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Fragment-Based Screening Identifies Novel Non-Amino Acid Inhibitors of the Sodium-Coupled Neutral Amino Acid
Sebastian Jakobsen1, Carsten Uhd Nielsen2
1Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.
Introduction:
Amino acid transporters like the sodium-coupled neutral amino acid transporter 2 (SNAT2, SLC38A2) have gained interest for their roles in, e.g., the central nervous system and in cancer. Efforts in discovering inhibitors against these transporters often result in amino acid-based inhibitors that lack selectivity and are likely to compete with amino acid substrates to bind their targets. To circumvent this, we aimed to discover novel non-amino acid inhibitors of SNAT2 by screening a library of fragment-sized compounds.
Methods:
320 fragment compounds were screened for their inhibition of 3H-Gly uptake in hyperosmotically upregulated SNAT2-expressing PC-3 cells. The top five hits were studied further for their inhibitory potency and structure-activity relationship (SAR). Their ability to be translocated by SNAT2 was studied using the FLIPR membrane potential (FMP) assay, as well as their mechanism of inhibition.
Results:
The screen revealed two similar scaffolds that showed SNAT2 inhibition, namely 1,3-benzothiazole-2-amine and 1,3-benzoxazole-2-amine. The SAR revealed how hydrophobic substituents at specific positions were needed for the structures to show SNAT2 inhibition. The best inhibitors inhibited SNAT2 with IC50s in the range of 0.64-1.08 mM. Many of the fragment compounds showed an apparent hyperpolarization in the FMP assay, making it difficult to determine their ability to be translocated by SNAT2. An allosteric mechanism of inhibition was implied for the thiazole and oxazole scaffolds, as these resulted in inhibition patterns that resembled non- or un-competitive inhibitors.
Conclusion:
In conclusion, we discovered multiple novel non-amino acid compounds that inhibited SNAT2 and can serve as starting points for the further development of SNAT2 inhibitors.
