Related Experiment Video
Updated: Jan 15, 2026
Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF
Published on: September 17, 2019
An intracellular recombinant single-chain variable antibody fragment as a new class of phosphodiesterase type 5
Kürsat Kirkgöz1,2, Sophie Sprenger1,2, Olga Schweigert2,3
1Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
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.
More Related Videos
10:33Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors
Published on: October 26, 2015
12:28Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments
Published on: October 15, 2016
Related Concept Videos
01:28Treatment for Pulmonary Arterial Hypertension: Phosphodiesterase Inhibitors
Among the PDE5 inhibitors, sildenafil (Revatio) stands out as a competitive and selective inhibitor. It operates by elevating cellular levels of cGMP and augmenting signaling through the cGMP-PKG pathway, promoting vasodilation. Upon oral...
01:23Dipeptidyl Peptidase 4 Inhibitors
01:27Transducer Mechanism: Enzyme-Linked Receptors
Major types that are helpful drug targets include:
01:01Phosphodiester Linkages
Phosphodiester bond forms when a phosphoric acid molecule (H3PO4) links with two hydroxyl groups (–OH) of two other molecules, forming two ester bonds. Two water molecules are released in this process. The phosphodiester bond is commonly found in nucleic acids (DNA and RNA) and plays a critical role in their structure and function.
Phosphodiester Bonds Link Nucleotides Together
DNA and RNA are polynucleotides or long chains of nucleotides that are linked together. A nucleotide is...
01:23Treatment for Pulmonary Arterial Hypertension: Prostacyclin Receptor Agonists
These agonists bind to the IPR receptor situated on the plasma membrane of the pulmonary artery smooth muscle cells. This binding triggers a cascade of reactions known as the GS-AC-cAMP-PKA pathway. This pathway results in the relaxation of smooth muscle...
01:42Phosphoinositides and PIPs
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...