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. Author manuscript; available in PMC: 2022 May 13.
Published in final edited form as: J Med Chem. 2021 Apr 22;64(9):5577–5592. doi: 10.1021/acs.jmedchem.0c02041

Figure 5: Illustration of the Results Obtained from the Penta-Amine Mixture-Based Positional Scan (TPI1952, 50 μg/mL).

Figure 5:

A mixture-based positional scan was conducted around a poly-amine template (inset) based on the reduced tetrapeptide scaffold R4-R3-R2-Pro-R1-NH2 where R1, R2, and R3 were based on 26 amino acid building blocks and R4 was based on 42 carboxylic acid derivatives. HEK293 cells stably transfected with the desired melanocortin receptor subtype were stimulated with 50 μg/mL a mixture in a CRE reporter β-galactosidase assay in a 96-well plate format. The results were normalized to well protein content and the maximal activity achieved by NDP-MSH. After mixture stimulation, the health of the cells was visually assessed under a microscope, and the fraction of wells which appeared stressed was approximated and reported above via a pie chart for the mMC3R expressing cells. Unlike the pyrrolidine bis-cyclic guanidine mixture-based positional scan, a substantial number of wells appeared stressed which is a possible indicator of cytotoxicity. The building blocks picked for follow up mixture deconvolution were indicated (*) above each of the selected building blocks, and from these results a combinatorial library of 54 (2x3x3x3) individual compounds was constructed.