A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems.

In this work, we present and discuss a comprehensive set of both newly and previously synthesized compounds belonging to 5 distinct molecular classes of linear aromatic N-polycyclic systems that efficiently inhibits bovine viral diarrhea virus (BVDV) infection. A coupled in silico/in vitro investigation was employed to formulate a molecular rationale explaining the notable affinity of all molecules to BVDV RNA dependent RNA polymerase (RdRp) NS5B. We initially developed a three-dimensional common-feature pharmacophore model according to which two hydrogen bond acceptors and one hydrophobic aromatic feature are shared by all molecular series in binding the viral polymerase. The pharmacophoric information was used to retrieve a putative binding site on the surface of the BVDV RdRp and to guide compound docking within the protein binding site. The affinity of all compounds towards the enzyme was scored via molecular dynamics-based simulations, showing high correlation with in vitro EC50 data. The determination of the interaction spectra of the protein residues involved in inhibitor binding highlighted amino acids R295 and Y674 as the two fundamental H-bond donors, while two hydrophobic cavities HC1 (residues A221, I261, I287, and Y289) and HC2 (residues V216, Y303, V306, K307, P408, and A412) fulfill the third pharmacophoric requirement. Three RdRp (K263, R295 and Y674) residues critical for drug binding were selected and mutagenized, both in silico and in vitro, into alanine, and the affinity of a set of selected compounds towards the mutant RdRp isoforms was determined accordingly. The agreement between predicted and experimental data confirmed the proposed common molecular rationale shared by molecules characterized by different chemical scaffolds in binding to the BVDV RdRp, ultimately yielding compound 6b (EC50 = 0.3 µM; IC50 = 0.48 µM) as a new, potent inhibitor of this Pestivirus.

Synthesis and anti-picornaviridae in vitro activity of a new class of helicase inhibitors the N,N'-bis[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl] alkyldicarboxamides.

A series N,N'-bis[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamides (3a-f and 5a-j) were prepared starting from their already known (1a-d) and (4a-c) or new (4d) amine parents. Because of the antiviral activity of several N-[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides previously reported, title compounds were evaluated in vitro for cytotoxicity and antiviral activity against viruses representative of Picornaviridae, [i.e. Enterovirus Coxsackie B2 (CVB-2) and Polio (Sb-1)] and of two of the three genera of the Flaviviridae [Bovine Viral Diarrhea Virus (BVDV) and Yellow Fever Virus (YFV)]. Furthermore, because of the in silico activity against the RNA-dependent RNA-helicase of Polio 1 previously reported, title compounds were evaluated against the 3D model of the Sb-1 helicase and against the 2D model of the CVB-2 helicase. As a reference we used the antiviral and in silico activities of an imidazo counterpart of the title compounds, N,N'-bis[4-(2-benzimidazolyl)phenyl]alkyldicarboxamides (III) that other authors reported to be able to inhibit the corresponding enzyme of Hepatitis C Virus (HCV). In cell-based antiviral assays, N,N'-bis[4-(1H-benzotriazol-1-yl)phenyl]alkyldicarboxamides (3a-f) resulted completely inactive whereas the bis-5,6-dimethyl-benzotriazol-2-yl derivatives (5d-f) exhibited good activity against the Enteroviruses, (EC(50)s ranged between 7 and 11 microM against CVB-2 and 19-52 against Sb-1). Interestingly, bis-5,6-dichloro-benzotriazol-2-yl derivatives (5h-j) showed very selective activity against CVB-2 (EC(50)s = 4-11 microM) whereas they resulted completely inactive against all the other viruses screened. In general, all title compounds showed a good cytotoxicity profile in MT-4 cells. Molecular modeling investigations showed that active compounds may interact with the binding site of the Sb-1 helicase and that their free binding energy values are in agreement with their EC(50)s values.

Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis.

Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.

Isolation of protease-free alcohol dehydrogenase (ADH) from Drosophila simulans and several homozygous and heterozygous Drosophila melanogaster variants.

The enzyme alcohol dehydrogenase (ADH) from several naturally occurring ADH variants of Drosophila melanogaster and Drosophila simulans was isolated. Affinity chromatography with the ligand Cibacron Blue and elution with NAD+ showed similar behavior for D. melanogaster ADH-FF, ADH-71k, and D. simulans ADH. Introduction of a second Cibacron Blue affinity chromatography step, with gradient elution with NAD+, resulted in pure and stable enzymes. D. melanogaster ADH-SS cannot be eluted from the affinity chromatography column at a high concentration of NAD+ and required a pH gradient for its purification, preceded by a wash step with a high concentration of NAD+. Hybrid Drosophila melanogaster alcohol dehydrogenase FS has been isolated from heterozygous flies, using affinity chromatography with first elution at a high concentration NAD+, directly followed by affinity chromatography elution with a pH gradient. Incubation of equal amounts of pure homodimers of Drosophila melanogaster ADH-FF and ADH-SS, in the presence of 3 M urea at pH 8.6, for 30 min at room temperature, followed by reassociation yielded active Drosophila melanogaster ADH-FS heterodimers. No proteolytic degradation was found after incubation of purified enzyme preparations in the absence or presence of SDS, except for some degradation of ADH-SS after very long incubation times. The thermostabilities of D. melanogaster ADH-71k and ADH-SS were almost identical and were higher than those of D. melanogaster ADH-FF and D. simulans ADH. The thermostability of D. melanogaster ADH-FS was lower than those of D. melanogaster ADH-FF and ADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constants with the ligand Cibacron Blue at pH 8.6, which are two times higher at pH 9.5. The Ki values for D. simulans ADH are three times lower at both pH values. D. melanogaster ADH-SS and ADH-FS have similar Ki values, which are lower than those for D. melanogaster ADH-FF at pH 8.6. But at pH 9.5 the Ki value for ADH-FS is the same as at pH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters of Drosophila melanogaster ADH-FF, ADH-SS, and ADH-71k and Drosophila simulans ADH, at pH 8.6 and 9.5, showed little or no variation in K(m)eth values. The K(m)NAD values measured at pH 9.5 for Drosophila alcohol dehydrogenases are all lower than those measured at pH 8.6. The rate constants (kcat) determined for all four Drosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D. melanogaster ADH-FS showed nonlinear kinetics.

Computerized system for nuclear emergency response in the ENEA Nuclear Research Center of Frascati.

At the National Committee for Research and Development of Nuclear Energy and Alternate Energy Sources (ENEA) Center of Frascati, there are several radiation-producing machines: two tokamaks and three electron accelerators; moreover, a neutron generator will begin to operate in a short time. A completely automatic monitoring system has been developed. Radiation control is performed by means of classical active and passive detectors. An automatic acquisition system has been developed: Measured quantities are acquired and stored in a specific data base; information regarding radioactivity levels, machines status, personnel dosimetry and meteorological parameters are available in real time. If any of the radiometric quantities exceeds appropriate reference levels, the following operations automatically activate: An automatic switch turns off the machines and an alarm signal is broadcast to the Health Physics group. In addition, the "Nuclear Emergency" software module starts if a radionuclide emission is detected. This module has been implemented to provide response to radiological emergencies in the ENEA nuclear research centers. The modularity of the computer-based system allows its utilization also in other nuclear centers, such as at nuclear power plants. When activated, the "Nuclear Emergency" displays an alarm signal and informs the Health Physics group about the monitor's location and characteristics and the measured data exceeding the reference level. If emission of radionuclides occurs, a preliminary evaluation of their diffusion in the atmosphere and an estimation of the population dose are performed. Statistical analysis of the event is also possible.