In vitro selection of RNA-based irreversible inhibitors of human neutrophil elastase.
BACKGROUND
We describe a new approach to drug discovery which joins the technologies of medicinal and combinatorial chemistry, allowing selection of the most active variant of a lead compound from a large (> 10(12)) pool. A small-molecule covalent inhibitor of elastase was coupled to a randomized pool of RNA, and this assembly was iteratively selected for oligonucleotide sequences that promote the covalent reaction of the inhibitor with the human neutrophil elastase (hNE) active site.


RESULTS
Incorporation of the covalent inhibitor into the randomized pool increases the second-order rate of inactivation of hNE by approximately 15-fold; sequences selected from this pool show an additional approximately 20-fold increase in activity. The relative rate of cross-reaction with another serine protease, cathepsin G, was reduced > 100-fold. Low doses of the inhibitor were found to prevent lung damage inflicted by human neutrophils in an isolated rat lung model of acute respiratory distress syndrome (ARDS).


CONCLUSIONS
This result supports the hypothesis that neutrophil elastase is a significant effector of inflammatory disease. More generally, our findings demonstrate that blending small molecules into combinatorial libraries is a feasible method of drug discovery.
Smith
D
D
NeXstar Pharmaceuticals, Inc., Boulder, CO 80301, USA.
Kirschenheuter
G P
GP
Charlton
J
J
Guidot
D M
DM
Repine
J E
JE
D000818:Animals; D001483:Base Sequence; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003429:Cross Reactions; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D009841:Oligonucleotides / Q000737:chemistry* / Q000494:pharmacology*; D009929:Organ Size; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry* / Q000494:pharmacology*; D051381:Rats; D012128:Respiratory Distress Syndrome / Q000473:pathology / Q000517:prevention & control; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology / Q000627:therapeutic use; D013379:Substrate Specificity
D009841:Oligonucleotides; D015842:Serine Proteinase Inhibitors; D012313:RNA; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534097:Ctsg protein, rat; D019272:Leukocyte Elastase