Vitamin K-dependent carboxylase. In vitro inhibitory activity of cyclopentane and cyclohexane-derived analogues of glutamic acid and their conformational study by NMR and molecular dynamics in aqueous solution.
The conformational analysis of four glutamic acid analogues containing a cyclopentyl or cyclohexyl ring, substituted in position 1 by a Boc-protected amino group and a methyl ester group and in position 3 by a free carboxylate group (6-9), has been carried out in an aqueous environment, by 1H and 13C NMR spectroscopy, and molecular dynamics (MD). These compounds have been shown to be weak competitive inhibitors (Ki approximately 20-65 mM) of the vitamin K-dependent carboxylation of Boc-Glu-OMe in rat liver microsomes independently of their ring size and stereochemical features. However, the cyclic trans isomers have been found more active than the cis ones, and Boc-trans-C5-OMe (9) is the most potent inhibitor in the series (cis and trans isomers are defined by the relative arrangement of the carboxyl functions). Such cyclic glutamyl derivatives may provide valuable informations on the preferred bioactive conformations of synthetic glutamyl substrates at the active site of the carboxylase. In aqueous solution, the Boc-cis- and trans-C6 esters exhibit chair conformations with exclusively equatorial and axial substituent positions, while the Boc-cis- and -trans-C5 compounds may display envelope E or 'twist' T conformations with the substituents in the following positions, equatorial; axial and isoclinal. For each compound, the conformations resulting from NMR and MD data were analyzed and classified according to the dihedral angles chi 1 and chi 2, the distances of functional groups, and the spatial charge distribution involving the free carboxyl group. A reduced number of conformational families were found to be in qualitative agreement with NMR and MD data. These results are discussed in relation with the carboxylase inhibitory activity of the analogues, and a spatial disposition of the glutamyl side chain that could be recognized by the carboxylase is deduced.
Larue
V
V
René Descartes-Paris V University, Laboratory of Pharmacological and Toxicological Chemistry and Biochemistry (URA 400 CNRS), Paris, France.
Gharbi-Benarous
J
J
Acher
F
F
Azerad
R
R
Girault
J P
JP
D019735:Carbon-Carbon Ligases; D002851:Chromatography, High Pressure Liquid; D003510:Cyclohexanes / Q000737:chemistry*; D003517:Cyclopentanes / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry*; D008025:Ligases / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D012996:Solutions
D003510:Cyclohexanes; D003517:Cyclopentanes; D012996:Solutions; D018698:Glutamic Acid; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase