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P D Cook

Publications and source records attributed to P D Cook.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis of novel 3'-C-methylene thymidine and 5-methyluridine/cytidine H-phosphonates and phosphonamidites for new backbone modification of oligonucleotides.

Novel 5'-O-DMT- and MMT-protected 3'-C-methylene-modified thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates 1-7 with O-methyl, fluoro, hydrogen, and O-(2-methoxyethyl) substituents at the 2'-position have been synthesized by a new effective strategy from the corresponding key intermediates 3'-C-iodomethyl nucleosides and intermediate BTSP, prepared in situ through the Arbuzov reaction. The modified reaction conditions for the Arbuzov reaction prevented the loss of DMT- and MMT-protecting groups, and directly provided the desired 5'-O-DMT- and/or MMT-protected 3'-C-methylene-modified H-phosphonates 1-6 although some of them were also prepared through the manipulation of protecting groups after the P-C bond formation. The modified Arbuzov reaction of 3'-C-iodomethyl-5-methylcytidine 53, prepared from its 5-methyluridine derivative 42, with BTSP provided the 5-methylcytidine H-phosphonate 54, which was further transferred to the corresponding 4-N-(N-methylpyrrolidin-2-ylidene)-protected H-phosphonate monomer 7. 5'-O-MMT-protected 3'-C-methylene-modified H-phosphonates 5, 3, and 7 were converted to the corresponding cyanoethyl H-phosphonates 50, 51, and 56 using DCC as a coupling reagent. One-pot three-step reactions of 50, 51, and 56 provided the desired 3'-C-methylene-modified phosphonamidite monomers 8-10. Some of these new 3'-methylene-modified monomers 1-10 have been successfully utilized for the synthesis of 3'-methylene-modified oligonucleotides, which have shown superior antisense properties including nuclease resistance and binding affinity to the target RNA.

Anti-Infective Agents↗
PubMed2000

Synthetic oligonucleotides as therapeutics: the coming of age.

Synthetic oligonucleotides (ODNs) are short nucleic acid chains that can act in a sequence specific manner to control gene expression. Significant progress has been made in the development of synthetic ODN therapeutics since the first demonstration of gene inhibition by antisense ODNs in a cell culture system two decades ago. This new class of therapeutic agents can potentially target any abnormally expressed genes in a broad range of diseases from viral infections to psychoneurological disorders. A number of "first" generation synthetic ODNs have entered into human clinical trials in the last few years. The eminent approval of the first ODN for the treatment of cytomaglovirus retinitis by the FDA in USA will provide much excitement that this new class of compounds holds great promise as a therapeutic "magic bullet". However, many obstacles still exist in the development of this technology. In this review, the current status of synthetic ODN chemistry, drug delivery methods, mechanisms of ODN action, potential clinical applications and its limitations in a wide range of human disorders will be described.

Animals↗
PubMed2000

Identification of ligands for RNA targets via structure-based virtual screening: HIV-1 TAR.

Binding of the Tat protein to TAR RNA is necessary for viral replication of HIV-1. We screened the Available Chemicals Directory (ACD) to identify ligands to bind to a TAR RNA structure using a four-step docking procedure: rigid docking first, followed by three steps of flexible docking using a pseudobrownian Monte Carlo minimization in torsion angle space with progressively more detailed conformational sampling on a progressively smaller list of top-ranking compounds. To validate the procedure, we successfully docked ligands for five RNA complexes of known structure. For ranking ligands according to binding avidity, an empirical binding free energy function was developed which accounts, in particular, for solvation, isomerization free energy, and changes in conformational entropy. System-specific parameters for the function were derived on a training set of RNA/ligand complexes with known structure and affinity. To validate the free energy function, we screened the entire ACD for ligands for an RNA aptamer which binds L-arginine tightly. The native ligand ranked 17 out of ca. 153,000 compounds screened, i.e., the procedure is able to filter out >99.98% of the database and still retain the native ligand. Screening of the ACD for TAR ligands yielded a high rank for all known TAR ligands contained in the ACD and suggested several other potential TAR ligands. Eight of the highest ranking compounds not previously known to be ligands were assayed for inhibition of the Tat-TAR interaction, and two exhibited a CD50 of ca. 1 microM.

Arginine↗
PubMed1999

Structural origins of the exonuclease resistance of a zwitterionic RNA.

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2'-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1 degrees C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3' ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3'-5' exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-A resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 A and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2'-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.

Base Sequence↗
PubMed1999

Structure-activity relationships of novel 2-substituted quinazoline antibacterial agents.

High-throughput screening of in-house compound libraries led to the discovery of a novel antibacterial agent, compound 1 (MIC: 12-25 microM against S. pyogenes). In an effort to improve the activity of this active compound, a series of 2-substituted quinazolines was synthesized and evaluated in several antibacterial assays. One such compound (22) displayed improved broad-spectrum antibacterial activity against a variety of bacterial strains. This molecule also inhibited transcription/translation of bacterial RNA, suggesting a mechanism for its antibiotic effects. Structure-activity relationship studies of 22 led to the synthesis of another 24 compounds. Although some of these molecules were found to be active in bacterial growth assays, none were as potent as 22. Compound 22 was tested for its ability to cure a systemic K. pneumonia infection in the mouse and displayed moderate effects compared with a control antibiotic, gentamycin.

Animals↗
PubMed1999

Site-specific cross-linking of nucleic acids using the abasic site.

[formula: see text] An efficient site-specific cross-linking reaction between two carbohydrate residues present in two complementary DNA sequences is described. One oligodeoxynucleotide, 5'd(GGCTGA*CTGCG)3', carries an amino nucleophile tethered to the 2'-hydroxyl of an adenosine residue (A*). The target electrophile is an abasic site generated in the complementary sequence, 5'd(CGCAGDCAGCC)3' (D represents the deoxyribose). The cross-linking reaction was carried out by a reductive amination reaction in > 95% yield.

Adenosine↗
PubMed1999

Crystal structure and improved antisense properties of 2'-O-(2-methoxyethyl)-RNA.

2'-O-(2-Methoxyethyl)-RNA (MOE-RNA) is a nucleic acid analog with promising features for antisense applications. Compared with phosphorothioate DNA (PS-DNA), the MOE modification offers improved nuclease resistance, enhanced RNA affinity, improved cellular uptake and intestinal absorption, reduced toxicity and immune stimulation. The crystal structure of a fully modified MOE-RNA dodecamer duplex (CGCGAAUUCGCG) was determined at 1.7 A resolution. In the majority of the MOE substituents, the torsion angle around the ethylene alkyl chain assumes a gauche conformation. The conformational preorganization of the MOE groups is consistent with the improved RNA affinity and the extensive hydration of the substituents could play a role in the improved cellular uptake of MOE-RNA. A specific hydration pattern that bridges substituent and phosphate oxygen atoms in the minor groove of MOE-RNA may explain its high nuclease resistance.

Base Pairing↗
PubMed1998

New piperazinyl polyazacyclophane scaffolds, libraries and biological activities.

Four novel unsymmetric piperazinyl polyazacyclophane scaffolds 1-4 were synthesized in high yields by an efficient cyclization strategy. Twenty-six libraries 12-37 (total 16,000 compounds) were generated by a solution-phase combinatorial approach from 1-4 and thirty-eight functional groups. Potent antibacterial and HIV-1 tat/TAR protein-RNA disrupting activities were discovered.

Anti-Bacterial Agents↗
PubMed1998

Discovery of novel pyridinopolyamines with potent antimicrobial activity: deconvolution of mixtures synthesized by solution-phase combinatorial chemistry.

A 1638-member pyridinopolyamine library, consisting of 13 sublibraries of 126 members prepared by a solution-phase approach, was completely deconvoluted from orthogonally protected intermediates by a combination of iterative and positional scanning procedures. Antibacterial assays against Streptococcus pyogenes and Escherichia coli imp- and a Candida albicans yeast specificity assay were employed to follow the activity of sublibraries. Screening of the 13 sublibraries, which were prepared by a synthetic method that places the differentiating functionality in a selected position A (secondary amine), at the end of the synthesis (fix last), provided several first-round activities. Subsequently, six single pyridinopolyamines (2-7) were prepared where the first-round winner, a hydrogen atom, is in the first deconvoluted position and the remaining three positions contained the same functionalities. The range of antibacterial and yeast activities of these single compounds suggested that a more active and selective compound may be discovered by completely deconvoluting the first-round active sublibraries. Pyridinopolyamine positions B (secondary benzylamine) and C (primary benzylamine) were then sequentially positionally scanned with a set of six meta-substituted benzyl functionalities to generate two sets of second/third-round sublibraries, containing 21 or 36 compounds in each sublibrary, respectively. High-throughput screening yielded sublibraries 15, 18, and 21 with MICs of 1-5 microM against S. pyogenes and E. coli imp-. Using rounds 1 and 2/3 screening data, two sets of single compounds (22-27) and (28-32) with the combination of m-(trifluoromethyl)-benzyl group at position C and m-(trifluoromethyl)benzyl or m-methylbenzyl group at position B with position D (primary benzylamine) fixed were synthesized in the fourth round deconvolution. Subsequently, broader screening of deconvoluted compounds against a tier II panel of wild-type bacteria identified eight compounds (5, 7, 27, and 29-32) with approximately 100-fold greater selectivity for Gram-positive than Gram-negative bacteria. Thus, S. pyogenes, S. pyogenes (wild-type), Streptomyces aureus, and Enterococcus faecalis were inhibited at MICs of 1-12 microM, whereas MICs for E. coli, Klebsiella pneumoniae, Proteus vulgaris, and Pseudomonas aeruginosa were > 100 microM. These eight compounds were not active (> 100 microM) against fungus C. albicans.

Anti-Bacterial Agents↗
PubMed1997

In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.

Animals↗
PubMed1996

2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

Exonucleases↗
PubMed1996

The role of deer as a possible reservoir host of potosi virus, a newly recognized arbovirus in the United States.

Potosi (POT) virus (Bunyaviridae) was isolated from Aedes albopictus, an introduced Asian mosquito species, collected at a used tire yard in Potosi, Missouri (USA), in August and September, 1989. In September, 1990, small animals were trapped at the tire yard and six cattle were sampled at an adjacent farm; in November 1990 and 1991, blood samples were collected with filter paper strips from 364 hunter-killed, white-tailed deer (Odocoileus virginianus) in the region to determine the possible reservoir hosts of the virus. Deer specimens from Arkansas (n = 70), Colorado (n = 29), and Iowa (n = 763) (USA) were also analyzed. Specimens from 33 small vertebrates captured at the tire yard were negative for viruses. Only one eastern chipmunk (Tamias striatus) and none of six cattle had neutralizing (N) antibody against POT virus by the plaque-reduction serum neutralization test in Vero cell culture but 45 (25%) of 178 deer specimens in 1990 and 55 (30%) of 186 in 1991 were antibody positive. The 186 deer sera from 1991 were tested further and 29 (16%) were also N antibody positive to Cache Valley (CV) virus. From the 763 deer specimens tested from Iowa in 1993, 114 (15%) had N antibody to POT virus. Of 70 serum specimens from Arkansas deer in 1990, 33 (47%) had N antibody to POT and 15 (21%) to CV viruses; two (7%) of 29 CV negative serum specimens from Colorado deer in 1981 were serologically positive to POT virus. Three eastern chipmunks were experimentally inoculated with POT virus to determine their reservoir potential; none became viremic but all developed N antibody. Thus we propose that POT virus may be another virus regularly infecting wild deer populations but its impact on the health of these animals is unknown.

Animals↗
PubMed1995

Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.

The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.

Base Sequence↗
PubMed1995

Follicular lymphoma adjacent to foreign body granulomatous inflammation and fibrosis surrounding silicone breast prosthesis.

Silicone lymphadenopathy has been associated with fracture and/or erosive breakdown of silastic implants in joint replacements and is also known to occur with cosmetic and reconstructive breast implant surgery. In the orthopedic literature rare malignant lymphomas have been reported in association with silicone granulomas in lymph nodes; whether silicone is a causative agent remains controversial. We report a single case of a 56-year-old woman who had painful capsular contractures and a 2-cm palpable nodule medial to her silicone mammary implant. Histologically the mass comprised an extra nodal follicular mixed lymphoma with surrounding granulomatous response to polarizable foreign body material. Paraffin immunophenotyping, bcl-2 protein staining, and gene rearrangement analysis verified this diagnosis.

Breast Implants↗