Gabriele Giliberti

Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole derivatives

Seventy-six 2-phenylbenzimidazole derivatives were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. The most commonly affected viruses were, in decreasing order, CVB-2, BVDV, Sb-1, HSV-1, and YFV, while HIV-1 and VSV were not affected, and RSV, VV and Reo-1 were only susceptible to a few compounds. Thirty-nine compounds exhibited high activity (EC(50)=0.1-10microM) against at least one virus, and four of them were outstanding for their high and selective activity against VV (24, EC(50)=0.1microM) and BVDV (50, 51, and 53 with EC(50)=1.5, 0.8, and 1.0microM, respectively). The last compounds inhibited at low micromolar concentrations the NS5B RdRp of BVDV and also of HCV, the latter sharing structural similarity with the former. The considered compounds represent attractive leads for the development of antiviral agents against poxviruses, pestiviruses and even HCV, which are important human and veterinary pathogens.

Acridine derivatives as anti-BVDV agents.

Twenty-six 9-aminoacridine derivatives were evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. While seven compounds (9, 10, 14, 19, 21, 22, 24) did not affect any virus and two (6, 11) were moderately active against CVB-5 or Reo-1, 17 compounds exhibited a marked specific activity against BVDV, prototype of pestiviruses which are responsible for severe diseases of livestock. Most anti-BVDV agents showed EC(50) values in the range 0.1-8 μM, thus comparing favorably with the reference drugs ribavirine and NM 108. Some compounds, particularly those bearing a quinolizidinylalkyl side chain, displayed pronounced cytotoxicity. Further studies are warranted in order to achieve still better anti-BVDV agents, and to explore the potential antiproliferative activity of this kind of compounds.

Quinoline tricyclic derivatives. Design, synthesis and evaluation of the antiviral activity of three new classes of RNA-dependent RNA polymerase inhibitors

In this study three new classes of linear N-tricyclic compounds, derived by condensation of the quinoline nucleus with 1,2,3-triazole, imidazole or pyrazine, were synthesized, obtaining triazolo[4,5-g]quinolines, imidazo[4,5-g]quinolines and pyrido[2,3-g]quinoxalines, respectively. Title compounds were tested in cell-based assays for cytotoxicity and antiviral activity against RNA viruses representative of the three genera of the Flaviviridae family, that is BVDV (Pestivirus), YFV (Flavivirus) and HCV (Hepacivirus). Quinoline derivatives were also tested against representatives of other RNA virus families containing single-stranded, either positive-sense (ssRNA(+)) or negative-sense (RNA(-)), and double-stranded genomes (dsRNA), as well as against representatives of two DNA virus families. Some quinolines showed moderate, although selective activity against CVB-5, Reo-1 and RSV. However, derivatives belonging to all classes showed activity against BVDV. Among the most potent were the bis-triazoloquinoline 1m, the imidazoquinolines 2e and 2h, and the pyridoquinoxalines 4h, 4j and 5n (EC(50) range 1-5 μM). When tested in a replicon assay, compound 2h was the sole derivative to also display anti-HCV activity (EC(50)=3.1 μM). In enzyme assays, 1m, 2h, 5m and 5n proved to be potent inhibitors of the BVDV RNA-dependent RNA polymerase (RdRp), while only 2h also inhibited the recombinant HCV enzyme.

Disubstituted thiourea derivatives and their activity on CNS: Synthesis and biological evaluation

A series of new thiourea derivatives of 1,2,4-triazole have been synthesized. The difference in structures of obtained compounds are directly connected with the kind of isothiocyanate (aryl/alkyl). The (1)H NMR, (13)C NMR, MS methods were used to confirm structures of obtained thiourea derivatives. The molecular structure of (1, 17) was determined by an X-ray analysis. Two of the new compounds (8 and 14) were tested for their pharmacological activity on animal central nervous system (CNS) in behavioural animal tests. The results presented in this work indicate the possible involvement of the serotonergic system in the activity of 8 and 14. In the case of 14 is also a possible link between its activity and the endogenous opioid system. All obtained compounds were tested for antibacterial activity against gram-positive cocci, gram-negative rods and antifungal activity. Compounds (1, 2, 5, 7, 9) showed significant inhibition against gram-positive cocci. Microbiological evaluation was carried out over 20 standard strains and 30 hospital strains. Selected compounds (1-13) were examined for cytotoxicity, antitumor, and anti-HIV activity.

Synergistic experimental/computational studies on arylazoenamine derivatives that target the bovine viral diarrhea virus RNA-dependent RNA polymerase

Starting from a series of arylazoenamine derivatives, shown to be selectively and potently active against the bovine viral diarrhea virus (BVDV), we developed a hierarchical combined experimental/molecular modeling strategy to explore the drug leads for the BVDV RNA-dependent RNA polymerase. Accordingly, BVDV mutants resistant to lead compounds in our series were isolated, and the mutant residues on the viral molecular target, the RNA-dependent RNA polymerase, were identified. Docking procedures upon previously identified pharmacophoric constraints and actual mutational data were carried out, and the binding affinity of all active compounds for the RdRp was estimated. Given the excellent agreement between in silico and in vitro data, this procedure is currently being employed in the design a new series of more selective and potent BVDV inhibitors.

Alteration of cell morphology and viability in a recA mutant of Streptococcus thermophilus upon induction of heat shock and nutrient starvation

We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation. Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses. Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S. thermophilus is independent from the intracellular accumulation of these stress-specific chaperones.

5-Acetyl-2-arylbenzimidazoles as antiviral agents. Part 4

Within a project aimed at discovering new Flaviviridae inhibitors, new variously substituted 2-phenylbenzimidazoles were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against viruses representatives of the three genera of the Flaviviridae family, i.e.: Pestivirus (BVDV), Flavivirus (YFV) and Hepacivirus (HCV). Title compounds were also tested against RNA viruses representative of other single-stranded, positive-sense (ssRNA(+)) negative-sense (RNA(-)), or double-stranded (dsRNA) genomes, as well as against representatives of two DNA virus families. Nine compounds showed activity against BVDV (EC(50) = 0.8-8.0 μM), compound 31 being the most potent (EC(50) = 0.80 μM) and selective (SI = CC(50)/EC(50) = >100). When tested in an HCV replicon assay, compound 31 resulted again the most potent, displaying an EC(50) value of 1.11 μM and an SI of 100. Besides inhibiting BVDV, two compounds (35 and 38) showed a moderate activity also against YFV (EC(50) = 13 μM). Interestingly, 35 was moderately active also against RSV (EC(50) = 25 μM).

Different molecular mechanisms of inhibition of bovine viral diarrhea virus and hepatitis C virus RNA-dependent RNA polymerases by a novel benzimidazole.

The virus-encoded RNA-dependent RNA polymerase (RdRp) has emerged as a primary target in the search for selective inhibitors of Flaviviridae. Recently, we reported on the selective inhibition, in cell-based assays, of both BVDV (EC50 = 0.80 ± 0.06 μM) and HCV (EC50 = 1.11 ± 0.15 μM) by 2-{1-[2-(2,4-dimethoxyphenyl)-1H-benzimidazol-5-yl]ethylidene}hydrazinecarbothioamide (227G). Here we show that, in enzyme assays with recombinant enzymes, 227G inhibits, in a dose-dependent manner, the RdRp of both BVDV (IC50 = 0.0020 ± 0.0004 μM) and HCV (IC50 = 0.40 ± 0.04 μM). Furthermore, we report on the selection and molecular analysis of a BVDV-resistant mutant, characterized by the presence of the I261M mutation. By applying a multilevel computational approach, we identified different 227G binding sites on the two RdRps. They were further validated by the good agreement between the calculated affinities and those extrapolated from IC50 values. Our findings suggest different molecular mechanisms of inhibition of the HCV and BVDV RdRps by 227G and indicate the importance of understanding ligand-enzyme interactions at the molecular level for the rational design of new and more potent leads.

Styrylbenzimidazoles. Synthesis and Biological Activity - Part 3

As a follow up of an anti-Flaviviridae project, a new series of variously substituted 2-styryl-benzimidazoles were synthesized and tested in vitro for biological activity. Compounds were tested in cell-based assays against viruses representative of: i) two of the three genera of the Flaviviridae family, i.e. Pestiviruses and Flaviviruses; ii) other RNA virus families, such as Retroviridae, Picornaviridae, Paramyxoviridae, Rhabdoviridae and Reoviridae; iii) two DNA virus families (Herpesviridae and Poxviridae) as well as for cytotoxicity tests, run in parallel with antiviral assays,against MDBK, BHK and Vero 76 cells. In the series examined, new leads emerged against BVDV, CVB-2 and RSV. Compounds 11, 12, 17, 18, 24, 31 exhibited anti-BVDV activity in the concentration range 1.7-16 microM; among them, compound 17 was the most active, with an EC(50) = 1.7 microM. Compounds 18 and 21 were equally active against CVB-2, with EC(50) values of 7 - 8 microM, while the derivative 30 was active against RSV with EC(50)= 1 microM and represents a new lead compound.

Transcriptional analysis of the recA gene of Streptococcus thermophilus

BackgroundRecA is a highly conserved prokaryotic protein that not only plays several important roles connected to DNA metabolism but also affects the cell response to various stress conditions. While RecA is highly conserved, the mechanism of transcriptional regulation of its structural gene is less conserved. In Escherichia coli the LexA protein acts as a recA repressor and is able, in response to DNA damage, of RecA-promoted self-cleavage, thus allowing recA transcription. The LexA paradigm, although confirmed in a wide number of cases, is not universally valid. In some cases LexA does not control recA transcription while in other RecA-containing bacteria a LexA homologue is not present.ResultsWe have studied the recA transcriptional regulation in S. thermophilus, a bacterium that does not contain a LexA homologue. We have characterized the promoter region of the gene and observed that its expression is strongly induced by DNA damage. The analysis of deletion mutants and of translational gene fusions showed that a DNA region of 83 base pairs, containg the recA promoter and the transcriptional start site, is sufficient to ensure normal expression of the gene. Unlike LexA of E. coli, the factor controlling recA expression in S. thermophilus acts in a RecA-independent way since recA induction was observed in a strain carrying a recA null mutation.ConclusionIn S. thermophilus, as in many other bacteria,recA expression is strongly induced by DNA damage, however, in this organism expression of the gene is controlled by a factor different from those well characterized in other bacteria. A small DNA region extending from 62 base pairs upstream of the recA transcriptional start site to 21 base pairs downstream of it carries all the information needed for normal regulation of the S. thermophilus recA gene.