Development of FAM20B Kinase Inhibitors as Inducers of Tooth Regeneration

Objectives: Family with sequence similarity 20, member B (FAM20B), is one of three atypical, disulfide-containing kinases that play key roles in posttranslational modification of proteins destined for secretion in Golgi vesicles. FAM20C (formerly known as DMP4) is a protein kinase that phosphorylates serine residues in a specific motif of most if not all secreted phosphoproteins, such as SIBLING and enamel matrix proteins. FAM20A was recently shown to be an inactive pseudokinase, which allosterically enhances the activity of FAM20C by formation of a heterotetrameric complex. Interestingly, FAM20B is a sugar kinase, which specifically phosphorylates a xylose in the common tetrasaccharide linker connecting glycosaminoglycan (GAG) chains with the protein core of proteoglycans. Phosphorylation of the first cyclic monomer serves as a molecular switch to regulate the biosynthesis of proteoglycans, hence is crucial for formation of cartilage, bone and connective tissue. Furthermore, one of our labs (Wang) recently reported that inactivation ofFam20B in dental epithelium leads to supernumerary maxillary and mandibular incisors. Thus inhibitors of FAM20B kinase might induce tooth regeneration via application in tissue culture and/or in vivo.
Methods: Over 640,000 commercially available, drug-like compounds from the UCSF ZINC library were docked in silico to a structure-based homology model of FAM20B through a supercomputer portal (https://drugdiscovery.tacc.utexas.edu/), and from the top 1000 hits, 50 selected for purchase from a commercial vendor. Compounds soluble at 500 micromolar in aqueous buffer were screened by fluorescence-based thermal shift (ThermoFluor) assays with purified recombinant protein.
Results: Compounds that appreciably increased the melting temperature (Tm) of the protein, a high-throughput assay common to the pharmaceutical industry, were sorted into functional group families that provide leads for development of ATP-competitive inhibitors.
Conclusions: Our array of leads can be further screened for inhibition of chondrogenesis in tissue culture in high-throughput and eventually in whole animals for induction of tooth.