Phosphodiesterase 5 (PDE5) is most familiar in connection to sildenafil (Viagra), a popular pharmaceutical PDE5 inhibitor. In response to nitric acid-activated soluble guanylyl cyclase production of cGMP, dimeric PDE5 binds and hydrolyzes cGMP in vascular smooth muscles cells, lung, kidney, brain, cardiac myocytes, and platelets to create 5′-GMP. This is accomplished by cGMP binding to PDE5’s N-terminal domain that, in turn, activates its C-terminal catalytic domain to hydrolyze cGMP’s phosphodiester bond. This increases intracellular calcium concentration and vasoconstriction, increasing blood pressure. Post-translational modifications of PDE5 can both activate (phosphorylation) and signal degradation (nitrosylation). While initially investigated as a remedy for angina and high blood pressure, PDE5 inhibitors have recently been approved for use against pulmonary arterial hypertension, cardiac hypertrophy, and lower urinary tract symptoms (such as benign prostatic hyperplasia). Recent studies indicated PDE5 inhibitors may even have therapeutic promise for treating obesity, cancer, cardiovascular disease, neurological disease, diabetes, and COVID-19.
The principal issue that continues to restrain greater use of PDE5 inhibitors is cross-inhibition of other PDEs, leading to off-target effects. The cGMP binding domain of all 11 human PDEs is highly conserved. Hence, finding inhibitors that exclusively and tightly bind PDE5 remains challenging.1
Identifying & Characterizing the Potent PDE5 Inhibitor
Pyridopyrazinones are naturally occurring substances with diverse biological activities. Modern medicinal chemistry takes advantage of pyridopyrazinone scaffolds to synthesize various small, easily absorbable drugs, such as CRF-R1 antagonists, PI3K inhibitors, and cancer therapies. Amin et al (2022) found their own synthesized range of substituted pyridopyrazinones shared structural similarities to those Pfizer previously characterized as PDE5 inhibitors. They created an in silico system (trained by a library of established PDE5 inhibitors) to perform 2D-QSAR and molecular docking analyses on their candidates to evaluate their docking scores, hydrophobic interactions, hydrogen bonding availability, and predicted IC50 values.2
Amin et al obtained actual IC50 values for the most promising candidates with an in vitro assay that measures PDE5 activity. Using cGMP as the reaction substrate, the Transcreener AMP2/GMP2 FP (Fluorescence Polarization) Assay detected GMP production and derived IC50 values.
Most often, the in silico predicted IC50 of the candidates did not match their actual in vitro inhibitory properties. In fact, the FP assay was crucial for finding the most potent candidate inhibitor (11b at IC50 = 18.13 nM). Further structural study of 11b informed a new structure-activity relationship for these pyridopyrazinone derivatives.2
11b’s terminal phenyl ring occupies the G pocket, its pyridopyrazinone ring lodges in the H pocket, its oxadiazole ring sits at the entrance to the catalytic site, and its acetyl group exposes to solvent when docked to PDE5. Molecular dynamic simulation revealed that 11b-PDE5 binding stabilizes the protein’s structure, employing 2-4 hydrogen bonding interactions in the process.2
Implications for this Inhibitor
Pyridopyrazinone scaffolds and substituted heterocyclic scaffolds, in general, are an expanding frontier in rational drug design. Amin et al. revealed a promising, new PDE5 inhibitor, derived from their own rational synthesis and vetted by a unique set of analytic techniques. Further characterization will be required to determine the inhibitor’s specificity for PDE5. This acute specificity is urgently needed for PDE5 inhibitors to finally enter routine use for the treatment of cancer, obesity, neurological disease, and other indications.
The Transcreener AMP2/GMP2 Assay is a technology of BellBrook Labs. The assay relies on direct immunodetection of AMP and GMP without using coupling enzymes, lowering the chance of interference. The assay is sensitive and robust, generating Z’ ≥ 0.7 at low substrate concentrations. It is a single-addition, mix-and-ready assay available with an FP or TR-FRET readout. To learn more about how you can use the Transcreener AMP2/GMP2 Assay, visit:
1. Ahmed, W.S. et al. (2020) Phosphodiesterase 5 (PDE5): Structure-function regulation and therapeutic applications of inhibitors. Biomedicine & Pharmacotherapy, 134: 111128. Review. https://doi.org/10.1016/j.biopha.2020.111128
2. Amin, K.M et al. (2022) Scaffold Repurposing Reveals New Nanomolar Phosphodiesterase Type 5 (PDE5) Inhibitors Based on Pyridopyrazinone Scaffold: Investigation of In Vitro and In Silico Properties. Pharmaceutics, 14(9), 1954. https://doi.org/10.3390/pharmaceutics14091954