Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC50 values of 18–100 nM in a standard TRAP assay.

On the acidity and reactivity of HNO in aqueous solution and biological systems

The gas phase and aqueous thermochemistry and reactivity of nitroxyl (nitrosyl hydride, HNO) were elucidated with multiconfigurational self-consistent field and hybrid density functional theory calculations and continuum solvation methods. The pKa of HNO is predicted to be 7.2 ± 1.0, considerably different from the value of 4.7 reported from pulse radiolysis experiments. The ground-state triplet nature of NO− affects the rates of acid-base chemistry of the HNO/NO− couple. HNO is highly reactive toward dimerization and addition of soft nucleophiles but is predicted to undergo negligible hydration (Keq = 6.9 × 10−5). HNO is predicted to exist as a discrete species in solution and is a viable participant in the chemical biology of nitric oxide and derivatives.

Cadherin Sequences That Inhibit β-Catenin Signaling: A Study in Yeast and Mammalian Cells

Drosophila Armadillo and its mammalian homologue β-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/β-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/β-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or β-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/β-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited β-catenin–mediated signaling. This suggests that the interaction between β-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

Mechanism of anti-HIV action of masked alaninyl d4T-MP derivatives.

So324 is a 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4T-MP) prodrug containing at the phosphate moiety a phenyl group and the methylester of alanine linked to the phosphate through a phosphoramidate linkage. So324 has anti-HIV activity in human CEM, MT4, and monocyte/macrophage cells that is superior to that of d4T. In contrast to d4T, So324 is also able to inhibit HIV replication in thymidine kinase-deficient CEM cells. After uptake of So324 by intact human lymphocytes, d4T-MP is released and subsequently converted intracellularly to d4T-TP. In addition, accumulation of substantial amounts of a novel d4T derivative has been found. This d4T metabolite has been characterized as alaninyl d4T-MP. The latter metabolite accumulates at approximately 13- to 200-fold higher levels than d4T-TP depending the experimental conditions. Alaninyl d4T-MP should be considered as an intra- and/or extracellular depot form of d4T and/or d4T-MP. These findings may explain the superior anti-retroviral activity of So324 over d4T in cell culture.

Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection.

The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.

To determine which features of retroviral vector design most critically affect gene expression in hematopoietic cells in vivo, we have constructed a variety of different retroviral vectors which encode the same gene product, human adenosine deaminase (EC 3.5.4.4), and possess the same vector backbone yet differ specifically in transcriptional control sequences suggested by others to be important for gene expression in vivo. Murine bone marrow cells were transduced by each of the recombinant viruses and subsequently used to reconstitute the hematopoietic system of lethally irradiated recipients. Five to seven months after transplantation, analysis of the peripheral blood of animals transplanted with cells transduced by vectors which employ viral long terminal repeats (LTRs) for gene expression indicated that in 83% (77/93) of these animals, the level of human enzyme was equal to or greater than the level of endogenous murine enzyme. Even in bone marrow transplant recipients reconstituted for over 1 year, significant levels of gene expression were observed for each of the vectors in their bone marrow, spleen, macrophages, and B and T lymphocytes. However, derivatives of the parental MFG-ADA vector which possess either a single base mutation (termed B2 mutation) or myeloproliferative sarcoma virus LTRs rather than the Moloney murine leukemia virus LTRs led to significantly improved gene expression in all lineages. These studies indicate that retroviral vectors which employ viral LTRs for the expression of inserted sequences make it possible to obtain high levels of a desired gene product in most hematopoietic cell lineages for close to the lifetime of bone marrow transplant recipients.

A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice.

A system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a tetracycline-controlled transactivator (tTA) fusion protein combining the tetracycline repressor and the transcriptional activation domain of VP16 [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. This system yielded only low levels of transactivator protein, probably because tTA is toxic. To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory. When used to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantially higher levels of variable (diversity) joining [V(D)J] recombination activity (70-fold on average) and inducible expression in a much larger fraction of transfected cells (autoregulatory, 90%, vs. constitutive, 18%). In addition, this system allowed the creation of transgenic mice in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined. Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system. With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice. This autoregulatory system represents an improved strategy for tetracycline-regulated gene expression both in cultured cells and in transgenic animals.

Suppression of the breakthrough of human immunodeficiency virus type 1 (HIV-1) in cell culture by thiocarboxanilide derivatives when used individually or in combination with other HIV-1-specific inhibitors (i.e., TSAO derivatives).

Five structurally related thiophene and furane analogues of the oxathiin carboxanilide derivative NSC 615985 (UC84) (designated UC10, UC68, UC81, UC42, and UC16) were identified as potent inhibitors of HIV-1 replication in cell culture and HIV-1 reverse transcriptase activity. These compounds were markedly active against a series of mutant HIV-1 strains, containing the Leu-100-->Ile, Val-106-->Ala, Glu-138-->Lys, or Tyr-181-->Cys mutations in their reverse transcriptase. However, the thiocarboxanilide derivatives selected for mutations at amino acid positions 100 (Leu-->Ile), 101 (Lys-->Ile/Glu), 103 (Lys-->Thr/Asp) and 141 (Gly-->Glu) in the HIV-1 reverse transcriptase. The compounds completely suppressed HIV-1 replication and prevented the emergence of resistant virus strains when used at 1.3-6.6 microM--that is, 10- to 25-fold lower than the concentration required for nevirapine and bis(heteroaryl)piperazine (BHAP) U90152 to do so. If UC42 was combined with the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)]-beta-D-pentofuranosyl (TSAO) derivative of N3-methylthymine (TSAO-m3T), virus breakthrough could be prevented for a much longer time, and at much lower concentrations, than if the compounds were used individually. Virus breakthrough could be suppressed for even longer, and at lower drug concentrations, if BHAP was added to the combination of UC42 with TSAO-m3T, which points to the feasibility of two- or three-drug combinations in preventing virus breakthrough and resistance development.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.