br Experimental br Acknowledgment This work was supported in
Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Glyoxalase I (GLO1) is a zinc enzyme that catalyzes the isomerization of a hemithioacetal, formed from glutathione (GSH) and methylglyoxal (MG), to lactic SP 141 thioester. The potential of GLO1 as a drug target for cancer and other diseases has been discussed over the last several decades. GSH analogues, flavonoids, curcumins, and a benzothiazole derivative are reported to show moderate to potent GLO1 inhibitory activity and are expected to be evaluated further for practical uses. We conducted a discovery program of non-peptidic GLO1 inhibitors through lead generation from high throughput screening (HTS) followed by rational inhibitor design using human GLO1/GSH analogue cocrystal structural data deposited in Protein Data Bank (PDB)., , We analyzed the ligand–protein interactions observed in those structures and found that the following interactions are commonly observed: (1) hydrophobic interactions of ligands’ substituents on the sulfur atom in a hydrophobic pocket; (2) a hydrogen bond between cysteine NH of ligands and a water molecule (hereafter referred to as W1) bound to Thr101A and Glu99A; (3) CH/π interaction between alkyl chain of γ-glutamic acid of ligands and Phe67A of the protein; (4) ionic interactions between the γ-glutamyl carboxylate/ammonium groups of ligands and Arg37A/Asn103A/Arg122B of the protein (). We conducted an HTS and identified a few hundred compounds whose GLO1 IC values were less than 10μM. The majority of them had apparent zinc binding functional groups, which was anticipated from the nature of the GLO1 catalytic site. Among them, 4,6-diphenyl--hydroxypyridone () was found to have more potent GLO1 inhibitory activity (IC=1.2μM) than a well-known GLO1 inhibitor, -(-bromobenzyl)glutathione (BBG) (IC=21μM, in-house data), as well as good stability in serum and against liver microsomal oxidation (). A binding model of compound was generated by its docking into the GLO1 catalytic site of a complex with zinc-coordinating -(-hydroxy-(-iodophenyl)carbamoyl)glutathione (PDB code:) in which the position of the two zinc-coordinating oxygen atoms was maintained. The probable binding mode is depicted in . Another mode of zinc-coordination with alternative positions for N–O and CO was denied because of severe steric repulsion between two phenyl groups and protein. In the model, 4-phenyl group of was situated in the hydrophobic pocket (composed of Phe62A, Cys60A, Ile88A, Leu92A, Met157B, Met179B, Leu182B and Met183B) making an edge-to-face aromatic interaction with Phe62A. 6-Phenyl group of was situated at the space which γ-glutamyl moiety of GSH analogues occupied (GSH binding pocket). Phe162B made an edge-to-face interaction with the 6-phenyl ring. No steric repulsion of with W1 was observed (). Since the zinc-coordination of -hydroxypyridone fixed the vector of 4- and 6-substituents, we thought structure-activity relationship (SAR) of each substituent is independent and the combination of each optimized substituent would results in the best optimized compound. The first step of our lead optimization was to find alternative groups for the 4-phenyl group of to enhance hydrophobic interaction in the hydrophobic pocket while still keeping the 6-phenyl group. Creighton et al. reported a positive correlation between competitive inhibition constant () and the Hansch hydrophobicity constant of aryl and alkyl group of -(-hydroxy--aryl/alkyl-carbamoyl)glutathiones. Therefore we prepared several 6-phenyl -hydroxypyridones bearing a hydrophobic substituent at 4-position. Among them (, , was the most potent. 3-Thienyl (), a bioisostere of the phenyl group, kept its activity but 3-pyridinyl () decreased in activity, presumably because of its hydrophilicity. 4-Substituted phenyl groups ( and ) also decreased activity indicating limited space around the 4-position of the phenyl group. Non-aromatic substituents, 1-butynyl and butyl group ( and , respectively) lost activity. This result indicates that the edge-to-face aromatic interaction between the phenyl group and Phe62A is important (). Accordingly, we kept the phenyl group at 4-position during the following modifications.