Formation of Trideuteromethylated Artifacts of Pyrrole-Containing Natural Products.

Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d6 during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d6 during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.

Recifin A, Initial Example of the Tyr-Lock Peptide Structural Family, Is a Selective Allosteric Inhibitor of Tyrosyl-DNA Phosphodiesterase I.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel ß-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDP1 in a FRET assay with an IC50 of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDP1 while not affecting the activity of a truncated catalytic domain of TDP1 lacking the N-terminal regulatory domain (Δ1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDP1. Recifin A represents both the first of a unique structural class of knotted disulfide-rich peptides and defines a previously unseen mechanism of TDP1 inhibition that could be productively exploited for potential anticancer applications.

Multiphase Assembly of Small Molecule Microcrystalline Peptide Hydrogel Allows Immunomodulatory Combination Therapy for Long-Term Heart Transplant Survival.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP.

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.

Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.

Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form.

The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.

Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP.

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.

Inhibition of ABCG2-mediated drug efflux by naphthopyrones from marine crinoids.

Five new naphthopyrones (1-5) along with the known compounds TMC-256A1, 5,8-dihydroxy-6-methoxy-2-propyl-4H-naphtho[2,3-b]pyran-4-one, TMC-256C1, comaparvin, 6-methoxycomaparvin, and 6-methoxycomaparvin 5-methyl ether (6-11) were isolated from crinoids of the family Comasteridae. All compounds were tested for their ability to inhibit the multidrug transporter ABCG2, which plays a role in drug resistance. Six of the seven angular naphthopyrones showed moderate activity with <60% inhibition of ABCG2-mediated transport as compared to the positive control fumitremorgin C. None of the linear naphthopyrones inhibited ABCG2-mediated efflux.

North- and south-bicyclo[3.1.0]hexene nucleosides: the effect of ring planarity on anti-HIV activity.

The syntheses of new conformationally locked North- and South-bicyclo[3.1.0]hexene nucleosides is reported. The North analogues were synthesized by a convergent approach from the known (1S,2R,5R)-5-[(tert-butyldiphenylsilyloxy)methyl]bicyclo[3.1.0]hex-3-en-2-ol by Mitsunobu coupling with the nucleobases. The South analogues were synthesized from their bicyclo[3.1.0]hexane nucleoside precursors by the selective protection of the primary hydroxy group, conversion of the secondary alcohol into a good leaving group, and base-catalyzed elimination to generate the olefin. The transformation of a bicyclo[3.1.0]hexane nucleoside into a bicyclo[3.1.0]hexene nucleoside flattens the five-membered ring of the bicyclic system and rescues anti-HIV activity for North-D4T, North-D4A, and South-D4C. The relationship between planarity and the anti/syn disposition of the nucleobase that is favored by a particular pseudosugar platform are proposed as key parameters in controlling biological activity.

Synthesis of a cross-bridged cyclam derivative for peptide conjugation and 64Cu radiolabeling.

The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo- tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H 2CB-TE2A), a member of the ethylene "cross-bridged" cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its nonbridged analogue, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications.

Conformationally constrained analogues of diacylglycerol (DAG). 27. Modulation of membrane translocation of protein kinase C (PKC) isozymes alpha and delta by diacylglycerol lactones (DAG-lactones) containing rigid-rod acyl groups.

Highly rigid and geometrically well-defined rods composed of ethynylene-substituted aromatic spacers [oligo(p-phenyleneethynylene), OPE] were incorporated as acyl moieties on diacylglycerol lactones (DAG-lactones) and investigated for their ability to bind to protein kinase C (PKC) and translocate PKC alpha and delta isoforms to plasma and internal membranes. The kinetics of PKC translocation were correlated with biological responses, viz. ERK phosphorylation, induction of IL-6 secretion, inhibition of cell proliferation, and induction of cellular attachment, that display very different time courses. Because OPE rods assemble through noncovalent forces and form stable films, they may influence the microdomain environment around the DAG-lactone membrane-binding site. A comparison of two DAG-lactones (1 and 10), one with two PE units (1) and the other with an equivalent flexible acyl chain (10) of matching lipophilicity, clearly demonstrated the effect of the rigid OPE chain in substantially prolonging the translocated state of both PKC alpha and delta.

Mining the NCI antiviral compounds for HIV-1 integrase inhibitors.

HIV-1 integrase (IN) is an essential enzyme for effective viral replication and is a validated target for the development of antiretroviral drugs. Currently, there are no approved drugs targeting this enzyme. In this study, we have identified 11 structurally diverse small-molecule inhibitors of IN. These compounds have been selected by mining the moderately active antiviral molecules from a collection of 90,000 compounds screened by the National Cancer Institute (NCI) Antiviral Program. These compounds, which were screened at the NCI during the past 20 years, resulted in approximately 4000 compounds labeled as 'moderately active.' In our study, chalcone 11 shows the most potent activity with an IC(50) of 2+/-1 microM against purified IN in the presence of both Mn(2+) and Mg(2+) as cofactors. Docking simulations using the 11 identified inhibitors as a training set have elucidated two unique binding areas within the active site: the first encompasses the conserved D64-D116-E152 motif, while the other involves the flexible loop region formed by amino acid residues 140-149. The tested inhibitors exhibit favorable interactions with important amino acid residues through van der Waals and H-bonding contacts.

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. The magic of its chemistry and biology.

1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.

Interleukin 6 supports the maintenance of p53 tumor suppressor gene promoter methylation.

A strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. Interleukin 6 (IL-6) is an inflammatory cytokine known to play a role in the growth and survival of many types of tumors, yet the mechanisms employed by this pleomorphic cytokine to accomplish this feat are still poorly understood. Another important factor in tumor development seems to be the hypermethylation of CpG islands located within the promoter regions of tumor suppressor genes. This common epigenetic alteration enables tumor cells to reduce or inactivate the expression of important tumor suppressor and cell cycle regulatory genes. Here we show that in the IL-6-responsive human multiple myeloma cell line KAS 6/1, the promoter region of p53 is epigenetically modified by methyltransferases, resulting in decreased levels of expression. Furthermore, cells treated with IL-6 exhibit an increase in the expression of the DNA maintenance methylation enzyme, DNMT-1. The DNA methyltransferase inhibitor zebularine reverses the methylation of the p53 promoter, allowing the resumption of its expression. However, when zebularine is withdrawn from the cells, the reestablishment of the original CpG island methylation within the p53 promoter does not occur in the absence of IL-6, and cells which do not receive IL-6 eventually die, as p53 expression continues unchecked by remethylation. Interestingly, this loss of viability seems to involve not the withdrawal of cytokine, but the inability of the cell to resilence the promoter. Consistent with this model, when cells that express IL-6 in an autocrine fashion are subjected to identical treatment, p53 expression is reduced shortly after withdrawal of zebularine. Therefore, it seems IL-6 is capable of maintaining promoter methylation thus representing one of the possible mechanisms used by inflammatory mediators in the growth and survival of tumors.

Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells.

Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5'-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2'-deoxycytidine and requires higher doses for equivalent inhibition of DNMT.

Synthesis and biological study of a flavone acetic acid analogue containing an azido reporting group designed as a multifunctional binding site probe.

Flavone-8-acetic acid (FAA) is a potent immunomodulatory small molecule that is uniquely characterized as being active on mouse but not human cells. Although FAA is a potent inducer of murine cytokine, chemokine and interferon gene expression, its mode of action remains unknown. In this report, we describe the synthesis of a new flavone acetic acid (FAA) analogue, (2-[2-(4-azidophenyl)-4-oxochromen-8-yl-]acetic acid (compound 2). We demonstrate that compound 2 is equally active as the parent FAA in inducing chemokine gene expression and that the azide functional group is capable of reacting with a reporter molecule, such as the FLAG peptide-phosphine, under mild conditions. This reaction will be useful for detecting the drug-bound protein active complex utilizing an anti-FLAG antibody.

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy.

1-(Beta-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) corresponds structurally to cytidine minus the exocyclic 4-amino group. The increased electrophilic character of its simple aglycon endows the molecule with unique biologic properties as a potent inhibitor of both cytidine deaminase and DNA cytosine methyltransferase. The latter activity makes zebularine a promising antitumor agent that is hydrolytically stable, preferentially targets cancer cells, and shows activity both in vitro and in experimental animals, even after oral administration.

Microarray analysis of epigenetic silencing of gene expression in the KAS-6/1 multiple myeloma cell line.

The epigenetic control of gene transcription in cancer has been the theme of many recent studies and therapeutic approaches. Carcinogenesis is frequently associated with hypermethylation and consequent down-regulation of genes that prevent cancer, e.g., those that control cell proliferation and apoptosis. We used the demethylating drug zebularine to induce changes in DNA methylation, then examined patterns of gene expression using cDNA array analysis and Restriction Landmark Genomic Scanning followed by RNase protection assay and reverse transcription-PCR to confirm the results. Microarray studies revealed that many genes were epigenetically regulated by methylation. We concluded that methylation decreased the expression of, or silenced, several genes, contributing to the growth and survival of multiple myeloma cells. For example, a number of genes (BAD, BAK, BIK, and BAX) involved in apoptosis were found to be suppressed by methylation. Sequenced methylation-regulated DNA fragments identified by Restriction Landmark Genomic Scanning were found to contain CpG islands, and some corresponded to promoters of genes that were regulated by methylation. We also observed that after the removal of the demethylating drug, the addition of interleukin 6 restored CpG methylation and re-established previously silenced gene patterns, thus implicating a novel role of interleukin 6 in processes regulating epigenetic gene repression and carcinogenesis.

Macrocyclization in the design of non-phosphorus-containing Grb2 SH2 domain-binding ligands.

Macrocyclization from the phosphotyrosyl (pTyr) mimetic's beta-position has previously been shown to enhance Grb2 SH2 domain-binding affinity of phosphonate-based analogues. The current study examined the effects of such macrocyclization using a dicarboxymethyl-based pTyr mimetic. In extracellular assays affinity was enhanced approximately 5-fold relative to an open-chain congener. Enhancement was also observed in whole-cell assays examining blockade of Grb2 binding to the erbB-2 protein-tyrosine kinase.

Caulibugulones A-F, novel cytotoxic isoquinoline quinones and iminoquinones from the marine bryozoan Caulibugula intermis.

An extract of the marine bryozoan Caulibugula intermis, collected in the Indo-Pacific off Palau, produced a distinct pattern of differential cytotoxicity in the National Cancer Institute's 60 cell line antitumor screen. Bioactivity-directed fractionation of the extract provided six new compounds, caulibugulones A-F (1-6). The structures of these novel metabolites were determined by spectrochemical analyses including LC-MS, HRFABMS, 1-D and 2-D NMR experiments, and by comparison with related compounds. The structures of compounds 2 and 3 were confirmed by chemical interconversion. The isolated compounds exhibited IC(50)'s of 0.03-1.67 microg/mL against murine tumor cells in an in vitro cytotoxicity assay.