Nick X. Wang

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.

Molecular Modeling of Sulfoxaflor and Neonicotinoid Binding in Insect Nicotinic Acetylcholine Receptors: Impact of the Myzus β1 R81T Mutation

BACKGROUND Sulfoxaflor (Isoclast™ active), a new sulfoximine-class insecticide, targets sap-feeding insect pests, including those resistant to neonicotinoids. Sulfoxaflor acts on the insect nicotinic acetylcholine receptor (nAChR) in a distinct manner relative to neonicotinoids. Unlike any of the neonicotinoids, sulfoxaflor has four stereoisomers. A homology model of Myzus persicae (green peach aphid) based on the ACh binding protein from Aplysia californica, overlaid with M. persicae nAChR sequence (α2 and β1 subunits) was used to investigate the interactions of the sulfoxaflor stereoisomers with WT and R81T versions of the nAChR. RESULTS Whole-molecule van der Waals interactions are highly correlated with the binding affinity for the neonicotinoids and correctly predict the rank order of binding affinity for neonicotinoids and sulfoxaflor. The R81T mutation in M. persicae nAChR is predicted to have much less effect on binding of sulfoxaflors stereoisomers than that of the neonicotinoids. CONCLUSION All four stereoisomers predictably contribute to the activity of sulfoxaflor. The WT and R81T nAChR homology models suggest that changes in a whole-molecule electrostatic energy component can potentially explain the effects of this target-site mutation on the pattern of reduced efficacy for the modeled neonicotinoids, and provide a basis for the reduced effect of this mutation on sulfoxaflor.

SAR studies directed toward the pyridine moiety of the sap-feeding insecticide sulfoxaflor (Isoclast™ active).

Sap-feeding insect pests constitute a major insect pest complex that includes a range of aphids, whiteflies, planthoppers and other insect species. Sulfoxaflor (Isoclast™ active), a new sulfoximine class insecticide, targets sap-feeding insect pests including those resistant to many other classes of insecticides. A structure activity relationship (SAR) investigation of the sulfoximine insecticides revealed the importance of a 3-pyridyl ring and a methyl substituent on the methylene bridge linking the pyridine and the sulfoximine moiety to achieving strong Myzus persicae activity. A more in depth QSAR investigation of pyridine ring substituents revealed a strong correlation with the calculated logoctanol/water partition coefficient (SlogP). Model development resulted in a highly predictive model for a set of 18 sulfoximines including sulfoxaflor. The model is consistent with and helps explain the highly optimized pyridine substitution pattern for sulfoxaflor.

Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK-2A: Mechanism of action of fenpicoxamid

Abstract BACKGROUND Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated. RESULTS UK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides. CONCLUSION Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A.

Studies toward understanding the SAR around the sulfoximine moiety of the sap-feeding insecticide sulfoxaflor.

BACKGROUND The discovery of sulfoxaflor (Isoclast™ active) stemmed from a novel scaffold-based approach toward identifying bioactive molecules. It exhibits broad-spectrum control of many sap-feeding insect pests, including aphids, whiteflies, hoppers and Lygus. Systematic modifications of the substituents flanking each side of the sulfoximine moiety were carried out to determine whether these changes would improve potency. RESULTS Structure-activity relationship (SAR) studies showed that, with respect to the methylene linker, both mono- and disubstitution with alkyl groups of varying sizes as well as cyclic analogs exhibited excellent control of cotton aphids. However, against green peach aphids a decrease in activity was observed with substituents larger than ethyl as well as larger cycloalkyl groups. At the terminal tail there appeared to be a narrow steric tolerance as well, with linear groups or small rings more active against green peach aphids than bulkier groups. CONCLUSION A novel series of compounds exploring the substituents flanking the sulfoximine moiety of sulfoxaflor were prepared and tested for bioactivity against cotton aphids and green peach aphids. SAR studies indicated that a decrease in green peach aphid potency was observed at the methylene linker as well as at the terminal tail with bulkier substituents. A quantitative structure-activity relationship analysis of the compounds revealed significant correlation of activity with two molecular descriptors, vol (volume of a molecule) and GCUT_SMR_3 (molar refractivity). This predictive model helps to explain the observed activity with the various substituents.

Safety considerations derived from Cry34Ab1/Cry35Ab1 structure and function.

Insecticidal proteins developed for in-plant protection against crop pests undergo extensive safety testing during the product development process. Safety considerations for insecticidal proteins expressed in crops follow recommended, science-based guidelines and specific studies are conducted on a case by case basis. Corn events expressing Bacillus thuringiensis (Bt) Cry34Ab1 and Cry35Ab1 were developed to protect maize from Diabrotica virgifera virgifera (western corn rootworm) feeding damage. The protein crystal structures of Cry34Ab1 and Cry35Ab1 are different from the more common three-domain Cry or Vip3 proteins expressed in insect resistant maize varieties. Cry34Ab1 is a single domain protein that folds into a beta sandwich structure that resembles membrane-active proteins, including several cytolysins, from a variety of natural sources. Cry35Ab1 has two domains, one domain with structural relatedness to sugar binding motifs and a second domain with an extended beta sheet structure that is clearly related to beta pore forming proteins, some of which are insecticidal, e.g. B. sphaericus BinA/BinB. In this review we discuss Cry34Ab1/Cry35Ab1 structure and function in the context of protein safety studies for insect resistant crops.

Physicochemical property guidelines for modern agrochemicals: Physicochemical properties for agrochemicals

The relentless need for the discovery and development of new agrochemicals continues as a result of driving forces such as loss of existing products through the development of resistance, the necessity for products with more favorable environmental and toxicological profiles, shifting pest spectra, and the changing agricultural needs and practices of the farming community. These new challenges underscore the demand for novel, high-quality starting points to accelerate the discovery of new agrochemicals that address market challenges. This article discusses the efforts to identify the optimum ranges of physicochemical properties of agrochemicals through analysis of modern commercial products. Specifically, we reviewed literature studies examining physicochemical property effects and analyzed the properties typical of successful fungicides, herbicides, and insecticides (chewing and sap-feeding pests). From the analysis, a new set of physicochemical property guidelines for each discipline, as well as building block class, are proposed. These new guidelines should significantly aid in the discovery of next-generation agrochemicals.

Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK‐2A

Abstract BACKGROUND Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated. RESULTS UK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides. CONCLUSION Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A.

Characterization of the mechanism of action of fenpicoxamid fungicide and its metabolite UK‐2A

Abstract BACKGROUND Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated. RESULTS UK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides. CONCLUSION Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A.