Fig 1.
Drug design of the new compounds.
Scheme 1.
Synthesis of the furoxan derivatives (4a-o).
Scheme 2.
Synthesis of benzofuroxan (4p-x) and N-acyl-hydrazones (14a-g) derivatives.
Table 1.
NO-released data for compounds (4a-x) and (14a-e).
Fig 2.
Analysis of CPB expression in P. pastoris.
(A) SDS-PAGE showing the supernatants of non- transformed Pichia pastoris X-33, and aliquots from 0 to 144 hours of the transformed clone pPIC-CPB2.8#25 induced with methanol. The arrow indicates the expected size for active (processed) CPB. The arrowhead indicates the unprocessed protein after 24 and 48 h induction. MW: molecular weight Bench Mark (Invitrogen). (B) Analysis of L. mexicana CPB after purification and dialysis. Coomassie-stained SDS-PAGE 15% showing in MW: molecular weight Page Ruler (Thermo Scientific); 1, the purified and dialyzed recombinant CPB. The arrow indicates the recombinant protein (about 26 kDa) and the arrowheads, the products of proteolysis. (C) Active site titration of the recombinant CPB using the irreversible protease inhibitor E-64. Residual activity % (Relative Fluorescence Units) was measured by the ratio between the enzyme activity in absence or presence of increasing concentrations of E-64.
Table 2.
Inhibition of CPB, biological activities, and safety by furoxan and benzofuroxan derivatives (μM).
Fig 3.
Ligand-CPB complex predicted by molecular docking.
Possible poses and interactions performed by compounds 14a (A), 4a (B) and 4e (C) against CPB. Superposition of 4a (beige), 14a (cyan) and 14e (pink). The yellow surface represents CYS26 and dark blue is HIS164.
Fig 4.
The LDU index (number of Leishmania amastigotes in 1,000 nucleated cells per organ weight) was determined in the spleen (A) and liver (B) of L. infantum infected BALB/c mice using. The data are expressed as average ± SD. Control: Infected and non-treated animals. MT: miltefosine. *: Statistically significant difference in all groups compared to the Infected and non-treated animals (p < 0.0001). #: Not statistically significant (p > 0.05).