Novel natural product 5,5-trans-lactone inhibitors of human alpha-thrombin: mechanism of action and structural studies.
High-throughput screening of methanolic extracts from the leaves of the plant Lantana camara identified potent inhibitors of human alpha-thrombin, which were shown to be 5,5-trans-fused cyclic lactone euphane triterpenes [O'Neill et al. (1998) J. Nat. Prod. (submitted for publication)]. Proflavin displacement studies showed the inhibitors to bind at the active site of alpha-thrombin and alpha-chymotrypsin. Kinetic analysis of alpha-thrombin showed tight-binding reversible competitive inhibition by both compounds, named GR133487 and GR133686, with respective kon values at pH 8.4 of 1.7 x 10(6) s-1 M-1 and 4.6 x 10(6) s-1 M-1. Electrospray ionization mass spectrometry of thrombin/inhibitor complexes showed the tight-bound species to be covalently attached, suggesting acyl-enzyme formation by reaction of the active-site Ser195 with the trans-lactone carbonyl. X-ray crystal structures of alpha-thrombin/GR133686 (3.0 A resolution) and alpha-thrombin/GR133487 (2.2 A resolution) complexes showed continuous electron density between Ser195 and the ring-opened lactone carbonyl, demonstrating acyl-enzyme formation. Turnover of inhibitor by alpha-thrombin was negligible and mass spectrometry of isolated complexes showed that reversal of inhibition occurs by reformation of the trans-lactone from the acyl-enzyme. The catalytic triad appears undisrupted and the inhibitor carbonyl occupies the oxyanion hole, suggesting the observed lack of turnover is due to exclusion of water for deacylation. The acyl-enzyme inhibitor hydroxyl is properly positioned for nucleophilic attack on the ester carbonyl and therefore relactonization; furthermore, the higher resolution structure of alpha-thrombin/GR133487 shows this hydroxyl to be effectively superimposable with the recently proposed deacylating water for peptide substrate hydrolysis [Wilmouth, R. C., et al. (1997) Nat. Struct.Biol. 4, 456-462], suggesting the alpha-thrombin/GR133487 complex may be a good model for this reaction.
Weir
M P
MP
Glaxo Wellcome Medicines Research Centre, Stevenage, Hertfordshire, U.K.
Bethell
S S
SS
Cleasby
A
A
Campbell
C J
CJ
Dennis
R J
RJ
Dix
C J
CJ
Finch
H
H
Jhoti
H
H
Mooney
C J
CJ
Patel
S
S
Tang
C M
CM
Ward
M
M
Wonacott
A J
AJ
Wharton
C W
CW
D000215:Acylation / Q000187:drug effects; D001665:Binding Sites / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D007783:Lactones / Q000737:chemistry* / Q000494:pharmacology; D013058:Mass Spectrometry; D008958:Models, Molecular; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014315:Triterpenes / Q000737:chemistry* / Q000494:pharmacology
C112219:GR 133686; C112220:GR 133687; D007783:Lactones; D015842:Serine Proteinase Inhibitors; D014315:Triterpenes; D013917:Thrombin