Inhibitors of Trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease
Fig 1
Trypanosoma cruzi Sir2rp1 characterization and inhibition by nicotinamide.
A) Purity analysis of 5 and 10 μg of TcSir2rp1 by SDS-PAGE stained with Coomassie brilliant blue (left panel). Western Blot analysis of 100 ng of the hexa-histidine tailed TcSir2rp1 recombinant protein with an anti-HisTag antibody. B) TcSir2rp1 deacetylase activity was measured with a fluorimetric kit in the presence or absence of NAD+ (+CTRL), and in presence of 1 μM of trichostatin A (TSA). Bars represent mean + standard deviation. Data from 3 independent experiments. C) Deacetylation reactions for the determination of the kinetic constants of Ac-peptide. NAD+ was fixed at 2000 μM while Ac-peptide was varied (0.63 to 40 μM). Plots of initial velocities versus [Ac-peptide] were fitted to the Michaelis-Menten equation, yielding the kinetic constants (see Table 1). D) Deacetylation reactions for the determination of the kinetic constants of NAD+. Ac-peptide was fixed at 40 μM while NAD+ was varied (15.63 to 2000 μM). Plots of initial velocities versus [NAD+] were fitted to the Michaelis-Menten equation, yielding the kinetic constants (see Table 1). Dots and error bars represent mean + standard deviation. C-D) Data from 3 independent experiments. E) Dose-response curve of TcSir2rp1 inhibition by nicotinamide. Data represents the average + standard deviation of three independent experiments. F) Differential inhibition of TcSir2rp1, TbSir2rp1, LiSir2rp1 and hSIRT1 by a dose of 200 μM of nicotinamide (NAM). Bars represent mean + standard deviation. Data from 2 independent experiments. Differences between the experimental groups were considered significant as follows: * p<0.05, ** p<0.005, *** p<0.001 and **** p<0.0001.
