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    • 3 methyladenine Additionally increased GLO gene expression p

    2021-09-28

    Additionally, increased GLO1 gene expression, protein expression, and activity have been reported in a variety of cancers, including breast,22, 23, 24 pancreatic, melanoma, and prostate.27, 28, 29 Vince and Daluge proposed that inhibitors of GLO1 could serve as anti-tumor agents, by increasing concentrations of MG in tumor cells. S-substituted glutathione derivatives have been shown to be competitive inhibitors of GLO1, with S-p-bromobenzylglutathione being the most potent of these inhibitors (Ki=0.08μM). Thornalley et al. and Sakamoto et al. reported that S-p-bromobenzylglutathione diesters had antitumor activity in human leukemia 60 3 methyladenine and human lung cancer cells, respectively. Murthy et al. reported the first transition state analogues of GLO1 with Ki values ranging from 14 to 160nM and Sharkey et al. reported that the diethyl ester prodrug of S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (CHG) was able to inhibit tumor growth in mice. More recently, a variety of GLO1 inhibitors have been synthesized, including the first bivalent-transition state analogues of GLO1 (Ki 0.96–84nM), in which two CHG molecules are covalently linked together, enhancing the effective concentration of inhibitor at the second active site. Additionally, More and Vince have reported GLO1 inhibitors with Ki values in the low micro/nano-molar range37, 38 that are metabolically stable and resistant to γ-glutamyltranspeptidase, which is capable of cleaving the γ-Glu-Cys bond of compounds such as S-p-benzoylglutathione and the S-(N-aryl-N-hydroxycarbamoyl)glutathione derivatives.39, 40 To date, all of the GLO1 inhibitors above have been either substrate or transition state analogues, and while some of these molecules show strong potency, the potential to increase potency and specificity should not be ignored. To directly address specificity, we have taken a novel approach in designing an active-site directed covalent inhibitor of GLO1. The covalent inhibitor was designed based on the X-ray crystallographic structures of the dimeric GLO1 with the substrate analogue S-p-bromobenzyl glutathione and the transition state analogue S-(N-4-iodophenyl-N-hydroxycarbamoyl)glutathione. These 3-dimensional structures indicate that there is a hydrophobic binding pocket composed of residues from both monomers (Met157, Leu160, Phe162, Leu174, Met179, Met183 from one monomer; Cys60, Phe62, Met65, Phe67, Leu69, Phe71, Ile88 from the other) adjacent to the enzyme active site, with the free sulfhydryl group of C60 sticking into this hydrophobic pocket. The goal of this study is to provide a proof of principle that GLO1 could be inhibited covalently at Cys60 within the enzyme active site and the aims of this study were threefold: (1) to synthesize a rudimentary GSH-analogue with a leaving group that could potentially modify GLO1 covalently, (2) characterize the kinetic parameters of this compound, and (3) determine the amino acid modified in the active site. Here we report the inhibitor 4-bromoacetoxy-1-(S-glutathionyl)-acetoxy butane (4BAB, Fig. 1) that is able to covalently modify Cys60 in the enzyme active site of GLO1.
    Experimental
    Results
    Discussion Although the synthesis of 4BAB is straightforward, the final yields of 4BAB only ranged from 9% to 25%. The reason for the low yields is not entirely clear, but possible sources of product loss could be the breakdown of product during washes, formation of the di-GSH substituted diester, or loss of product during purification. The starting dibromo-diester is in 5-fold excess over GSH, which should prevent, or at least limit, the formation of the di-GSH substituted diester. However, 1H NMR of the HPLC peak preceding the product peak (data not shown) shows that this side product is formed in the reaction mixture, which in turn decreases the overall yield of desired product. Additionally, it is possible that the leaving group is hydrolyzed during the wash cycles. The pH of the GSH solution used in the reaction was ∼9.5 with the reaction stopped by the addition of formic acid until the pH is 3.5. This should prevent the hydrolysis of the leaving group, although the low pH could make the ester bonds susceptible to hydrolysis. There is also the possibility of other side reactions that have not been accounted for here.