CL387626 exhibits marked and unusual antiviral activity against respiratory syncytial virus in tissue culture and in cotton rats.

CL387626 (4,4'-Bis[4,6-di[3-aminophenyl-N,N-bis(2-carbamoylethyl)-sulfon ilimino]-1,3,5-triazine-2-ylamino-bi-phenyl-2,2'-disulfonic acid, disodium salt), a compound synthesized by Wyeth-Ayerst Research Laboratories, was tested for its cytotoxicity and antiviral activity against respiratory syncytial virus (RSV) in tissue culture and in cotton rats. The median cell inhibitory (IC50) and median efficacious (EC50) concentrations of CL387626 against RSV in proliferating HEp2 or Vero tissue culture cells were determined to be 375 and 0.25 microg/ml, respectively, giving the compound an apparent selective index (S.I.) of 1500. This compound also exhibited uncommon antiviral activity against RSV in cotton rats. In multiple experiments, a single 30 mg/kg dose of CL387626 administered intranasally 4 or 5 days prior to virus challenge, significantly inhibited pulmonary replication of RSV compared to that seen in control animals inoculated similarly with placebo (i.e. water). In contrast to these results, most lots of CL387626 failed to significantly inhibit pulmonary RSV replication when administered utilizing therapeutic administration schedules. Although some cytotoxicity was noted in tissue culture assays, no overt toxic effects were noted in any test animal, including those inoculated with > 300 mg CL387626/kg, a dose approximately 150 times the apparent minimal efficacious dose (i.e. 1.9 mg/kg).

Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation.

Among the most potent inhibitors of human cytomegalovirus protease identified by random screening of a chemical library was 1,4-dihydro-7,8-dimethyl 6H-pyrimido[1,2-b]-1,2,4,5-tetrazin-6-one (1) (PTH2). The oxidized form (2), PT, which is present in solutions of PTH2, was shown to be the actual inhibitory species which irreversibly inactivates the protease; recycling of PTH2 by dissolved oxygen results in complete inhibition of the protease at substoichiometric amounts of compound. No evidence for a covalent adduct between the protease and the inhibitor was obtained, and protease activity was restored by incubation of the inactivated enzyme with the reducing agent bismercaptoethyl sulfone, suggesting that disulfide bond formation was responsible for the observed inhibition. The five cysteines of the protease are normally in the reduced state; analysis of tryptic peptides from inhibited protease indicated that disulfide bonds Cys84-Cys87 and Cys138-Cys161 were formed. Using site-directed mutagenesis, the disulfide pair induced between Cys138 and Cys161 disulfide is dependent upon interaction of PT with the protease and does not form spontaneously, unlike that of the Cys84-Cys87 pair which can form in the absence of inhibitor. The inhibitor's redox chemistry is analogous to that of flavin, and, in fact, flavin inhibits the protease by the same mechanism, causing formation of a disulfide bond between Cys138 and Cys161. That the cysteines are dispensable, but can regulate protease activity by formation of a unique disulfide pair, suggests a plausible mechanism for control of proteolysis during the viral life cycle.

Ultrastructural observation on macrophage-lymphocyte interactions in semen from Tripterygium wilfordii Hook.f. takers.

Sixty-nine specimens from Tripterygium Wilfordii Hook.f. (TWH) users were investigated by electron microscopy. No macrophages were demonstrated in the 21 specimens collected prior to the administration of TWH. However, it was found in 23 out of the 48 semen specimens obtained following the TWH administration. The macrophages were functionally active as shown by the presence of a large number of cytoplasmic processes and pseudopodia on the surface, and primary and secondary lysosomes in the cytoplasm. The macrophages phagocytized sperm debris and degenerated or dead spermatids with formation of specific phagosomes. Around those macrophages, lymphocytes were commonly noted. The cytoplasmic processes of the two cell types could come into contact or even fuse with each other, leading to tight junction-like structure; in some of the contacts, the plasma membranes were found dissolved so as to form direct cytoplasmic linkage.

Cleavage behavior of calicheamicin gamma 1 and calicheamicin T.

Calicheamicin gamma 1 is a potent antitumor antibiotic that cleaves DNA with a high degree of specificity; there is much interest in the recognition process. We have investigated the DNA-cleaving properties of calicheamicin T, a truncated derivative of calicheamicin. We show that calicheamicin T cleaves DNA in a double-stranded fashion, indicating that the first two sugars are sufficient to facilitate binding of the aglycone in the minor groove. However, calicheamicin T cleaves DNA nonselectivity. This result suggests that cyclization kinetics do not determine the cleavage specificity of the intact drug. Instead, cleavage specificity probably reflects binding specificity. Because of the recognition sites reported in the original cleavage paper, calicheamicin has been assumed to recognize oligopyrimidine DNA sequences containing G-C base pairs. We show here that calicheamicin also cuts efficiently at A.T tracts, sometimes in preference to G.C-rich homopyrimidine tracts. Crystallographic data and experiments with chemical probes indicate that DNA sequences including G.C base pairs have significantly different local conformations from DNA sequences containing several (four or more) sequential A.T base pairs. This difference makes it unlikely that calicheamicin simply senses inherent groove conformation and suggests that there is some degree of "induced fit." The ability to recognize both A.T- and G.C-rich oligopyrimidine sequences with a high degree of specificity makes calicheamicin an unusual minor-groove binder.

Identification of the leucine-rich amelogenin peptide (LRAP) as the translation product of an alternatively spliced transcript.

The polymerase chain reaction was used to amplify bovine tooth amelogenin cDNA, resulting in several products which were separated by agarose gel electrophoresis. Sequence determination of one of the products revealed that it encoded an amino acid sequence identical to that of a small leucine-rich amelogenin polypeptide (LRAP) previously characterized by protein sequencing. Comparison of the nucleotide sequence of this cDNA with that determined for the cloned bovine amelogenine gene strongly suggested that the LRAP transcript resulted from alternative splicing of the primary transcript of this gene, thus explaining the origin of the puzzling LRAP sequence. Analysis of the structure of LRAP suggests that the polypeptide might exhibit interesting properties relative to hydroxy apatite crystal formation.

Photoaffinity labeling of bacteriorhodopsin.

14C-Labeled optically pure 3S- and 3R-(diazoacetoxy)-all-trans-retinals were incorporated separately into bacterioopsin to reconstitute functional bacteriorhodopsin (bR) analogues, 3S- and 3R-diazo-bRs. UV irradiation at 254 nm generated highly reactive carbenes, which cross-linked the radiolabeled retinals to amino acid residues in the vicinity of the beta-ionine ring. The 3S- and 3R-diazo analogues were found to cross-link, respectively, to cyanogen bromide fragments CN 7/CN9 and CN 8/CN 9. More specifically, Thr121 and Gly122 in fragment CN 7 were found to be cross-linked to the 3S-diazo analogue. The identification of cross-linked residues and fragments favors assignments of the seven helices A-G-F-E-D-C-B or B-C-D-E-F-G-A to helices 1-2-3-4-5-6-7 in the two-dimensional electron density map (Henderson et al., 1975, 1986; Mogi et al., 1987). The present results show that the chromophore chain is oriented with the ionone ring inclined toward the outside of the membrane (the 9-methyl group also faces the extracellular side of the membrane).

Determination of guanidino compounds by anion-exchange chromatography and amperometric detection.

Guanidino compounds were separated and determined by anion-exchange chromatography and electrochemical detection using a basic aqueous eluent and a nickel working electrode. It was found necessary to use a sample clean-up procedure prior to chromatographic analysis of uremic dialysate and serum samples. The effect of eluent hydroxide concentration on the retention of guanidino compounds was studied. Quantitative calibration showed that working curves were non-linear. Electrochemical detection for guanidino compounds with a nickel working electrode, while not selective, has high detection sensitivity. Detection limits for guanidino compounds ranged from 3 to 12 pmol.