Figure 8

Comparison between Z-FL (Millipore) and Z-FL-hydrate (Adooq Bioscience). (A) Derived from a dipeptide of L-phenylalanine and L-leucine, Z-FL has two chiral centers (red arrow). In addition, Z-FL has additional modifications to the amino and carboxyl terminus. The studies in the main text were prepared using Z-FL (from Millipore, molecular weight = 424.5 g/mol); however, a second set of studies were performed using Z-FL-hydrate (from Adooq Bioscience, molecular weight = 442.5 g/mol). The increased molecular weight reflects a hydrate of the carboxyterminal aldehyde (Blue square); (B) To confirm relative equivalence of these two forms of Z-FL, Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) was used to evaluate both in comparison to vehicle alone (25% DMSO + 75% Acetonitrile). Z-FL showed a retention time at 8.03 min, while Z-FL-hydrate showed retention time at 10.32 min. Both peaks indicated by the black arrow were collected and confirmed to have an inhibitory effect on 12.5 nM human recombinant CTSS with 96.51% and 96.26% from Z-FL and Z-FL-hydrate, respectively; (C) The half maximal inhibitory concentration (IC₅₀) of Z-FL from both forms toward human recombinant CTSS was assessed. The IC₅₀ were generally equivalent, with values of 172.5 ± 32.7 nM and 261.9 ± 59.3 nM for the batches of Z-FL and Z-FL-hydrate respectively; (D) The CTSS inhibitory potential of Z-FL and Z-FL-hydrate at 20 µM was also assessed in spleen lysates obtained from 16-week male NOD mice. Z-FL-hydrate showed less potent CTSS inhibition in spleen lysates compared to Z-FL (n = 4, P = 0.0007, data represent mean ± SD, a two-tailed, unpaired Student’s t-test was used to compare between groups).