The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 μM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 μM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. © 2013 Tavis et al

PHOTOINDUCED ELECTRON-TRANSFER (PET) IN ORGANIC-SYNTHESIS - [3+2)-TYPE CYCLOADDITION, CYCLIZATION AND C-C BOND-CLEAVAGE REACTIONS

ALBRECHT E, AVERDUNG J, BISCHOF EW, et al. PHOTOINDUCED ELECTRON-TRANSFER (PET) IN ORGANIC-SYNTHESIS - [3+2)-TYPE CYCLOADDITION, CYCLIZATION AND C-C BOND-CLEAVAGE REACTIONS. Journal of Photochemistry and Photobiology A Chemistry. 1994;82(1-3):219-232.An area of growing interest, in organic photochemistry in particular, involves the use of light to induce an electron transfer from a donor (D) to an acceptor (A) molecule, designated photoinduced electron transfer (PET). Excitation of A or D leads to well-defined changes in their redox properties, i.e. A (D) becomes a stronger acceptor (donor). In general, the feasibility of producing radical ions can be predicted using the well-known Weller equation and, moreover, polar solvents and salts effects support their formation. Following a brief introduction, we focus on the synthetic applications of PET, and discuss [3+2] cycloadditions, reductive and oxidative cyclizations and C-C bond cleavage reactions. The PET ring cleavage of azirines results in the formation of 2-azaallenyl radical cations which may be trapped by dipolarophiles to give heterocyclic compounds. Using this method, we have developed a new route to heterophanes. Porphyrins can also be prepared by this procedure. Further studies aim ot reveal the scope and limitations of these new reactions. Radical cations and radical anions can attack pi bonds within the same molecule. Depending on the reaction conditions chosen to generate the radical ions, five- or six-membered ring systems can be obtained. Under PET reductive conditions, radical anions arise which preferentially form five-membered ring molecules (Baldwin rule). However, using PET oxidative conditions, radical cations are generated which preferentially form six-membered ring molecules due to their electrophilic character. In addition, we have developed a new reductive C-C bond cleavage reaction with annular cyclopropanes and cyclobutanes and have applied this method to a new short synthesis of the hirsutene ring system

Photoinduced cycloadditions and cyclizations. Mechanistic studies and synthetic applications

Mattay J, Albrecht E, Fagnoni M, et al. Photoinduced cycloadditions and cyclizations. Mechanistic studies and synthetic applications. JOURNAL OF INFORMATION RECORDING. 1996;23(1-2):23-29.Synthetic applications and mechanistic studies from three areas of photoinduced cycloadditions and cyclizations are reported: First it is shown that unsaturated silyl enol ethers preferentially cyclisize in a 6-endo mode via their photochemically generated radical cations. In the following chapter we discuss fragmentation-cyclization reactions of unsaturated alpha-cyclopropyl ketones by photoinduced electron transfer leading to polycyclic compounds. Finally a kinetic and theoretical study of [2+3] cycloadditions of nitrile ylides generated from 2H-azirines is presented

Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors

<div><p>Influenza polymerase is a heterotrimer composed of polymerase acidic protein A (PA) and basic proteins 1 (PB1) and 2 (PB2). The endonuclease active site, located in the PA subunit, cleaves host mRNA to prime viral mRNA transcription, and is essential for viral replication. To date, the human influenza A endonuclease activity has only been studied on the truncated active-site containing N-terminal domain of PA (PA_N) or full-length PA in the absence of PB1 or PB2. In this study, we characterized the endonuclease activity of recombinant proteins of influenza A/PR8 containing full length PA, PA/PB1 dimer, and PA/PB1/PB2 trimer, observing 8.3-, 265-, and 142-fold higher activity than PA_N, respectively. Using the PA/PB1/PB2 trimer, we developed a robust endonuclease assay with a synthetic fluorogenic RNA substrate. The observed <i>K</i>_m (150 ± 11 nM) and <i>k</i>_<i>cat</i> [(1.4 ± 0.2) x 10^-3s^-1] values were consistent with previous reports using virion-derived replication complex. Two known influenza endonuclease phenylbutanoic acid inhibitors showed IC₅₀ values of 10–20 nM, demonstrating the utility of this system for future high throughput screening.</p></div