Fig 1. Anti-PPIase and immunosuppressive activities of STG-175.

A. Structures of cyclophilin inhibitors. Inhibition of CypA (B) and CypD (C) isomerase activities were assessed using the α-chymotrypsin-coupled assay [24]. Each test compound was prepared at 10 concentrations (0.05–1000 nM). All solutions were equilibrated, and reactions conducted at 5°C. Reactions were initiated by mixing 95 μL reaction mix with 5 μL of the peptide to be cleaved and measuring OD405 nm at 6-sec intervals for 6 min. Data were fitted with Graphpad Prism 6.0 to obtain first-order rate constants. Enzyme catalyzed rate constants were calculated by subtracting the rate constant from uncatalyzed reactions (no CypA/D), and catalytic rate constants plotted as a function of inhibitor concentration to obtain IC₅₀s. Data are representative of two independent experiments. D. IL-2 promoter activation upon T-cell stimulation was determined using an engineered Jurkat T-cell line, which expresses betagalactosidase. Jurkat cells (1 million) were activated with PMA (10 ng/mL). DMSO, STG-175, CsA, ALV or SCY-635 were added together with PMA and enzymatic activity in cell lysates quantified after 24 h. The Y-axis values are expressed as percentage of activity. Data expressed as EC₅₀s are representative of two independent experiments.