Fig 1.
Structure-based drug design including cavity selection and dynamics of the enzyme target.
(A) Flowchart illustrating the global strategy for developing allosteric CD73 inhibitors. (B) Five cavities detected using Fpocket on the closed dimeric form of CD73 (4H2G) and shown in colored mesh representations. (C) Top view of superimposed structures of CD73 during the TMD simulation highlighting the large rotating motion of N-domains (centers of mass depicted as spheres in arc shape). (D) Volumes changes and mean local hydrophobic densities observed during TMD for the blue cavity from panel “B” located at the dimerization interface. (E) Target cavity (mesh representation) outside the substrate binding site (AMP and Zn ions are depicted in cyan sticks). (F) Illustration of the target binding site in complex with one hit compound (green sticks) obtained by docking (Glu543 residues are depicted as spheres).
Fig 2.
Selected hit compounds identified by docking at the dimerization interface of CD73 (full list in S1 Table).
MolPort code and chemical structure of hit compounds ranked by docking score. In addition to clog P value, different metrics for ligand efficiency were computed: LE, LLE, BEI and SEI (see Materials and Methods section for details). Enzymatic inhibition by RR compounds (at 5 μM) performed with the purified recombinant CD73 Inhibition constants (Ki) and mode are indicated (NC for non-competitive). *means +/- SD of three independent experiments.
Fig 3.
Enzymatic inhibition assay in the presence of RR compounds using the purified recombinant enzyme.
Red bars indicate the most active RR compounds promoting an enzyme inhibition as efficiently as APCP (5 μM) used as a positive control (green bar). Values of inhibitions are means from three independent experiments ± SD and negative values reflect enzyme activation.
Fig 4.
Comparison of hit compounds by using conventional metrics used in drug design.
Inhibition constants (Ki) are expressed as pKi (A) and ligand (B), ligand-lipophilicity (C), binding and surface (D) efficiencies correspond to LE, LLE, BEI and SEI, respectively. Note that for compounds exhibiting a mixed inhibition mode, two inhibition constants (“a” and “b”) were determined as for RR2 and RR16.
Fig 5.
Determination of the kinetics inhibition profiles for the most representative compounds.
Secondary plots and double-reciprocal of steady state rate constants as a function of AMP concentration in the absence (circles) or with increasing concentrations of hit compounds (squares, triangles and stars). (A): RR3 at 0, 0.4, 0.8 and 1.6 μM; (B): RR4 at 0, 0.3, 0.6 and 1.2 μM; (C): RR6 at 0, 0.25, 0.5 and 1.0 μM; (D): RR20 at 0, 0.3, 0.6 and 1.2 μM.
Fig 6.
Detailed analysis of the binding mode for best-ranked hit compounds.
(A) Overlay of the docking poses from all selected hits at the dimerization interface. Compounds are depicted in sticks and CD73 as solvent accessible surface (yellow and pink for differentiating the two monomers). (B) Overlay of the three most active compounds, RR3 (green) RR6 (cyan) and RR16 (purple). Main polar interactions involved in the binding of RR3 (C), RR6 (D) and RR16 (E) viewed in the same orientation. (F) Binding pose of hit compound RR11 (blue) holding an extended and dimeric structure.
Fig 7.
Hydrophobic contacts involved in the binding of RR compounds.
Docking poses obtained for the two structurally-related compounds, (A) RR4 (yellow) and (B) RR6 (cyan) highlighting the binding differences (thick sticks correspond to residues that are inversely involved). Binding mode of compound having a stretched structure as for RR11 (C) and for RR20 (D) depicted as blue and orange sticks, respectively. Comparison of the binding mode for the inhibitory compound RR3 (E) and the activator RR28 (F) assuming a common binding site for both. Residues making halogen bonds are depicted in yellow sticks. All residues contributing to hydrophobic contacts (either with backbone or sidechain atoms) are depicted in solvent accessible surface and in thin sticks (all compounds are not oriented identically).
Fig 8.
Binding mode of hit compound RR28 linking both enzyme monomers.
Residues are depicted in yellow or pink thin sticks according to the monomer they belong and RR28 in thick pink sticks.