Inhibition of Escherichia coli glutamine synthetase by .alpha.- and .gamma.-substituted phosphinothricins

Abstract

We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)], a naturally occurring inhibitor of GS. These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range. At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity. ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins. The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds. Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds. Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT. 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution. The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor.

Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

“…[23][24][25][26][27] However, none of the 2-amino-phosphono-carboxylic acids (16)(17)(18) identified in the virtual screening showed any inhibitory activity, despite the fact that the 9 phosphono group of docked 2-amino-6-phosphonohexanoic acid (17) overlaps well with the phosphono group of MSO-P. This is in accordance with results for DL-homophosphinothricin, which did not show any inhibition of sheep brain GS.…”

“…In fact a number of inhibitors, including MSO, 8 or its diastereomeric mixture L-(SR), 8,11,13,23 PPT, 8,19,20,[22][23][24][25]28 3, 19,20 and 4 29,35 have shown inhibitory activity when tested on both bacterial and eukaryotic GSs. Therefore a literature search was performed to identify known GS inhibitors for both eukaryotic and prokaryotic enzymes.…”

“…Several functional groups, including a carboxylic acid, a thioether, a sulfoxide, a sulfonamide and a phosphonate ester, were investigated as R 2 substituents. The compounds were synthesized from glycine (24)(25)(26)(27)(28) or from L- (29)(30)(31)(32)(33) or D-serine (34)(35)(36)(37)(38), as described in the experimental section. The desired products were prepared via a CuI catalyzed N-arylation of glycine, D-and L-serine, using K 2 CO 3 as a base, with an appropriately substituted arylbromide (Scheme 2).…”

“…4.4.12 3-(Carboxymethylamino)benzoic acid (24) Following the general procedure, 3-bromobenzoic acid (0. The value is an average from three independent experiments, reported together with the standard deviation.…”

“…[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] Much effort has been invested in developing GS inhibitors not only for antibacterial research, but also for weed control, 20,25,[30][31][32] since GS is a key enzyme in ammonia assimilation in plants. However, no inhibitor has shown improved activity compared to MSO or PPT.…”