Self-assembly of peptide nanocapsules by a solvent concentration gradient.

Biological systems can create materials with intricate structures and specialized functions. In comparison, precise control of structures in human-made materials has been challenging. Here we report on insect cuticle peptides that spontaneously form nanocapsules through a single-step solvent exchange process, where the concentration gradient resulting from the mixing of water and acetone drives the localization and self-assembly of the peptides into hollow nanocapsules. The underlying driving force is found to be the intrinsic affinity of the peptides for a particular solvent concentration, while the diffusion of water and acetone creates a gradient interface that triggers peptide localization and self-assembly. This gradient-mediated self-assembly offers a transformative pathway towards simple generation of drug delivery systems based on peptide nanocapsules.

A Core Structural Protein That Builds the Locust Mandible with a Mechanical Gradient.

Natural materials, such as locust mandibles and squid beaks, define significant mechanical gradients that have been attributed to the chemical gradients of their specialized structural proteins (SPs). However, the mechanism by which SPs form chemical gradients in these materials remains unknown. In this study, a highly abundant histidine-rich structural protein (LmMHSP) was identified in the mandible of a migratory locust (Locusta migratoria). LmMHSP was proven by both in vivo and in vitro evidence to act as a core building block of the mandible with a variety of synergistic functions including chitin binding, matrix formation via liquid-liquid phase separation, chemical cross-linking, and metal coordination. Furthermore, we found that the SP gradient in the locust mandible stems from the chitin-binding activity of LmMHSP and different microstructures of chitin scaffolds in different regions. These findings advance our understanding of the formation mechanisms of natural biomaterials and have implications for the fabrication of biomimetic materials.

Discovery of Natural Products as Multitarget Inhibitors of Insect Chitinolytic Enzymes through High-Throughput Screening.

Small-molecule inhibitors of insect chitinolytic enzymes are potential insecticides. However, the reported inhibitors that target one enzyme usually exhibit unsatisfactory bioactivity. On the basis of the multitarget strategy, we performed a high-throughput screening of a natural product library to find insecticide leads against four chitinolytic enzymes from the Asian corn borer Ostrinia furnacalis (OfChtI, OfChtII, OfChi-h, and OfHex1). Several phytochemicals were discovered to be multitarget inhibitors of these enzymes and were predicted to occupy the -1 substrate-binding subsite and engage in polar interactions with catalytically important residues. Shikonin and wogonin, which had good inhibitory activities toward all four enzymes, also exhibited significant insecticidal activities against lepidopteran agricultural pests. This study provides the first example of using a multitarget high-throughput screening strategy to exploit natural products as insecticide leads against chitin biodegradation during insect molting.

Discovery of Kasugamycin as a Potent Inhibitor of Glycoside Hydrolase Family 18 Chitinases.

Kasugamycin, a well-known aminoglycoside antibiotic, has been used widely in agriculture and medicine to combat microbial pathogens by binding the ribosome to inhibit translation. Here, kasugamycin was discovered to be a competitive inhibitor of glycoside hydrolase family 18 (GH18) chitinases from three different organisms (bacterium, insect and human). Results from tryptophan fluorescence spectroscopy and molecular docking revealed that kasugamycin binds to the substrate-binding clefts in a similar mode as the substrate. An electrostatic interaction between the amino group of kasugamycin and the carboxyl group of a conserved aspartate in GH18 chitinase (one of the catalytic triad residues) was found to be vital for the inhibitory activity. This paper not only reports new molecular targets of kasugamycin, but also expands our thinking about GH inhibitor design by using a scaffold unrelated to the substrate.

Molecular Insights into the Insensitivity of Lepidopteran Pests to Cycloxaprid.

Cycloxaprid (CYC) is effective in the control of hemipteran pests, but its bioactivity against lepidopteran pests is still unclear. Here, the bioactivity of CYC against lepidopteran pests was found to be much worse than that against hemipteran insects. To reveal the mechanism, the transcriptomes of CYC-treated and untreated Ostrinia furnacalis larvae were compared. Among the top 20 differentially expressed genes, 11 encode proteins involved in cuticle formation, while only one encodes a detoxifying enzyme. Thus, the cuticle appears to be important for the insensitivity of O. furnacalis to CYC. A pretreatment of O. furnacalis larvae with methoprene enhanced the bioactivity of CYC by 1.12-fold. Moreover, mixtures of CYC with graphene oxide increased the bioactivity of CYC by 1.88-fold. Because lepidopteran and hemipteran insects often harm crops at the same time, the work can help make full use of CYC and reduce the environmental impacts of using multiple pesticides.

Design and synthesis of thiazolylhydrazone derivatives as inhibitors of chitinolytic N-acetyl-ß-d-hexosaminidase.

N-acetyl-ß-d-hexosaminidase (Hex) is potential target for pesticide design. Here, a series of thiazolylhydrazone derivatives were designed, synthesized and evaluated as competitive inhibitors of OfHex1, a Hex from the agricultural pest Ostrinia furnacalis. The derivative 3k, with a (benzyloxy)methyl group at the N3 atom, demonstrated greater potency with a Ki of 10.2 µM. Molecular docking analysis indicated that the (benzyloxy)methyl group of 3k was bound to a previously unexplored pocket formed by Loop478-496. Then further optimization around naphthalene ring led to find the more potency substituent phenyl. The derivative 7, with phenoxyethyl group at R1 and a phenyl group at R2, demonstrated an augmented potency with a Ki of 2.1 µM. Molecular docking analysis indicated that 7 was bound to the active pocket of OfHex1 more favorably than 3k. This work suggests a novel scaffold for developing specific Hex inhibitors.