Structural Elucidation of Relevant Gibberellic Acid Impurities and <i>In Silico</i> Investigation of Their Interaction with Soluble Gibberellin Receptor GID1

Gibberellin derivatives are a family of tetracyclic diterpenoid plant hormones used in agriculture as plant growth regulators included in the European Directive 91/414. In the pesticide peer review process and to assess their toxicological relevance and product chemical equivalence, the European Food Safety Authority (EFSA) highlighted data gaps such as the identification of hydrolysis products and unknown impurities. The aspect of impurity characterization and quantitation is challenging and requires the use of hyphenated analytical techniques. In this regard, we used an LC-QTOF/MS and NMR analysis for the characterization of gibberellic acid impurities found in technical products. Gibberellic acid impurities such as gibberellin A1 (GA1), 3-isolactone gibberellic acid (iso-GA3), gibberellenic acid, 1α,2α-epoxygibberellin A3 (2-epoxy- GA3), and (1α,2β,3α,4bβ,10β)-2,3,7-trihydroxy-1-methyl-8-methylenegibb-4-ene-1,10-dicarboxylic acid were identified and successfully characterized. Moreover, an in silico investigation on selected gibberellic acid impurities and derivatives and their interactions with a gibberellin insensitive dwarf1 (GID1) receptor has been carried out by means of induced fit docking (IFD), generalized-Born surface area (MM-GBSA), and metadynamics (MTD) experiments. A direct HPLC method with DAD and MS for the detection of gibberellic acid and its impurities in a technical sample has been developed. Moreover, by means of the in silico characterization of the GID1 receptor-binding pocket, we investigated the receptor affinity of the selected gibberellins, identifying compounds (2) and (4) as the most promising hit to lead compounds

El Correo gallego : diario político de la mañana: Ano LVIII Número 20110 - 1936 xullo 26

<p>Reverse transcriptase (RT)-associated DNA polymerase (RDDP) and ribonucleaser H (RNase H) functions are both essential for HIV-1 genome replication, and the identification of new inhibitors to block both of them is a goal actively pursued by the scientific community. In this field, natural extracts have shown a great potential as source of new antivirals. In the present work, we investigated the effect of <i>Uvaria angolensis</i> extracts on the HIV-1 reverse transcriptase-associated DNA polymerase and ribonuclease H activities. The <i>U. angolensis</i> stem bark methanol extract inhibit both HIV-1 RNase H function and RDDP activity with IC₅₀ values of 1.0 ± 0.2 and 0.62 ± 0.15 μg/mL, respectively and, after been fractionated with different solvents, its solid residue showed an IC₅₀ of 0.10 ± 0.03 and of 0.23 ± 0.04 μg/mL against RNase H and RDDP, respectively, hence laying the bases for further studies for identification of single active components.</p

Exploiting Drug-Resistant Enzymes as Tools To Identify Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)­thieno­[2,3-<i>d</i>]­pyrimidin-4­(3<i>H</i>)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3′,4′-dihydroxyphenyl (catechol) substituted thienopyrimidinones with submicromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones <i>destabilize</i> the enzyme, in some instances reducing the <i>T</i>_m by 5 °C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthen the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy