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D R Corey

Publications and source records attributed to D R Corey.

At least 19 recordsLinked to original sources
PubMed2002

Telomerase-dependent reactivation of DNA synthesis in macrophages implies alteration of telomeres.

In previous work we demonstrated that various types of cultured cells with a limited life span could not reactivate DNA synthesis in the nuclei of mouse peritoneal macrophages in heterokaryons. We now investigate the role of telomerase in the process of the macrophage nucleus reactivation in heterokaryons with immortal telomerase-positive 3T3 Swiss mouse fibroblasts and human fibroblasts with introduced hTERT gene. We report that introduction of the hTERT gene into human diploid fibroblasts results in emergence of telomerase activity in these cells and the ability to induce the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. Inhibition of telomerase activity in heterokaryons by reverse transcriptase inhibitors (azidothymidine and guanosine polyphosphonate analogues) and by a 2'-O-methyl-RNA oligonucleotide anti-sense to the template region of telomerase RNA, block reactivation of DNA synthesis in macrophage nuclei without inhibiting DNA synthesis in the nuclei of fibroblasts. Our results suggest alterations (shortening or damage) in the macrophage telomere structure. As far as we know, heterokaryons with macrophages are the first cellular model for rapid investigation of the effects of telomerase inhibitors.

3T3 Cells↗
PubMed2001

Engineering orthogonal ligand-receptor pairs from "near drugs".

Cell-permeable small molecules are powerful tools for unraveling complex cellular pathways. We demonstrate that nuclear hormone receptors can be engineered through mutagenesis to create orthogonal ligand-receptor pairs to control transcription. Mutated residues in the retinoid X receptor (RXR) were chosen from structural analysis of RXR and the retinoic acid receptor (RAR) ligand binding domains. The potential ligands screened for activation of variant receptors are "near drugs"--compounds synthesized during structure-activity studies that are structurally similar to an approved drug yet inactive on the wild-type receptor. One variant, Q275C;I310M;F313I, is poorly activated by ligands for the wild-type receptor but is activated by a "near drug", fulfilling the criteria of an orthogonal ligand-receptor pair. These experiments demonstrate that nuclear hormone receptors are well suited to supply orthogonal ligand-receptor pairs for experimental biology, biotechnology, and gene therapy. Our findings also demonstrate the general principle that inactive compounds synthesized during drug discovery can be combined with mutant proteins to rapidly create new tools for controlling cellular processes.

Alitretinoin↗
PubMed2001

Liver cell specific targeting of peptide nucleic acid oligomers.

Chimeric molecules consisting of peptide nucleic acid (PNA) and lactose have been synthesized to test the hypothesis that lactose moieties can promote cell-specific uptake of PNAs. We find that lactose modified PNAs rapidly enter liver-derived HepG2 cells while unmodified PNAs do not and that lactose modified PNAs can inhibit cellular telomerase.

Antineoplastic Agents↗
PubMed2001

Inhibition of telomerase by 2'-O-(2-methoxyethyl) RNA oligomers: effect of length, phosphorothioate substitution and time inside cells.

2'-O-(2-methoxyethyl) (2'-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and selective inhibitors. Here we report inhibition of human telomerase by 2'-MOE RNA oligomers that are complementary to the RNA template region. Fully complementary oligomers inhibited telomerase in a cell extract with IC(50) values of 5-10 nM at 37 degrees C. IC(50) values for mismatch-containing oligomers varied with length and phosphorothioate substitution. After introduction into DU 145 prostate cancer cells inhibition of telomerase activity persisted for up to 7 days, equivalent to six population doublings. Inside cells discrimination between complementary and mismatch-containing oligomers increased over time. Our results reveal two oligomers as especially promising candidates for initiation of in vivo preclinical trials and emphasize that conclusions regarding oligonucleotide efficacy and specificity in cell extracts do not necessarily offer accurate predictions of activity inside cells.

Animals↗
PubMed2001

Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length.

Genome sequencing has revealed thousands of novel genes, placing renewed emphasis on chemical approaches for controlling gene expression. Antisense oligomers designed directly from the information generated by sequencing are one option for achieving this control. Here we explore the rules governing the inhibition of gene expression by peptide nucleic acids (PNAs) inside cells. PNAs are a DNA/RNA mimic in which the phosphate deoxyribose backbone has been replaced by uncharged linkages. Binding to complementary sequences is not hindered by electrostatic repulsion and is characterized by high rates of association and elevated affinities. Here we test the hypothesis that the favorable properties of PNAs offer advantages for recognition of mRNA and antisense inhibition of gene expression in vivo. We have targeted 27 PNAs to 18 different sites throughout the 5'-untranslated region (5'-UTR), start site, and coding regions of luciferase mRNA. PNAs were introduced into living cells in culture as PNA-DNA-lipid complexes, providing a convenient high throughput method for cellular delivery. We find that PNAs targeted to the terminus of the 5'-UTR are potent and sequence-specific antisense agents. PNAs fifteen to eighteen bases in length were optimal inhibitors. The introduction of one or two mismatches abolished inhibition, and complementary PNAs targeted to the sense strand were also inactive. In striking contrast to effective inhibition by PNAs directed to the terminal region, PNAs complementary to other sites within the 5'-UTR do not inhibit gene expression. We also observe no inhibition by PNAs complementary to the start site or rest of the coding region, nor do we detect inhibition by PNAs that are highly C/G rich and possess extremely high affinities for their target sequences. Our results suggest that PNAs can block binding of the translation machinery but are less able to block the progress of the ribosome along mRNA. The high specificity of antisense inhibition by PNAs emphasizes both the promise and the challenges for PNAs as antisense agents and provides general guidelines for using PNAs to probe the molecular recognition of biological targets inside cells.

5' Untranslated Regions↗
PubMed2001

Editorial.

Explore the source record for details and available documents.

Gene Library↗
PubMed2001

Synthesis, analysis, purification, and intracellular delivery of peptide nucleic acids.

Peptide nucleic acids (PNAs) are nonionic DNA mimics. Their novel chemical properties may facilitate the development of selective and potent antisense and antigene strategies for regulating intracellular processes. Described herein are procedures for the synthesis, purification, handling, and characterization of PNAs. A simple protocol for the lipid-mediated introduction of PNAs into in vitro cultures of mammalian cells is provided.

Animals↗
PubMed2001

Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.

Locked nucleic acid is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. This conformation restriction increases binding affinity for complementarity sequences and provides an exciting new chemical approach for the control of gene expression and optimization of microarrays.

Animals↗
PubMed2001

A bridge between the RNA and protein worlds? Accelerating delivery of chemical reactivity to RNA and DNA by a specific short peptide (AAKK)(4).

BACKGROUND: RNA can catalyze diverse chemical reactions, leading to the hypothesis that an RNA world existed early in evolution. Today, however, catalysis by naturally occurring RNAs is rare and most chemical transformations within cells require proteins. This has led to interest in the design of small peptides capable of catalyzing chemical transformations. RESULTS: We demonstrate that a short lysine-rich peptide (AAKK)(4) can deliver a nucleophile to DNA or RNA and amplify the rate of chemical modification by up to 3400-fold. We also tested similar peptides that contain ornithine or arginine in place of lysine, peptides with altered stereochemistry or orientation, and peptides containing eight lysines but with different spacing. Surprisingly, these similar peptides function much less well, suggesting that specific combinations of amino acids, charge distribution, and stereochemistry are necessary for the rate enhancement by (AAKK)(4). CONCLUSIONS: By appending other reactive groups to (AAKK)(4) it should be possible to greatly expand the potential for small peptides to directly catalyze modification of DNA or RNA or to act as cofactors to promote ribozyme catalysis.

Amino Acid Sequence↗
PubMed2001

The structure of a Michaelis serpin-protease complex.

Serine protease inhibitors (serpins) regulate the activities of circulating proteases. Serpins inhibit proteases by acylating the serine hydroxyl at their active sites. Before deacylation and complete proteolysis of the serpin can occur, massive conformational changes are triggered in the serpin while maintaining the covalent linkage between the protease and serpin. Here we report the structure of a serpin-trypsin Michaelis complex, which we visualized by using the S195A trypsin mutant to prevent covalent complex formation. This encounter complex reveals a more extensive interaction surface than that present in small inhibitor-protease complexes and is a template for modeling other serpin-protease pairs. Mutations of several serpin residues at the interface reduced the inhibitory activity of the serpin. The serine residue C-terminal to the scissile peptide bond is found in a closer than usual interaction with His 57 at the active site of trypsin.

Amino Acid Sequence↗
PubMed2001

Application of PNA and LNA oligomers to chemotherapy.

Peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers bind to complementary sequences with extremely high affinity. This high-affinity binding supports the hypothesis that they have advantages for targeting cellular nucleic acids and provide a better route for the development of oligonucleotide-based antiproliferative drugs. This article reviews the properties of PNA and LNA oligomers and describes the challenges that confront their application to cancer therapy.

Animals↗
PubMed2000

Strand invasion by mixed base PNAs and a PNA-peptide chimera.

Peptide nucleic acid oligomers (PNAs) have a remarkable ability to invade duplex DNA at polypurine-polypyrimidine target sequences. Applications for PNAs in medicine and biotechnology would increase if the rules governing their hybridization to mixed base sequences were also clear. Here we describe hybridization of PNAs to mixed base sequences and demonstrate that simple chemical modifications can enhance recognition. Easily synthesized and readily soluble eight and 10 base PNAs bind to plasmid DNA at an inverted repeat that is likely to form a cruciform structure, providing convenient tags for creating PNA-plasmid complexes. PNAs also bind to mixed base sequences that cannot form cruciforms, suggesting that recognition is a general phenomenon. Rates of strand invasion are temperature dependent and can be enhanced by attaching PNAs to positively charged peptides. Our results support use of PNAs to access the information within duplex DNA and demonstrate that simple chemical modifications can make PNAs even more powerful agents for strand invasion. Simple strategies for enhancing strand invasion should facilitate the use of PNAs: (i) as biophysical probes of double-stranded DNA; (ii) to target promoters to control gene expression; and (iii) to direct sequence-specific mutagenesis.

Base Pair Mismatch↗
PubMed2000

Negative selectivity and the evolution of protease cascades: the specificity of plasmin for peptide and protein substrates.

BACKGROUND: Understanding the networks of selective proteolysis that regulate complex biological systems requires an appreciation of the molecular mechanisms used to maintain substrate specificity. Human plasmin, a serine protease that promotes the dissolution of blood clots and is essential in maintaining normal hemostasis, is usually described as having broad substrate specificity. Recent evidence that plasmin also plays a key role in a variety of other important biological and pathological processes, however, has suggested that this description might need to be re-evaluated. RESULTS: We used substrate phage display to elucidate optimal subsite occupancy for substrates of plasmin. We identified a peptide substrate that is cleaved 710,000-fold more efficiently by plasmin than a peptide containing the activation sequence of plasminogen. Plasmin achieves this unexpected, large differential activity even though both target sequences possess an arginine residue in the P1 position. We also demonstrate that proteolysis by plasmin can be targeted to an engineered protein substrate and that introduction of substrate sequences identified by phage display into plasminogen increases plasmin-mediated cleavage of the mutant 2000-fold. CONCLUSIONS: The specificity of plasmin is more tightly controlled than previously recognized; interactions with substrates at all subsites between S4 and S2' contribute to catalysis. Furthermore, in contrast to most enzymes that exhibit positive selectivity for substrate, the evolution of substrate specificity by plasmin has apparently been dominated by a strong negative selection against development of autoactivation activity. This 'negative selectivity' avoids short-circuiting regulation of the fibrinolytic system and other important biological processes, and might be an important general mechanism for controlling protease cascades.

Amino Acid Sequence↗
PubMed2000

Modifying ligand specificity of gene regulatory proteins.

Nuclear receptors contain a conserved hydrophobic ligand binding pocket that is particularly amenable to structure-based protein engineering. Thus, site-directed mutagenesis of the ligand binding pocket has resulted in the creation of nuclear receptors with novel ligand specificities. Such proteins are now being used to control gene expression in vivo in a ligand-dependent manner.

Animals↗
PubMed1999

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death.

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2'-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.

Antineoplastic Agents↗
PubMed1999

Telomerase inhibition by peptide nucleic acids reverses 'immortality' of transformed human cells.

Telomerase activity, the ability to add telomeric repeats to the ends of chromosomes, has been detected in most immortal cell lines including tumor cells, but is low or absent in most diploid, mortal cells such as those of somatic tissues. Peptide nucleic acids (PNAs), analogs of DNA or RNA which bind to complementary nucleic acids with very high affinity, were co-electroporated into immortal human cells along with a selectable plasmid. Introduction of PNAs inverse-complementary to telomerase RNA effectively inhibited telomerase activity in intact cells, shortened telomeres, reduced colony size, and arrested cell proliferation after a lag period of 5-30 cell generations, consistent with suppression of their 'immortality'. Electroporation of selection plasmid alone had no effect, while PNAs of altered sequence were markedly less effective in each assay. This constitutes the first demonstration of cell growth arrest through telomerase inhibition, upon treatment of intact cells with an exogenous compound which can be efficiently delivered in vivo. The phenotype of telomerase-inhibited transformed cells differs from senescence of normal diploid fibroblasts, but rather resembles the crisis state of incompletely transformed cells.

Amino Acid Metabolism, Inborn Errors↗