Fragments are small molecules typically with a molecular mass between 150–250 Da and binds to certain subpockets of the binding site.
‘Fragment hits are weak binders therefore non-conventional procedures such as thermal shift assays are required to identify such hits.’
Glutaminyl cyclase (QC) catalyzes the intramolecular cyclization of N-terminal glutaminyl (Gln) and glutamyl (Glu) residues to form pyroglutamic (pGlu) peptides or proteins and play a role in the pathogenesis of Alzheimer disease. The fragment library was generated by disconnection of the structure of known QC inhibitors and extensive 2D similarity search of commercial fragment libraries. The fragment library (204 compounds) was acquired from commercial suppliers and screened first by thermal shift assay (DSF: differential scanning fluorimetry) followed by in vitro enzyme assays.
DSF was performed in a 96-well format in a BioRad CFX96 Real-Time PCR equipment using Sypro Orange as a fluorescent label. Glutaminyl cyclase (QC) was heterologously expressed in Pichia pastoris X33 yeast strain. The enzymatic activity of QC was measured in a pyroglutamyl aminopeptidase (PGPEP) coupled fluorescent end point assay.
The in vitro hits (10 compounds) were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions.
‘Two fragments bound to different regions in a complementary manner, thus, linking those fragments offers a rational strategy to generate novel QC inhibitors.’
For further details read our publication:
Szaszkó, M., Hajdú, I., Flachner, B., Dobi, K., Magyar, C., Simon, I., Lőrincz, Z., Kapui, Z., Pázmány, T., Cseh, S., Dormán, G. (2017). Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening. Molecular Diversity, 21, 175-86. IF:2,229
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