Novel design of nonpeptide AVP V(2) receptor agonists: structural requirements for an agonist having 1-(4-aminobenzoyl)-2,3,4, 5-tetrahydro-1H-1-benzazepine as a template.

The discovery of a series of nonpeptide arginine vasopressin V(2) receptor agonists is described. After identifying the aniline derivative 8 as our lead compound from the metabolites of compound 7 that showed antidiuretic activity by po administration to Brattleboro rats, improvements in the in vitro potency involving evaluations of the structural requirements for agonist action and optimizing the structure of the benzoyl moiety have been intensively undertaken. These studies led to compounds 16g, 19a, and 23b,h,i that show potent agonist activity for the V(2) receptor.

Characterization of a novel nonpeptide vasopressin V(2)-agonist, OPC-51803, in cells transfected human vasopressin receptor subtypes.

We discovered the first nonpeptide arginine-vasopressin (AVP) V(2)-receptor agonist, OPC-51803. Pharmacological properties of OPC-51803 were elucidated using HeLa cells expressing human AVP receptor subtypes (V(2), V(1a) and V(1b)) and compared with those of 1-desamino-8-D-arginine vasopressin (dDAVP), a peptide V(2)-receptor agonist. OPC-51803 and dDAVP displaced [(3)H]-AVP binding to human V(2)- and V(1a)-receptors with K(i) values of 91.9+/-10.8 nM (n = 6) and 3.12+/-0.38 nM (n = 6) for V(2)-receptors, and 819+/-39 nM (n = 6) and 41.5+/-9.9 nM (n = 6) for V(1a)-receptors, indicating that OPC-51803 was about nine times more selective for V(2)-receptors, similar to the selectivity of dDAVP. OPC-51803 scarcely displaced [(3)H]-AVP binding to human V(1b)-receptors even at 10(-4) M, while dDAVP showed potent affinity to human V(1b)-receptors with the K(i) value of 13.7+/-3.2 nM (n = 4). OPC-51803 concentration-dependently increased cyclic adenosine 3', 5'-monophosphate (cyclic AMP) production in HeLa cells expressing human V(2)-receptors with an EC(50) value of 189+/-14 nM (n = 6). The concentration-response curve for cyclic AMP production induced by OPC-51803 was shifted to the right in the presence of a V(2)-antagonist, OPC-31260. At 10(-5) M, OPC-51803 did not increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in HeLa cells expressing human V(1a)-receptors. On the other hand, dDAVP increased [Ca(2+)](i) in HeLa cells expressing human V(1a)- and V(1b)-receptors in a concentration-dependent fashion. From these results, OPC-51803 has been confirmed to be the first nonpeptide agonist for human AVP V(2)-receptors without agonistic activities for V(1a)- and V(1b)-receptors. OPC-51803 may be useful for the treatment of AVP-deficient pathophysiological states and as a tool for AVP researches.

Origin of an alkaline protease associated with the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta (Haworth).

The larval digestive juice of the armyworm, Pseudaletia unipuncta (Haworth), has extremely high alkaline protease activity. No protease or negligible protease activity is present in the hemolymph and in extracts of the body wall. The digestive juice protease is absorbed by the capsules of a granulosis virus under low ionic condition. The protease degrades the capsule matrix proteins at alkaline conditions. The absorption of digestive juice protease by capsules occurs when capsules are obtained by triturating the whole infected larvae. On the other hand, capsules, which have been collected from the triturated body walls of infected larvae whose digestive tracts have been removed, are not degraded when the incubation in an alkaline solution is for less than 24 hours at 25 degrees C. The results suggest that the protease associated with the capsule is derived from the digestive juice of the insect.

Physicochemical properties and location of capsule components, in particular the synergistic factor, in the occlusion body of a granulosis virus of the armyworm, Pseudaletia unipuncta.

A synergistic strain of granulosis virus (GVH) enhances the infection of larvae of the armyworm, Pseudaletia unipuncta, with a nuclear polyhedrosis virus (NPV). The matrix of the capsule (occlusion body) of GVH contains two major components, the structural protein and the synergistic factor. The structure of the capsule matrix of GVH was analyzed with a primary amine-coupling reagent, 2,4,6,-trinitrobenzene 1-sulfonic acid (TNBS). When undissolved capsules are incubated with TNBS, only the structural protein is labeled by TNBS. This indicates that the synergistic factor is not accessible to TNBS. Tests with the isolated capsule components show that the synergistic factor is more soluble than the structural protein at neutral pH. The synergistic factor has a strong affinity for viral envelopes. The attachment of about eight molecules of the synergistic factor to each enveloped NPV greatly enhances the infectivity of the virus.

Two 2-dimensional electrophoreses: a combination of crossed immunoelectrophoresis and dodecyl sulfate polyacrylamide gel electrophoresis for protein analysis.

An improved method of two 2-dimensional electrophoreses that combined crossed immunoelectrophoresis and 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis was developed with a horizontal gel-electrophoretic apparatus.

Comparative analysis of the enveloped virions of two granulosis viruses of the armyworm, Pseudaletia unipuncta.

The enveloped virions of two strains of a granulosis virus, a synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain which infect the armyworm, Pseudaletia unipuncta, were liberated from their capsules with 0.02 N NaOH and purified by filtration through membrane filters. The envelopes were solubilized with 0.1% Triton X-100. The enveloped virions, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), had 9 to 12 polypeptides, 3 of which were associated with the naked virion. The two strains differed in their electrophoretic patterns mainly in the presence of two heavy polypeptides (89,000 and 97,000 daltons) and a 52,000-dalton polypeptide in GVH and in the absence of the heavy polypeptides and the presence of a 57,000-dalton polypeptide in GVO. The two heavy polypeptides were sensitive to proteinase. Two two-dimensional electrophoreses (first dimension with agarose gel electrophoresis and second dimension with SDS-PAGE and immunoelectrophoresis) of the envelope of GVH resolved two polypeptides, 37,000 and 48,000 daltons, which were serologically related to the synergistic factor present in the capsule of GVH; however their molecular weights differed from that of the GVH synergistic factor. In GVO, no polypeptide serologically related to the synergistic factor was detected in the envelope. The enveloped virions purified by differential filtration were infectious when fed to armyworm larvae, but the naked virions, free of envelopes, were not infectious.

Characterization of a Ribonucleic Acid Polymerase Activity Associated with Purified Cytoplasmic Polyhedrosis Virus of the Silkworm Bombyx mori.

Purified cytoplasmic-polyhedrosis virus has been found to have associated with it a polymerase activity capable of catalyzing the synthesis of virus-specific, single-stranded ribonucleic acid (RNA) from the double-stranded RNA genome.