A conserved hymenopteran-specific family of cytochrome P450s protects bee pollinators from toxic nectar alkaloids.

Many plants produce chemical defense compounds as protection against antagonistic herbivores. However, how beneficial insects such as pollinators deal with the presence of these potentially toxic chemicals in nectar and pollen is poorly understood. Here, we characterize a conserved mechanism of plant secondary metabolite detoxification in the Hymenoptera, an order that contains numerous highly beneficial insects. Using phylogenetic and functional approaches, we show that the CYP336 family of cytochrome P450 enzymes detoxifies alkaloids, a group of potent natural insecticides, in honeybees and other hymenopteran species that diverged over 281 million years. We linked this function to an aspartic acid residue within the main access channel of CYP336 enzymes that is highly conserved within this P450 family. Together, these results provide detailed insights into the evolution of P450s as a key component of detoxification systems in hymenopteran species and reveal the molecular basis of adaptations arising from interactions between plants and beneficial insects.

Insight into the Binding Mode of Agonists of the Nicotinic Acetylcholine Receptor from Calculated Electron Densities.

Insect nicotinic acetylcholine receptors (nAChRs) are among the most prominent and most economically important insecticide targets. Thus, an understanding of the modes of binding of respective agonists is important for the design of specific compounds with favorable vertebrate profiles. In the case of nAChRs, the lack of available high-resolution X-ray structures leaves theoretical considerations as the only viable option. Starting from classical homology and docking approaches, binding mode hypotheses are created for five agonists of the nAChR, covering insecticides in the main group 4 of the Insecticide Resistance Action Committee (IRAC) mode of action (MoA) classification, namely, neonicotinoids, nicotine, sulfoxaflor, and butenolides. To better understand these binding modes, the topologies of calculated electron densities of small-model systems are analyzed in the framework of the quantum theory of atoms in molecules. The theoretically obtained modes of binding are very much in line with the biology-driven IRAC MoA classification of the investigated ligands.

Flupyradifurone (Sivanto™) and its novel butenolide pharmacophore: Structural considerations.

Flupyradifurone (4-[(2,2-difluoroethyl)amino]-2(5H)-furanone), a member of the new class of butenolide insecticides, contains a novel bioactive scaffold as pharmacophore. It is very versatile in terms of application methods to a variety of crops, exhibits excellent and fast action against a broad spectrum of sucking pest insects including selected neonicotinoid resistant pest populations such as whiteflies and aphids expressing metabolic resistance mechanisms. As a partial agonist flupyradifurone reversibly binds to insect nicotinic acetylcholine receptors (nAChRs) and lacks metabolization by CYP6CM1, a cytochrome P450 over-expressed in cotton whiteflies resistant to imidacloprid and pymetrozine. The butenolide insecticides exhibit structure-activity relationships (SAR) that are different from other nAChR agonists such as the classes of neonicotinoids and sulfoximines. The paper briefly reviews the discovery of the butenolide insecticide flupyradifurone, its SAR differentiating it from established nAChR agonists and a molecular docking approach using the binding site model of CYP6CM1vQ of Bemisia tabaci known to confer metabolic resistance to neonicotinoid insecticides.

Flupyradifurone: a brief profile of a new butenolide insecticide.

BACKGROUND: The development and commercialisation of new chemical classes of insecticides for efficient crop protection measures against destructive invertebrate pests is of utmost importance to overcome resistance issues and to secure sustainable crop yields. Flupyradifurone introduced here is the first representative of the novel butenolide class of insecticides active against various sucking pests and showing an excellent safety profile. RESULTS: The discovery of flupyradifurone was inspired by the butenolide scaffold in naturally occurring stemofoline. Flupyradifurone acts reversibly as an agonist on insect nicotinic acetylcholine receptors but is structurally different from known agonists, as shown by chemical similarity analysis. It shows a fast action on a broad range of sucking pests, as demonstrated in laboratory bioassays, and exhibits excellent field efficacy on a number of crops with different application methods, including foliar, soil, seed treatment and drip irrigation. It is readily taken up by plants and translocated in the xylem, as demonstrated by phosphor imaging analysis. Flupyradifurone is active on resistant pests, including cotton whiteflies, and is not metabolised by recombinantly expressed CYP6CM1, a cytochrome P450 conferring metabolic resistance to neonicotinoids and pymetrozine. CONCLUSION: The novel butenolide insecticide flupyradifurone shows unique properties and will become a new tool for integrated pest management around the globe, as demonstrated by its insecticidal, ecotoxicological and safety profile.

Targeting GSK3 from Ustilago maydis: type-II kinase inhibitors as potential antifungals.

Protein kinases are key enzymes in the complex regulation of cellular processes in almost all living organisms. For this reason, protein kinases represent attractive targets to stop the growth of eukaryotic pathogens such as protozoa and fungi. However, using kinase inhibitors to fight against these organisms bears several challenges since most of them are unselective and will also affect crucial host kinases. Here we present the X-ray structure of glycogen synthase kinase 3 from the fungal plant pathogen Ustilago maydis (UmGSK3) and its inhibition by type-II kinase inhibitors. Despite the high sequence homology between the human and the fungal variant of this vital kinase, we found substantial differences in the conformational plasticity of their active sites. Compounds that induced such conformational changes could be used to selectively inhibit the fungal kinase. This study serves as an example of how species-specific selectivity of inhibitors can be achieved by identifying and addressing the inactive state of a protein kinase. In addition to this, our study gives interesting insights into the molecular plasticity of UmGSK3 by revealing a previously unknown inactive conformation of this important kinase family.

Identification of Ustilago maydis Aurora kinase as a novel antifungal target.

Infestation of crops by pathogenic fungi has continued to have a major impact by reducing yield and quality, emphasizing the need to identify new targets and develop new agents to improve methods of crop protection. Here we present Aurora kinase from the phytopathogenic fungus Ustilago maydis as a novel target for N-substituted diaminopyrimidines, a class of small-molecule kinase inhibitors. We show that Aurora kinase is essential in U. maydis and that diaminopyrimidines inhibit its activity in vitro. Furthermore, we observed an overall good correlation between in vitro inhibition of Aurora kinase and growth inhibition of diverse fungi in vivo. In vitro inhibition assays with Ustilago and human Aurora kinases indicate that some compounds of the N-substituted diaminopyrimidine class show specificity for the Ustilago enzyme, thus revealing their potential as selective fungicides.

Do Fukui function maxima relate to sites of metabolism? A critical case study.

The usefulness of local reactivity descriptors for understanding drug metabolism is investigated. Electrophilic Fukui functions are calculated for 18 drugs and 11 agrochemicals and their relation to experimentally observed metabolites is discussed. Maxima of the Fukui functions correspond to major sites of metabolic attack in many examples, facilitating a posteriori understanding of experimental findings. In the second part of the paper, the nature of the electrophilic oxidant species in cytochromes, called "Compound I" (Cpd I), is investigated within the Fukui framework. Nucleophilic Fukui functions are calculated involving the relevant spin states of Cpd I, allowing a more qualitative, intuitive understanding of its reactivity.

Implementation of a general multireference configuration interaction procedure with analytic gradients in a semiempirical context using the graphical unitary group approach.

The graphical unitary group approach has been applied in an efficient implementation of a general multireference configuration interaction (MRCI) method for use with small active molecular orbital spaces in a semiempirical framework. Gradients can be computed analytically for molecular orbitals from a closed-shell or a half-electron open-shell Hartree-Fock calculation. CPU times for single point energy and gradient calculations are reported. The code allows MRCI geometry optimizations of large molecules, as illustrated for the singlet ground state and the four lowest triplet states of fullerene C(76).

First Principles Calculations of Aqueous pKa Values for Organic and Inorganic Acids Using COSMO-RS Reveal an Inconsistency in the Slope of the pKa Scale.

The COSMO-RS method, a combination of the quantum chemical dielectric continuum solvation model COSMO with a statistical thermodynamics treatment for more realistic solvation (RS) simulations, has been used for the direct prediction of pKa constants of a large variety of 64 organic and inorganic acids. A highly significant correlation of r(2) = 0.984 with a standard deviation of only 0.49 between the calculated values of the free energies of dissociation and the experimental pKa values was found, without any special adjustment of the method. Thus, we have a theoretical a priori prediction method for pKa, which has the regression constant and the slope as only adjusted parameters. Such a method can be of great value in many areas of physical chemistry, especially in pharmaceutical and agrochemical industry. To our surprise, the slope of pKa vs ΔGdiss is only 58% of the theoretically expected value of 1/RTln(10). A careful analysis with respect to different contributions as well as a comparison with the work of other authors excludes the possibility that the discrepancy is due to weaknesses of the calculation method. Hence, we must conclude that the experimental pKa scale depends differently on the free energy of dissociation than generally assumed.

Prediction of aqueous solubility of drugs and pesticides with COSMO-RS.

The COSMO-RS method, originally developed for the prediction of liquid-liquid and liquid-vapor equilibrium constants based on quantum chemical calculations, has been extended to solid compounds by addition of a heuristic expression for the Gibbs free energy of fusion. By this addition, COSMO-RS is now capable of a priori prediction of aqueous solubilities of a wide range of typical neutral drug and pesticide compounds. Only three parameters in the heuristic expression have been fitted on a data set of 150 drug-like compounds. On these data an rms deviation of 0.66 log-units was achieved. Later, the model was tested on a set of 107 pesticides, which have been critically selected based on two experimental data sources and by a crosscheck with an independent HQSAR model. On this data set an rms of 0.61 log-units was achieved, without any adjustments to the structurally extremely diverse pesticides. This result verifies the ability of this extended COSMO-RS to predict aqueous solubilities of drugs and pesticides of almost arbitrary structural classes. The new method is COSMO-RSol.