Identification of 1‐phenyl‐4‐cyano‐5‐aminopyrazoles as novel ecdysone receptor ligands by virtual screening, structural optimization, and biological evaluations

Abstract

Ecdysteroids initiate the molting process in insects by binding to the ecdysone receptor (EcR), which is a promising target for identifying insect growth regulators. This paper presents an in silico/in vitro screening procedure for identifying new EcR ligands. The three-step virtual screening procedure uses a three-dimensional pharmacophore model, docking and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) rescoring routine. A novel hit (VS14) with good binding activity against Plutella xylostella EcR was identified from a library of over 200,000 chemicals. Subsequently, the 1-phenyl-4-cyano-5-aminopyrazole scaffold and twelve EcR ligands were synthesized. Their IC 50 values against Plutella xylostella EcR ranged from 0.64 to 23.21 μm. Furthermore, a preliminary analysis of the structure-activity relationship for novel scaffolds provided a basis for designing new ligands with improved activity.

In Vitro Binding Effects of the Ecdysone Receptor−Binding Domain and PonA in Plutella xylostella

“…After testing the activity of the purified target protein and the binding activity of the natural ecdysteroid PonA, we also tested the in vitro activity of a typical ecdysteroid analog control insecticide, tebufenozide, using the radioligand binding method by incubating the reaction with different concentrations of tebufenozide at a target protein concentration of 300 µg·mL −1 and [ 3 H]PonA 10 nmol·L −1 and performing a competitive binding assay. As the concentration of tebufenozide increased, its ability to inhibit [ 3 H]PonA increased such that the detectable [ 3 H]PonA bound to Px GST−EcR/USP decreased; the results are shown in Figure 3 D. The IC 50 value of tebufenozide for this heterodimer was 80.58 nmol·L −1 , which is approximately ten-fold lower than our previous results with transcriptional translation of the target protein (IC 50 = 0.85 µM) [ 25 ], indicating that the sensitivity of our experimental method for target in vitro activity detection showed a significant improvement over that in our previous work. The high sensitivity facilitates the selection of relatively less active but more structurally novel potential compounds in the subsequent screening of candidates, which are often reported to be micromolar concentrations [ 33 , 34 ].…”

“…To date, in vitro activity testing methods for ecdysteroids and their analogs have included radioligand binding and emerging reporter gene assays [ 21 , 22 , 23 ], of which radioligand binding remains the “gold standard” and an indispensable tool for characterizing the structure-activity relationship of new test compounds [ 24 ]. The in vitro transcription-translation protein system combined with the radioligand assay was also used in our laboratory to test the in vitro activity of the commercial insecticide tebufenozide and ecdysone agonists with EcR [ 25 ]. However, the purity of the target proteins obtained by this method was quite low, and there were many heterogeneous proteins [ 26 ], which interfered with the sensitivity and accuracy of the in vitro activity test at the protein level.…”

Reporter gene assays for screening and identification of novel molting hormone- and juvenile hormone-like chemicals

“…Since synthetic ecdysone agonists and JHAs act on insectspecific endocrine systems, extensive efforts have been made to obtain novel compounds. 1,121) Regarding ecdysone agonists, derivatives of imidazothiadiazole, 124) 1-phenyl-4-cyano-5-aminopyrazoles, 125) heptacyclic pyrazolamide, 126) methylene lactams, 127) oxadiazolines, 128) and imidazoles 129) have been lined up by (Q) SAR studies and/or virtual screening methods. These compounds have potential as candidates of new classes of EcRtargeting insecticides.…”

TB-IECS: an accurate machine learning-based scoring function for virtual screening

“…The binding site for each target was determined based on the position of the co-crystallized ligand [ 2 , 38 ] Then, all the prepared ligands were docked into the corresponding structures by Glide SP docking or Smina docking, and only the top-1 scored binding pose was retained for each ligand. Although the top-1 scored binding pose may not be the correct binding conformation, retaining the top-1 scored binding pose for modelling has the following advantages: (1) it significantly enhances the computational efficiency, which is crucial when screening large ligand libraries [ 14 , 39 – 41 ], (2) it aligns with common practice in the field as evidenced by previous research [ 23 , 24 , 42 , 43 ], and (3) it may improve the generalization ability of the machine learning model by training on docked poses rather than crystalized structures, thereby potentially improving its performance.…”