Design of inhibitors against guanase: Synthesis and biochemical
evaluation of analogues of azepinomycin
Ravi K. Ujjinamatada, Anila Bhan
and Ramachandra S. Hosmane
Laboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry,
University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
Received 8 July 2006; revised 4 August 2006; accepted 7 August 2006
Available online 22 August 2006
Abstract—As part of a program to design rational, mechanism-based inhibitors of guanase, we report here the synthesis and bio-
chemical screening of two analogues of azepinomycin (1 and 2), a naturally occurring inhibitor of guanase, known to mimic the
transition-state of the enzyme-catalyzed reaction. Our biochemical results show that compounds 1 and 2 are competitive inhibitors
of 2.01 ± 0.16 · 10
and 5.36 ± 0.14 · 10
Ó 2006 Elsevier Ltd. All rights reserved.
Guanine deaminase (GDA) or guanase (EC 126.96.36.199) is
an enzyme that catalyzes the hydrolytic deamination
of guanine to xanthine. This enzyme has been found in
human liver, brain, and kidney.
There have been re-
ports of abnormally high levels of serum guanase activ-
ity in patients with liver diseases,
and so, the elevated
enzyme activity has been suggested as a marker of hep-
atitis and hepatoma.
Furthermore, such a high guanase
activity is believed to be a biochemical indicator of rejec-
tion in liver transplant recipients.
Increased levels of
guanase have also been detected in cancerous kidney
and breast cancer tissues.
In addition, patients with
multiple sclerosis exhibit signiﬁcantly elevated levels of
guanase activity in their cerebral spinal ﬂuids.
observations suggest that a potent guanase inhibitor is
necessary for exploring the biochemical mechanisms of
the above metabolic disorders as well to understand
the speciﬁc physiological role played by guanase, and
not to mention its potential therapeutic use in treating
these disorders. While many studies on guanase inhibi-
tion have been reported in the literature,
guanase inhibitor with a submicromolar or nanomolar
has yet to be discovered. We report here the design,
synthesis, and guanase inhibitory activity of two
analogues (1 and 2) of the natural product azepinomy-
cin, a moderate inhibitor of guanase, which is believed
to mimic the transition state of the enzyme-catalyzed
1, R = -CH
2, R = H
Guanase catalyzes the hydrolysis of guanine (3) to xan-
thine (4)(Scheme 1) via the tetrahedral intermediate
The recently solved crystal structure of guanase
from Bacillus subtilis suggests that the enzyme-catalyzed
reaction is assisted by an active site zinc metal ion
), which forms a tetrahedral complex with His-
53, Cys-83, and Cys-86 of the protein, as well as with
an isolated water molecule. Glutamate-55 serves as a
proton shuttle, abstracting a proton from the zinc-acti-
vated water to form the necessary hydroxide nucleo-
phile, while also enabling protonation at the N-3 site
of guanine, thus resulting in the formation of the inter-
mediate 5, as shown. Glu-55 also assists in protonation
of the NH
group of 5, facilitating the elimination of a
molecule of ammonia to form the ﬁnal product
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Guanase (guanine deaminase); Inhibitors; Azepinomycin
analogues; Synthesis; Biochemical studies.
Corresponding author. Tel.: +1 410 455 2520; fax: +1 410 455
1148; e-mail: firstname.lastname@example.org
Present address: G.E Healthcare, BioSciences, Protein Separations,
800 Centennial Ave, Piscataway, NJ 08855, USA.
Bioorganic & Medicinal Chemistry Letters 16 (2006) 5551–5554