A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides.

A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different chemically tuned succinylfluorescein dye distinguished by its fluorescent emission is described. Avian myeloblastosis virus reverse transcriptase is used in a modified dideoxy DNA sequencing protocol to produce a complete set of fluorescence-tagged fragments in one reaction mixture. These DNA fragments are resolved by polyacrylamide gel electrophoresis in one sequencing lane and are identified by a fluorescence detection system specifically matched to the emission characteristics of this dye set. A scanning system allows multiple samples to be run simultaneously and computer-based automatic base sequence identifications to be made. The sequence analysis of M13 phage DNA made with this system is described.

Non-peptide corticotropin-releasing hormone antagonists: syntheses and structure-activity relationships of 2-anilinopyrimidines and -triazines.

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist alpha-helical CRH(9-41) (Ki = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated adenylate cyclase activity in the same tissue, but it was less potent than alpha-helical CRH(9-41) (IC50 = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1 receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH1 Ki = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines.

The synthesis and CRF receptor binding affinities of several new series of N-aryltriazolo- and -imidazopyrimidines and -pyridines are described. These cyclized systems were prepared from appropriately substituted diaminopyrimidines or -pyridines by nitrous acid, orthoester, or acyl halide treatment. Variations of amino (ether) pendants and aromatic substituents have defined the structure-activity relationships of these series and resulted in the identification of a variety of high-affinity agents (Ki's < 10 nM). On the basis of this property and lipophilicity differences, six of these compounds (4d,i,n,x, 8k, 9a) were initially chosen for rat pharmacokinetic (PK) studies. Good oral bioavailability, high plasma levels, and duration of four of these compounds (4d,i,n,x) prompted further PK studies in the dog following both iv and oral routes of administration. Results from this work indicated 4i,x had properties we believe necessary for a potential therapeutic agent, and 4i1 has been selected for further pharmacological studies that will be reported in due course.

Improvements in the chain-termination method of DNA sequencing through the use of 7-deaza-2'-deoxyadenosine.

Significant improvements in the quality of DNA sequencing data have been shown when deoxyadenosine triphosphate (dATP) is replaced by 7-deaza-2'-deoxyadenosine triphosphate (c7dATP). The use of c7dATP in conjunction with 7-deaza-2'-deoxyguanosine triphosphate (c7dGTP) further decreases anomalies in electrophoretic mobility which are caused by compressions involving G and/or A residues. This effect is observed for both isotope-based and fluorescence-based sequencing approaches. Replacing dATP with c7dATP also results in a higher degree of uniformity in the frequency of chain termination reactions, when such terminations involve the incorporation of fluorescence-labeled dideoxynucleotides by T7 polymerase. These improvements in the gel-resolution and distribution of chain-terminated DNA products result in higher accuracy in both manual and automated base assignment.

Use of the Suzuki reaction for the synthesis of aryl-substituted heterocycles as corticotropin-releasing hormone (CRH) antagonists.

The Suzuki reaction has been used to synthesize a variety of aryl-substituted heterocyclic antagonists of the CRH1 receptor. Examples with several different heterocyclic cores are potent CRH receptor ligands.

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate.

Biodistribution and metabolism of oligonucleotides were determined using a 3H-labeled 20-nucleotide phosphodiester and its phosphorothioate analog. The oligonucleotides were radiolabeled by 3H-methylation of an internal deoxyctidine with HhaI methylase and S- [3H]adenosylmethionine. Biodistribution studies were conducted after intravenous injection of 6 mg/kg (5 muCi) oligonucleotide. Metabolism of the oligonucleotides was determined by paired-ion high performance liquid chromatography. After phosphodiester injections, radiolabel rapidly cleared the blood. Relative initial concentrations were as follows: kidney > blood > heart > liver > lung > spleen. Radiolabel in spleen peaked at 1 hr and remained elevated for 24 hr. At 2 hr the concentration in all organs, except spleen, was equal to that in blood. High performance liquid chromatographic analysis of the kidney, liver, and spleen extracts and urine indicated extremely rapid metabolism to monomer. Results of studies after the injection of phosphorothioate oligonucleotide differed from those using the phosphodiester. Despite its rapid clearance from blood, phosphorothioate accumulated rapidly in all tissues, especially the kidney. Kidney uptake increased over time, remaining very high for 24 hr. Ratios of organ to blood concentrations at 2 hr for all organs were 5:1 or greater. Kidney and liver ratios were 84:1 and 20:1, respectively. Analysis of the kidney and liver extracts and urine indicated that slow metabolism occurred. These data suggest that phosphodiester oligonucleotides would have limited therapeutic utility. The stability and organ distribution of the phosphorothioate oligonucleotide imply that such oligonucleotides may have therapeutic potential.

Conjugation of a monoclonal antibody with a DTPA modified random copolymer of hydroxyethyl methylacrylate and methyl methacrylate.

A new approach for covalent coupling diethylenetriaminepentaacetic acid (DTPA) molecules to a partially reduced monoclonal antibody utilizes a malemide modified copolymer of hydroxyethyl methylacrylate and methyl methacrylate (DTPA copolymer) prepared by the group transfer polymerization (GTP) method. An average of 6 DTPA molecules were incorporated per mol maleimeide DTPA copolymer and 1.5 mol maleimide DTPA copolymer per mol antibody. Maleimide DTPA copolymer modified antibody was intramolecularly cross-linked, reduced immunoactivity and had a high in vivo liver uptake.

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. II. 3',5'-blocked oligonucleotides.

The pharmacokinetics and metabolism of four radiolabeled phosphodiester oligonucleotides with 3'- and 5'-blocked ends were studied in mice and compared with previously studied, unblocked, all-phosphodiester and all-phosphorothioate oligonucleotides. The radiolabel was a tritiated methyl group enzymatically attached at an internal cytidine. The ends of the blocked phosphodiester oligonucleotides were protected by cyclization or by incorporation of either phosphorothioate or methylphosphonate linkages. Although these modifications protected the blocked oligonucleotides from degradation by exonucleases present in mouse serum, degradation initiated by endonucleases was 50% complete in 0.5-5 hr. After intravenous injection, the blocked oligonucleotides were much less stable than the all-phosphorothioate oligonucleotide and only marginally more stable than the previously studied, unblocked phosphodiester oligonucleotide. Even a "chimeric" blocked oligonucleotide with 16 phosphorothioate linkages and eight contiguous phosphodiester linkages was rapidly degraded. Despite the favorable serum binding, tissue accumulation, and stability observed with phosphorothioate oligonucleotides, these properties did not provide the chimeric oligonucleotide access to a compartment where its phosphodiester linkages were stable. In other respects, the blocked and chimeric phosphodiester oligonucleotides also resembled the unblocked phosphodiester oligonucleotide; radiolabel was cleared rapidly from the blood, there was little evidence of tissue accumulation, high performance liquid chromatographic analysis of tissue extracts showed extremely rapid degradation to mononucleotides, and only mononucleotide metabolites were present in urine. In summary, blocked phosphodiester oligonucleotides are rapidly attacked by endonucleases present in mice. Unless this problem is less serious in primates, such blocked oligonucleotides will be relatively unattractive candidates for drug development.

4-Aryl-2-anilinopyrimidines as corticotropin-releasing hormone (CRH) antagonists.

A series of 4-aryl-2-(N-ethylanilino)pyrimidines has been synthesized as corticotropin-releasing hormone (CRH) inhibitors. The effect of substitution on each aromatic ring on receptor binding was investigated.

Synthesis and evaluation of efavirenz (Sustiva) analogues as HIV-1 reverse transcriptase inhibitors: replacement of the cyclopropylacetylene side chain.

Two series of efavirenz analogues have been developed: one in which the cyclopropane ring has been replaced by small heterocycles and another in which the entire acetylenic side chain has been replaced by alkyloxy groups. Several members of both series show equivalent potency to efavirenz against both wild-type virus and the key K103N mutant.

4,1-Benzoxazepinone analogues of efavirenz (Sustiva) as HIV-1 reverse transcriptase inhibitors.

A series of 4,1-benzoxazepinone analogues of efavirenz (Sustiva) as potent NNRTIs has been discovered. The cis-3-alkylbenzoxazepinones are more potent then the trans isomers and can be synthesized preferentially by a novel stereoselective cyclization. The best compounds are potent orally bioavailable inhibitors of both wild-type HIV-1 and its clinically relevant K103N mutant virus, but are highly protein-bound in human plasma.