An intracellular recombinant single-chain variable antibody fragment as a new class of phosphodiesterase type 5 inhibitors.

BACKGROUND AND PURPOSE: Cyclic guanosine monophosphate (cGMP) is a ubiquitous second messenger involved in human (patho-)physiology. Phosphodiesterase 5 (PDE5) is a major cGMP hydrolyzing enzyme in many cell types including vascular smooth muscle cells (VSMCs). Several highly selective PDE5 inhibitors are in clinical use. However, there are currently no tissue/cell-specific PDE5 inhibitors with precise action lacking systemic side effects. We sought to develop a new non-chemical PDE5 inhibitor which could be used in specific tissues.

EXPERIMENTAL APPROACH: The single-chain variable fragment (scFv) construct was generated by sequencing heavy and light chains of the inhibitory antibody against GAF-A domain of PDE5. The scFv was purified from bacteria and tested by competitive binding assays, and its sequence was expressed in aortic VSMCs using adenoviral gene transfer. Functional effects on cGMP levels were tested using live cell imaging of cGMP, phosphorylation of cGMP-dependent protein kinase (PKG) substrate and by measuring vasorelaxation in mouse aortic rings following svFv expression.

KEY

RESULTS

scFv expressed as a 29-kDa protein and could specifically bind to the GAF-A domain of PDE5. Live cell imaging demonstrated that scFv increased NO-donor stimulated intracellular cGMP, whereas subsequent effects of the PDE5 inhibitor sildenafil were diminished, as was the PKG-dependent VASP phosphorylation in response to sildenafil. Lastly, adenoviral expression of scFv in mouse aortic rings potentiated NO-donor-induced vasorelaxation.

CONCLUSION AND IMPLICATION: Our data suggest that recombinant scFv could represent a new class of intracellular PDE5 inhibitors. Treatment of chronic conditions with scFv could provide tissue-/cell-specific therapy, minimizing potential side effects.