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.
Table 1.
Kinetic parameters determined for TcSir2rp1.
Fig 2.
Chemical structures of the bisnaphthalimidopropyl derivatives used in the present study.
R = H in all compounds except for compound 13.
Fig 3.
Enzymatic inhibition of TcSir2rp1 by BNIP derivatives.
A-B) The percentage of inhibition of the NAD+-dependent deacetylase activity of TcSir2rp1 by BNIP derivatives is represented in the y axis. Positive (no drug) and negative (NAM, nicotinamide at 2 mM) controls are represented. Bars represent the average + standard deviation of at least two independent experiments. Differences between the experimental groups were considered significant as follows: * p<0.05, *** p<0.001 and **** p<0.0001.
Table 2.
EC50 values (μM) of BNIP derivatives and nifurtimox against T. cruzi epimastigotes, wild-type (WT) and cells overexpressing TcSir2rp1 (OE) with the pTcINDEX.
Fig 4.
In vitro activity of BNIP derivatives.
A) Primary screening of BNIP derivatives by an in vitro assay for intracellular T. cruzi amastigotes at a single dose of 2.5 μM. Values for both anti-parasitic activity and cell ratio are represented in the dot plot graph. The blue square represents the zone of selection for active, low toxicity hits to be evaluated by dose-response curve analysis. The green square represents the zone of selection for compounds to be tested at a higher dose. B) Primary screening of the compounds selected for testing at a higher dose of 10 μM. Values for both anti-parasitic activity and cell ratio are represented in the dot plot graph. The blue square represents the zone of selection for active, low toxicity hits to be evaluated by dose-response curve analysis. C) Representation of the selectivity indexes of the compounds analysed by dose-response curve analysis, with the determined anti-parasitic activity (EC50) in the y axis, and the cell ratio (CC50) in the x axis. Dots represent the average of three independent experiments.
Fig 5.
In vitro hepatocytes and neurons injury scores for BNIPSpd (9) by HCS.
The score was calculated as the sum of individual scores obtained from a panel of in vitro cytotoxicity assays that include: mitochondrial dysfunction measured by TMRM probe dynamics in cells; membrane integrity assayed by lactate dehydrogenase quantification; DNA damage by imaging with H2AX antibody; apoptosis by caspase 3/7 activation; and neurite outgrowth as imaged with an anti-tubulin III antibody. Nimesulide (400 μM), an approved drug with a mild toxicological profile, was included as a toxicity control.
Table 3.
Anti-parasitic activity, cytotoxicity and selectivity index of BNIP derivatives selected for dose-response curve analysis.
Table 4.
Cytotoxicity and selectivity of BNIPSpd (9).
Fig 6.
TcSir2rp1 structural model and docking with compound 9.
A) The 3.5 Å structural model shows a large Rossmann-fold domain (composed of 6 parallel β-strands, sandwiched between 2 layers of α-helices), and a small zinc binding domain. The substrate acetylated peptide p53 is bound to the cleft between the small and the large domains. B-C) The substrate p53 peptide was removed from the structure and docking studies conducted with compound 9. Several conformations (only 2 shown here) of compound 9 were possible in a putative ligand binding site close to the NAD binding site.