The two competitive bivalent GlxI inhibitors polyBHG nM and

The two competitive bivalent GlxI inhibitors polyBHG2-62 (=1nM) and polyBHG2-54 (=0.3nM) () were designed based on the transition-state analog -(-bromophenyl--hydroxycarbamoyl) glutathione (BHG) by an examination of the X-ray crystal structure of human GlxI in complex with one CHG at each active site (, PDB code 1QIN). CHG is a stable transition-state analog that chelates the active site zinc and mimics the transition-state. We chose a central scaffold of poly β-ala and suberic sodium fluoride bis(-hydroxysuccinimide ester) for the linkage of the two binding sites. In our previous work, amide groups were used to link the CHG units to the spacer groups. However, amidation of CHG decreases the affinity of the inhibitor, as illustrated in . Both compounds A (=330nM) and B (=130nM) have inhibition constants that are significantly higher than that of CHG itself (46nM). Addition of an ethylcarbonyl substituent to give compound C (=46nM), however, does not decrease the inhibition constant relative to CHG itself. These results indicate that the carbonyl group on the substituent is crucial. On the other hand, the unfavorable effect of a carbonyl group near the nitrogen may be rationalized by the weakened hydrogen bonds/electrostatic interactions observed in the X-ray structure (PDB code 1QIN). The bivalent transition state analogs incorporate a symmetric scaffold based on and (), in which the terminal carbonyls are protected by pyrrolidine-2,5-dione. The intermediate was used as a flexible linker with ,-(dipropylamine)diethyleneglycol, and suberic acid bis-(-hydroxysuccinimide ester) was used to adjust the linker spacing. To synthesize two inhibitors with different distances between the active groups, a building block strategy was used (, ). PolyBHG2-62 was synthesized in five steps, as outlined in , and synthesis of polyBHG2-54 followed a similar strategy (). In , ,-(dipropylamine)diethyleneglycol was replaced by poly β-ala to generate the more rigid intermediate , which was used to prepare polyBHG2-54, in which the length of the linker is different from polyBHG2-62. For these inhibitors -(-bromophenyl--hydroxy-carbamoyl) glutathione (BHG, =14nM), is used instead of CHG because of its better binding to GlxI. The bivalent inhibitors polyBHG2-62 and polyBHG2-54 exhibit much tighter binding ability than BHG itself, with values of 1.0nM and 0.3nM, respectively. The tightest binding compound (polyBHG2-54) is more than 50-fold more potent than BHG, and three-fold more potent than the best bivalent inhibitor we obtained previously. Modification of the linker by using ethylcarbonyl groups instead of amide groups on the BHG results in the strongest inhibitor yet reported for hGlxI. Importantly, use of the PEG linker in polyBHG2-62 retains most of the inhibition toward GlxI, while providing enhanced water solubility. It should be noted that, compounds polyBHG2-62 and polyBHG2-54 have similar solubilities (26mg/mL and 32mg/mL, respectively). This combination of enhanced water solubility and strong binding is an important step toward the development of inhibitors for GlxI that can cross cell membranes. , shows the selectivities of the newly synthesized compounds with different glyoxalase I enzymes, including hGlxI, yeast GlxI (yGlxI), and bovine liver GlxII (bGlxII). CHG was used as a positive control. Surprisingly, the two target compounds exhibited higher levels of inhibition than CHG in the hGlxI control. Poly BHG2-62 ( 1.0nM) and poly BHG2-54 ( 0.3nM) are almost 50-fold more active and 156-fold more active than CHG, respectively. In addition, CHG binds 78-fold less tightly to yGlxI than to hGlx1. Thus, cross-linking increases the inhibitor selectivity by approximately 158-fold, as CHG binds 78-fold more tightly to hGlxI than to yGlx, while poly BHG2-54 binds about 12,300-fold more tightly. A comparison of the inhibition constants of CHG and poly BHG2-54 for hGlxI versus bGlxII shows that cross-linking increases binding selectivity 8.6-fold, from 37-fold to 320-fold.