Pharmacokinetics and toxicity of the novel oral demethylating agent zebularine in laboratory and tumor bearing dogs.

The purpose of this study was to determine the plasma pharmacokinetics (PK) and toxicity of zebularine, an oral cytidine analog with demethylating activity, in dogs. Plasma zebularine concentrations were determined by HPLC-MS/MS following an oral zebularine dose of 8 or 4 mg kg-1 . Plasma zebularine clearance was constant. Mean maximum concentration (Cmax ) was 23 ± 4.8 and 8.6 ± 1.4 µM following 8 and 4 mg kg-1 , respectively. Mean half-life was 5.7 ± 0.84 and 7.1 ± 2.1 following 8 and 4 mg kg-1 , respectively. A single 8 mg kg-1 dose was well tolerated. Daily 4 mg kg-1 treatment in three laboratory dogs resulted in grade 4 neutropenia (n = 3), grade 1 anorexia (n = 2) and grade 1 or 2 dermatologic changes (n = 2). All adverse events resolved with supportive care. A 4 mg kg-1 dose every 21 days was well tolerated. A follow-up dose escalation study is in progress with a lower starting dose.

Batch and Pulsed Fed-Batch Cultures of Aspergillus flavipes FP-500 Growing on Lemon Peel at Stirred Tank Reactor.

Aspergillus flavipes FP-500 grew up on submerged cultures using lemon peel as the only carbon source, developing several batch and pulsed fed-batch trials on a stirred tank reactor. The effect of carbon source concentration, reducing sugar presence and initial pH on exopectinase and endopectinase production, was analyzed on batch cultures. From this, we observed that the highest substrate concentration favored biomass (X max) but had not influence on the corresponding specific production (q p) of both pectinases; the most acid condition provoked higher endopectinase-specific productions but had not a significant effect on those corresponding to exopectinases; and reducing sugar concentrations higher than 1.5 g/L retarded pectinase production. On the other hand, by employing the pulsed fed-batch operation mode, we observed a prolonged growth phase, and an increase of about twofold on endopectinase production without a significant raise on biomass concentration. So, pulsed fed-batch seems to be a good alternative for obtaining higher endopectinase titers by using high lemon peel quantities without having mixing and repression problems to the system. The usefulness of unstructured kinetic models for explaining, under a theoretic level, the behavior of the fungus along the batch culture with regard to pectinase production was evident.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Disruption of the MYC-miRNA-EZH2 loop to suppress aggressive B-cell lymphoma survival and clonogenicity.

c-MYC (hereafter MYC) overexpression has been recognized in aggressive B-cell lymphomas and linked to adverse prognosis. MYC activation results in widespread repression of micro-RNA (miRNA) expression and associated with lymphoma aggressive progression. Our recent study identified a MYC-miRNA-EZH2 feed-forward loop linking overexpression of MYC, EZH2 and miRNA repression. Here, using a novel small-molecule BET bromodomain inhibitor, JQ1, and the EZH2 inhibitor, DZNep, we demonstrated that combined treatment of JQ1 and DZNep cooperatively disrupted MYC activation, resulting in a greater restoration of miR-26a expression and synergistically suppressed lymphoma growth and clonogenicity in aggressive lymphoma cells. Furthermore, CHIP assay demonstrated that MYC recruited EZH2 to miR-26a promoter and cooperatively repressed miR-26a expression in aggressive lymphoma cell lines, as well as primary lymphoma cells. Loss- or gain-of-function approaches revealed that miR-26a functioned as a tumor suppressor miRNA and mediated the combinatorial effects of JQ1 and DZNep. These findings represent a novel promising approach for silencing MYC-miRNA-EZH2 amplification loop for combinatorial therapy of aggressive B-cell lymphomas.

Silencing of Kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer.

The Kruppel-like factor (KLF) proteins are multitasked transcriptional regulators with an expanding tumor suppressor function. KLF2 is one of the prominent members of the family because of its diminished expression in malignancies and its growth-inhibitory, pro-apoptotic and anti-angiogenic roles. In this study, we show that epigenetic silencing of KLF2 occurs in cancer cells through direct transcriptional repression mediated by the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2). Binding of EZH2 to the 5'-end of KLF2 is also associated with a gain of trimethylated lysine 27 histone H3 and a depletion of phosphorylated serine 2 of RNA polymerase. Upon depletion of EZH2 by RNA interference, short hairpin RNA or use of the small molecule 3-Deazaneplanocin A, the expression of KLF2 was restored. The transfection of KLF2 in cells with EZH2-associated silencing showed a significant anti-tumoral effect, both in culture and in xenografted nude mice. In this last setting, KLF2 transfection was also associated with decreased dissemination and lower mortality rate. In EZH2-depleted cells, which characteristically have lower tumorigenicity, the induction of KLF2 depletion 'rescued' partially the oncogenic phenotype, suggesting that KLF2 repression has an important role in EZH2 oncogenesis. Most importantly, the translation of the described results to human primary samples demonstrated that patients with prostate or breast tumors with low levels of KLF2 and high expression of EZH2 had a shorter overall survival.

Hypermethylation of specific microRNA genes in MLL-rearranged infant acute lymphoblastic leukemia: major matters at a micro scale.

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is the most aggressive type of childhood leukemia. To develop more suitable treatment strategies, a firm understanding of the biology underlying this disease is of utmost importance. MLL-rearranged ALL displays a unique gene expression profile, partly explained by erroneous histone modifications. We recently showed that t(4;11)-positive infant ALL is also characterized by pronounced promoter CpG hypermethylation. In this study, we investigated whether this widespread hypermethylation also affected microRNA (miRNA) expression. We identified 11 miRNAs that were downregulated in t(4;11)-positive infant ALL as a consequence of CpG hypermethylation. Seven of these miRNAs were re-activated after exposure to the de-methylating agent Zebularine. Interestingly, five of these miRNAs are associated either with MLL or MLL fusions, and for miR-152 we found both MLL and DNA methyltransferase 1 (DNMT1) as potential targeted genes. Finally, a high degree of methylation of the miR-152 CpG island was strongly correlated with a poor clinical outcome. Our data suggests that inhibitors of methylation have a potential beyond re-expression of hypermethylated protein-coding genes in t(4;11)-positive infant ALL. In this study, we provide additional evidence that they should be tested for their efficacy in MLL-rearranged infant ALL in in vivo models.

Wealth of opportunity - the C1 domain as a target for drug development.

The diacylglycerol-responsive C1 domains of protein kinase C and of the related classes of signaling proteins represent highly attractive targets for drug development. The signaling functions that are regulated by C1 domains are central to cellular control, thereby impacting many pathological conditions. Our understanding of the diacylglycerol signaling pathways provides great confidence in the utility of intervention in these pathways for treatment of cancer and other conditions. Multiple compounds directed at these signaling proteins, including compounds directed at the C1 domains, are currently in clinical trials, providing strong validation for these targets. Extensive understanding of the structure and function of C1 domains, coupled with detailed insights into the molecular details of ligand - C1 domain interactions, provides a solid basis for rational and semi-rational drug design. Finally, the complexity of the factors contributing to ligand - C1 domain interactions affords abundant opportunities for manipulation of selectivity; indeed, substantially selective compounds have already been identified.

Synthesis, stability, and protonation studies of a self-complementary dodecamer containing the modified nucleoside 2'-deoxyzebularine.

The nucleoside 2'-deoxyzebularine (K) was incorporated into the self-complementary dodecamer 5'-CGTACGKGTACG-3' by solid-phase 2-cyanoethylphosphoramidite chemistry using dimethoxytrityl (DMT) as the 5'-hydroxyl protecting group. Standard synthesis cycles using trichloroacetic acid and short ammonia treatment (50 degrees C for 30 min) were found to be the optimal conditions to obtain the desired dodecamer with minimum acid and basic degradation of the acid- and base-sensitive 2-pyrimidinone residue. The protonation equilibria of the K nucleoside and of the dodecamer at 37 degrees C were studied by means of spectroscopically monitored titrations. For the K nucleoside, a pK(a) value of 3.13 +/- 0.09 was obtained. For the dodecamer, four acid-base species were found in the pH range 2-12, with pK(a) values of 9.60 +/- 0.07, 4.46 +/- 0.16, and 2.87 +/- 0.19. Melting experiments were carried out to confirm the proposed acid-base concentration profiles. Finally, kinetic experiments were also carried out at several pH values to evaluate the stability of the K nucleoside and of the dodecamer. An increased stability was shown by the K nucleoside when incorporated into the dodecamer. Multivariate methods based on both hard- and soft-modeling were applied for the analysis of spectroscopic data, allowing the estimation of concentration profiles and pure spectra.

Conformationally constrained analogues of diacylglycerol. 18. The incorporation of a hydroxamate moiety into diacylglycerol-lactones reduces lipophilicity and helps discriminate between sn-1 and sn-2 binding modes to protein kinase C (PK-C). Implications for isozyme specificity.

An approach to reduce the log P in a series of diacylglycerol (DAG)-lactones known for their high binding affinity for protein kinase C (PK-C) is presented. Branched alkyl groups with reduced lipophilicity were selected and combined with the replacement of the ester or lactone oxygens by NH or NOH groups. Compound 6a with an isosteric N-hydroxyl amide arm represents the most potent and least lipophilic DAG analogue known to date.

Ribose modified nucleosides and nucleotides as ligands for purine receptors.

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3',5'-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs.

Inhibition of (cytosine C5)-methyltransferase by oligonucleotides containing flexible (cyclopentane) and conformationally constrained (bicyclo[3.1.0]hexane) abasic sites.

Pseudorotationally locked sugar analogues based on bicyclo[3.1.0]-hexane templates were placed in DNA duplexes as abasic target sites in the M. HhaI recognition sequence. The binding affinity of the enzyme increases when the abasic site is constrained to the South conformation and decreases when it is constrained to the North conformation. A structural understanding of these differences is provided.

Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings.

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5'-position with an N-methyluronamide or with phosphate groups.

Ring-Constrained (N)-methanocarba nucleosides as adenosine receptor agonists: independent 5'-uronamide and 2'-deoxy modifications.

Novel methanocarba adenosine analogues, having the pseudo-ribose northern (N) conformation preferred at adenosine receptors (ARs), were synthesized and tested in binding assays. The 5'-uronamide modification preserved [N6-(3-iodobenzyl)] or enhanced (N6-methyl) affinity at A3ARs, while the 2'-deoxy modification reduced affinity and efficacy in a functional assay.

5-acyloxy-5-hydroxymethyltetrahydro-2-furancarboxylate as a novel template for protein kinase C (PKC) binding.

A series of alkyl tetrahydrofuran-2-carboxylates (1-4) bearing a new set of three pharmacophoric groups were tested as protein kinase C (PKC) ligands. The compounds were synthesized from commercially available glycidyl 4-methoxyphenyl ether. The correlation between their binding affinities for PKC-alpha and a conformational fit to phorbol ester indicates they mimic a pharmacophore model comprising the C20-OH, C3-C=O and C9-OH rather than that including the C13-C=O moiety.

An optimized protein kinase C activating diacylglycerol combining high binding affinity (Ki) with reduced lipophilicity (log P).

A small, focused combinatorial library encompassing all possible permutations of acyl branched alkyl chains-small and large, saturated and unsaturated-was generated from the active diacylglycerol enantiomer (S-DAG) to help identify the analogue with the highest binding affinity (lowest Ki) for protein kinase C (PK-C) combined with the minimum lipophilicity (log P). The selected ligand (3B) activated PK-C more effectively than sn-1,2-dioctanoylglycerol (diC8) despite being 1.4 log units more hydrophilic. Compound 3B indeed represents the most potent, hydrophilic DAG ligand to date. With the help of a green fluorescent protein (GFP)-tagged PK-Calpha, 3B was able to translocate the full length protein to the membrane with an optimal dose of 100 microM in CHO-K1 cells, while diC8 failed to achieve translocation even at doses 3-fold higher. Molecular modeling of 3B into an empty C1b domain of PK-Cdelta clearly showed the existence of a preferred binding orientation. In addition, molecular dynamic simulations suggest that binding discrimination could result from a favorable van der Waals (VDW) interaction between the large, branched sn-1 acyl group of 3B and the aromatic rings of Trp252 (PK-Cdelta) or Tyr252 (PK-Calpha). The DAG analogue of 3B in which the acyl groups are reversed (2C) showed a decrease in binding affinity reflecting the capacity of PK-C to effectively discriminate between alternative orientations of the acyl chains.

Role of hydrophobic residues in the C1b domain of protein kinase C delta on ligand and phospholipid interactions.

The C1 domains of conventional and novel protein kinase C (PKC) isoforms bind diacylglycerol and phorbol esters with high affinity. Highly conserved hydrophobic residues at or near the rim of the binding cleft in the second cysteine-rich domain of PKC-delta (PKC-deltaC1b) were mutated to probe their roles in ligand recognition and lipid interaction. [(3)H]Phorbol 12,13-dibutyrate (PDBu) binding was carried out both in the presence and absence of phospholipids to determine the contribution of lipid association to the ligand affinity. Lipid dependence was determined as a function of lipid concentration and composition. The binding properties of a high affinity branched diacylglycerol with lipophilicity similar to PDBu were compared with those of PDBu to identify residues important for ligand selectivity. As expected, Leu-20 and Leu-24 strongly influenced binding. Substitution of either by aspartic acid abolished binding in either the presence or absence of phosphatidylserine. Mutation of Leu-20 to Arg or of Leu-24 to Lys caused a dramatic (340- and 250-fold, respectively) reduction in PDBu binding in the presence of lipid but only a modest reduction in the weaker binding of PDBu observed in the absence of lipid, suggesting that the main effect was on C1 domain -phospholipid interactions. Mutation of Leu-20 to Lys or of Trp-22 to Lys had modest (3-fold) effects and mutation of Phe-13 to Tyr or Lys was without effect. Binding of the branched diacylglycerol was less dependent on phospholipid and was more sensitive to mutation of Trp-22 to Tyr or Lys, especially in the presence of phospholipid, than was PDBu. In terms of specific PKC isoforms, our results suggest that the presence of Arg-20 in PKC-zeta may contribute to its lack of phorbol ester binding activity. More generally, the results emphasize the interplay between the C1 domain, ligand, and phospholipid in the ternary binding complex.

N-(3-Acyloxy-2-benzylpropyl)-N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives as potent vanilloid receptor agonists and analgesics.

A series of N-(3-acyloxy-2-benzylpropyl)-N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives were investigated as vanilloid receptor ligands in an effort to discover a novel class of analgesics. The proposed pharmacophore model of resiniferatoxin. which includes the C20 homovanillic moiety, the C3-carbonyl and the orthoester phenyl ring as key pharmacophoric groups, was utilized as a guide for drug design. The compounds were synthesized after several steps from diethylmalonate and evaluated in vitro in a receptor binding assay and in a capsaicin-activated channel assay. Additional evaluation of analgesic activity, anti-inflammatory activity and pungency was conducted in animal models by the writhing test, the ear edema assay, and the eye-wiping test, respectively. Among the new compounds, 23 and 28 were found to be the most potent receptor agonists of the series with Ki values of 19 nM and 11 nM, respectively. Their strong in vitro potencies were also reflected by an excellent analgesic profile in animal tests with ED50 values of 0.5 microg kg for 23 and 1.0 microg/kg for 28. Relative to capsaicin these compounds appear to be ca. 600 and 300 times more potent. Both 23 and 28 were found to be less pungent than capsaicin based on the eye-wiping test. However, the compounds did not show significant anti-inflammatory activity. A molecular modeling study comparing the energy-minimized structures of resiniferatoxin and 35 demonstrated a good correlation in the spatial disposition of the corresponding key pharmacophores. The thioureas described in this investigation, which were designed as simplified resiniferatoxin surrogates, represent a novel class of potent vanilloid receptor agonists endowed with potent analgesic activity and reduced pungency.

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites.

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2'-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.

A remarkably simple chemicoenzymatic approach to structurally complex bicyclo[3.1.0]hexane carbocyclic nucleosides.

[reaction: see text] Intramolecular cyclopropanation of a carbene engendered from the corresponding diazo beta-ketoester produced the desired bicyclo[3.1. 0]hexane pseudosugar. Purine nucleosides obtained via Mitsunobu coupling were resolved with adenosine deaminase. The requisite beta-ketoester was assembled in one step from ethyl acetoacetate and acrolein.

Conformationally constrained analogues of diacylglycerol (DAG). 17. Contrast between sn-1 and sn-2 DAG lactones in binding to protein kinase C.

In previous work, we have obtained potent protein kinase C (PK-C) ligands with low-namomolar binding affinities by constructing diacylglycerol (DAG) mimetics in which the sn-2 carbonyl of DAG was constrained into a lactone ring. An additional structural element that helped achieve high binding affinity was the presence of branched acyl or alpha-alkylidene chains. In the present study, the effects of similarly branched chains on a different lactone system, where the lactone carbonyl is now equivalent to the sn-1 carbonyl of DAG, are investigated. In this new lactone template, the two chiral centers must have the S-configuration for enzyme recognition. As with the sn-2 DAG lactones, the branched chains were designed to optimize van der Waals contacts with a group of conserved hydrophobic amino acids located on the rim of the C1 domain of PK-C. The acyl and alpha-alkylidene chains were also designed to be lipophilically equivalent (8 carbons each). Eight new compounds (7-14) representing all possible combinations of linear and branched acyl and alpha-alkylidene were synthesized and evaluated. The sn-1 DAG lactones were less effective as PK-C ligands than the sn-2 DAG lactones despite having a similar array of linear or branched acyl and alpha-alkylidene chains