Fig 1: Effect of compound 3 and 7 treatment on intracellular GMP levels and on PDE6C activity.(A) Relative GMP levels in the human cell line BJ-hTERT after treatment with 20 µM sildenafil, 3, or 7. Circles are values from individual samples, grey bars are means, and error bars are SEM. Three independent assays were conducted. ***p<0.001 vs control (one-way ANOVA with Tukey’s multiple comparisons test). Range of average GMP concentration in control samples (no drug treatment, 4E5 cells): 0.1 ± 0.001 to 3 ± 0.2 µM. (B) Recombinant human PDE6C activity (measured as GMP production) when treated with sildenafil, 3, or 7 at 50x the PDE5 IC50 concentration. % PDE6C activity remaining was calculated as described in the materials and methods section. Circles are values from three replicates, grey bars are mean, and error bars areSEM. #p<0.01 vs hypothetical mean of 100%; sildenafil and compound 3 were not significant (one sample t test). †p<0.05 vs sildenafil (p = 0.02) and vs compound 3 (p<0.001); sildenafil vs compound 3 was not significant (two-sample t test). Average untreated control reaction velocity: 1E6 ± 0.2E6 µmol min-1 U-1.
Fig 2: Molecular modeling of PDE5 with icariin analogs using PDB ID: 2H44 [20] as the starting model.(A) Crystal structure of compound 1 (grey) bound to PDE5 (PDB ID: 2H44) [20], highlighting ligand orientation and nearby interacting residues. Residues shared with panel B are white while residues shared with panel C are blue (interacts with 7-O position) and yellow (hydrophobic pocket; interacts with 3-O position). (B) Ligand docking model of compound 9 (pink) bound to PDE5 illustrates residues (white) that interact with conserved elements of the icariin-derived compounds, such as the flavone backbone and prenyl group. (C) Ligand docking model of compound 3 (white) bound to PDE5 illustrates residues that form hydrogen bonds with the 7-O-glucose (blue) and that form a hydrophobic pocket surrounding the 3-O-hydroxyethyl group (yellow). Hydrogen bonding is indicated with dashed yellow lines.
Fig 3: The 3-O and 7-O positions of icariin pharmacophore act synergistically to influence icariin analog PDE5 inhibition.IC50 values for icariin and icariin analogs 1–9 were obtained via in vitro inhibition assays using purified recombinant human PDE5. IC50 values represent mean (of at least three replicates) ± SEM. Fold improved potency refers to fold change in IC50 compared to icariin.
Fig 4: Lineweaver-Burk plots from in vitro PDE5 kinetics assays with compound 3 (A) or 7 (B).(A) Compound 3 acts as a competitive inhibitor with an inhibition constant (Ki) in the mid-nanomolar range. (B) Compound 7 inhibition may be more complicated. The Ki for a competitive inhibition model is also in the mid-nanomolar range. ? is no inhibitor, ? is 0.015 µM inhibitor, ¦ is 0.05 µM inhibitor, and ? is 0.2 µM inhibitor. Each data point and Ki, Km, and Vmax values represent means (from at least three replicates) ± SEM. Ki, Km, and Vmax values were determined by nonlinear regression analysis.
Fig 5: The natural product icariin inhibits phosphodiesterase-5 (PDE5).PDE5 hydrolyzes cGMP (cyclic guanosine monophosphate) to GMP (guanosine monophosphate). Icariin is a flavonoid known to competitively inhibit PDE5 with an IC50 around 1–6 µM [16–18].
Supplier Page from Abcam for Recombinant human PDE5A/PDE5 protein