Abstract
Purpose :
Mutations in the transforming growth factor beta induced (TGFBI) gene encoding the TGFBI protein (TGFBIp) are associated with inherited corneal diseases including various types of lattice corneal dystrophies (LCDs) and granular corneal dystrophies (GCDs) characterized by corneal deposition of TGFBIp amyloid fibers and granular non-amyloid TGFBIp aggregates, respectively. To date, there are no drugs available for the treatment of these visual conditions. We have performed an early stage drug discovery study for identifying TGFBIp-ligand interactions using recombinantly produced wild type (WT) and mutant TGFBIp domain targets and native mass spectrometry (MS)-based high-throughput screening (HTS). Previously isolated variants of TGFBIp Fasciclin (FAS)1-4 domains mimics the structural stabilities of the full-length TGFBIp variants and is therefore used in this research.
Methods :
TGFBIp FAS1-4 wild type (WT) and mutant domains (R555W and A546T) were expressed in E. coli and purified by nickel-affinity and anion-exchange chromatography. A library of 1762 compounds, primary composed of natural products and a small fraction of commercially available small molecules, were screened against the three FAS1-4 domain variants. Each of the domains was mixed with the compounds in a 1:5 molar ratio. The samples were screened in a 384 well-plate format. 10% methanol was used to improve the solubility of the tested compounds. Control samples of protein in 10% methanol were also included in the screening. A total of 0.8 mg of each protein was used to complete the screening. The obtained MS spectra were used to identify hits of protein-ligand complexes, observed as a shift in the mass (m/z) of the original protein peak. Only peaks with relative intensity above 10% were considered as hits.
Results :
The achieved hit rate from the screening is 5%, comprising 52 compounds interacting with all FAS1-4 domain variants and 38 compounds interacting with one or two of the protein domains. The relative peak intensity of the identified protein-ligand complexes varies amongst the tested compounds and the different domain variants.
Conclusions :
The identified TGFBIp-ligand interactions in our study show that natural product compounds complexing with TGFBIp FAS1-4 should be further investigated for their potential use as drugs for TGFBIp-associated corneal dystrophies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.