Table 1.
Crystallographic parameters, data and refinement statistics.
Fig 1.
Comparison of the TcPRAC structures in complex with BrOxoPA and PYC.
(A) Superimposed TcPRAC structures: the complex with PYC (yellow); TcPRAC monomers in complex with BrOxoPA (green and light blue) and BrOxoPA (shown as orange spheres). (B) Superposition of the ligands and key residues of the catalytic site. BrOxoPA is shown in yellow and PYC in gray (C) 2Fo-Fc electron density omitmap contoured at 3σ of BrOxoPA after reaction, showing the covalent bonds to Cys130 and Cys300. Atom numbering is displayed on the left.
Fig 2.
Chemical structures and activity of potential TcPRAC inhibitors.
(A) Key compounds obtained by modulation of OxoPA and BrOxoPA. (B, C, E) Kinetic curves of 40 mM L-proline racemization catalyzed by 15 μg of TcPRAC in the presence of 5 μM of compounds 1 to 6 or 9 (B and E) or in the presence of 5 μM of OxoPA and BrOxoPA (C); Kinetic curves using 100 mM L-proline racemization catalized by 10 μM of TcPRAC in the presence of 10 μM of compounds 7 or 8 (D).
Fig 3.
Main panel: Racemisation curves of 40mM of L-Proline in the presence of 5 μg of TcPRAC and 0.0, 0.3125, 0.625, 1.25, 2.5 and 5 μM of NG-P27 inhibitor are reported by continuous lines from top to bottom respectively. Single exponential fits are given by underlining dashed curves. Insert: Rates of the exponential fits are given for NG-P27 (diamonds), OxoPA (triangles), and PYC (squares). Xmgrace was used to generate the graphics [http://plasma-gate.weizmann.ac.il].
Fig 4.
Structure of NG-P27 after reaction in the TcPRAC binding site.
(A) Left, ligand atom numbering of NG-P27 bound with the catalytic Cys300, oxygen atoms are shown in red, nitrogen in blue, and carbon in yellow for NG-P27 and green for the Cys300; Right, two views of NG-P27 bound to Cys300 with a difference electron density omitmap contoured at 3σ. (B) Orientation of the inhibitor and key residues in the catalytic site. The electron density map is calculated as in (A).
Fig 5.
Projections on the first two Principal Components (PCs), (A) of the whole dimers coordinates and symmetric forms, (B) of chains A or B isolated, and, (C) of the binding pocket of chain A or B. Triangles pointing right/left mark 1W62/its-symmetric or chain A/B. Diamonds mark 1W61; "+", BrOxoPA complex; "x", OxoPA; and "o", NG-P27, respectively. The 49 transition path intermediates are connected by lines and conformations 1, 4, 10 and 49 used for the virtual screening are shown by "*". The 49 intermediates of chain B are connected by dashed lines.
Fig 6.
Close views of the binding sites showing conformational changes for (A) the crystal co-complexes with PYC (1W62-A, pink), OxoPA (chain B, white), and NG-P27 (chain B, cyan), and for (B) for transitional model intermediates, conf1A (pink), conf4A (white) and conf10A (cyan). Main chains are presented by ribbons, sidechains, thin sticks for amino acids of the binding site (see list in Materials and Methods). Catalytic cysteines, the ligand and residues making hydrogen bonds (dashed lines) with the ligand carboxylic moiety (C130 G131, H132, C300, G301, T302) are displayed in bolder sticks, and non carbon atoms as blue, red, and yellow spheres for nitrogen, oxygen and sulfur, respectively. The crystallographic ligands are also represented with lines in the transitional models for reference. Amino acids C130, N218, F290 are labeled, and regions 127–130 and 289–291 of the backbone are labeled with * and Ø signs.
Table 2.
Volume of the binding site cavities.
Fig 7.
Activity of potential TcPRAC inhibitors with cysteine proteases.
Residual activity (%) of papain (A) and bromelain (B) after incubation with different stoichiometric ratios of OxoPA, Br-OxoPA and NG-P27. Data are expressed as mean ± SD. Residual activities after incubation with the E-64 cysteine protease inhibitor (gray hatched area) or with DMSO (light-yellow area) are shown.
Fig 8.
Determination of IC50 for epimastigote forms of Trypanosoma cruzi.
Trypanocydal/trypanostatic effect after 72h of incubation of bioluminescent parasites of CL and Y strains with different concentrations of the reference drug benznidazole (BNZ; A) and the NG-P27 TcPRAC inhibitor (B) added once (1x, complete lines) or three times, once a day for three days (3x, dashed lines).
Fig 9.
Proposed mechanism explaining (A) regioselectivity and (B) stereoselectivity in the inhibition of TcPRAC by oxopentenoic derivatives.
Fig 10.
(A) Volume available as seen in the 1W62 structure; protein atoms, lines; key residues, sticks; PYC, bold sticks; and cavity volume containing PYC, mesh of transparent spheres. (B) BrOxoPA after reaction showing bonds with the cysteines and bending of the originally flat molecule following C2 = C3 saturation.