Selective delivery of nitric oxide to a cellular target: a pseudosubstrate-coupled dinitrosyl-iron complex inhibits the enteroviral protease 2A.
Nitric oxide (NO) regulates multiple biological processes. To use NO as a potential therapeutic substance, a more selective modulation of individual NO targets is desirable. Here, we tested whether peptide conjugation of the dinitrosyl-iron complex (DNIC), a potent NO donor, confers targeted NO delivery. As target, we used the protease 2A of Coxsackie-B-viruses (2A(pro)), which can cause dilated cardiomyopathy. Through S-nitrosylation, NO inhibits this protease, which is essential for viral replication. The tetrapeptide Leu-Ser-Thr-Cys (LSTC) (based on the 2A(pro) substrate recognition motif) and DNIC generated LSTC-DNIC in vitro by S-nitrosylation as evidenced by reverse-phase chromatography. In vitro, LSTC-DNIC (IC(50) 510 nM) dose-dependently inhibited purified 2A(pro) 4.7-fold more effectively than DNIC (IC(50) 2.4 microM), whereas LSTC alone had no effect. In intact cells, expression of Coxsackievirus protease 2A by transient transfection led to eIF4G-I-cleavage. LSTC-DNIC (IC(50) 23 microM) dose-dependently inhibited eIF4G cleavage in 2A(pro)-transfected cells 3.8-fold more effectively than DNIC (IC(50) 88 microM). To test the specificity of the DNIC-conjugated LSTC peptide part, we investigated its influence on Caspase-3, a known target for S-nitrosylation. LSTC-DNIC and DNIC inhibited purified Caspase-3 in vitro (IC(50) 3.7 microM) and in intact cells similarly. LSTC conjugation of DNIC enhances its fidelity for inhibition of 2A(pro) in vitro and intracellularly. Peptide-DNIC may be useful to selectively modulate cellular processes by NO, i.e., to enhance its antiviral properties.
Badorff
Cornel
C
Molecular Cardiology Unit, Department of Medicine, Goethe-University, 60590 Frankfurt, Germany.
Fichtlscherer
Birgit
B
Muelsch
Alexander
A
Zeiher
Andreas M
AM
Dimmeler
Stefanie
S
D003546:Cysteine Endopeptidases / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007501:Iron / Q000378:metabolism / Q000494:pharmacology*; D009569:Nitric Oxide / Q000008:administration & dosage*; D009589:Nitrogen Oxides / Q000378:metabolism / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D013379:Substrate Specificity; D014764:Viral Proteins
D009589:Nitrogen Oxides; D011480:Protease Inhibitors; D014764:Viral Proteins; D009569:Nitric Oxide; C073661:dinitrosyl iron complex; D007501:Iron; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus