Dependence of the size of a protein-SDS complex on detergent and Na+ concentrations.

Sodium dodecyl sulfate (SDS) micelles provide ideal mimetic media for high-resolution NMR studies of membrane proteins and proteins or peptides interacting with micellar aggregates. (15)N NMR relaxation of the backbone amides of a protein-SDS complex has been measured under different experimental conditions. The rotational diffusion time of this complex has been found highly sensitive to detergent and NaCl concentrations. A comparison with calculated rotational diffusion times of protein-free SDS micelles under the same conditions suggests that the size of both aggregates must follow a similar functional dependence on detergent/NaCl concentration.

Synthesis and biological activity of 1H-benzotriazole and 1H-benzimidazole analogues--inhibitors of the NTpase/helicase of HCV and of some related Flaviviridae.

To improve anti-helical activity of analogues of 1H-benzotriazole and 1H-benzimidazole their N-alkyl derivatives were synthesized and tested for antihelicase activity against enzymes of selected Flaviviridae including hepatitis C virus (HCV), West Nile virus (WNV), Dengue virus (DENV) and Japanese encephalitis virus (JEV). 1- and 2-alkyl derivatives of 4,5,6,7-tetrabromo-1H-benzotriazole were obtained by direct alkylation of 4,5,6,7-tetrabromo-1H-benzotriazole with the use of respective alkyl halides in the presence of KOH in methanol, to give a mixture of 1- and 2- isomers, which was separated by flash column chromatography in good yield. The proportion of isomers strongly depended on the reaction time and temperature. 1- and 2-hydroxyethyl and 1- and 2-chloroethyl derivatives of the tetrabromobenzo-triazole were synthesized with the use of 2-bromoethanol and 1-bromo-2-chloroethane respectively as alkylating agents. N-alkylation of this benzotriazole compound enhanced inhibitory activity and selectivity towards the helicase activity of HCV NTPase/helicase. The most active were the 2-methyl, 2-ethyl and 2-propyl derivatives (IC50 approximately 6.5 microM in the presence of DNA as a substrate). Derivatives of the benzotriazole in which hydroxyethyl or chloroethyl replaced the alkyl substituents lost their inhibitory activity. Brominated or methylated benzotriazole N(1) ribosides also did not exert helicase inhibitory activity. Although a number of N(1) and N(2) alkyl derivatives exerted good HCV and WNV helicase inhibitory activity when DNA was used as substrate, the activity was strongly decreased or even disappeared when RNA was used as substrate. The cytotoxicity tests in Vero and HeLa Tat cells showed a substantial decrease of cytotoxicity of N-alkyl derivatives as compared to the parent benzotriazole.

Inhibitors of the NTPase/helicases of hepatitis C and related Flaviviridae viruses.

Abstract: In the search for inhibitors of the non-structural protein 3 (NS3)-associated NTPase/helicase activities of the hepatitis C virus (HCV), and of the related West Nile Virus (WNV), and Japanese Encephalitis Virus (JEV), random screening of a broad range of unrelated low-molecular weight compounds revealed that 4,5,6,7-tetrabromo-1H-benzotriazole (TBBT) is a good inhibitor of the helicase activity of HCV and WNV NTPase/helicases (IC50 >> 20 mM and 1.7 mM with a DNA substrate), but a very weak inhibitor of the JEV enzyme (IC50 >> 200 mM). The synthesis of new TBBT derivatives was undertaken and their inhibitory activities against HCV, WNV, and JEV NTPase/helicases and cytotoxicities were examined. The N-alkyl derivatives showed good activity and lower cytotoxicity than TBBT.

Experimental (13C NMR) and theoretical (ab initio molecular orbital calculations) studies on the prototropic tautomerism of benzotriazole and some derivatives symmetrically substituted on the benzene ring.

The prototropic tautomerism in anhydrous DMSO of benzotriazole and six derivatives symmetrically substituted on the benzene ring (5,6-dichloro, tetrachloro, 4,7-dibromo, tetrabromo, 5,6-dimethyl, and tetramethyl), was followed by both experimental (13C NMR and UV spectroscopy) and theoretical methods. In all of the analyzed systems, predominance of the asymmetric form, N(1)/N(3) protonated, was found. The rates of the N(1)-H<-->N(3)-H prototropic equilibrium, estimated by 13C NMR techniques, were in the medium exchange regime of 300-3000 s(-1), and are correlated with the spectroscopically determined pKa values in aqueous medium, and the anionic forms are the putative rate-limiting intermediate states.

Searching for a new anti-HCV therapy: synthesis and properties of tropolone derivatives.

Hepatitis C virus (HCV) is considered one of the most dangerous pathogens since about 3% of the world population is HCV-infected and the virus is a major cause of hepatitis, cirrhosis, and liver carcinoma. A need for a more efficient therapy prompted us to investigate new class of compounds, such as tropolone derivatives that possess antiviral, antibacterial, and antifungal activities. To synthesize bromo- and morpholinomethyl-analogues of tropolone, the previously reported methods were modified. The influence of new derivatives on the activity of the helicase and NTP-ase of HCV was investigated. The most potent inhibitory effect in the fluorometric helicase assay was exerted by 3,7-dibromo-5-morpholinomethyltropolone, for which the IC50 value was at low micromolar range. All the morpholino-derivatives had inhibitory activities higher than those of the non-modified analogues. Low toxicity in a yeast-based toxicity assay indicates that these compounds could be further modified to develop potent inhibitors of the HCV helicase and of viral replication.

Synthesis and activity of 1H-benzimidazole and 1H-benzotriazole derivatives as inhibitors of Acanthamoeba castellanii.

Chloro-, bromo- and methyl- analogues of 1H-benzimidazole and 1H-benzotriazole and their N-alkyl derivatives have been synthesized and tested in vitro against the protozoa Acanthamoeba castellanii. The results indicate that 5,6-dimethyl-1H-benzotriazole (11) and 5,6-dibromo-1H-benzotriazole (14) have higher efficacy than the antiprotozoal agent chlorohexidine.