Fig 2: 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 3: 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 4: 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 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].