A suicide inhibitor of nematode trehalose-6-phosphate phosphatases

Abstract

Protein-based drug discovery strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors. Currently, there are no known trehalose-6-phosphate phosphatase (TPP) inhibitors that possess reasonable inhibition constants and chemical scaffolds amenable to convenient modification. In the present study, we subjected recombinant TPPs to a two-tiered screening approach to evaluate several diverse compound groups with respect to their potential as TPP inhibitors. From a total of 5452 compounds tested, N-(phenylthio)phthalimide was identified as an inhibitor of nematode TPPs with apparent Ki values of 1.0 μM and 0.56 μM against the enzymes from the zoonotic roundworms Ancylostoma ceylanicum and Toxocara canis, respectively. Using site-directed mutagenesis, we demonstrate that this compound acts as a suicide inhibitor that conjugates a strictly conserved cysteine residue in the vicinity of the active site of nematode TPPs. The anthelmintic properties of N-(phenylthio)phthalimide were assessed in whole nematode assays using larvae of the ascaroids T. canis and T. cati, as well as the barber’s pole worm Haemonchus contortus. The compound was particularly effective against each of the ascaroids with an IC50 value of 9.3 μM in the survival assay of T. cati larvae, whereas no bioactivity was observed against H. contortus.

Inhibition of Trehalose Synthesis in Lepidoptera Reduces Larval Fitness

“…According to previous reports, the active site consists of conserved CYS-171, LYS-132, and ASP-211 residues. [17] From an earlier report, we observed effective TPP inhibition by eight phthalimide ring candidates (Table S2, Supporting Information). [17] We evaluated these eight candidates as possible HaTPP inhibitors and carried out a docking analysis (Figure S2, Supporting Information).…”

Pathway Driven Target Selection in Klebsiella pneumoniae: Insights Into Carbapenem Exposure

“…Bacteria can synthesize large amounts of this disaccharide to protect the integrity of the cell against a variety of environmental injuries ( Argüelles, 2000 ), and targeting enzymes involved in this process has been extensively studied for M. tuberculosis ( Thanna and Sucheck, 2016 ), particularly informed by the absence of these proteins in vertebrates. Trehalose-6-phosphate synthase (OtsA; DS = 0.71) and trehalose-6-phosphate phosphatase (OtsB; DS = 0.86) participate in this process and are conserved across a range of clinically relevant species ( Cross et al., 2017 ), fueling recent high-throughput screening efforts that led to the discovery of specific inhibitors of these proteins ( Cross et al., 2019 ).…”

Dual-Specificity Inhibitor Targets Enzymes of the Trehalose Biosynthesis Pathway

“…9,28−31 For TPP, N-(phenylthio)phthalimide has been found to be an inhibitor of nematode TPP. 32 However, all of these compounds target only one enzyme, TPS or TPP.…”

“…For TPS, compound Lead 25 has good binding affinity to MoTps1 as evaluated in silico; closantel inhibits Tps1 of both C. albicans and Aspergillus fumigatus; silver-tetrylene and bis-silver-tetrylene complexes could be inhibitors of MoTps1 as evaluated with docking; and validoxylamine A and its derivative validoxylamine A 7′-phosphate inhibit the activity of Tps1/OtsA. ,− For TPP, N-(phenylthio)­phthalimide has been found to be an inhibitor of nematode TPP . However, all of these compounds target only one enzyme, TPS or TPP.…”