Non-steroidal inhibitors of <i>Drosophila melanogaster</i> steroidogenic glutathione <i>S</i>-transferase Noppera-bo

Abstract

Insect growth regulators (IGRs) can be developed by elucidating the molecular mechanisms of insect-specific biological events. Because insect molting, and metamorphosis are controlled by ecdysteroids, their biosynthetic pathways can serve as targets for IGR development. The glutathione S-transferase Noppera-bo (Nobo), which is conserved in dipteran and lepidopteran species, plays an essential role in ecdysteroid biosynthesis. Our previous study using 17β-estradiol as a molecular probe revealed that Asp113 of Drosophila melanogaster Nobo (DmNobo) is essential for its biological function. However, to develop IGRs with a greater Nobo inhibitory activity than 17β-estradiol, further structural information is warranted. Here, we report five novel non-steroidal DmNobo inhibitors. Analysis of crystal structures of complexes revealed that DmNobo binds these inhibitors in an Asp113-independent manner. Among amino acid residues at the substrate-recognition site, conformation of conserved Phe39 was dynamically altered upon inhibitor binding. Therefore, these inhibitors can serve as seed compounds for IGR development.

Transcriptional Regulators of Ecdysteroid Biosynthetic Enzymes and Their Roles in Insect Development

“…Consistent with the fact that Nobo is a GST, gclc , encoding an enzyme required for the de novo synthesis of GSH, was shown to partly phenocopy nobo mutants in D. melanogaster ( Enya et al, 2017 ). Although the endogenous substrates of Nobo remain unclear, a high-throughput chemical screen and X-ray crystallography revealed that several small chemical compounds can be inserted into the putative ligand-binding pocket of Nobo, including the mammalian female sex hormone, 17β-estradiol ( Fujikawa et al, 2015 ; Koiwai et al, 2020 , 2021 ). This implies that a steroid could also be an endogenous substrate of Nobo, but future research is needed to clarify this point.…”

Molecular action of larvicidal flavonoids on ecdysteroidogenic glutathione S-transferase Noppera-bo in Aedes aegypti

“…To reveal the molecular mechanism through which these flavonoids inhibit AeNobo enzymatic activity, we conducted an X-ray crystallographic analysis of AeNobo. Gel filtration chromatography revealed that AeNobo forms a homodimer in solutions (Additional file 1 : Figure S4 A ), suggesting that its dimeric structure is a biological unit similar to DmNobo and other canonical GSTs [ 30 , 31 , 36 ]. Subsequently, we determined the crystal structure of the AeNobo protein in the presence of GSH at 1.51 Å resolution by the molecular replacement method using the structure of DmNobo (PDB ID: 6KEM) [ 37 ] as an initial model (Additional file 1 : Figure S4 B ).…”

“…Our group has developed a high-throughput screening system to identify chemical compounds that inhibit in vitro enzymatic activity of recombinant purified Nobo proteins [ 29 ]. Using this system, we previously isolated multiple inhibitors of D. melanogaster Nobo (DmNobo) and succeeded in an integrated structure biological analysis to partly reveal the mode of action of these inhibitors, including the vertebrate female sex hormone 17β-estradiol [ 30 , 31 ]. In this study, we expanded our strategy to the AeNobo recombinant protein to identify potential AeNobo inhibitors and demonstrate their mode of action.…”

New ways and new hopes for IGR development

“…The ongoing target-specific inhibitor design is aided by ample X-ray structural information on FPPSs, including those from Lepidoptera. 13) The team of Ryusuke Niwa presents high-quality original research 14) leading to inhibitors of a steroidogenic glutathione Stransferase that they had previously discovered as a product of the essential Drosophila noppera-bo (Nobo) gene. 15) A HTS of a chemical library yielded five non-steroidal Nobo inhibitors acting at micromolar concentrations.…”