Synthesis and evaluation of potential inhibitors of human and Escherichia coli histidine triad nucleotide binding proteins

Article abstract of DOI:10.1016/j.bmcl.2011.10.082

Based on recent substrate specificity studies, a series of ribonucleotide based esters and carbamates were synthesized and screened as inhibitors of the phosphoramidases and acyl-AMP hydrolases, Escherichia coli Histidine Triad Nucleotide Binding Protein (ecHinT) and human Histidine Triad Nucleotide Binding Protein 1 (hHint1). Using our established phosphoramidase assay, K_i values were determined. All compounds exhibited non-competitive inhibition profiles. The carbamate based inhibitors were shown to successfully suppress the Hint1-associated phenotype in E. coli, suggesting that they are permeable intracellular inhibitors of ecHinT.

Full text of DOI:10.1016/j.bmcl.2011.10.082

Bioorganic & Medicinal Chemistry Letters 22 (2012) 558–560
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of potential inhibitors of human and Escherichia coli
histidine triad nucleotide binding proteins
⇑
Sanaa K. Bardaweel, Brahma Ghosh, Carston R. Wagner
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on recent substrate specificity studies, a series of ribonucleotide based esters and carbamates were
synthesized and screened as inhibitors of the phosphoramidases and acyl-AMP hydrolases, Escherichia
coli Histidine Triad Nucleotide Binding Protein (ecHinT) and human Histidine Triad Nucleotide Binding
Protein 1 (hHint1). Using our established phosphoramidase assay, Ki values were determined. All com-
pounds exhibited non-competitive inhibition profiles. The carbamate based inhibitors were shown to
successfully suppress the Hint1-associated phenotype in E. coli, suggesting that they are permeable intra-
cellular inhibitors of ecHinT.
Received 18 August 2011
Revised 22 October 2011
Accepted 25 October 2011
Available online 31 October 2011
Keywords:
Histidine triad binding protein
Hint1
Ó 2011 Elsevier Ltd. All rights reserved.
Inhibitor
Phosphoramidase
Histidine triad nucleotide binding protein (Hint) is named after
the highly conserved motif related to the sequence His-X-His-X-
His-XX in the active site, where X is a hydrophobic amino acid.
Hint is considered to be the ancestor of the histidine triad protein
superfamily and can be found in both prokaryotes and eukaryotes.1
While eukaryotes generally express multiple forms of Hint (Hint1,
Hint2 and Hint3), prokaryote genomes, including gram negative
and gram-positive bacteria, typically encode one Hint gene.2
Recently, evidence has begun to emerge that Hint1 may function
as a tumor suppressor through multiple molecular mechanisms
involving triggering apoptosis,3 regulating gene transcription and
cell cycle modulation.4,5 Hint1 deleted mice studies have revealed
that an increased susceptibility to the carcinogenic DMBA induced
ovarian and mammary tumors, as well as an increased occurrence
of spontaneous tumors.6 Eukaryotic Hint1 has been shown to asso-
ciate with, and suppress the b-catenin Wnt signaling pathway tran-
scriptional activity by direct interactions with Reptin and Pontin.7
Hint1 has also been associated with transcription factors such as,
TFIIH,8 MITF,4,9 and USF2.5 In addition, the interactions between
MITF and USF2 appear to be mediated by LysRS.4,5,10 Recently, we
have demonstrated that lysyl-adenylate (lysyl-AMP) generated by
lysyl-tRNA synthetases (LysRS) are also substrates for Hints.11
Despite such a wide variety of possible biological functions in
eukaryotes, the cellular function and rationale for the evolutionary
conservation of Hint in bacteria has remained a mystery. Moreover,
none of the previously proposed functions has been successfully
linked to the enzymatic activity of Hints. Recently, we have shown
that the expression of the catalytically active Hint is essential for
the activity of the enzyme D-amino acid dehydrogenase (DadA)
in Escherichia coli DadA.12 Consequently, we have chosen to syn-
thesize novel Hint1 inhibitors, which can be used as chemical
probes to study the connection between the newly discovered
Hint1-associated phenotype in E. coli and its enzymatic activity.
Hint1 has been found to be a nucleoside phosphoramidase and
acyl-AMP hydrolase.1 Probing Hint1 base recognition site, the sub-
strate specificity for both purine and pyrimidine phosphorami-
dates was determined.13 Structure–activity relationship studies
revealed that Hint1 prefers purine over pyrimidine phosphorami-
dates. In addition, based on kinetic and crystallographic studies,
the 2- and 3-hydroxyl groups of the ribose ring were shown to
be required for maximal phosphoramidase efficiency.13
Based on the previous findings and knowledge of the general out-
line of substrate specificity requirements, compounds 1 and 2 were de-
signed and synthesized following Scheme S1 (Supplementary data).
Both compounds were titrated into a phosphoramidase assay using
the fluorogenic substrate tryptamine 50-adenosine phosphoramidate
(TpAd) (Fig. 1a) as described previously.13 In short, in the absence of
inhibitors, turnover rates were measured by following the hydrolysis
of TpAd by ecHinT and hHint1 proteins. Excitation wavelength was
set at 280 nm, and fluorescence emission was measured at 360 nm.
The rate of hydrolysis of the substrate was determined by measuring
the increase of fluorescence intensity upon the addition of the enzyme
over two minutes. The Michaelis–Menten constants, kcat (sꢀ1) and Km
⇑
Corresponding author. Tel.: +1 612 625 2614.
E-mail address: wagne003@umn.edu (C.R. Wagner).
(lM), were determined by nonlinear regression analysis of the initial
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.082

S. K. Bardaweel et al. / Bioorg. Med. Chem. Lett. 22 (2012) 558–560
559
NH2
(a)
(b)
N
N
N
O
N
H
N P O
O-
O
HN
OH OH
TpAd
H
N
O
n
R1
O
O
R3 R2
1 n= 1
2 n= 2
Figure 2. ecHinT inhibition resulted in impaired phenotype. ⁄Measurements were
carried out in duplicates and the standard deviation was 2%. Results for compound
6 taken from Ref. 12.
H
N
(c)
O
O
R1
N
H
O
the guanosine carbamate analogue ranged between 42
ecHinT and 34 M for hHint1. All compounds exhibited non-com-
petitive inhibition profiles.
lM for
l
R3
R2
Figure 1. Structures of (a) tryptamine 50-adenosine phosphoramidate and its (b)
ester and (c) carbamate analogues.
Recently, we have been able to connect ecHinT to an established
phenotype in bacteria. Using active site mutants, we demonstrated
that the catalytically active ecHinT is required for E. coli to utilize D-
alanine as a carbon source. To assess the ability of our compounds
to inhibit ecHinT in vivo, we tested the ability of E. coli BW25113
strain to grow on D-alanine in the presence of 100 lM of either
compound 3, 5 or 6. Cultures were grown in M9 media, supple-
mented with either 22 mM D,L-alanine or 10 mM glucose, at 37 °C
for 40 h before OD600 was measured. Consistent with our previous
finding, that the catalytic activity of ecHinT is required for E. coli to
velocity versus concentration using JMP IN 7 software. In the presence
of inhibitors, the proteins were pre-incubated with the tested inhibitor
for 30 s at 25 °C before the addition of the substrate. The reaction was
followed under the same condition as of in the absence of inhibitor.
While Hints did not show any appreciable esterase activity
against compound 1 and 2, kinetic analysis revealed a significant
and dose-dependent decrease in the apparent Vmax of Hint1 in the
presence of either compound 1 or 2 (Supplementary Fig. S1). In con-
trast, the Km value for TpAd was unaltered by the presence of the
inhibitor. Thus, the mechanism of inhibition was clearly of a non-
competitive type, suggesting that the inhibitor and TpAd substrate
do not share a common binding site on the enzyme. Ki values were
estimated from the Dixon plot and are summarized in Table 1.
A second series of carbamate analogues was prepared following
Schemes 2S and S3 (Supplementary data). As shown in Table 1,
compound 3, a thymidine carbamate analogue in which 3-hydro-
xyl group of the ribose ring was replaced with a primary amine
grow on
impaired growth when
D
-alanine, inhibition of ecHinT in BW25113 resulted in its
-alanine is the sole carbon source (Fig. 2).
D
In addition to the cell permeability properties of compounds 3, 5
and 6, no toxicity was observed with either inhibitor when glucose
is the sole carbon source in the growth medium.12
In conclusion, we have designed and synthesized the first gen-
eration of cell-permeable Hint inhibitors. Previously, we have
shown that
DhinT E. coli, a mutant strain in which hinT was deleted,
exhibited impaired growth when
D-lanine is the sole carbon
source.12 Using the developed inhibitors as chemical probes, this
work has demonstrated that inhibition of ecHinT resulted in simi-
group, inhibited Hint with a Ki value of 122
139 M for hHint1. Replacement of the amine group with an azido
moiety at the 30 position, compound 4, resulted in an inhibitor with
a Ki value of 565 M for ecHinT and 715 M for hHint1. The five-
lM for ecHinT and
lar impaired growth observed for
DhinT in the presence of D-ala-
l
nine as a sole carbon source. Guanosine carbamate, 6,12 one of
the best inhibitors reported in this study, may serve as structural
template for the design and development of more potent inhibitors
with enhanced inhibition constants. Ongoing crystallographic
studies of the inhibitor bound form of Hint should provide a struc-
tural foundation for understanding the mechanism of inhibition
and for the design of selective Hint inhibitors.
l
l
fold enhancement in equilibrium binding conferred by the amine
group at the 30 position is most likely explained by the formation
of favorable hydrogen bonding interactions between the inhibitor
and its binding site in the protein structure. Adenosine carbamate,
compound 5, and guanosine carbamate,12 compound 6, were the
most potent inhibitors prepared in this study. As shown in Table
1, Ki values for the adenosine carbamate analogue ranged between
Acknowledgment
73 lM for ecHinT and 103 lM for hHint1 while the Ki values for
This work was supported by a grant from the University of Min-
nesota AHC.
Table 1
Inhibition constants determined in HEPEs buffer (pH 7.2) at 25 °C
Supplementary data
Compds
R1
R2
R3
EcHinT Ki (
l
M)a
hHint1 Ki (l
M)a
1
2
3
4
5
6
Adenine
Adenine
Thymine
Thymine
Adenine
Guanine
OH
OH
H
H
OH
OH
OH
OH
NH2
N3
OH
OH
375 ( 13)
393 ( 17)
122 ( 10)
565 ( 23)
73 ( 4)
400 ( 27)
457 ( 32)
139 ( 17)
715 ( 35)
103 ( 18)
34 ( 4)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.082.
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a
Values are means of three experiments, standard deviation is given in
parentheses.
b
Value was taken from Ref. 12.

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