DOI: 10.1002/chem.201600883
Communication
&
Medicinal Chemistry
An Immucillin-Based Transition-State-Analogous Inhibitor of
tRNA–Guanine Transglycosylase (TGT)
[
a]
[a]
[b]
[a]
Christoph Hohn, Adrian Härtsch, Frederik Rainer Ehrmann, Toni Pfaffeneder,
[a]
[a]
[b]
[a]
Nils Trapp, Oliver Dumele, Gerhard Klebe,* and FranÅois Diederich*
ence provides a basis of selectivity for the planned bacterial in-
hibitors.
Abstract: Shigellosis is one of the most severe diarrheal
diseases worldwide without any efficient treatment so far.
The enzyme tRNA–guanine transglycosylase (TGT) has
been identified as a promising target for small-molecule
drug design. Herein, we report a transition-state analogue,
a small, immucillin-derived inhibitor, as a new lead struc-
ture with a novel mode of action. The complex inhibitor
synthesis was accomplished in 18 steps with an overall
yield of 3%. A co-crystal structure of the inhibitor bound
to Z. mobilis TGT confirmed the predicted conformation of
the immucillin derivative in the enzyme active site.
The catalyzed reaction of bacterial TGT proceeds through
[6]
a ping-pong mechanism beginning with the excision of the
nucleobase in a substitution reaction with Asp280 via transi-
tion-state TS1 (Figure 1a), which is similar to the transition
state of the depurination reaction (Figure 1b and Figure S1 in
the Supporting Information) catalyzed by human purine nu-
cleoside phosphorylase (PNP, EC 2.4.2.1). The enzyme catalyzes
the phosphorylation of purine nucleosides and follows a S 1-
N
type mechanism.
Immucillins are known as potent transition-state analogues
(
TSA) against human PNP and derivatives are currently in clini-
cal trials for the treatment against B-cell acute lymphoblastic
[
7]
The diarrheal disease shigellosis caused by Shigella bacteria is
leukemia and autoimmune disorders. The designed immucil-
lin inhibitors mimic the ribooxacarbenium ion-like cationic
transition state, while the modified nucleobase cannot be
cleaved due to the unnatural, noncleavable glycosidic CÀC
[1]
a severe, life-threatening burden worldwide. In 2013, the
annual mortality caused by shigellosis was estimated to be
7
3,900 deaths, whereas 45% of all Shigella-related deaths are
[1]
[8]
observed for children younger than five years old. Emerging
antimicrobial resistance against common therapies indicates
the urgent need for different treatments with new modes of
bond.
Both PNP and TGT belong to the same enzyme class (trans-
ferase) with related reaction mechanisms but very different
natural substrates. This fact encouraged us to synthesize the
immucillin derivative 1 (Figure 1c) to inhibit TGT in a transi-
tion-state-analogous way and to introduce this class of inhibi-
tors as leads towards a potential treatment of shigellosis. The
designed and ultimately prepared transition-state-analogous
inhibitor features a protonated azasugar moiety (Section S4,
Supporting Information), mimicking the ribooxacarbenium ion-
[2]
action against shigellosis. The enzyme tRNA–guanine trans-
glycosylase (TGT, EC 2.4.2.29) was identified in mutational stud-
[
3]
ies as a possible target for small-molecule drug design. In
prokaryotes and eukaryotes, the enzyme post-transcriptionally
modifies tRNA by exchanging guanine with a hypermodified
nucleobase at position 34 (wobble position) in the anticodon
of certain tRNAs. Bacteria use TGT to insert preQ into tRNA,
1
[
9]
which is further transformed to queuine in a downstream pro-
like cationic character of TS1. The second protonation of
1 occurs on the 2-aminopyrimidinone ring at N(1), similar to
what was observed for lin-benzoguanines in complex with
[
4]
cess. In contrast, eukaryotes lack the ability to synthesize
queuine de novo, but rely on the uptake from nutrition and
[5]
[10,11]
direct incorporation into tRNA catalyzed by TGT. This differ-
TGT.
An overlay of the available crystal structure of a preQ -modi-
1
[
a] Dr. C. Hohn, A. Härtsch, Dr. T. Pfaffeneder, Dr. N. Trapp, Dr. O. Dumele,
Prof. Dr. F. Diederich
Laboratorium für Organische Chemie, ETH Zurich
Vladimir-Prelog-Weg 3, HCI, 8093 Zurich (Switzerland)
E-mail: diederich@org.chem.ethz.ch
fied tRNA·TGT complex (Zymomonas (Z.) mobilis, PDB code
[
12]
1
Q2S; 3.2 )
and the immucillin derivative 1, which was
energy-minimized within the active site using the program
[
13]
MOLOC,
clearly indicates the desired binding mode
[
b] F. R. Ehrmann, Prof. Dr. G. Klebe
(
Figure 2). We assume that the void between N(5) of the pyr-
Institut für Pharmazeutische Chemie, Philipps Universität Marburg
Marbacher Weg 6, 35032 Marburg (Germany)
E-mail: klebe@mailer.uni-marburg.de
role moiety and the backbone Leu231–Ala232 region of the
enzyme is filled by water molecules.
Previous inhibitors based on 2-amino-lin-benzoguanine (6-
amino-2-(methylamino)imidazo[4,5g]quinazolin-8(7H)-one) as
1
13
characterization of all new compounds and their H and C NMR spectra;
conformational analysis; small-molecule crystal structure data for com-
pounds 3, 8b, and 1; co-crystal structure of 1 bound to Z. mobilis TGT; and
biological experiments. The coordinate file of the complex with 1 was de-
posited in the PDB with the following access code: 5EGR.
a central ligand scaffold bind with inhibition constants in the
[14]
low nanomolar range to TGT (K =2–58 nm), but show insuf-
i
[
10]
ficient physicochemical properties. This drawback of the lin-
benzoguanine-based inhibitors could possibly be avoided by
Chem. Eur. J. 2016, 22, 6750 – 6754
6750
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