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Two enzymes involved in the synthesis of pyrimidine and purine nucleotides, CTP synthase (CTPS) and IMP dehydrogenase (IMPDH), can assemble into a single or very few large filaments called rods and rings (RR) or cytoophidia. Most recently, asymmetric cytoplasmic distribution of organelles during cell division has been described as a decisive event in hematopoietic stem cell fate. We propose that cytoophidia, which could be considered as membrane-less organelles, may also be distributed asymmetrically during mammalian cell division as previously described for Schizosaccharomyces pombe. Furthermore, because each type of nucleotide intervenes in distinct processes (e.g., membrane synthesis, glycosylation, and G protein-signaling), alterations in the rate of synthesis of specific nucleotide types could influence cell differentiation in multiple ways. Therefore, we hypothesize that whether a daughter cell inherits or not CTPS or IMPDH filaments determines its fate and that this asymmetric inheritance, together with the dynamic nature of these structures enables plasticity in a cell population.

Drugging the p53 pathway has been a goal for both academics and pharmaceutical companies since the designation of p53 as the ‘guardian of the genome’. Through growing understanding of p53 biology, we can see multiple routes for activation of both wild-type p53 function and restoration of mutant p53. In this review, we focus on small molecules that activate wild-type p53 and that do so in a non-genotoxic manner. In particular, we will describe potential approaches to targeting proteins that alter p53 stability and function through posttranslational modification, affect p53’s subcellular localization, or target RNA synthesis or the synthesis of ribonucleotides. The plethora of pathways for exploitation of p53, as well as the wide-ranging response to p53 activation, makes it an attractive target for anti-cancer therapy.

Summary Dihydroorotate dehydrogenase (DHODH) is essential for the de novo synthesis of pyrimidine ribonucleotides, and as such, its inhibitors have been long used to treat autoimmune diseases and are in clinical trials for cancer and viral infections. Interestingly, DHODH is located in the inner mitochondrial membrane and contributes to provide ubiquinol to the respiratory chain. Thus, DHODH provides the link between nucleotide metabolism and mitochondrial function. Here we show that pharmacological inhibition of DHODH reduces mitochondrial respiration, promotes glycolysis, and enhances GLUT4 translocation to the cytoplasmic membrane and that by activating tumor suppressor p53, increases the expression of GDF15, a cytokine that reduces appetite and prolongs lifespan. In addition, similar to the antidiabetic drug metformin, we observed that in db/db mice, DHODH inhibitors elevate levels of circulating GDF15 and reduce food intake. Further analysis using this model for obesity-induced diabetes revealed that DHODH inhibitors delay pancreatic β cell death and improve metabolic balance., Graphical abstract, Highlights • DHODH inhibitors impair respiration and increase glycolysis. • They promote the expression and secretion of GDF15 in a p53-dependent manner. • In hyperphagic db/db mice, DHODH inhibitors increase circulating GDF15. • They protect pancreatic β cells and improve glucose balance in these mice., Biological sciences ; Physiology ; Cellular physiology ; Endocrinology ; Diabetology

Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the de novo pyrimidine synthesis [Löffler et al., 1997]. Initial clinical studies with the DHODH inhibitor brequinar in solid tumors showed limited efficacy due to dose limiting adverse effects [Peters et al, 2018]. Recently, there is renewed interest in the use of more potent and selective DHODH inhibitors for the treatment of myeloid and lymphoid malignancies [Sykes et al, 2016]. Here we report the identification of the novel potent and highly selective DHODH inhibitor GTX-0196, with excellent physicochemical properties. GTX-0196 exhibited a biochemical IC50 of 3.7 nM in a DHODH biochemical assay and an antiproliferative IC50 of 1.1 nM in the MOLM-13 cell line, with brequinar showing IC50 values of 6.9 and 50 nM, respectively. The activity in the MOLM-13 proliferation assay was uridine-dependent, indicating absence of general toxicity of the compound and confirming DHODH-mediated efficacy. Profiling of GTX-0196 in the BioPrint panel (Eurofins/CEREP, at 10 µM), showed 2000-fold selectivity versus other targets tested). Furthermore, no inhibition was observed in a human CYP panel (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4, at 30 µM) and in a panel of transporters (at 10 µM). High metabolic stability in human liver microsomes and hepatocytes was observed with t½ > 6h and 10h, respectively. Compared to an earlier lead compound, GTX-0196 demonstrated enhanced potency and metabolic stability (particularly in preclinical species). Potent and efficacious inhibition of proliferation through DHODH was confirmed in a large number of solid tumor and hematologic malignancy cell lines, illustrating the broad potential use of this chemical series. Furthermore, tumor growth inhibition was observed in the MOLM-13 xenograft mouse model. Due to its favorable properties, GTX-0196 has progressed towards IND-enabling studies. In parallel, we are exploring mono and combination therapy opportunities in hematological malignancies as well as combination therapy opportunities in solid tumors to investigate the full therapeutic potential in oncology for this optimized DHODH inhibitor. Löffler et al. Mol. Cell. Biochem. 1997Peters GJ et al, Nucleosides, Nucleotides and Nucleic Acids 2018Sykes DB et al. Cell 2016 Citation Format: Allard Kaptein, Sonia Lain, Diede Brunen, Edward van Wezel, Tjeerd Barf. Preclinical pharmacology profile of GTX-0196: A novel, potent and highly selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4049.

Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound

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

Congenital neutropenia is characterized by low absolute neutrophil numbers in blood, leading to recurrent bacterial infections, and patients often require life-long granulocyte CSF (G-CSF) support. X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich syndrome protein (WASp). To understand the pathophysiology in XLN and the role of WASp in neutrophils, we here examined XLN patients and 2 XLN mouse models. XLN patients had reduced myelopoiesis and extremely low blood neutrophil number. However, their neutrophils had a hyperactive phenotype and were present in normal numbers in XLN patient saliva. Murine XLN neutrophils were hyperactivated, with increased actin dynamics and migration into tissues. We provide molecular evidence that the hyperactivity of XLN neutrophils is caused by WASp in a constitutively open conformation due to contingent phosphorylation of the critical tyrosine-293 and plasma membrane localization. This renders WASp activity less dependent on regulation by PI3K. Our data show that the amplitude of WASp activity inside a cell could be enhanced by cell-surface receptor signaling even in the context in which WASp is already in an active conformation. Moreover, these data categorize XLN as an atypical congenital neutropenia in which constitutive activation of WASp in tissue neutrophils compensates for reduced myelopoiesis. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:128 issue:9 pages:4115-4131 ispartof: location:United States status: published

Pathogenic germline TP53 variants predispose to a wide range of early onset cancers, often recognized as the Li-Fraumeni syndrome (LFS). They are also identified in 1% of families with hereditary breast cancer (HrBC) that do not fulfill the criteria for LFS. In this study, we present a total of 24 different TP53 variants identified in 31 Swedish families with LFS or HrBC. Ten of these variants, nine exonic and one splice, have previously not been described as germline pathogenic variants. The nine exonic variants were functionally characterized and demonstrated partial transactivation activity compared to wild-type p53. Some show nuclear localization similar to wild-type p53 while others possess cytoplasmic or perinuclear localization. The four frameshift variants (W91Gfs*32, L111 Wfs*12, S227 Lfs*20 and S240Kfs*25) had negligible, while F134 L and T231del had low level of p53 activity. The L111 Wfs*12 and T231del variants are also deficient for induction of apoptosis. The missense variant R110C retain p53 effects and the nonsense E349* shows at least partial transcription factor activity but has reduced ability to trigger apoptosis. This is the first functional characterization of novel germline TP53 pathogenic or likely pathogenic variants in the Swedish cohort as an attempt to understand its association with LFS and HrBC, respectively.

Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine- N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine- N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.

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

SummaryThe FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes.

Side effects of chemotherapy are a major impediment in the treatment of cancer. Cyclotherapy is an emerging therapeutic strategy for protecting normal cells from the side effects of chemotherapy. Low, non-genotoxic doses of known p53 activators can be used to induce p53-dependent cell cycle arrest in normal cells bearing wild-type p53. This cytostatic effect of p53 can protect normal cells from the toxicity of S- or M-phase poisons. Here, we have reviewed existing cyclotherapy regimens using two well-known p53 activators, nutlin-3 and actinomycin D. We have highlighted an exemplar clinical perspective for cyclotherapy in breast cancer. The recent development of novel stapled peptides as activators of p53 without the corresponding cytotoxicity holds great promise for cyclotherapy to enhance the therapeutic window of existing chemotherapy drugs.

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

Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.

Pharmacological activation of wild-type p53 has been found to protect normal cells in culture from cytotoxicity and nuclear aberrations caused by conventional cancer therapeutics. Hence, small-molecule p53 activators could have clinical benefits as chemoprotectants for cancer patients bearing p53-mutant tumors. We have evaluated 16 p53-based cyclotherapy regimes combining p53 activators tenovin-6, leptomycin B, nutlin-3 and low dose actinomycin D, with clinically utilized chemotherapeutic agents (S- and M-phase poisons), vinblastine, vinorelbine, cytosine arabinoside and gemcitabine. All the p53 activators induce reversible cell-cycle arrest in primary human fibroblasts and protect them from both S- and M-phase poisons. Furthermore, studies with p53-mutant cancer cell lines show that nutlin-3 and low dose actinomycin D do not affect the sensitivity of these cells to any of the chemotherapeutics tested. Thus, these two small molecules could be suitable choices for cyclotherapy regimes involving S- or M-phase poisons. In contrast, pre-incubation of p53-mutant cells with tenovin-6 or leptomycin B reduces the efficacy of vinca alkaloids, suggesting that these p53 activators could be effective as chemoprotectants if combined with S- but not M-phase poisons. Discrepancies were observed between the levels of protection detected immediately after treatment and following recovery in fresh medium. This highlights the need to assess both short- and long-term effects when evaluating compounds as potential chemoprotectants for cancer therapy.

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.

Received: September 22, 2010 , Accepted: October 29, 2010 , Published: October 30, 2010 // p53-based cyclotherapy is proving to be a promising approach to palliate undesired effects of chemotherapy in patients with tumours carrying p53 mutations. For example, pre-treatment of cell cultures with Nutlin-3, a highly-selective inhibitor of the p53-mdm2 interaction, has been successfully used as a cytostatic agent to protect normal cells, but not p53-defective cells, from subsequent treatment with mitotic poisons or S-phase specific drugs. Here we sought to evaluate whether low doses of Actinomycin D (LDActD), a clinically-approved drug and potent p53 activator, could substitute Nutlin-3 in p53-based cyclotherapy. We found that pre-treatment with LDActD before adding the aurora kinase inhibitor VX-680 protects normal fibroblasts from polyploidy and nuclear morphology abnormalities induced by VX-680. However, and although to a lower extent than normal fibroblasts, tumour cell lines bearing p53 mutations were also protected by LDActD (but not Nutlin-3) from VX-680-induced polyploidy. We also report that a difference between the response of p53 wild-type cells and p53-defective cells to the LDActD/VX-680 sequential combination is that only the former fail to enter S-phase and therefore accumulate in G1/G0. We propose that drugs that incorporate into DNA during S-phase may perform better as second drugs than mitotic poisons in cyclotherapy approaches using LDActD as a cytostatic agent.

Summary The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins., Graphical Abstract, Highlights • Mass spectrometry captures intact complexes of the peripheral membrane protein DHODH • Detergent removal in the gas phase reveals lipid and co-factor binding • DHODH attaches to the membrane by binding charged phospholipids • Lipids stabilize the flexible substrate- and drug-binding site, The combination of mass spectrometry and molecular dynamics simulations provides insights into the relationship between lipid and substrate binding to the peripheral membrane protein dehydroorotate dehydrogenase, revealing ligand-induced stabilization of the flexible membrane-binding region.

Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development, and several original approaches to drug discovery that could have wide applications to drug development are being used. In one approach, molecules that activate p53 by blocking protein-protein interactions with MDM2 are in early clinical development. Remarkable progress has also been made in the development of p53-binding molecules that can rescue the function of certain p53 mutants. Finally, cell-based assays are being used to discover compounds that exploit the p53 pathway by either seeking targets and compounds that show synthetic lethality with TP53 mutations or by looking for non-genotoxic activators of the p53 response.

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.

In this paper we investigate the role of spatial effects in determining the dynamics of a subclass of signalling pathways characterised by their ability to demonstrate oscillatory behaviour. To this end, we formulate a simple spatial model of the p53 network that accounts for both a neg- ative feedback and a transcriptional delay. We show that the formation of protein density patterns can depend on the shape of the cell, position of the nucleus, and the protein diffusion rates. The temporal changes in the total amounts of protein are also subject to spatial influences. The level of DNA damage required to induce sustained oscillations, for instance, depends on the morphology of the cell. The model also provides a new interpretation of experimentally observed undamped oscillations in p53 levels in single cells. Our simulations reveal that alternate sequences of high- and low-amplitude oscillations can occur. We propose that the digital pulses may correspond to snap-shots of our high-amplitude sequences. Shorter waiting-times between subsequent time-lapse fluorescence microscopy images in combination with lower detection thresholds may reveal the ir- regular high-frequency oscillations suggested by our spatial model.

Promoting p53's function, without causing damage to the genome is widely accepted as a way to improve treatment of cancer. In the case of tumours where the p53 gene is not mutated or absent, there have been important advances in the recent past. Small molecules that, like the nutlins discovered by Luo Vassilev and colleagues,1 efficiently activate wild type p53 by inhibiting the function of p53's major negative regulator mdm2 and show promising results in preclinical models as single agents or in combination with classic chemotherapeutics.2 Finding compounds that instead of binding to mdm2, interact with p53 and prevent its degradation whilst still allowing its function, is also an approach under investigation.3 Restoring p53 effects in tumours that contain point mutations in the p53 gene is in some ways a bigger challenge. One possible way to achieve this is through peptides or small molecules that directly interact with mutant p53.4,5 Because, unlike wild type p53, mutant p53 is expressed at very high l...

Since the discovery of p53, a vast wealth of knowledge on its function and regulation has been accumulated. It is known that it is a key tumour suppressor and that its function is lost in many types of cancers, either by mutation or by excessive negative regulation. Recently, several discoveries have re-energised P53 as a therapeutic target as it has been shown that reintroduction of functional p53 into tumours has a therapeutic benefit. These encouraging results clearly justify the search for small molecules that diminish negative regulation of P53 in tumour cells, where P53 is not mutated as well as compounds that reactivate mutant P53. Important findings have been made to deal with both situations. Additionally, some of the small molecules identified may also help reduce the side effects of commonly used cancer therapeutics. These studies are still in their infancy and require further therapeutic validation, but the future appears bright for finally harnessing p53's tumour suppressing ability.

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

Human papillomavirus (HPV) infection is strongly associated with the development of anogenital neoplasia, particularly cervical cancer. It has been estimated that 99.7% of all cervical carcinomas are attributable to infection with HPV, and types 16 and 18 account for the vast majority of such cases. Both of these ‘high risk’ HPV types encode the oncoproteins E6 and E7, which exert multiple effects on many proteins involved in cell-cycle regulation, including p53. The nuclear export protein inhibitor leptomycin B (LMB) has been shown to cause the nuclear sequestration of p53 in cervical carcinoma cells. We demonstrate that LMB induces apoptosis selectively at nanomolar concentrations in primary human keratinocytes (PHKs) expressing HPV oncogenes. Both monolayer and organotypic raft cultures of transduced PHKs were highly susceptible to treatment with LMB. By contrast, although LMB stimulated p53 accumulation in normal PHKs, no significant induction of apoptosis was detected on Western blots or immunostained monolayer/raft cells, or following pulsed exposure to the drug. Furthermore, topical application of μM concentrations of LMB to mouse skin was non-toxic. These data suggest that the topical application of LMB to HPV-infected intra-epithelial lesions may represent a specific and effective therapeutic strategy against HPV-associated anogenital neoplasia. © 2007 Wiley-Liss, Inc.

The tumor suppressor ARF plays an important role as an inhibitor of the Mdm2-mediated degradation of p53. Here we demonstrate that human ARF (p14ARF) can form homo-oligomers. The stability of the oligomers is favored by oxidizing agents in a reversible fashion and involves all three cysteine residues in p14ARF. Furthermore, the effect of p14ARF in clonogenic assays is moderately but reproducibly increased by the mutation of its cysteine residues. We also observed that altering the amino terminus of p14ARF resulted in the appearance of remarkably stable oligomers. This indicates that the amino terminus of p14ARF interferes with the ability of the protein to form multimeric complexes. These observations suggest that p14ARF activity may be linked to its oligomerization status and sensitive to the redox status of the cell.

Inactivation of the p53 function is a common event in cancer. Approx. 50% of human tumours express mutant p53 and there is evidence that in others, including many childhood tumours, p53 function is impaired in other ways. These defects on p53 function may be due to the alteration of cellular factors that modulate p53 or to the expression of viral oncoproteins. Radiotherapy and many of the chemotherapeutic drugs currently used in cancer treatment are potent activators of p53. However, most of these therapies have a serious drawback; that is, the long-term consequences of their DNA-damaging effects. Understanding the mechanisms regulating p53 stability is crucial for the development of new strategies to activate p53 non-genotoxically. Here we describe the effect of a potent activator of the p53 response, the nuclear export inhibitor leptomycin B, on Mdm2 degradation and we provide evidence for the oligomerization of the p14ARF tumour suppressor and Mdm2 inhibitor in response to oxidative stress.

Ingeborg M.M. van Leeuwen 1 and Sonia Lain 1,2 1 Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels vag 16, 171 77 Stockholm, Sweden 2 Centre for Oncology and Molecular Medicine, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, UK Commentary on: Apontes et al. "Exploring long-term protection of normal human fibroblasts and epithelial cells from chemotherapy in cell culture" Oncotarget 2010; 2: 222-233. Received: April 12, 2011; Accepted: April 20, 2011; Published: April 20, 2011; Correspondence: Sonia Lain, e-mail: // //

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

Background: The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy. Results: Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intrac ellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells. Conclusions: Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML. publishedVersion

Sirtuins are NAD+ dependent deacetylases and/or ADP-ribosyl transferases active on histone and non-histone substrates. The first sirtuin was discovered as a transcriptional repressor of the mating-type-loci (Silent Information Regulator sir2) in the budding yeast, where it was shown to extend yeast lifespan. Seven mammalian sirtuins (SIRT1-7) have been now identified with distinct subcellular localization, enzymatic activities and substrates. These enzymes regulate cellular processes such as metabolism, cell survival, differentiation, DNA repair and they are implicated in the pathogenesis of solid tumors and leukemias. The purpose of the present study was to investigate the role of sirtuin expression, activity and inhibition in the survival of pediatric sarcoma cell lines.We have analyzed the expression of SIRT1 and SIRT2 in a series of pediatric sarcoma tumor cell lines and normal cells, and we have evaluated the activity of the sirtuin inhibitor and p53 activator tenovin-6 (Tv6) in synovial sarcoma and rhabdomyosarcoma cell lines. We show that SIRT1 is overexpressed in synovial sarcoma biopsies and cell lines in comparison with normal mesenchymal cells. Tv6 induced apoptosis as well as impaired autophagy flux. Using siRNA to knock down SIRT1 and SIRT2, we show that the expression of both proteins is crucial for the survival of rhabdomyosarcoma cells and that the loss of SIRT1 expression results in a decreased LC3II expression. Our results show that SIRT1 and SIRT2 expressions are crucial for the survival of synovial sarcomas and rhabdomyosarcomas, and demonstrate that the pharmacological inhibition of sirtuins impairs the autophagy process and induces tumor cell death.

Peutz-Jeghers syndrome is an inherited cancer syndrome that results in a greatly increased risk of developing tumors in those affected. The causative gene is a protein kinase termed LKB1, predicted to function as a tumor suppressor. The mechanism by which LKB1 is regulated in cells is not known. Here, we demonstrate that stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induced phosphorylation of endogenously expressed LKB1 at Ser(431). We present pharmacological and genetic evidence that p90(RSK) mediated this phosphorylation in response to agonists that activate ERK1/2 and that cAMP-dependent protein kinase mediated this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and we demonstrate that full-length LKB1 expressed in 293 cells was prenylated by addition of a farnesyl group to Cys(433). Our data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) did not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated was still able to suppress the growth of cells.

The p53 phosphoprotein acts as a tumor-suppressor gene product through the inhibition of cell growth and induction of apoptosis in a transcription-dependent manner. These functions require p53 activation through different biochemical postranslational modifications. Given the relevance of this protein in ultraviolet light-induced carcinogenesis, whose targets are primarily skin keratinocytes, we studied the functions of p53 in epidermal cell differentiation. We selected HaCaT cells, a human keratinocyte cell line bearing point-mutated, transcriptionally inactive, but highly stable p53, which facilitates immunochemical and biochemical analysis. In addition, a reliable in vitro differentiation system has been developed with these cells (Paramio et al. Oncogene 17:949, 1998). We report that during HaCaT differentiation there is a loss of immunoreactivity of p53 against antibodies that specifically recognize epitopes located at the carboxyl terminus of the protein. Because treatment with phosphatase restores this immunoreactivity, we conclude that p53 is phosphorylated at the carboxyl terminus during keratinocyte differentiation. This biochemical modification has been associated with the transcriptional activation of the molecule, and because p53 is involved in differentiation processes in other cell types, we investigated the potential functions of p53 during epidermal differentiation. To this end, we generated HaCaT clones expressing a murine temperature-sensitive p53 (Mp53ts) by transfection because the endogenous p53 is not functional even with phosphorylation. We characterized the expression and effects of the transfected protein in different selected clones. The ultraviolet-light response of these clones was restored, demonstrating the functionality of Mp53ts in these cells. We also observed that, with induction of differentiation, Mp53ts transfected cells differentiate faster than the parental or vector-transfected control cells, demonstrating that p53 promotes epidermal differentiation. The sustained expression of p53 in differentiating cells leads to massive cell death and detachment, a phenomenon that may be similar to epidermal terminal differentiation. In addition, we observed that the expression of p53-dependent genes such as p21waf/cip1 and mdm2 (which are known to participate in epidermal differentiation) increases during HaCaT differentiation, i.e., in a p53-independent manner.

In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21 WAF1/CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.

p53 tumour suppressor protein levels and p53-dependent transcriptional activity have been recently shown to increase in cells treated with leptomycin B (LMB), an inhibitor of nuclear export. Experiments presented here show that LMB treatment leads to growth arrest and a senescence-like phenotype in human normal fibroblast cultures. This effect is reversible after removal of the drug and further passage by trypsinization. Instead, LMB has a strong cytotoxic effect on human neuroblastoma cell lines even at nanomolar concentrations. In both these cell types the effects of LMB are attenuated when the activity of the endogenous wild type p53 protein is abrogated by overexpression of a dominant negative p53 mutant. We conclude that the induction of the p53 response by LMB plays an important role in the effects of this drug on cultured cells.

Leptomycin B is a cytotoxin which directly interacts with and inhibits the action of CRM1, an essential mediator of the nuclear exit of proteins containing nuclear export signals (NES) of the HIV1 REV type. We show that addition of leptomycin B to human primary fibroblasts increased the levels of the p53 tumor suppressor protein. This was accompanied by the induction of p53-dependent transcriptional activity in cultured cells and an increase in the levels of the products of two p53-responsive genes, the p21(CIP1/WAF1) and HDM2 proteins. Leptomycin B induced the accumulation of p53 and HDM2 in the nucleus and the appearance of discrete nuclear aggregates containing both proteins. It has been reported that the transcriptional activity of p53 is modulated by its interaction with the HDM2 protein which also targets p53 for rapid degradation. Using a model cell line conditionally expressing MDM2, the murine analogue of HDM2, we present evidence indicating that leptomycin B abrogates MDM2's role in p53 degradation and that the accumulation of p53 in distinct nuclear bodies is mediated by MDM2. Since HDM2 has recently been shown to contain a functional NES of the REV type, the most likely explanation for our results is that the effect of leptomycin B on HDM2 and p53 is due to the inhibition of nuclear export. The ability to visualize sites where p53 and HDM2 colocalize provides a new approach to study the association between the two proteins in vivo. These p53/HDM2-positive nuclear foci were found to also contain the U1A snRNP A and to be juxtaposed to the PML oncogenic domains.

Nutlin-3 selectively activates p53 by inhibiting the interaction of this tumor suppressor with its negative regulator murine double minute 2 (mdm2), while trichostatin A (TSA) is one of the most potent histone deacetylase (HDAC) inhibitors currently available. As both Nutlin-3 and TSA increase the levels of the cell cycle inhibitor p21(cip1/waf1) in cells, we investigated whether a combination of these compounds would further augment p21 levels. Contrary to expectations, we found that short-term exposure to Nutlin-3 and TSA in combination did not have an additive effect on p21 expression. Instead, we observed that activation of p53 prevented the ability of TSA to increase p21 levels. Furthermore, TSA inhibited Nutlin-3-induced expression of p53-dependent mRNAs including P21. This negative effect of TSA on Nutlin-3 was significantly less pronounced in the case of hdm2, another p53 downstream target. Aside from suggesting a model to explain these incompatible effects of Nutlin-3 and TSA, we discuss the implications of our findings in cancer therapy and cell reprogramming.

The authors acknowledge TENOVUS Tayside for funding the study Tenovin-6 (Tnv-6) is a bioactive small molecule with anti-neoplastic activity. Inhibition of the Sirtuin class of protein deacetylases with activation of p53 function is associated with the pro-apoptotic effects of Tnv-6 in many tumors. Here, we demonstrate that in chronic lymphocytic leukemia (CLL) cells, Tnv-6 causes non-genotoxic cytotoxicity, without adversely affecting human clonogenic hematopoietic progenitors in vitro, or murine hematopoiesis. Mechanistically, exposure of CLL cells to Tnv-6 did not induce cellular apoptosis or p53-pathway activity. Transcriptomic profiling identified a gene program influenced by Tnv-6 that included autophagy-lysosomal pathway genes. The dysregulation of autophagy was confirmed by changes in cellular ultrastructure and increases in the autophagy-regulatory proteins LC3 (LC3-II) and p62/Sequestosome. Adding bafilomycin-A1, an autophagy inhibitor to Tnv-6 containing cultures did not cause synergistic accumulation of LC3-II, suggesting inhibition of late-stage autophagy by Tnv-6. Thus, in CLL, the cytotoxic effects of Tnv-6 result from dysregulation of protective autophagy pathways. Publisher PDF

Background: The CDKN2/INK4A tumour suppressor gene is deleted or mutated in a large number of human cancers. Overexpression of its product, p16, has been shown to block the transition through the G 1 /S phase of the cell cycle in a pRb-dependent fashion by inhibiting the cyclin D-dependent kinases cdk4 and cdk6. Reconstitution of p16 function in transformed cells is therefore an attractive target for anti-cancer drug design. Results We have identified a 20-residue synthetic peptide — corresponding to amino acids 84–103 of p16 – that interacts with cdk4 and cdk6, and inhibits the in vitro phosphorylation of pRb mediated by cdk4–cyclin D1. The amino-acid residues of p16 important for its interaction with cdk4 and cdk6 and for the inhibition of pRb phosphorylation were defined by an alanine substitution series of peptides. In normal proliferating human HaCaT cells and in cells released from serum starvation, entry into S phase was blocked by the p16-derived peptide when it was coupled to a small peptide carrier molecule and applied directly to the tissue culture medium. This cell-cycle block was associated with an inhibition of pRb phosphorylation in vivo . Conclusion These results demonstrate that a p16-derived peptide can mediate three of the known functions of p16: firstly, it interacts with cdk4 and cdk6; secondly, it inhibits pRb phosphorylation in vitro and in vivo ; and thirdly, it blocks entry into S phase. The fact that one small synthetic peptide can enter the cells directly from the tissue culture medium to inhibit pRb phosphorylation and block cell-cycle progression makes this an attractive approach for future peptidometic drug design. Our results suggest a novel and exciting means by which the function of the p16 suppressor gene can be restored in human tumours.

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

In recent years, our knowledge of the molecular biology of plum pox potyvirus (PPV) and of potyviruses in general has greatly increased. The genome of PPV consists of a unique single-strand RNA molecule with messenger polarity. A terminal protein (VPg) and a poly-(A) tail are present at the 5’and 3’ends, respectively, of the genomic RNA. Four complete and other partial nucleotide sequences of PPV isolates have been reported. According to the levels of homology three different PPV strains can be defined. PPV RNA is translated into a unique polyprotein starting at the second AUG codon of the large open-reading frame that covers most of the genome. The polyprotein is proteolytically processed by three virus encoded proteases (P1Pro, HCPro and NIaPro). While P1Pro and HCPro cut at their own carboxyl termini, NIaPro recognizes seven cleavage sites characterized by conserved heptapeptides. These sites differ in their susceptibility to cis and trans processing and in their reaction profiles. Immunoelectron microscopy has shown NIa and NIb proteins to form nuclear inclusions, but also dense aggregates in the cytoplasm, and CI protein to make typical pinwheel cytoplasmic inclusions. On the basis of sequence comparisons, NIb protein has been proposed to be the viral RNA replicase. CI protein has been purified from infected leaves and from Escherichia coli harbouring a plasmid that encodes it. It has nucleic acid-stimulated NTPase and RNA helicase activities. Virus infection has been achieved by inoculation with a PPV full-length cDNA clone and with transcripts synthesized using another one as template. These clones can be used to apply genetic engineering techniques to the in vitro manipulation of the PPV genome.