Design, Synthesis and Characterization of Cyclic NU172 Analogues: A Biophysical and Biological Insight

NU172-a 26-mer oligonucleotide able to bind exosite I of human thrombin and inhibit its activity-was the first aptamer to reach Phase II clinical studies as an anticoagulant in heart disease treatments. With the aim of favoring its functional duplex-quadruplex conformation and thus improving its enz...

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Published in:International journal of molecular sciences 2020-05, Vol.21 (11), p.3860
Main Authors: Riccardi, Claudia, Meyer, Albert, Vasseur, Jean-Jacques, Cavasso, Domenico, Russo Krauss, Irene, Paduano, Luigi, Morvan, François, Montesarchio, Daniela
Format: Article
Language:English
Subjects:
Analytical chemistry
Anticoagulants - chemical synthesis
Aptamers, Nucleotide - chemical synthesis
biophysical characterization
Chemical Sciences
Circular Dichroism
cyclization
Cycloaddition Reaction - methods
DNA aptamers
duplex/quadruplex
Fibrinogen - chemistry
G-quadruplex
G-Quadruplexes
Medicinal Chemistry
NU172
Organic chemistry
Oximes - chemistry
Ultraviolet Rays
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Summary:NU172-a 26-mer oligonucleotide able to bind exosite I of human thrombin and inhibit its activity-was the first aptamer to reach Phase II clinical studies as an anticoagulant in heart disease treatments. With the aim of favoring its functional duplex-quadruplex conformation and thus improving its enzymatic stability, as well as its thrombin inhibitory activity, herein a focused set of cyclic NU172 analogues-obtained by connecting its 5'- and 3'-extremities with flexible linkers-was synthesized. Two different chemical approaches were exploited in the cyclization procedure, one based on the oxime ligation method and the other on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC), affording NU172 analogues including circularizing linkers with different length and chemical nature. The resulting cyclic NU172 derivatives were characterized using several biophysical techniques (ultraviolet (UV) and circular dichroism (CD) spectroscopies, gel electrophoresis) and then investigated for their serum resistance and anticoagulant activity in vitro. All the cyclic NU172 analogues showed higher thermal stability and nuclease resistance compared to unmodified NU172. These favorable properties were, however, associated with reduced-even though still significant-anticoagulant activity, suggesting that the conformational constraints introduced upon cyclization were somehow detrimental for protein recognition. These results provide useful information for the design of improved analogues of NU172 and related duplex-quadruplex structures.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms21113860