Synthesis and evaluation of indenoisoquinoline topoisomerase I inhibitors substituted with nitrogen heterocycles.

In connection with an ongoing investigation of indenoisoquinoline topoisomerase I (Top1) inhibitors as potential therapeutic agents, the pharmacophore possessing di(methoxy) and methylenedioxy substituents was held constant, and new derivatives were synthesized with nitrogen heterocycles appended to the lactam side chain. Compounds were evaluated for Top1 inhibition and for cytotoxicity in the National Cancer Institute's human cancer cell screen. Some of the more potent derivatives were also screened for in vivo activity in a hollow fiber assay.

Controlling bacteriophage phi29 DNA-packaging motor by addition or discharge of a peptide at N-terminus of connector protein that interacts with pRNA.

Bacteriophage phi29 utilizes a motor to translocate genomic DNA into a preformed procapsid. The motor contains six pRNAs, an enzyme and one 12-subunit connector with a central channel for DNA transportation. A 20-residue peptide containing a His-tag was fused to the N-terminus of the connector protein gp10.

Decomposition of protein experimental compressibility into intrinsic and hydration shell contributions.

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces.

Simultaneous downregulation of CDK inhibitors p18(Ink4c) and p27(Kip1) is required for MEN2A-RET-mediated mitogenesis.

Multiple endocrine neoplasia type 2A (MEN2A) is predisposed by mutations in the RET proto-oncogene. Low expression of the cyclin-dependent kinase inhibitor (CDKI) p27(Kip1) is present in thyroid tumors, and recent evidence demonstrates p27 downregulation by the active RET mutant, RET/PTC1, found in papillary thyroid carcinoma. This implicates decreased p27 activity as an important event during thyroid tumorigenesis.

Replacement of the metabolically labile methyl esters in the alkenyldiarylmethane series of non-nucleoside reverse transcriptase inhibitors with isoxazolone, isoxazole, oxazolone, or cyano substituents.

The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors that have potential value in the treatment of HIV/AIDS. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties. A series of novel ADAMs were therefore designed and synthesized in order to replace the metabolically labile methyl ester moieties of the existing ADAM lead compounds with hydrolytically stable, fused isoxazolone, isoxazole, oxazolone, or cyano substituents on the aromatic rings.

Synthesis and biological evaluation of bisindenoisoquinolines as topoisomerase I inhibitors.

The indenoisoquinolines represent a class of non-camptothecin topoisomerase I (Top1) inhibitors that exert cytotoxicity by trapping the covalent complex formed between DNA and Top1 during relaxation of DNA supercoils. As an ongoing evaluation of Top1 inhibition and anticancer activity, indenoisoquinolines were linked via their lactam side chains to provide polyamines end-capped with intercalating motifs. The resulting bisindenoisoquinolines were evaluated for cytotoxicity in the National Cancer Institute's human cancer cell screen and for Top1 inhibition.

Synthesis and activity of a folate peptide camptothecin prodrug.

A folate receptor targeted camptothecin prodrug was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. The conjugate was found to possess high affinity for folate receptor-expressing cells and inhibited cell proliferation in human KB cells with an IC(50) of 10nM. Activity of the prodrug was completely blocked by excess folic acid, demonstrating receptor-mediated uptake.

Migration and function of FoxP3+ regulatory T cells in the hematolymphoid system.

FoxP3+ T cells play critical roles in regulation of the hematolymphoid system and prevention of autoimmunity. Many FoxP3+ T cells, generated in thymus as the result of T cell receptor (TCR) recognition of self antigens, preferentially migrate to secondary lymphoid tissues such as lymph nodes and spleen in a manner similar to conventional naïve T cells. FoxP3+ T cells differentiated in the periphery acquire homing phenotype to bone marrow and nonlymphoid tissues.

Selective identification of Valpha14i T cells using slide-immobilized, CD1d-antigen complexes.

The ability to correlate changes in antigen-reactive lymphocytes with disease will provide information needed to develop strategies for combating illness. One critical group of lymphocytes are the CD1-restricted T cells. It is desirable to use CD1 molecules in an array format to query CD1-restricted lymphocytes in humans.

Regulation of trafficking receptor expression in human forkhead box P3+ regulatory T cells.

Forkhead Box P3(+) (FOXP3(+)) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3(+) T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3(+) T cells in neonatal cord blood, adult peripheral blood, and tonsils.

The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding.

Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells.

Synthesis and anticancer activity of brefeldin A ester derivatives.

Ester derivatives of brefeldin A (BFA) were synthesized to determine which of its two hydroxyl groups could be modified while still maintaining biological activity. The compounds were tested for antiproliferative activity in the National Cancer Institute's 60 cancer cell line screen. Monoderivatization at the C4 and C7 alcohols was tolerated, yielding biologically active compounds, whereas the analogues derivatized at both positions were the least active in the series.

Evaluation of indenoisoquinoline topoisomerase I inhibitors using a hollow fiber assay.

The indenoisoquinolines are a novel class of non-camptothecin topoisomerase I (Top1) inhibitors whose mechanism of action involves trapping the covalent complex formed between DNA and Top1 during cellular processes. As an ongoing evaluation of the indenoisoquinolines for Top1 inhibition and anticancer activity, indenoisoquinoline analogs have been screened in the National Cancer Institute's hollow fiber assay (HFA). Some of the derivatives demonstrated significant activity at intraperitoneal and subcutaneous fiber placement sites, along with net cancer cell kill in one or more cell lines.

Identification of a basic helix-loop-helix transcription factor expressed in mammary gland alveolar cells and required for maintenance of the differentiated state.

The development of mammary glands relies on complicated signaling pathways that control cell proliferation, differentiation, and apoptotic events through transcriptional regulatory circuits. A key family of transcription factors used in mammary gland development is the helix-loop-helix/basic helix-loop-helix (HLH/bHLH) protein family. In this study, we identify Mist1 as a tissue-restricted Class II bHLH transcription factor expressed in lactating mammary glands.

Current strategies and future directions for eluding adenoviral vector immunity.

Adenoviral (Ad) vectors can efficiently transduce a broad range of cell types and have been used extensively in preclinical and clinical studies for gene delivery applications. The presence of preexisting Ad immunity in the majority of human population and a rapid development of immune response against the Ad vector backbone following the first inoculation with the vector have impeded clinical use of these vectors. In addition, a number of animal inoculation studies have demonstrated that high systemic doses of Ad vectors invariably lead to initiation of acute inflammatory responses.

Src kinase activation: A switched electrostatic network.

Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A-loop); and movement of an alpha-helix (alphaC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down-regulated conformation of CD for the Src-kinase family member Lyn kinase, and to gain insight into the interdependence of these changes.

Cobalt(II) chloride catalyzed one-pot synthesis of alpha-aminonitriles.

A simple and efficient method has been developed for the synthesis of alpha-aminonitriles by a one-pot three- component condensation of aldehydes, amines, and potassium cyanide in acetonitrile in the presence of a catalytic amount of CoCl2 at room temperature.

Regioselective synthesis of N-beta-hydroxyethylaziridines by the ring-opening reaction of epoxides with aziridine generated in situ.

[reaction: see text] Biologically important N-beta-hydroxyethylaziridine intermediates were conveniently prepared by regioselective ring-opening reactions of diversely substituted epoxides. Ethyleneimine generated in situ under basic conditions from beta-chloroethylamine was used as a nucleophile to open the epoxides in an aqueous environment.

Total synthesis and biological evaluation of 22-hydroxyacuminatine.

A total synthesis of 22-hydroxyacuminatine, a cytotoxic alkaloid isolated from Camptotheca acuminata, is reported. The key step in the synthesis involves the reaction of 2,3-dihydro-1H-pyrrolo[3,4-b]quinoline with a brominated phthalide to generate a substituted pentacyclic 12H-5,11a-diazadibenzo[b,h]fluoren-11-one intermediate. Despite its structural resemblance to camptothecin and luotonin A, a biological evaluation of 22-hydroxyacuminatine in a topoisomerase I-deficient cell line P388/CPT45 has confirmed that the observed cytotoxicity is not due to topoisomerase I inhibition, even though 22-hydroxyacuminatine has a hydroxyl group that can theoretically hydrogen bond to Asp533.

RNA nanotechnology: engineering, assembly and applications in detection, gene delivery and therapy.

Biological macromolecules including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. RNA is unique in nanoscale fabrication due to its amazing diversity of function and structure. RNA molecules can be designed and manipulated with a level of simplicity characteristic of DNA while possessing versatility in structure and function similar to that of proteins.

Cell cycle requirements in assembling silent chromatin in Saccharomyces cerevisiae.

The establishment of silencing at the silent mating-type locus, HMR, in Saccharomyces cerevisiae requires that yeast pass through S phase of the cell cycle, yet requires neither the initiation of DNA replication at the locus destined to become silenced nor the passage of a replication fork through that locus. We tested whether this S-phase requirement reflects a window within the cell cycle permissive for recruitment of Sir proteins to HMR. The S-phase-restricted event necessary for silencing occurred after recruitment of Sir proteins to HMR.

Structural basis for the requirement of two phosphotyrosine residues in signaling mediated by Syk tyrosine kinase.

The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen.

Direct manipulation of activator protein-1 controls thymocyte proliferation in vitro.

B cell activating transcription factor (BATF) belongs to the activator protein-1 (AP-1) superfamily of basic leucine zipper transcription factors and forms heterodimers with Jun that possess minimal transcriptional activity. Mice carrying a p56(lck)HA-BATF transgene were created to observe the effects of constitutive expression of this well-characterized AP-1 inhibitor on T cell proliferation. Consistent with the role of AP-1 in promoting the proliferation of many cell types, BATF-transgenic thymocytes proliferate poorly in vitro when stimulated with anti-CD3epsilon and anti-CD28 antibodies or with Concanavalin A.

Synthesis and anti-HIV activity of new alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors (NNRTIs) incorporating benzoxazolone and benzisoxazole rings.

The HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) constitute a large and structurally diverse set of compounds, several of which are currently used in the treatment of AIDS. A series of novel alkenyldiarylmethanes (ADAMs) were designed and synthesized as part of an ongoing investigation to replace the metabolically labile methyl ester moieties found in the ADAM pharmacophore with stable modifications that retain the potent anti-HIV activity of the parent compounds. Unsurprisingly, the rat plasma half-lives of the new ADAMs were not improved when compared to the parent compounds, but all of the synthesized ADAMs inhibited the cytopathic effect of HIV-1 in cell culture.

Effect of E-ring modifications in camptothecin on topoisomerase I inhibition: a quantum mechanics treatment.

Camptothecins (CPTs) are the prototypical class of topoisomerase I (Top1) inhibitors with significant anticancer activities. Structure-activity relationship studies have demonstrated that inverting the stereochemistry at C-20 (R-CPT) or changing the E-ring lactone to a lactam (CPT-lactam) abolishes the Top1 inhibitory activity. The explanations that have been advanced for these effects are that there is either a failure of hydrogen bond formation involving the C-20 hydroxyl group of R-CPT or a failure of E-ring opening of the lactam, which have been proposed to be required for Top1 inhibition.