Inhibitors and inhibition studies of mammalian glyoxalase II activity

Abstract

545The glyoxalase-enzyme system, which has been known since 1913 [l, 21, is widely distributed in nature and catalyses the conversion of methylglyoxal (or other a-ketoaldehydes) to i)-lactate (or other a-hydroxy acids) with reduced glutathione serving as the cofactor [3-71. The system consists of two enzymes, glyoxalase I and glyoxalase I1 [8]. Glyoxalase I (EC 4.4.1.5) acts upon the equilibrium adduct of methylglyoxal and glutathione, a hemimercaptal, to form the thioester, S-i Aactoylglutathione (SLG). Glyoxalase I1 (EC 3.1.2.6) hydrolyses the thioester to regenerate glutathione and to liberate free iAactic acid. Many suggestions for the function(s) of the glyoxalase system have been made and include protection against a-ketoaldehyde toxicity [8], regulation of cell growth [9, 101 and a glycolytic bypass from dihydroxyacetone phosphate to I )-lactate, via methylglyoxal synthase and the glyoxalases [ 111. Several years ago the glyoxalase system was reported to affect cell-free microtubule assembly [ 121. Synthesis of competitive inhibitorsOne of the approaches employed in exploring possible functions of the glyoxalases has been with the use of competitive inhibitors of glyoxalase I and glyoxalase 11. From 1957 onwards, a number of glyoxalase I inhibitors, derived from Flutathione, have been synthesized [ 13-17]. More recently competitive inhibitors of glyoxalase 11, also derived from glutathione, have been prepared and studied [ 18-21 1 ; these inhibitors are typically thiocarbonate or thiocarbonate-carbamate derivatives of glutathione. Those derivatives which have been found to be effective glyoxalase I1 inhibitors are summarized in the generalized structure: OOCHCI lICHLCONHCHCONHCH,COO -I I NI I S I I K' R Abbreviations used: CH-G, S-carboxybenzoxylglutathione; DiFMOC-G, N,S-fluorenylmethoxycarbonylglutathione; FMOC-G, S-fluorenylmethoxycarbonylglutathione; 0-, m-, p-NCH-G, S-(0, m-, p-nitrocarbobenzoxyglutathione); SLG, S-1)-lactoylglutathione. This report is concerned mainly with the synthesis of new glyoxalase I1 inhibitors (in particular N,S-bisfluorenylmethoxycarbonylglutathione [DiFMOC-GI and some of its diesters) and their effects on purified mammalian glyoxalase I1 and on mammalian cells in culture. We will touch on the subject of the inhibition of purified preparations of glyoxalase I1 by a variety of purine nucleotides as well. W e have prepared S-fluorenylmethoxycarbonylglutathione (FMOC-G) and N,S-biscarbobenzoxy glutathione (DiCB-G) previously and have found them to be quite potent and specific competitive inhibitors of glyoxalase I1 purified from mammals and plants 1201. Recently we have prepared and studied N,S-bisfluorenylmethoxycarbonyl glutathione (DiFMOC-G). Table 1 lists a number of glyoxalase I1 inhibitors that have been reported, as well as the newly synthesized DiFMOC-G. The latter compound is the most potent competitive inhibitor of glyoxalase I1 known to us that has been synthesized ( K , = 0.75 pM, calfliver glyoxalase 11). The thiocarbonate derivatives of glutathione, which bind ve...

Explaining the inhibition of glyoxalase II by 9-fluorenylmethoxycarbonyl-protected glutathione derivatives

“…Nonetheless, the relatively tight binding of glutathione derivatives with bulky hydrophobic substituents is somewhat surprising. It is also difficult to reconcile the reported tight binding of D F G to calf liver GLX2 (K i values of 80-750 nM) [8,26] compared to the relatively weaker binding of the S-FMOC glutathione derivative (K i value of 2.1 lM). Moreover, there are no glutathione compounds, that contain substituents only on the N terminus of glutathione that can be used to test this hypothesis directly.…”

“…Previous inhibition studies using D F G were conducted on GLX2 purified from calf liver [8,26] and Aloe vera [27]; however, the K i values reported differed by several orders of magnitude. To determine whether D F G inhibits GLX2 from A. thaliana, the di-FMOC compound, D F G 1, was synthesized as previously described [26] and tested as an inhibitor of cytosolic GLX2 (GLX2-2) from A. thaliana [20].…”

“…However, inhibition studies on GLX2 from different sources often have resulted in inconsistent or contradictory findings. For example, in different studies the bulky N,S-linked glutathione derivative 1 (D F G) was reported to be a competitive inhibitor of calf liver GLX2 with a K i of 80 or 750 nM [8,26]. The S-FMOC glutathione derivative 3 was also reported to inhibit GLX2 from A. vera with a K i of 4.6 lM [28].…”

“…The glyoxalase system is made up of two enzymes: glyoxalase I (GLX1), which is a Zn(II)-or Ni(II)-dependent enzyme that isomerizes the equilibrium adduct of methylglyoxal and glutathione to form S-D Dlactoylglutathione (SLG) [2,3], and glyoxalase II (GLX2), which is a Fe-and Zn-containing enzyme that catalyzes the hydrolysis of the thioester to liberate D Dlactic acid and regenerate GSH [4,5]. Previous studies have implicated the glyoxalase system in cell cycle control, cancer, malaria, and diabetes, and inhibitors of GLX1 and GLX2 have been designed as antiprotozoal and antitumor agents [6][7][8][9][10][11][12][13].…”

“…1) [8,26,27] and S-phenylmethoxycarbonylglutathione (S-Cbz-glutathione or Z S G, 2, K i values 13-88 lM) [28][29][30][31] have been reported in the literature. However, inhibition studies on GLX2 from different sources often have resulted in inconsistent or contradictory findings.…”