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Int. J. Mol. Sci. 2008, 9(6), 1008-1023;

Effective DNA Inhibitors of Cathepsin G by In Vitro Selection

Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy
Gentium S.p.A., Piazza XX Settembre 2, 22079 Villa Guardia, Italy
Author to whom correspondence should be addressed.
Received: 5 May 2008 / Revised: 6 June 2008 / Accepted: 10 June 2008 / Published: 20 June 2008
(This article belongs to the Special Issue Nucleic Acid Derivatives in Emerging Technologies)
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Cathepsin G (CatG) is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions. View Full-Text
Keywords: Cathepsin G; defibrotide; Selex; TG repeats; alternating polynucleotides Cathepsin G; defibrotide; Selex; TG repeats; alternating polynucleotides
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Gatto, B.; Vianini, E.; Lucatello, L.; Sissi, C.; Moltrasio, D.; Pescador, R.; Porta, R.; Palumbo, M. Effective DNA Inhibitors of Cathepsin G by In Vitro Selection. Int. J. Mol. Sci. 2008, 9, 1008-1023.

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