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PDF file - 227K, Hdm2 promotes the binding of acetylated p53 to chromatin when p14ARF is present

PDF file - 178K, Tenovin-D3 causes cell cycle arrest in SirT2 wild type but not knockout cells.

PDF file - 38K, Inhibition of purified SirT1 and SirT2 by tenovin-D3 using FdL substrates at 15 μM.

Acetylation of C-terminal lysine residues in the p53 tumor suppressor is associated with increased stability and transcription factor activity. The function, protein level, and acetylation of p53 are downregulated by mdm2, which in its turn is inhibited by the p14ARF tumor suppressor. Here, we show that p14ARF increases the level of p53 acetylated at lysine 382 in a nuclear chromatin-rich fraction. Unexpectedly, this accumulation of p53AcK382 is dramatically enhanced in the presence of ectopic mdm2. In light of these observations, we propose that p14ARF increases the binding of p53–mdm2 complexes to chromatin, thereby limiting the access of protein deacetylases to p53. Supporting this notion, we show that p53AcK382 can be deacetylated in the cytoplasm and that sirtuin SirT2 catalyzes this reaction. These results help understand why inhibition of both SirT1 and SirT2 is needed to achieve effective activation of p53 by small-molecule sirtuin inhibitors. Mol Cancer Ther; 12(4); 471–80. ©2013 AACR.

PDF file - 179K, Ectopic hdm2 does not increase p53-dependent transcription in the presence of p14ARF

PDF file - 114K, SirT1 downregulates total and nuclear acetylated p53

PDF file - 18K, Retention time of tenovin-D3 = 0.61 minutes.

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PDF file - 42K, MDAMB468 cells were treated with the indicated amounts of TSA for 6 hours.

PDF file - 129K, Co-expression mdm2 and p14ARF increases acetylated p53 in the nuclear fraction without affecting the level of SirT1 protein

PDF file - 139K, Cell lines with differing p53 status were treated with TSA for 6 hours.

PDF file - 30K, Tenovin-6 and tenovin-D3 are poor inhibitors of HDAC8 and SirT3.

While small-molecule inhibitors of class I/II histone deacetylases (HDAC) have been approved for cancer treatment, inhibitors of the sirtuins (a family of class III HDACs) still require further validation and optimization to enter clinical trials. Recent studies show that tenovin-6, a small-molecule inhibitor of sirtuins SirT1 and SirT2, reduces tumor growth in vivo and eliminates leukemic stem cells in a murine model for chronic myelogenous leukemia. Here, we describe a tenovin analogue, tenovin-D3, that preferentially inhibits sirtuin SirT2 and induces predicted phenotypes for SirT2 inhibition. Unlike tenovin-6 and in agreement with its weak effect on SirT1 (a p53 deacetylase), tenovin-D3 fails to increase p53 levels or transcription factor activity. However, tenovin-D3 promotes expression of the cell-cycle regulator and p53 target p21WAF1/CIP1 (CDKN1A) in a p53-independent manner. Structure–activity relationship studies strongly support that the ability of tenovin-D3 to inhibit SirT2 contributes to this p53-independent induction of p21. The ability of tenovin-D3 to increase p21 mRNA and protein levels is shared with class I/II HDAC inhibitors currently used in the clinic and therefore suggests that SirT2 inhibition and class I/II HDAC inhibitors have similar effects on cell-cycle progression. Mol Cancer Ther; 12(4); 352–60. ©2013 AACR.

PDF file - 163K, Supplementary Figure S1A. 1H NMR spectrum of tenovin-D3. Supplementary Figure S1B. 13C NMR spectrum of tenovin-D3.

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PDF file - 55K, Cell lines with differing p53 status were treated with tenovin-D3 for 8 hours.

ML, CD, IvL, GP, TM, SD, MS, APF, CT, DL, MAH, KL and SL: project grants from the Swedish Research Council, the Swedish Cancer Society and the Swedish Childhood Cancer Foundation. MHi and JC: Cancer Research UK (C8/A6613). MC, EP and WE: Wellcome Trust (073915). MN and BV: projects MEYS-NPS-LO1413 and GACR P206/12/G151. EMC, MP, MMS, ZF and PG: Norwegian Cancer Society (182735, 732200) and Helse Vest (911884, 911789). RB and SC: NIH (R01 CA95684), the Leukemia and Lymphoma Society and the Waxman Foundation. NW, AH, Ad’H: Cancer Research UK (C21383/A6950) and Engineering and Physical Sciences Research Council Doctoral Training Program. JL and YZ: Cancer Research UK (C240/A15751). MH and BW: SARomics Biostructures ABUY, KF: DDDP SciLife, Sweden. LJ, MHa, RS and A-LG: CBCS, Sweden. VP: SciLife fellowship. AT: Breast Cancer Research Scotland. The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect. Publisher PDF

The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect., Activation of the tumor suppressor p53 is a promising approach in cancer therapy. Here, the authors discover a series of small molecule dihydroorotate dehydrogenase (DHODH) inhibitors that increase p53 synthesis and reduce tumor growth in synergy with the common mdm2 inhibitor nutlin3.

// Marijke C.C. Sachweh 1,* , William C. Stafford 2,* , Catherine J. Drummond 1 , Anna R. McCarthy 1 , Maureen Higgins 3 , Johanna Campbell 3 , Bertha Brodin 4 , Elias S.J. Arner 2 and Sonia Lain 1 1 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 2 Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 3 Centre for Oncology and Molecular Medicine, University of Dundee, Ninewells Hospital and Medical School, Dundee, Tayside, United Kingdom 4 Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden * These authors have contributed equally to this work Correspondence to: Marijke C.C. Sachweh, email: // Keywords : malignant melanoma, auranofin, vemurafenib, thioredoxin reductase 1, p53 Received : December 15, 2014 Accepted : April 23, 2015 Published : May 12, 2015 Abstract Malignant melanoma is the most dangerous type of skin cancer. Although recent progress in treatment has been achieved, lack of response, drug resistance and relapse remain major problems. The tumor suppressor p53 is rarely mutated in melanoma, yet it is inactive in the majority of cases due to dysregulation of upstream pathways. Thus, we screened for compounds that can activate p53 in melanoma cells. Here we describe effects of the small molecule MJ25 (2-{[2-(1,3-benzothiazol-2-ylsulfonyl)ethyl]thio}-1,3-benzoxazole), which increased the level of p53-dependent transactivation both as a single agent and in combination with nutlin-3. Furthermore, MJ25 showed potent cytotoxicity towards melanoma cell lines, whilst having weaker effects against human normal cells. MJ25 was also identified in an independent screen as an inhibitor of thioredoxin reductase 1 (TrxR1), an important selenoenzyme in the control of oxidative stress and redox regulation. The well-characterized TrxR inhibitor auranofin, which is FDA-approved and currently in clinical trials against leukemia and a number of solid cancers, displayed effects comparable with MJ25 on cells and led to eradication of cultured melanoma cells at low micromolar concentrations. In conclusion, auranofin, MJ25 or other inhibitors of TrxR1 should be evaluated as candidate compounds or leads for targeted therapy of malignant melanoma.

This work was supported by five grants to Sonia Laín: Vetenskapsrådet (VR) 521-2014-3341, Cancerfonden (Swedish Cancer Society) 150393, CAN 2014/702, Association for International Cancer Research (AICR) 130086, Barncancerfonden (Swedish Childhood Cancer Foundation) TJ-2014-0038, Barncancerfonden (Swedish Childhood Cancer Foundation) PR-2014-0038; two grants to Ravi Bhatia: Leukemia and Lymphoma Society (LLS) 6137-14 and NIH R01 CA95684; one grant to David P Lane: Vetenskapsrådet (VR) 538-2013-8807; one grant to Marcus J G W Ladds: Karolinska Institute KID Doctoral Student Funding; one grant to Gergana Popova: Karolinska Institutet KID Doctoral Student Funding; two grants to Nicholas J Westwood: Cancer Research UK C21383 and Cancer Research UK A6950; two grants to Gerald McInerney: Vetenskapsrådet (VR) 621-2014-4718 and Cancerfonden (Swedish Cancer Society) 150393, CAN 2015/751; and four grants to Emmet McCormack: Kreftforeningen 182735, Kreftforeningen 732200, Halse Vest 911884, Halse Vest 911789. Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors. Publisher PDF

While small-molecule inhibitors of class I/II histone deacetylases (HDAC) have been approved for cancer treatment, inhibitors of the sirtuins (a family of class III HDACs) still require further validation and optimization to enter clinical trials. Recent studies show that tenovin-6, a small-molecule inhibitor of sirtuins SirT1 and SirT2, reduces tumor growth in vivo and eliminates leukemic stem cells in a murine model for chronic myelogenous leukemia. Here, we describe a tenovin analogue, tenovin-D3, that preferentially inhibits sirtuin SirT2 and induces predicted phenotypes for SirT2 inhibition. Unlike tenovin-6 and in agreement with its weak effect on SirT1 (a p53 deacetylase), tenovin-D3 fails to increase p53 levels or transcription factor activity. However, tenovin-D3 promotes expression of the cell-cycle regulator and p53 target p21WAF1/CIP1 (CDKN1A) in a p53-independent manner. Structure–activity relationship studies strongly support that the ability of tenovin-D3 to inhibit SirT2 contributes to this p53-independent induction of p21. The ability of tenovin-D3 to increase p21 mRNA and protein levels is shared with class I/II HDAC inhibitors currently used in the clinic and therefore suggests that SirT2 inhibition and class I/II HDAC inhibitors have similar effects on cell-cycle progression. Mol Cancer Ther; 12(4); 352–60. ©2013 AACR.

Acetylation of C-terminal lysine residues in the p53 tumor suppressor is associated with increased stability and transcription factor activity. The function, protein level, and acetylation of p53 are downregulated by mdm2, which in its turn is inhibited by the p14ARF tumor suppressor. Here, we show that p14ARF increases the level of p53 acetylated at lysine 382 in a nuclear chromatin-rich fraction. Unexpectedly, this accumulation of p53AcK382 is dramatically enhanced in the presence of ectopic mdm2. In light of these observations, we propose that p14ARF increases the binding of p53–mdm2 complexes to chromatin, thereby limiting the access of protein deacetylases to p53. Supporting this notion, we show that p53AcK382 can be deacetylated in the cytoplasm and that sirtuin SirT2 catalyzes this reaction. These results help understand why inhibition of both SirT1 and SirT2 is needed to achieve effective activation of p53 by small-molecule sirtuin inhibitors. Mol Cancer Ther; 12(4); 471–80. ©2013 AACR . See related article by McCarthy et al., [p. 352][1] [1]: /lookup/volpage/12/352?iss=4

The tenovins are small molecule inhibitors of the NAD(+)-dependent family of protein deacetylases known as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible applications in both cancer and neurodegenerative disease therapy. Through the synthesis of novel tenovin analogues, further insights into the structural requirements for activity against the sirtuins in vitro are provided. In addition, the activity of one of the analogues in cells led to an improved understanding of the function of SirT1 in cells.

The tenovins and cambinol are two classes of sirtuin inhibitor that exhibit antitumor activity in preclinical models. This report describes modifications to the core structure of cambinol, in particular by incorporation of substitutents at the N1-position, which lead to increased potency and modified selectivity. These improvements have been rationalized using molecular modeling techniques. The expected functional selectivity in cells was also observed for both a SIRT1 and a SIRT2 selective analog.

We have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest. Postprint

4′-Substituted bicyclic pyridones were prepared and evaluated as non-steroidal inhibitors of type 1 and 2 steroid 5α-reductase (SR). A range of 4′-substituents were incorporated into the bicyclic scaffold to investigate SAR within and across different classes of non-steroidal inhibitors of SR. Bicyclic pyridones containing a 4′-benzoyl or long carbon chain tether showed more potent inhibition against type 1 SR than inhibitors with N-substituted acetamide groups in the 4′-position. SAR derived from 4′-substituted bicyclic pyridones reported here do not correlate with SAR derived from known potent 4′-substituted biaryl acid SR inhibitors. A 4′-benzoyl group is favoured by the active site in both isozymes.

There is concern that insecticides are able to mimic the action of 17beta-estradiol by interaction with the human estrogen receptor. Pyrethroids are commonly used insecticides and several have been assessed for potential endocrine disrupting activity by various methods. It has been noted that some metabolites of pyrethroids, in particular, permethrin and cypermethrin, have chemical structures that are more likely to interact with the cellular estrogen receptor than the parent pyrethroid. For this study permethrin and cypermethrin metabolites 3-(4-hydroxy-3-phenoxy)benzyl alcohol, 3-(4-hydroxy-3-phenoxy)benzoic acid, and N-3-(phenoxybenzoyl)glycine were synthesised, and together with the commercially available 3-phenoxybenzyl alcohol, 3-phenoxybenzaldehyde, and 3-phenoxybenzoic acid, were studied in a recombinant yeast assay expressing human estrogen receptors (YES). Three metabolites, 3-phenoxybenzyl alcohol, 3-(4-hydroxy-3-phenoxy)benzyl alcohol, and 3-phenoxybenzaldehyde, showed estrogenic activity of approximately 10(5) less than that of 17beta-estradiol. No activity was observed in the yeast assay for 3-phenoxybenzoic acid, 3-(4-hydroxy-3-phenoxy)benzoic acid, and N-3-(phenoxybenzoyl)glycine. The results from this study show that pyrethroid metabolites are capable of interacting with the human estrogen receptor, and so might present a risk to human health and environmental well being. The impact would be expected to be small, but still add to the overall environmental xenoestrogen load.

This work was supported by the Hallas Møller Investigator grant from the Novo Nordisk Foundation to C.C. S.A.W. and P.B. were supported by individual postdoctoral grants from the Danish Research Council (FSS: 10-085134, FSS: 12-12610). C.C. is supported by the EMBO Young Investigator program. J.E.B. is supported by a grant from the Doris Duke Charitable Foundation. Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases. Postprint

The search for potent and selective sirtuin inhibitors continues as chemical tools of this type are of use in helping to assign the function of this interesting class of deacetylases. Here we describe SAR studies starting from the unselective sirtuin inhibitor tenovin-6. These studies identify a sub-micromolar inhibitor that has increased selectivity for SIRT2 over SIRT1 compared to tenovin-6. In addition, a H-1-NMR-based method is developed and used to validate further this class of sirtuin inhibitors. A thermal shift analysis of SIRT2 in the presence of tenovin-6, -43, a control tenovin and the known SIRT2 inhibitor AGK2 is also presented. Publisher PDF

Recent advances in the field of pharmacological activation of the p53 tumor suppressor are beginning to be translated into the clinic. In addition, small molecules that activate p53 through established mechanisms of action are proving invaluable tools for basic research. Here we analyze and compare the effects of nutlin-3, tenovin-6 and low doses of actinomycin-D on p53 and its main negative regulator, mdm2. We reveal striking differences in the speed at which these compounds increase p53 protein levels, with nutlin-3 having a substantial impact within minutes. We also show that nutlin-3 is very effective at increasing the synthesis of mdm2 mRNA, mdm2 being not only a modulator of p53 but also a transcriptional target. In addition, we show that nutlin-3 stabilizes mdm2's conformation and protects mdm2 from degradation. These strong effects of nutlin-3 on mdm2 correlate with a remarkable rate of recovery of p53 levels upon removal of the compound. We discuss the potential application of our results as molecular signatures to assess the on-target effects of small-molecule mdm2 inhibitors. To conclude, we discuss the implications of our observations for using small-molecule p53 activators to reduce the growth of tumors retaining wild-type p53 or to protect normal tissues against the undesired side effects of conventional chemotherapy.

The reactivation of mutant forms of the transcriptional regulator p53 or artificially raising activated p53 levels in a controlled nongenotoxic manner are seen as two of the grand challenges in anti-cancer drug discovery. Recent reports suggest that these demanding goals are achievable. This review article focuses on the use of cell-based high-throughput screening to discover novel nongenotoxic activators of endogenous p53. This challenging approach to the early phases of drug discovery prioritises the discovery of compounds with activity in cells in the hope that the compounds discovered will ultimately be of more direct relevance to therapeutic development. However, this approach also requires that protein target identification studies are carried out. We, and others, have shown that whilst a sometimes daunting proposition, it is possible to identify the targets of compounds that activate p53.

A robust p53 cell-based assay that exploits p53's function as a transcription factor was used to screen a small molecule library and identify bioactive small molecules with potential antitumor activity. Unexpectedly, the majority of the highest ranking hit compounds from this screen arrest cells in mitosis and most of them impair polymerization of tubulin in cells and in vitro. One of these novel compounds, JJ78:1, was subjected to structure-activity relationship studies and optimized leading to the identification of JJ78:12. This molecule is significantly more potent than the original hit JJ78:1, as it is active in cells at two-digit nanomolar concentrations and shows clear antitumor activity in a mouse xenograft model as a single agent. The effects of nocodazole, a well established tubulin poison, and JJ78:12 on p53 levels are remarkably similar, supporting that tubulin depolymerization is the main mechanism by which JJ78:12 treatment leads to p53 activation in cells. In summary, these results identify JJ78:12 as a potential cancer therapeutic, demonstrate that screening for activators of p53 in a cell-based assay is an effective way to identify inhibitors of mitosis progression and highlights p53's sensitivity to alterations during mitosis.