoptosis, even at concentrations up to 1µM RG3788. In primary CLL cells, Cpd 1 reduced cell viability (48 h) with an average LC50 of 131 + 46 nM (n = 4), compared to 400 + 55 nM for RG7388 (n =23). We also quantified mRNA levels in 25 primary CLL samples and found that RG7388 induced a predominantly pro-apoptotic gene signature: following 6h treatment, 1µM RG7388 resulted in an average 8-fold induction of PUMA and 3.5-fold increase in BAX compared to a 1.6-fold induction of CDKN1A (p21). Our data confirm that targeting the MDM2-p53 interaction is an effective strategy to inhibit growth and viability of tumor cells, and that some cells, including those expressing high levels of MDM2 or those derived from hematological malignancies, display a striking apoptotic response. Citation Format: Elaine Willmore, Yan Zhao, Carmela Ciardullo, Laura Woodhouse, Huw D. Thomas, Maria Ahn, Lynsey Fazal, Martin E. Noble, Ian Hardcastle, Steven Howard, Gianni Chessari, John Lunec, Steve Wedge. The anti-proliferative and pro-apoptotic effect of MDM2-p53 antagonists evaluated in human tumor cells lines and chronic lymphocytic leukemia patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1870.

Cyclin-dependent kinases (CDK) play a crucial role in the control of the cell cycle. Aberrations in the control of cell cycle progression occur in the majority of human malignancies; hence, CDKs are promising targets for anticancer therapy. Here, we define the cellular effects of the novel CDK inhibitor NU6140, alone or in association with paclitaxel, with respect to inhibition of cell proliferation and cell cycle progression and induction of apoptosis in HeLa cervical carcinoma cells and in comparison with purvalanol A. Both CDK inhibitors induced a concentration-dependent cell cycle arrest at the G2-M phase and an increase in the apoptotic rate, with a concomitant down-regulation of the antiapoptotic protein survivin, a member of the inhibitors of apoptosis protein family. Notably, the addition of NU6140 to paclitaxel-treated cells resulted in markedly increased cytotoxic effect and apoptotic response in comparison with the paclitaxel-purvalanol A combination (86 ± 11% and 37 ± 8%, respectively). Similarly, the extent of caspase-9 and caspase-3 activation in paclitaxel-NU6140–treated cells was ∼4-fold higher than after the paclitaxel-purvalanol A combination. Moreover, an almost complete abrogation of the expression of the active, Thr34-phosphorylated form of survivin was observed in cells exposed to the paclitaxel-NU6140 combination. A synergistic effect of the paclitaxel-NU6140 combination, as a consequence of survivin inhibition and increased activation of caspase-9 and caspase-3, was also observed in OAW42/e ovarian cancer line but not in the derived OAW42/Surv subline ectopically expressing survivin. Results from this study indicate that NU6140 significantly potentiates the apoptotic effect of paclitaxel, with inhibition of survivin expression/phosphorylation as the potential mechanism.

A series of 2-N-alkyl-3-aryl-3-alkoxyisoindolinones has been synthesised and evaluated as inhibitors of the MDM2-p53 interaction. The most potent compound, 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (NU8231), exhibited an IC50 of 5.3 +/- 0.9 microM in an ELISA assay, and induced p53-dependent gene transcription in a dose-dependent manner, in the SJSA human sarcoma cell line.

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and these arise through metabolism at the alpha-position to give an ester which reacts with DNA. (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-but-1-en e (idoxifene) is an analogue of tamoxifen in which formation of DNA adducts is greatly reduced; we could not detect any adducts in the DNA of cultured rat hepatocytes treated with 10 microM idoxifene, after analysis by the 32P-post-labelling method. The metabolite (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3-but en-2-ol (alpha-hydroxyidoxifene) gave adducts in rat hepatocytes, but far fewer than the corresponding tamoxifen metabolite. In human hepatocytes, neither idoxifene nor tamoxifen induced detectable levels of DNA adducts. We prepared the alpha-acetoxy ester of idoxifene as a model for the ultimate reactive metabolite formed in rat liver. It was less reactive than alpha-acetoxytamoxifen, as might be expected on mechanistic grounds. It reacted with DNA in the same way, to give adducts which were probably N2-alkyldeoxyguanosines, but to a lower extent. All these results indicate that idoxifene is much less genotoxic than tamoxifen, and should therefore be a safer drug.

Evaluation of the effects of purine C-8 substitution within a series of CDK1/2-selective O(6)-cyclohexylmethylguanine derivatives revealed that potency decreases initially with increasing size of the alkyl substituent. Structural analysis showed that C-8 substitution is poorly tolerated, and to avoid unacceptable steric interactions, these compounds adopt novel binding modes. Thus, 2-amino-6-cyclohexylmethoxy-8-isopropyl-9H-purine adopts a "reverse" binding mode where the purine backbone has flipped 180°. This provided a novel lead chemotype from which we have designed more potent CDK2 inhibitors using, in the first instance, quantum mechanical energy calculations. Introduction of an ortho-tolyl or ortho-chlorophenyl group at the purine C-8 position restored the potency of these "reverse" binding mode inhibitors to that of the parent 2-amino-6-cyclohexylmethoxy-9H-purine. By contrast, the corresponding 8-(2-methyl-3-sulfamoylphenyl)-purine derivative exhibited submicromolar CDK2-inhibitory activity by virtue of engineered additional interactions with Asp86 and Lys89 in the reversed binding mode, as confirmed by X-ray crystallography.

The metabolic activation of tamoxifen and its metabolite alpha-hydroxytamoxifen in primary cultures of rat, mouse and human hepatocytes has been compared. The extent of formation of DNA adducts in these cells was measured by 32P-postlabelling, using either nuclease P1 digestion or sorbent extraction of DNA digests to enhance the sensitivity of the assay. DNA adducts were readily detected in rat hepatocytes treated with 1 or 10 microM tamoxifen (mean levels 18.2 and 89.8 adducts/10(8) nucleotides respectively). Similar levels of adducts were formed by mouse hepatocytes (15.0 +/- 1.8 adducts/10(8) nucleotides, 10 microM tamoxifen). However DNA adducts were not detected in tamoxifen-treated human hepatocytes with a detection limit for the assay of 4 adducts/10(10) nucleotides. Treatment of rat cells with alpha-hydroxytamoxifen resulted in 15- to 63-fold higher levels of adducts than with comparable concentrations of tamoxifen. A similar level of adducts was also seen in mouse hepatocytes treated with alpha-hydroxytamoxifen at the 1 microM concentration (173.9 +/- 4.1 adducts/10(8) nucleotides). Treatment of human cells with alpha-hydroxytamoxifen resulted in DNA adduct formation at levels (1.94 +/- 0.89 and 18.9 +/- 17.9 adducts/10(8) nucleotides at 1 and 10 microM respectively) approximately 300-fold lower than those in rat hepatocytes. The presence of alpha-hydroxytamoxifen in the culture medium from experiments where cells were incubated with tamoxifen was monitored by mass spectrometry. Concentrations were found to be approximately 50-fold lower in the medium from human hepatocytes than from rat and mouse hepatocytes.

Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an in vitro HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC(50) 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8-27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer.

The small-molecule compound pifithrin-α (PFT-α) has been reported to inhibit p53 function and protect against a variety of genotoxic agents. We show here that PFT-α is unstable in tissue culture medium and is rapidly converted to its condensation product PFT-β. Both compounds showed limited solubility with PFT-α precipitating out of tissue culture medium at concentrations >30 μmol/L. PFT-α and -β exhibited cytotoxic effects in vitro towards two human wild-type p53–expressing tumor cell lines, A2780 ovarian and HCT116 colon (IC50 values for both cell lines were 21.3 ± 8.1 μmol/L for PFT-α and 90.3 ± 15.5 μmol/L for PFT-β, mean ± SD, n = 4). There was no evidence of protection by clonogenic assay with either compound in combination with ionizing radiation. Indeed, there was some evidence that PFT-α enhanced cytotoxicity, particularly at higher concentrations of PFT-α. Neither compound had any effect on p53, p21, or MDM-2 protein expression following ionizing radiation exposure and there was no evidence of any abrogation of p53-dependent, ionizing radiation–induced cell cycle arrest. Similarly, there was no evidence of cellular protection, or of effects on p53-dependent gene transcription, or on translation of MDM-2 or p21 following UV treatment of these human tumor cell lines. In addition, there was no effect on p53 or p21 gene transactivation or p38 phosphorylation after UV irradiation of NIH-3T3 mouse fibroblasts. In conclusion, neither PFT-α nor -β can be regarded as a ubiquitous inhibitor of p53 function, and caution should be exercised in the use of these agents as specific p53 inhibitors.

Pharmacological antagonism of the interaction between MDM2 and/or MDMX with p53 has been pursued as a therapeutic strategy to restore p53 tumour suppressor activity in wild type TP53 tumours with amplification or over-expression of these negative regulators. The majority of small molecule approaches described to date inhibit MDM2 binding to p53, which has E3 ligase activity and through ubiquitination targets p53 for degradation. However, MDMX, although devoid of E3 ligase activity itself, forms heterodimers with MDM2 and augments its activity to induce polyubiquitination of p53. Further complexity in this regulatory network arises from the fact that MDM2 is a target gene of p53 transcription, and that MDM2 can also control the stability of MDMX. Since MDMX is widely reported to confer resistance to established MDM2:p53 antagonists, this study aimed to examine the activity of MDM2:p53 selective inhibitors across a panel of wild-type TP53 cell lines with variable MDM2 and MDMX protein expression, and to contrast this with recently described inhibitors with mixed MDM2:p53 and MDMX:p53 activity. Growth inhibition studies (72h) were performed using XTT assays, but with verification by orthogonal methods (SRB, clonogenic assays). Gene expression was determined at the level of mRNA and protein. The selectivity of all inhibitors was confirmed initially in p53 wild-type and non-functional paired cell lines. As anticipated, MDM2:p53 selective inhibitors (nutlin-3a, MI-773, RG7112) were active in MDM2 amplified cell lines with low MDMX expression (SJSA-1 and T778; GI50 range 0.2 to 1 uM). However, a similar level of activity was also evident in cell lines with low/medium MDM2 in the presence of medium/high MDMX expression (Pre B 697, A375, HCT116, MRK-NU1, and NGP cells: GI50 range 0.2 to 2 uM). Appreciable resistance was only evident in Jeg3 and MCF-7 cells (low MDM2 and high MDMX), averaged compound GI50 values ranging from 9 - 10 uM in Jeg3 and 6 - 14 uM in MCF-7. Whilst a concentration dependent degradation of MDMX was observed in some sensitive cell lines with medium/high MDMX expression, in others no change was evident, suggesting that there are multiple determinants of cellular sensitivity to such inhibitors. Importantly, the mixed MDM2:p53 and MDMX:p53 antagonist RO-5963, displayed a different spectrum of activity, with least growth inhibition evident in SJSA-1 and T778 MDM2 amplified cell lines, and activity being retained in MDMX amplified Jeg3 cells. These studies support the concept that targeting MDMX may provide a different spectrum of antitumour activity to selective MDM2:p53 inhibition. However, MDMX protein levels per se do not constitute a universal resistance factor to selective MDM2:p53 inhibitors and, beyond MDM2 amplification, the identification of additional markers of sensitivity is warranted for this class of agent. Citation Format: Sue Tudhope, Yan Zhao, Anita Wittner, Elaine Wilmore, Annalisa Bertoli, Santosh Adhikari, Suzannah J. Harnor, Fabio Del Bello, Alessandro Piergentili, John Lunec, Ian R. Hardcastle, Roger J. Griffin, Bernard R. Golding, Celine Cano, David R. Newell, Stephen R. Wedge. Profiling inhibitors of MDM2:p53 and MDMX:p53 in relation to MDMX protein levels. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2014-5451

The drug tamoxifen shows evidence of genotoxicity and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen and these arise, at least in part, from its metabolite alpha-hydroxytamoxifen. This probably undergoes conjugation in the liver tissue to give an ester, which alkylates DNA. We have prepared alpha-acetoxytamoxifen as a model for this reactive intermediate and studied its reaction with DNA in vitro. The products of this reaction were chromatographically identical to DNA adducts found in the liver of rats treated with tamoxifen. We have isolated three of these products as the nucleosides TG1, TG2 and TA1 and identified them by ultraviolet, mass and proton magnetic resonance spectroscopy. TG1 and TG2 were tamoxifen-deoxyguanosine adducts in which the alpha-position of tamoxifen was linked to the amino group of guanine; TG1, (E)-4-[4-[2-(dimethylamino)ethoxy]phenyl]-3,4-diphenyl-2-(9beta-de oxyribofuranosyl-6-oxopurin-2-ylamino)-3-butene; TG2, (Z) isomer of TG1. In TG2, the tamoxifen group had undergone trans-cis isomerization. The minor product TA1 was a tamoxifen-deoxyadenosine adduct, where linkage was through the amino group of adenine: (E)-4-[4-[2-(dimethylamino) ethoxy]phenyl]-3,4-diphenyl-2-(9beta-deoxyribofuranosylpurin -6-ylamino)-3-butene. These three adducts accounted for >90% of the reaction products (approximately 67% TG1, 18% TG2 and 7% TA1); trace products included other stereoisomers of these and dinucleotide adducts which resisted enzymatic digestion.

Screening identified 2-(3-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile as an MDM2–p53 inhibitor (IC50 = 12.3 μM). MDM2–p53 and MDMX–p53 activity was seen for 5-((1-(4-chlorophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (MDM2 IC50 = 0.11 μM; MDMX IC50 = 4.2 μM) and 5-((1-(4-nitrophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (MDM2 IC50 = 0.15 μM; MDMX IC50 = 4.2 μM), and cellular activity consistent with p53 activation in MDM2 amplified cells. Further SAR studies demonstrated the requirement for the triarylpyrrole moiety for MDMX–p53 activity but not for MDM2–p53 inhibition.

The potent and selective MDM2-p53 antagonist 4-chloro-3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one 1 displays promising activity in cellular tumour models; however, poor solubility precludes in vivo investigations.(Bioorg. Med. Chem. Letters 2011, 21, 5916-5919) A water-soluble succinate ester of 1 was designed as a pro-drug, based on an understanding of the interaction of the parent compound 1 with MDM2, and the cellular pharmacology and in vivo pharmacokinetics of the ester 2 were defined. Compound 2 was synthesised and evaluated as an antagonist of MDM2- and MDMX-p53 using ELISA assays. Cell growth inhibition induced by 2 and 1 were compared in paired p53 functional and p53 non-functional HCT116, A2780 and SJSA-1 cell lines. Conversion of 2 into 1 was studied in human and mouse plasma in vitro, as was uptake into tumour cells, by HPLC analysis. The pharmacokinetics of 2 was studied in vivo in tumour-bearing mice. Unexpectedly, the succinate ester 2 was 7-fold more potent than the parent compound 1 as an MDM2-p53 binding antagonist in cell-free assays (IC50 values 20 ± 13 nM versus 95 ± 96 nM, respectively) and maintained >100-fold selectivity for MDM2 over MDMX antagonism. In cell growth inhibition assays (GI50) 2 was 2.3-, 6.1- and 8.6-fold more potent in paired p53 functional versus non-functional HCT116, A2780 and SJSA-1 cell lines, respectively. As anticipated, the solubility of 2 (65 µM) was greatly improved over 1 (6.5 µM), and the succinate ester was converted to the parent compound in mouse plasma (>90% in 60 min), although interestingly not in human plasma. Following incubation of tumour cell lines with 2, both the pro-drug and parent compound could be detected. Administration of 2 to A2780 tumour-bearing mice i.v. at 10 mg/kg generated plasma concentrations approaching those required for cell growth inhibition (≥ 3 μM), and oral administration at 100 mg/kg resulted in 2 bio-availability of 7%, and an 1 AUC that was estimated to be 21% of the equivalent i.v. dose of 2. However, a major unidentified metabolite was observed which compromised the in vivo utility of the succinate ester 2. Protein-protein interaction inhibitors as exemplified by MDM2-p53 binding antagonists are a major new class of antitumour agent. However, targeting hydrophobic protein-protein interactions can result in molecules with poor physicochemical properties. A cleavable succinate ester approach can be applied to address this problem without compromising target interaction or cellular activity, as in the current study. Although additional metabolite pathways may limit the utility of this approach, the improved potency gained by addition of a solubilising group provides valuable information for further lead optimisation. (This work was supported by the CR-UK) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 919. doi:1538-7445.AM2012-919

The cellular response to DNA double-strand break (DSB) formation is an essential component of normal cell survival, following exposure to DNA-damaging chemicals and ionising radiation. The serine/threonine kinase DNA-dependent protein kinase (DNA-PK) is a member of the phosphatidylinositol (PI) 3-kinase related kinase (PIKK) family of enzymes, and plays an important role in DNA DSB repair via the non-homologous end-joining (NHEJ) pathway. DNA-PK inhibitors may, therefore, be useful as agents to improve the activity of radio- and chemo-therapy in the treatment of cancer. Identification of the lead benzo[h]chromen-4-one DNA-PK inhibitor NU7026 (IC50 = 0.23 uM) guided the development of the potent and selective ATP-competitive chromenone NU7441 (DNA-PK IC50 = 30 nM). Structure-activity relationship studies for DNA-PK inhibition by chromenone-derivatives were conducted in conjunction with homology modelling. Library synthesis was undertaken employing a solution multiple-parallel approach, by O-alkylation or N-acylation of the appropriately substituted NU7441 derivatives, respectively, followed by reaction with a range of amines to afford the target compounds. These studies resulted in the identification of the highly potent inhibitor KU-0060648 (IC50 = 8.6 nM). The further development of KU-0060648 and analogues will be described. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5780.

The cellular response to DNA double-strand break (DSB) formation is an essential component of normal cell survival, following exposure to DNA-damaging chemicals and ionising radiation. The serine/threonine kinase DNA-dependent protein kinase (DNA-PK) is a member of the phosphatidylinositol (PI) 3-kinase related kinase (PIKK) family of enzymes, and plays an important role in DNA DSB repair via the non-homologous end-joining (NHEJ) pathway. DNA-PK inhibitors may, therefore, be useful as agents to improve the activity of radio- and chemo-therapy in the treatment of cancer. Identification of the lead benzo[h]chromen-4-one DNA-PK inhibitor NU7026 (IC50 = 0.23 uM) guided the development of the potent and selective ATP-competitive chromenone NU7441 (DNA-PK IC50 = 30 nM). Structure-activity relationship studies for DNA-PK inhibition by chromenone-derivatives were conducted in conjunction with homology modelling. Library synthesis was undertaken employing a solution multiple-parallel approach, by O-alkylation or N-acylation of the appropriately substituted NU7441 derivatives, respectively, followed by reaction with a range of amines to afford the target compounds. These studies resulted in the identification of KU-0060648, which combines high potency as a DNA-PK inhibitor (IC50 = 8.6 nM) with good aqueous solubility as an acid salt. The further development of KU-0060648 and analogues will be described. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A138.

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.

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 [Science 2004, 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.