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Identification of small molecule compounds with higher binding affinity to guanine deaminase (cypin) than guanine.

Bioorganic & medicinal chemistry (2010-08-19)
José R Fernández, Eric S Sweet, William J Welsh, Bonnie L Firestein
ABSTRACT

Guanine deaminase (GDA; cypin) is an important metalloenzyme that processes the first step in purine catabolism, converting guanine to xanthine by hydrolytic deamination. In higher eukaryotes, GDA also plays an important role in the development of neuronal morphology by regulating dendritic arborization. In addition to its role in the maturing brain, GDA is thought to be involved in proper liver function since increased levels of GDA activity have been correlated with liver disease and transplant rejection. Although mammalian GDA is an attractive and potential drug target for treatment of both liver diseases and cognitive disorders, prospective novel inhibitors and/or activators of this enzyme have not been actively pursued. In this study, we employed the combination of protein structure analysis and experimental kinetic studies to seek novel potential ligands for human guanine deaminase. Using virtual screening and biochemical analysis, we identified common small molecule compounds that demonstrate a higher binding affinity to GDA than does guanine. In vitro analysis demonstrates that these compounds inhibit guanine deamination, and more surprisingly, affect GDA (cypin)-mediated microtubule assembly. The results in this study provide evidence that an in silico drug discovery strategy coupled with in vitro validation assays can be successfully implemented to discover compounds that may possess therapeutic value for the treatment of diseases and disorders where GDA activity is abnormal.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Guanine, 98%
Sigma-Aldrich
Guanine, BioUltra
Supelco
Caffeine solution, analytical standard, 1.0 mg/mL in methanol
Supelco
Melting point standard 235-237°C, analytical standard
Sigma-Aldrich
Anti-GDA antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Caffeine, anhydrous, tested according to Ph. Eur.
Supelco
Mettler-Toledo Calibration substance ME 18872, Caffeine, analytical standard, for the calibration of the thermosystem 900, traceable to primary standards (LGC)
Sigma-Aldrich
Xanthine, ≥99.5% (HPLC), purified by recrystallization
Sigma-Aldrich
Caffeine, BioXtra
Sigma-Aldrich
Caffeine, powder, ReagentPlus®
Sigma-Aldrich
Caffeine, meets USP testing specifications, anhydrous
Sigma-Aldrich
Caffeine, Sigma Reference Standard, vial of 250 mg
Sigma-Aldrich
Xanthine, ≥99%
Sigma-Aldrich
Xanthine, BioUltra, ≥99%
Sigma-Aldrich
Caffeine, anhydrous, 99%, FCC, FG