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While c-MYC is well established as a proto-oncogene, its structure and function as a transcription factor have made c-MYC a difficult therapeutic target. To identify small-molecule inhibitors targeting c-MYC for anticancer therapy, we designed a high-throughput screening (HTS) strategy utilizing cellular assays. The novel approach for the HTS was based on the detection of cellular c-MYC protein, with active molecules defined as those that specifically decreased c-MYC protein levels in cancer cells. The assay was based on a dual antibody detection system using Forster/fluorescence resonance energy transfer (FRET) and was utilized to detect endogenous c-MYC protein in the MYC amplified cancer cell lines DMS273 and Colo320 HSR. The assays were miniaturized to 1536-well plate format and utilized to screen the GlaxoSmithKline small-molecule collection of approximately 2 million compounds. In addition to the HTS assay, follow-up assays were developed and used to triage and qualify compounds. Two cellular assays used to eliminate false-positive compounds from the initially selected HTS hits were (1) a cellular toxicity assay and (2) an unstable protein reporter assay. Three positive selection assays were subsequently used to qualify compounds: (1) 384-well cell cycle flow cytometry, (2) 384-well cell growth, and (3) c-MYC gene signature reverse transcription quantitative PCR (RT-qPCR). The HTS and follow-up assays successfully identified three compounds that specifically decreased c-MYC protein levels in cancer cells and phenocopied c-MYC siRNA in terms of cell growth inhibition and gene signatures. The HTS, triage, and three compounds identified are described.

// Kathleen A. Bridges 1 , Carlo Toniatti 2 , Carolyn A. Buser 3 , Huifeng Liu 1 , Thomas A. Buchholz 4 , and Raymond E. Meyn 1 1 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 2 IRBM/Merck Research Laboratories Rome, Italy 3 Merck Sharp & Dohme Corp., Upper Gwynedd, Pennsylvania 4 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas Correspondence: Raymond E. Meyn, email: // Keywords : Radiation, PARP, MK-4827, DNA damage, niraparib Received : May 5, 2014 Accepted : June 7, 2014 Published : June 9, 2014 Abstract The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H2AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H 2 O 2 and converted H 2 O 2 -induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation.

Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series.

The poly-(ADP-ribose) polymerase (PARP) inhibitor, MK-4827, is a novel potent, orally bioavailable PARP-1 and PARP-2 inhibitor currently in phase I clinical trials for cancer treatment. No preclinical data currently exist on the combination of MK-4827 with radiotherapy. The current study examined combined treatment efficacy of MK-4827 and fractionated radiotherapy using a variety of human tumor xenografts of differing p53 status: Calu-6 (p53 null), A549 (p53 wild-type [wt]) and H-460 (p53 wt) lung cancers and triple negative MDA-MB-231 human breast carcinoma. To mimic clinical application of radiotherapy, fractionated radiation (2 Gy per fraction) schedules given once or twice daily for 1 to 2 weeks combined with MK-4827, 50 mg/kg once daily or 25 mg/kg twice daily, were used. MK-4827 was found to be highly and similarly effective in both radiation schedules but maximum radiation enhancement was observed when MK-4827 was given at a dose of 50 mg/kg once daily (EF = 2.2). MK-4827 radiosensitized all four tumors studied regardless of their p53 status. MK-4827 reduced PAR levels in tumors by 1 h after administration which persisted for up to 24 h. This long period of PARP inhibition potentially adds to the flexibility of design of future clinical trials. Thus, MK-4827 shows high potential to improve the efficacy of radiotherapy.

Hepatocellular carcinoma (HCC) is a heterogeneous and highly aggressive malignancy, for which there are no effective cures. Identification of a malignant stemlike subtype of HCC may offer patients with a dismal prognosis a potential targeted therapy using c-MET and Wnt pathway inhibitors. MicroRNAs (miRNAs) show promise as diagnostic and prognostic tools for cancer detection and stratification. Using a TRE-c-Met-driven transgenic HCC mouse model, we identified a cluster of 23 miRNAs that is encoded within the Dlk1-Gtl2 imprinted region on chromosome 12qF1 overexpressed in all of the isolated liver tumors. Interestingly, this region is conserved among mammalian species and maps to the human DLK1-DIO3 region on chromosome 14q32.2. We thus examined the expression of the DLK1-DIO3 miRNA cluster in a cohort of 97 hepatitis B virus-associated HCC patients and identified a subgroup (n = 18) of patients showing strong coordinate overexpression of miRNAs in this cluster but not in other cancer types (breast, lung, kidney, stomach, and colon) that were tested. Expression levels of imprinted gene transcripts from neighboring loci in this 14q32.2 region and from a subset of other imprinted sites were concomitantly elevated in human HCC. Interestingly, overexpression of the DLK1-DIO3 miRNA cluster was positively correlated with HCC stem cell markers (CD133, CD90, EpCAM, Nestin) and associated with a high level of serum α-fetoprotein, a conventional biomarker for liver cancer, and poor survival rate in HCC patients. In conclusion, our findings suggest that coordinate up-regulation of the DLK1-DIO3 miRNA cluster at 14q32.2 may define a novel molecular (stem cell-like) subtype of HCC associated with poor prognosis.

Elevated expression of the c-MYC oncogene (either due to gene amplification, translocation, abnormality in upstream signaling pathways and/or protein stabilization) is one of the most common abnormalities in human cancers. Efforts to identify direct pharmacological inhibitors of c-MYC function have not yet yielded drug-like molecules. Therefore, we sought to pursue alternative screening strategies for this classically “undruggable” transcription factor. We developed a novel antibody-based high-throughput HTRF screening assay that specifically detects endogenous c-MYC protein levels in a MYC amplified cancer cell line. Taking advantage of the short half-life of c-MYC, both at the protein and mRNA level, we conducted a cell-based screen of the GSK screening collection to identify compounds that can rapidly decrease c-MYC protein levels. Elimination of false positive hits using stringent triage assays successfully identified two valid hit series exemplified by GSK970 and GSK417. Molecular mode of action studies revealed these molecules inhibit MYC transcription by binding to the minor groove of DNA with AT sequence specificity. Unfortunately, this mechanism of c-MYC inhibition demonstrated poor in vivo translatability as tissue DNA acts as a molecular sink, effectively sequestering compound, and limiting its pharmacodnyamic response. Citation Format: Biju Mangatt, Anthony D. Pietra, Anna Waszkiewicz, Jon-Paul Jaworski, Sonja Ghidelli-Disse, Thomas J. Berrodin, Christian S. Sherk, Derrick W. Meinhold, Anna Rutkowska-Klute, Shanker K. Sundaram, Gopinath Ganji, Wendy S. Halsey, George P. Livi, William Li, James Mack, Stuart P. Romeril, Elisabeth A. Minthorn, Rakesh Kumar, Gerard C. Drewes, Dirk A. Heerding, Lorena A. Kallal, Carolyn A. Buser, Jesus R. Medina. Discovery of small-molecule compounds targeting c-MYC using a novel cell-based screen [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 2919.

ERBB2/neu and Notch signaling are known to be deregulated in many human cancers. However, pathway cross-talk and dependencies are not well understood. In this study, we use an ERBB2-transgenic mouse model of breast cancer (neuT) to show that Notch signaling plays a critical role in tumor maintenance. Inhibition of the Notch pathway with a γ-secretase inhibitor (GSI) decreased both the Notch and the mammalian target of rapamycin/AKT pathways. Antitumor activity resulting from GSI treatment was associated with decreased cell proliferation as measured by Ki67 and decreased expression of glucose transporter Glut1. Positron emission tomography (PET) imaging showed that the functional consequences of decreased Glut1 translated to reduced glucose uptake and correlated with antitumor effects as measured by micro-computed tomography imaging. The decrease of Glut1 in neuT tumors was also observed in several human breast cancer cell lines following GSI treatment. We provide evidence that ∼27% of ERBB2-positive human breast cancer specimens display high expression of HES1, phospho-S6RP, and GLUT1. Together, these results suggest that pathways downstream of Notch signaling are, at least in part, responsible for promoting tumor growth in neuT and also active in both neuT and a subset of human breast cancers. These findings suggest that GSI may provide therapeutic benefit to a subset of ERBB2-positive breast cancers and that [18F]FDG-PET imaging may be useful in monitoring clinical response. Cancer Res; 70(6); 2476–84

A number of inhibitors of kinesin spindle protein (KSP) have been described, which are known from X-ray crystallography studies to bind to an induced fit pocket defined by the L5 loop. We describe the characterization of eight mutant forms of KSP in which six residues that line this pocket have been altered. Mutants were analyzed by measuring rates of enzyme catalysis, in the presence and absence of six KSP inhibitors of four diverse structural classes and of varied ATP-competition status. Our analysis was in agreement with the model of binding established by the structural studies and suggests that binding energy is well distributed across functional groups in these molecules. The majority of the mutants retained significant enzymatic activity while diminishing inhibitor binding, indicating potential for the development of drug resistance. These data provide detailed information on interactions between inhibitor and binding pocket at the functional group level and enable the development of novel KSP inhibitors.

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.

Purpose: We determined the effects of vorinostat (suberoylanalide hydroxamic acid) and/or MK-0457 (VX-680), an Aurora kinase inhibitor on the cultured human (HL-60, OCI-AML3, and K562) and primary acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML), as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and mutant forms of Bcr-Abl. Experimental Design: Following exposure to MK-0457 and/or vorinostat, apoptosis, loss of viability, as well as activity and levels of Aurora kinase and Bcr-Abl proteins were determined. Results: Treatment with MK-0457 decreased the phosphorylation of Aurora kinase substrates including serine (S)10 on histone H3 and survivin, and led to aberrant mitosis, DNA endoreduplication as well as apoptosis of the cultured human acute leukemia HL-60, OCI-AML3, and K562 cells. Combined treatment with vorinostat and MK-0457 resulted in greater attenuation of Aurora and Bcr-Abl (in K562) kinase activity and levels as well as synergistically induced apoptosis of OCI-AML3, HL-60, and K562 cells. MK-0457 plus vorinostat also induced synergistic apoptosis of BaF3 cells with ectopic overexpression of wild-type or mutant Bcr-Abl. Finally, cotreatment with MK-0457 and vorinostat induced more loss of viability of primary AML and imatinib-refractory CML than treatment with either agent alone, but exhibited minimal toxicity to normal CD34+ progenitor cells. Conclusions: Combined in vitro treatment with MK-0457 and vorinostat is highly active against cultured and primary leukemia cells. These findings merit in vivo testing of the combination against human AML and CML cells, especially against imatinib mesylate–resistant Bcr-AblT315I–expressing CML Cells.

Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.

From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.

Inspired by previous efforts in the pyrazolobenzoxazine class of KSP inhibitors, the design and synthesis of 1,4-diaryl-4,5-dihydropyrazole inhibitors of KSP are described. Crystallographic evidence of binding mode and in vivo potency data is also highlighted.

Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.

Activation of the inositol-requiring enzyme-1 alpha (IRE1α) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1α dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1α-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1α. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1α RNase activity appears to be caused by a conformational change, whereby the αC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1α, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1α RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.

The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.

Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I (HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for potency by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines, PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several of these compounds.

Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.

2,4-Diaryl-2,5-dihydropyrroles have been discovered to be novel, potent and water-soluble inhibitors of KSP, an emerging therapeutic target for the treatment of cancer. A potential concern for these basic KSP inhibitors (1 and 2) was hERG binding that can be minimized by incorporation of a potency-enhancing C2 phenol combined with neutral N1 side chains. Aqueous solubility was restored to these, and other, non-basic inhibitors, through a phosphate prodrug strategy.

The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s10nM. Ancillary hERG activity was evaluated for this series of inhibitors.

Pyrimidino-thiazolyl carbonitriles were prepared that are potent VEGFR-2 (KDR) kinase inhibitors. The modification of lead structures resulted in 3m which exhibited the best overall profile in KDR inhibitory activity, iv/po pharmacokinetics, and reduced hERG affinity.

Modifications to the basic side-chain of early lead structures of the indolyl quinolinone class of KDR kinase inhibitors resulted in improved pharmacokinetic and ancillary profiles. Specifically, compounds bearing 5-amido- and 5-sulphonamido-indolyl substituents exhibited lower plasma clearance and weaker binding affinity for the I(Kr) potassium channel hERG.

We report here the first inhibitor-bound structure of a mitotic motor protein. The 1.9 {angstrom} resolution structure of the motor domain of KSP, bound with the small molecule monastrol and Mg{sup 2+} {center_dot} ADP, reveals that monastrol confers inhibition by 'induced-fitting' onto the protein some 12 {angstrom} away from the catalytic center of the enzyme, resulting in the creation of a previously non-existing binding pocket. The structure provides new insights into the biochemical and mechanical mechanisms of the mitotic motor domain. Inhibition of KSP provides a novel mechanism to arrest mitotic spindle formation, a target of several approved and investigative anti-cancer agents. The structural information gleaned from this novel pocket offers a new angle for the design of anti-mitotic agents.

We have synthesized 3-t-butylchrysene and measured the Larmor frequency ω/2π (= 8.50, 22.5, and 53.0 MHz) and temperature T (110–310 K) dependence of the proton spin–lattice relaxation rate R in the polycrystalline solid [low-frequency solid state nuclear magnetic resonance (NMR) relaxometry]. We have also determined the molecular and crystal structure in a single crystal of 3-t-butylchrysene using x-ray diffraction, which indicates the presence of a unique t-butyl group environment. The spin-1/2 protons relax as a result of the spin–spin dipolar interactions being modulated by the superimposed reorientation of the t-butyl groups and their constituent methyl groups. The reorientation is successfully modeled by the simplest motion; that of random hopping describable by Poisson statistics. The x-ray data indicate near mirror-plane symmetry that places one methyl group nearly in the aromatic plane and the other two almost equally above and below the plane. The NMR relaxometry data indicate that the nearly in...

A series of novel diaryl ether lactams have been identified as very potent dual inhibitors of protein farnesyltransferase (FTase) and protein geranylgeranyltransferase I (GGTase-I), enzymes involved in the prenylation of Ras. The structure of the complex formed between one of these compounds and FTase has been determined by X-ray crystallography. These compounds are the first reported to inhibit the prenylation of the important oncogene Ki-Ras4B in vivo. Unfortunately, doses sufficient to achieve this endpoint were rapidly lethal.

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.

A series of macrocyclic 3-aminopyrrolidinone farnesyltransferase inhibitors (FTIs) has been synthesized. Compared with previously described linear 3-aminopyrrolidinone FTIs such as compound 1, macrocycles such as 49 combined improved pharmacokinetic properties with a reduced potential for side effects. In dogs, oral bioavailability was good to excellent, and increases in plasma half-life were due to attenuated clearance. It was observed that in vivo clearance correlated with the flexibility of the molecules and this concept proved useful in the design of FTIs that exhibited low clearance, such as FTI 78. X-ray crystal structures of compounds 49 and 66 complexed with farnesyltransferase (FTase)-farnesyl diphosphate (FPP) were determined, and they provide details of the key interactions in such ternary complexes. Optimization of this 3-aminopyrrolidinone series of compounds led to significant increases in potency, providing 83 and 85, the most potent inhibitors of FTase in cells described to date.

Compound 1 has been shown to be a dual prenylation inhibitor with FPTase (IC50=2 nM) and GGPTase-I (IC50=95 nM). Analogues of 1, which replaced the cyanophenyl group with various biaryls, led to the discovery of highly potent dual FPTase/GGPTase-I inhibitors. 4-Trifluoromethylphenyl, trifluoropentynyl, and trifluoropentyl were identified as good p-cyano replacements.

The synthesis, structure-activity relationships, and biological properties of a novel series of imidazole-containing inhibitors of farnesyltransferase are described. Starting from a 3-aminopyrrolidinone core, a systematic series of modifications provided 5h, a non-thiol, non-peptide farnesyltransferase inhibitor with excellent bioavailability in dogs. Compound 5h was found to have an unusually favorable ratio of cell potency to intrinsic potency, compared with other known FTIs. It exhibited excellent potency against a range of tumor cell lines in vitro and showed full efficacy in the K-rasB transgenic mouse model.

The 92nd Annual Meeting of the AACR comprised over 5000 abstracts, 12 plenary and award lectures and numerous talks in educational sessions, symposia and mini-symposia. Given the wealth of information presented, we narrowed our coverage to the area of prenyltransferase and protein kinase inhibitors. Many rationally designed drugs are now in clinical trials and exciting results were presented for the Bcr-Abl inhibitor STI-571. The cancer community is beginning to envision new ways to evaluate and administer these well-tolerated drugs which do not fit the traditional anticancer drug profile. There is an emphasis in developing surrogate markers for evaluating the mechanism-based effectiveness as well as identifying off-target toxicities. In addition, there is a large effort in investigating effective drug combinations and the use of these new agents as radiosensitisers. Here we present specific examples of these issues as applied to prenylation and protein kinase inhibitors.

A series of aryloxy substituted piperazinones with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to have potent inhibitory activity in vitro and are promising agents for the inhibition of Ki-Ras signaling.

A series of 2-arylindole-3-acetamide farnesyl protein transferase inhibitors has been identified. The compounds inhibit the enzyme in a farnesyl pyrophosphate-competitive manner and are selective for farnesyl protein transferase over the related enzyme geranylgeranyltransferase-I. A representative member of this series of inhibitors demonstrates equal effectiveness against HDJ-2 and K-Ras farnesylation in a cell-based assay when geranylgeranylation is suppressed.

We have measured the proton spin-lattice relaxation rate as a function of temperature in polycrystalline 3-ethylchrysene at nuclear magnetic resonance Larmor frequencies of 53.0 and 22.5 MHz and in polycrystalline 3-isopropylchrysene at 53.0, 22.5 and 8.50 MHz. The syntheses of these new compounds are presented. The relatively large chrysene backbone creates an ideal and unique environment for the alkyl groups such that methyl group rotation is the only motion on the nuclear magnetic resonance Larmor frequency timescale over a large temperature range. The relaxation rate data are interpreted in terms of the simplest possible dynamical model; that of random hopping for the methyl group(s), all of which are equivalent in the solid state. The barriers of 11–12 kJ mol−1 are typical for methyl groups in `isolated' ethyl and isopropyl groups.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (

Several proteins that are important components of the calcium/phospholipid second messenger system (e.g. phospholipase C, protein kinase C, myristoylated alanine-rich C kinase substrate (MARCKS) and pp60src) contain clusters of basic residues that can interact with acidic lipids on the cytoplasmic surface of plasma membranes. We have studied the membrane binding of MARCKS and pp60src, peptides that mimic the basic regions of these proteins, and simple model peptides. Specifically, we determined how the binding of these model peptides depends on (1) the number of basic residues in the peptide (2) the fraction of acidic lipids in the membrane (3) the ionic strength of the solution (4) the chemical nature of the basic residues (Arg versus Lys) and the acidic phospholipids [phosphatidylglycerol (PG) versus phosphatidylserine (PS)] (5) the pressure and (6) the temperature. The results are consistent with a simple theoretical model: each basic residue in a peptide binds independently to an acidic lipid with an intrinsic microscopic association constant of 1-10 M-1 (binding energy congruent to 1 kcal/mol). The binding is described with a mass action formalism and the non-specific electrostatic accumulation of the peptides in the aqueous diffuse double layer is described with the Gouy-Chapman theory. This Gouy-Chapman/mass action model accounts surprisingly well for the sigmoidal dependence of binding on the percentage of acidic lipids in the membrane (apparent co-operativity or Hill coefficient1); the model assumes that the multivalent basic peptides bind1 acidic lipids and thus induce or stabilize domain formation.

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥ 4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.

Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation.

Membrane association is required for cell transformation by pp60v-src (v-Src), the product of the v-src oncogene of Rous sarcoma virus. Previous experiments have identified two NH2-terminal membrane-binding motifs: a myristate (14-carbon acyl chain) attached to the NH2-terminal glycine and three basic residues at positions 5, 7, and 9 of Src. We examined the membrane binding of each motif using myristylated (myr-src) and nonmyristylated (nonmyr-src) peptides corresponding to the NH2 terminus of Src. All myristylated peptides partitioned equally well onto electrically neutral phosphatidylcholine vesicles (K1 = 10(4) M-1). Identical binding has been observed for simple myristylated peptides (e.g., myr-Gly) and arises from the hydrophobic insertion of the myristate into the bilayer. A nonmyristylated peptide corresponding to residues 2-16 of Src [nonmyr-src(2-16), net charge = +5] bound to vesicles containing 33% monovalent acidic phospholipids with K1 = 10(3) M-1. Penta(lysine) (+5 net charge) exhibits the same binding behavior, which is due to the electrostatic interaction between basic residues and acidic lipids. The corresponding myristylated peptide, myr-src(2-16), binds 3 orders of magnitude more strongly to vesicles containing 33% acidic lipids than to neutral vesicles. The resulting apparent association constant, K1 = 10(7) M-1, is approximately equal to the product of the partition coefficients for the two individual interactions. This 10(7) M-1 binding is sufficiently strong to anchor the Src protein to biological membranes. We propose a simple model that explains the observed synergism between the two peptide-membrane interactions.

Biomarkers derived from gene expression profiling data may have a high false-positive rate and must be rigorously validated using independent clinical data sets, which are not always available. Although animal model systems could provide alternative data sets to formulate hypotheses and limit the number of signatures to be tested in clinical samples, the predictive power of such an approach is not yet proven. The present study aims to analyze the molecular signatures of liver cancer in a c-MET-transgenic mouse model and investigate its prognostic relevance to human hepatocellular carcinoma (HCC). Tissue samples were obtained from tumor (TU), adjacent non-tumor (AN) and distant normal (DN) liver in Tet-operator regulated (TRE) human c-MET transgenic mice (n = 21) as well as from a Chinese cohort of 272 HBV- and 9 HCV-associated HCC patients. Whole genome microarray expression profiling was conducted in Affymetrix gene expression chips, and prognostic significances of gene expression signatures were evaluated across the two species. Our data revealed parallels between mouse and human liver tumors, including down-regulation of metabolic pathways and up-regulation of cell cycle processes. The mouse tumors were most similar to a subset of patient samples characterized by activation of the Wnt pathway, but distinctive in the p53 pathway signals. Of potential clinical utility, we identified a set of genes that were down regulated in both mouse tumors and human HCC having significant predictive power on overall and disease-free survival, which were highly enriched for metabolic functions. In conclusions, this study provides evidence that a disease model can serve as a possible platform for generating hypotheses to be tested in human tissues and highlights an efficient method for generating biomarker signatures before extensive clinical trials have been initiated. © 2011 Ivanovska et al., published_or_final_version

Purpose: Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells. Experimental Design: Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G2 block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice. Results: Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G2 block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors. Conclusions: These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation. Clin Cancer Res; 17(17); 5638–48. ©2011 AACR.

Background: In hepatocellular carcinoma (HCC) genes predictive of survival have been found in both adjacent normal (AN) and tumor (TU) tissues. The relationships between these two sets of predictive genes and the general process of tumorigenesis and disease progression remains unclear. Methodology/Principal Findings: Here we have investigated HCC tumorigenesis by comparing gene expression, DNA copy number variation and survival using ~250 AN and TU samples representing, respectively, the pre-cancer state, and the result of tumorigenesis. Genes that participate in tumorigenesis were defined using a gene-gene correlation meta-analysis procedure that compared AN versus TU tissues. Genes predictive of survival in AN (AN-survival genes) were found to be enriched in the differential gene-gene correlation gene set indicating that they directly participate in the process of tumorigenesis. Additionally the AN-survival genes were mostly not predictive after tumorigenesis in TU tissue and this transition was associated with and could largely be explained by the effect of somatic DNA copy number variation (sCNV) in cis and in trans. The data was consistent with the variance of AN-survival genes being rate-limiting steps in tumorigenesis and this was confirmed using a treatment that promotes HCC tumorigenesis that selectively altered AN-survival genes and genes differentially correlated between AN and TU. Conclusions/Significance: This suggests that the process of tumor evolution involves rate-limiting steps related to the background from which the tumor evolved where these were frequently predictive of clinical outcome. Additionally treatments that alter the likelihood of tumorigenesis occurring may act by altering AN-survival genes, suggesting that the process can be manipulated. Further sCNV explains a substantial fraction of tumor specific expression and may therefore be a causal driver of tumor evolution in HCC and perhaps many solid tumor types. © 2011 Lamb et al., published_or_final_version

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)

Lanthanides and calcium compete for binding sites on the oxidizing side of Photosystem II. When the trivalent ions replace calcium, the oxygen evolving complex is unable to proceed to the higher oxidation states. Oxygen-evolution activity can be reconstituted by removing the lanthanide and adding back calcium. The number of lanthanide atoms bound to PS II preparations with and without the 17 and 23 kDa species is the same. This is taken as evidence that the extrinsic 17 and 23 kDa do not bind lanthanide/calcium ions. A low-temperature EPR study, with both diamagnetic and paramagnetic lanthanide ions, has demonstrated a close interaction between the lanthanides and the tyrosine radical YD.