High-affinity aptamers selectively inhibit human nonpancreatic secretory phospholipase A2 (hnps-PLA2).
A family of sequence-related 2'-aminopyrimidine, 2'-hydroxylpurine aptamers, developed by oligonucleotide-based combinatorial chemistry, SELEX (systematic evolution of ligand by exponential enrichment) technology, binds human nonpancreatic secretory phospholipase A2 (hnps-PLA2) with nanomolar affinities and inhibits enzymatic activity. Aptamer 15, derived from the family, binds hnps-PLA2 with a Kd equal to 1.7 +/- 0.2 nM and, in a standard chromogenic assay of enzymatic activity, inhibits hnps-PLA2 with an IC50 of 4 nM, at a mole fraction of substrate concentration of 4 x 10(-6) and a calculated Ki of 0.14 nM. Aptamer 15 is selective for hnps-PLA2, having a 25- and 2500-fold lower affinity, respectively, for the unrelated proteins human neutrophil elastase and human IgG. Contractions of guinea pig lung pleural strips induced by hnps-PLA2 are abolished by 0.3 microM aptamer 15, whereas contractions induced by arachidonic acid are not altered. The structure that is essential for binding and inhibition appears to be a 40-base hairpin/loop motif with an asymmetrical internal loop. The affinity and activity of the aptamers demonstrate the ability of the SELEX process to isolate antagonists of nonnucleic-acid-binding proteins from vast oligonucleotide combinatorial libraries.
Bridonneau
P
P
NeXstar Pharmaceuticals, Inc., Boulder, Colorado 80301, USA.
Chang
Y F
YF
O'Connell
D
D
Gill
S C
SC
Snyder
D W
DW
Johnson
L
L
Goodson
T
T
Herron
D K
DK
Parma
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D015723:Gene Library; D054501:Group II Phospholipases A2; D006168:Guinea Pigs; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000187:drug effects; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054467:Phospholipases A2; D010994:Pleura / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D012313:RNA / Q000737:chemistry
D004791:Enzyme Inhibitors; D007211:Indoles; C095553:LY 311727; D009841:Oligonucleotides; D012313:RNA; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2