We next considered the effect of alkyl chain
We next considered the effect of alkyl chain length on the inhibitory activity. As can be seen from , it is obvious that there is no proportional relationship between the methylene chain length and activity. The activity decreased when the chain length increased from 1 to 3 and increased with elongation of the chain length until 5. The cause of this behavior is unclear, but it is likely that the flexible unbranched alkyl chains fold themselves to fit the whole structure appropriate to the enzyme’s active site, especially when the chain length is 5. We also examined the pterin conjugated with Gly-Phe (), which has a chain length close to that of , and found it to be ineffective, probably because of the hydrogen-bondable amide functionality and the branched large benzyl group.
Overall, the simplest was the most potent ALR2 inhibitor among the compounds tested.
Next, in order to obtain a better insight into the interaction of the pterins with the active site, docking simulations were conducted. In these experiments, the protein structure coded as 2IKG in the Protein Data Bank was employed because the structural features of our pterins resemble the original ligand of 2IKG. All the docking experiments were carried out with the GOLD Suite software package., ChemPLP scoring function was used for docking pose prediction, and the obtained poses were rescored with GOLDscore. The best pose for each ligand was determined by considering the results obtained with both of these scoring functions.
First, we performed the docking simulation for . shows the predicted pose of in 2IKG structure. Obviously, tightly binds to the active site of ALR2 through hydrogen-bonding networks. The carboxylic Liproxstatin-1 moiety was well inserted in the anion-binding pocket to hydrogen bond with His110 and Tyr48. It should be noted that a water molecule assists the interaction of the amide NH of the inhibitor with Trp111. The pterin ring makes a π−π interaction with Trp111 and a hydrogen bond with Thr113 in the specificity pocket. The most noteworthy thing is that the carbonyl oxygen of the pterin makes a hydrogen bond with Leu300, which is significant for selective inhibition toward ALR2. The predicted docking poses for and are shown in (a) and (b). It can be seen from these figures that the carboxylic acid moiety of makes a hydrogen bond with Tyr48, while does not. In addition, had higher docking score than . Conceivably, this explains why compounds that have -amino acid residues have higher inhibitory activity than the -counterparts.
(c) and (d) show the predicted docking poses for and . As can be seen from these figures, hydroxy groups of the serine residues in both of the compounds form hydrogen bonds with His110 to misdirect the carboxylate group to the opposite side of the anion-binding site, while the pterin ring is accommodated in the specificity pocket by making the same interactions with Trp111, Thr113, and Leu300 as those observed for . This type of misdirection possibly comes about with , which has a phenolic hydroxy group, and is likely to give rise to decrease the activity of these inhibitors.
In conclusion, we explored the synthesis and h-ALR2 inhibitory activity of pterin-7-carboxamides. From the results of the aldose reductase inhibitory activity evaluation, it was found that is the most effective inhibitor (IC=1.97μM). This value is nearly the same as that of sorbinil, a highly potent ARI (IC∼2μM). From the docking study for , it was suggested that tightly interacts with amino acid residues composing the active site of ALR2. Notably, the specific interaction with Leu300 that is important for ALR2 selective inhibition was observed with . Thus, is a potential drug for treating diabetic complications that exhibit fewer side effects by only inhibiting the relevant h-ALR2.
Conflicts of interest
Acknowledgements This research was supported by the Japan Society for the Promotion of Science (grant number 25410179); and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (2012–2016). The authors would like to thank Editage (www.editage.jp) for English language editing.