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  • The calculations for the R substrate showed that like

    2022-05-18

    The calculations for the R substrate showed that, like the S substrate, the coordination shell of the Zn UNC 3230 sale is more symmetric than in the HIC-SG crystal structure. This symmetry indicates that the enzyme could use the same reaction mechanisms as for the S substrate, but with exchanged roles of Glu-99 and Glu-172. Consequently, our results show that the R variant of the RKCH mechanism gives a higher barrier than the HS mechanism and that an alternative mechanism leading to the L-P product gives the same overall barrier as the HS mechanism. Thus, our calculations do not reflect the experimentally observed stereospecificity of the enzyme, owing to the too symmetric model QM-cluster model used in this study. On the other hand, our calculations show some asymmetry, caused by the positions of the fixed atoms, which are influenced by the different coordination states of the glutamate residues in the starting crystal structure.
    Acknowledgements This investigation has been supported by grants from the Swedish research council (project 2014-5540). The computations were performed on computer resources provided by the Swedish National Infrastructure for Computing (SNIC) at Lunarc at Lund University.
    Human glyoxalase I (GlxI) is a 42kDa dimeric Zn metalloenzyme that detoxifies methylglyoxal in vivo by converting it into --lactoylglutathione, which is then converted to -lactate by glyoxalase II., Since high activities of GlxI are present in tumor tissues, inhibitors of GlxI increase the accumulation of cytotoxic methylglyoxal, which results in significant anti-tumor activity both in vitro and in vivo. A potent and selective GlxI inhibitor could therefore potentially result in an adjuvant to restore chemotherapy sensitivity in tumor cells., Linking two identical binding groups by a spacer unit has been proposed to improve both the selectivity and the activity of inhibitors compared with the corresponding univalent ligands, and this approach has been verified experimentally., , However, optimization of the spacer unit between the binding groups remains a challenge, and is crucial in fragment-based drug design. In this Letter, we extend our previous work on bivalent transition-state analog inhibitors of human glyoxalase I (hGlxI) to include two new competitive bivalent GlxI inhibitors in which symmetric ligands are linked by linkers that differ in flexibility, length, and water solubility. We previously developed a new class of competitive inhibitors of homodimeric human glyoxalase I by cross-linking two molecules of the transition state analog -(-chlorophenyl--hydroxycarbamoyl)glutathione (CHG) through their γ-glutamyl-NH groups with poly-β-alanyl tethers of different length: [CHG(β-ala)] suberate diamide (=17). The strongest inhibitors of this antitumor target enzyme likely bind simultaneously to the active site on each subunit, and give values as low as 0.96nM (=6), a 50-fold tighter binding than the monomer inhibitor CHG (=46nM)., Cross-linking not only improves the binding affinity, but also improves the selectivity by almost 100-fold for human GlxI (hGlxI) relative to yeast GlxI (yGlxI). In the X-ray crystal structure of the hGlxI complex with CHG, the γ-glutamyl-NH groups are exposed to solvent, and are about 30Å apart. However, a 7080Å tether length was found to give the best inhibition. Nevertheless, these inhibitors have two drawbacks that need to be addressed: amidation of the γ-glutamyl-NH groups decreases the binding affinity by 7 to 13-fold, and the bivalent inhibitors have low solubility, making them unable to cross the cell membrane. In this work, we modify the linker by replacing the amide groups with ethylcarbonyl groups, which do not affect the binding affinity of CHG. Two new inhibitors based on -(-bromophenyl--hydroxycarbamoyl) glutathione (BHG), exhibit inhibition constants of 1nM or less, and one of these compounds has a PEG linker, which increases water solubility and should result in an enhanced ability to cross cell membranes., ,