Small-Molecule Inhibitors of the Tumor Suppressor Fhit

Article abstract of DOI:10.1002/cbic.201700226

The tumor suppressor Fhit and its substrate diadenosine triphosphate (Ap₃A) are important factors in cancer development and progression. Fhit has Ap₃A hydrolase activity and cleaves Ap₃A into adenosine monophosphate (AMP) and adenosine diphosphate (ADP); this is believed to terminate Fhit-mediated signaling. How the catalytic activity of Fhit is regulated and how the Fhit?Ap₃A complex might exert its growth-suppressive function remain to be discovered. Small-molecule inhibitors of the enzymatic activity of Fhit would provide valuable tools for the elucidation of its tumor-suppressive functions. Here we describe the development of a high-throughput screen for the identification of such small-molecule inhibitors of Fhit. Two clusters of inhibitors that decreased the activity of Fhit by at least 90 % were identified. Several derivatives were synthesized and exhibited in vitro IC₅₀ values in the nanomolar range.

Full text of DOI:10.1002/cbic.201700226

Accepted Article
Title: Small molecule inhibitors of the tumor suppressor Fhit
Authors: Andreas Marx, Sandra Lange, Stephan Hacker, Philipp
Schmid, and Martin Scheffner
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Small molecule inhibitors of the tumor suppressor Fhit
Sandra Lange,[a] Stephan M. Hacker,[a, b] Philipp Schmid,[a] Martin Scheffner,[c] and Andreas Marx*[a]
Abstract: The tumor suppressor Fhit and its substrate diadenosine
triphosphate (Ap3A) are important factors in cancer development and
progression. Fhit has Ap3A hydrolase activity and cleaves Ap3A into
adenosine monophosphate (AMP) and adenosine diphosphate
(ADP) which is believed to terminate Fhit-mediated signaling. How
the catalytic activity of Fhit is regulated and how the Fhit-Ap3A
complex exerts its growth-suppressive function remain to be
discovered. Small molecule inhibitors of the enzymatic activity of Fhit
would provide valuable tools for the elucidation of its tumor-
suppressive functions. Here, we describe the development of a high
throughput screen for the identification of such small molecule
inhibitors of Fhit. Two clusters of inhibitors were identified that
decreased activity of Fhit by at least 90%. Several derivatives were
synthesized and exhibited IC50 values in vitro in the nM range.
mediated signaling pathway are acting and regulated, however,
still remain to be discovered. Small molecule inhibitors of the
enzymatic activity of Fhit would provide tools that could assist in
the elucidation of its functions in this context. Only a few
molecules have been reported to date that are able to inhibit
Fhit’s enzymatic activity. Among these, Suramin[15] and non-
cleavable ApnA analogues[16–18] have been presented over the
years next to transition metal ions like Cu2+ and Zn2+.[19] The
latter, however, inhibit Fhit rather unspecific as they have many
other functions and targets within cells. Suramin also inhibits
several different classes of enzymes.[20, 21] Reported IC50 values
of ZnCl2 and Suramin towards Fhit are in the micromolar rather
than the nanomolar range.[22] Overall, small-molecule inhibitors
of Fhit are of considerable interest and not described so far.
To discover potent small-molecule inhibitors of Fhit, we set out
to develop
a high-throughput screening assay to identify
The fragile histidine triad protein (Fhit) is a tumor suppressor
and the respective gene is often mutated in lung,[1] kidney,[2]
esophagus,[3] breast[4] and many other types of human cancer.[5]
The gene is located on chromosome 3p14.2 which spans one of
the most fragile sites of the genome, FRA3B.[3] Upon complex
formation with two molecules of diadenosine triphosphate (Ap3A)
the tumor suppressive function of Fhit and hence the signaling
pathway towards apoptosis are activated.[5, 6] Besides, Fhit also
comprises Ap3Aase activity and cleaves Ap3A into adenosine
monophosphate (AMP) and adenosine diphosphate (ADP). This
is believed to terminate Fhit-induced signaling[7] since it was
previously reported that Fhit mutants without hydrolase activity
inhibitors against the enzymatic activity of Fhit from small
molecule libraries. Recently, we reported the synthesis and
application of a doubly labeled Ap3A probe 1 (Figure 1A),
capable of undergoing Förster Resonance Energy Transfer
(FRET), whose fluorescence characteristics change drastically
upon enzymatic cleavage by Fhit.[22] The cleavage of the FRET
pair Sulfo-Cy3/Sulfo-Cy5 can be readily followed by measuring
the fluorescence intensity at 590 nm, the maximum emission of
the donor Sulfo-Cy3. Therefore, this approach is ideally suited
as a high-throughput format for the identification of effectors of
Fhit’s enzymatic activity.
still show tumor suppressive function, whereas Ap3A binding
8]
mutants are inactive in this respect.[6,
Furthermore,
reinstatement of the FHIT gene in Fhit-negative tumor cells
resulted in significant inhibition of cell proliferation and increased
apoptosis in vitro[9–11] and in decreased tumorigenicity in vivo.[6]
Because the lifetime of the Fhit-Ap3A complex is determining the
level of tumor suppression it is suggested that stress conditions
in vivo result in reduced Ap3Aase activity.[12] Following this
hypothesis, it was discovered that Fhit might be regulated by Src
kinase-mediated phosphorylation.[12, 13] The importance of Fhit
and its enzymatic activity for cellular function has been further
demonstrated by the observation that a non-hydrolyzable ApnA
analogue triggers Fhit-dependent and caspase-directed
apoptosis.[14]
The exact mechanisms of how Fhit activity and the Fhit-Ap3A-
[a]
S. Lange, Dr. S. M. Hacker, P. Schmid, Prof. Dr. A. Marx
Department of Chemistry, Konstanz Research School Chemical
Biology, University of Konstanz, Universitätsstraße 10, 78457
Konstanz (Germany)
Figure 1. A) Structure of the previously reported doubly fluorescently labeled
Ap3A FRET probe 1. B) Schematic overview of the screening assay. Potential
inhibitors were added to Fhit and incubated for 30 min at 25 °C. Fluorescence
intensity with excitation at 535 nm and emission at 590 nm was measured
upon FRET-Ap3A (1) addition at t0 = 0 min and t1 = 30 min.
E-mail: andreas.marx@uni-konstanz.de
Dr. S. M. Hacker
Department of Chemical Physiology, The Skaggs Institute for
Chemical Biology, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, California 92037 (United States)
Prof. Dr. M. Scheffner
[b]
[c]
Department of Biology, University of Konstanz, Universitätsstraße
10, 78457 Konstanz (Germany)
Supporting information for this article is given via a link at the end of
the document.
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Figure 2. A) The best initial hit found in the screening assay (2). B) FRET assay to determine the inhibitory potential of the best compounds found in the
screening. Increasing concentrations of small molecules were incubated with Fhit before FRET-Ap3A was added. Time points were taken every 3 min over 1 h
and the fluorescence intensity of the donor emission was measured at 590 nm. The resulting plot of fluorescence intensity (FI) against time shows a decrease of
FI when inhibitor is added in increasing concentrations. (C) Dose response curve of 2 against Fhit.
Using the assay shown in Figure 1B, we screened 15.136
compounds for inhibition of Fhit’s enzymatic activity from
libraries from ChemBioNet, ChemDiv and Maybridge that were
available in the screening facility at the University of Konstanz.
For this purpose, Fhit (6 nM) and screening buffer were placed in
384-well plates and incubated with the respective compounds
(10 µM) at 25 °C for 30 min. FRET-probe 1 (500 nm) was added
and fluorescence intensity with emission at 590 nm (535 nm
excitation) was measured after t0 = 0 min and t1 = 30 min. The
difference in fluorescence intensity was then calculated relative
to a DMSO control in the absence of any small molecule. In total,
we found 45 compounds that inhibited the enzymatic activity of
Fhit by at least 90% (Figure S1, S2). The statistical parameters
of the high throughput screening were excellent with an average
z-score over all plates of 0.860 ± 0.032 and a SSMD value of
−27.244 ± 6.572.
The synthesis of both clusters was carried out in a one-pot
manner with microwave-assisted heating (see Schemes 1C and
3D). In case of cluster I, the commercially available starting
materials dimedone, 3-amino-5-methyl-pyrazole and the
respective aldehydes were mixed in a 1:1:1 ratio and were
heated in aqueous medium with 1.2 eq. of NEt3 to 170 °C in a
microwave for 15 min. Precipitation with EtOH/H2O (1:1) and
subsequent filtration yielded the products. The synthesis of
compounds 2-10 was previously reported, except for compound
4 that was synthesized in analogy to the literature.[23, 24]
The best initial hit of the screening was compound 2 (Figure 2A)
that reduced Fhit activity to less than 2% in comparison to the
DMSO control. To validate this result, compound
2 was
synthesized according to a previously reported microwave-
assisted procedure from dimedone, 3-amino-5-methyl-pyrazole
and 4-chlorobenzaldehyde.[23] 2 was then employed at different
concentrations in an in vitro dose response FRET assay, similar
to the screening procedure but with time points taken every
3 min over 60 min (see Figure 2B). Corresponding to linear time
response of the cleavage of 1, a concentration of 1 nM was
applied in the dose response assay (see Figure S3). In this way,
the IC50 value of 2 was determined as 199 ± 10 nM (Figure 2C).
Overall we identified one main structural motive, dimethyl-
hexahydro-quinolinone (Scheme 1), in the screening that was
found with several derivatives among the best 45 hits. Besides
the best inhibitor of the screening (2) with a methyl-pyrazolo
group bridging positions R1 and R2, we discovered inhibitors that
have an indeno residue substituted at R1 and R2 (Scheme 1B).
The screening results further indicated that bulky groups at R3
significantly compromise activity indicating that this part of the
molecule should be tightly bound to the protein. We therefore
chose to improve the inhibitory properties of these initial
scaffolds by synthesizing derivatives differing in position R3.
Hence, we set out to synthesize derivatives of the methyl-
pyrazolo-motive (cluster I, 2–10) and the indeno motive (cluster
II, 11–19) (Scheme 1B). Lead compound 2 was the only one
containing a methyl-pyrazolo group at R1 and R2 in the
screening. In case of the cluster II compounds, toluyl (11), 4-
ethylphenyl (12) and 2-thiophenyl-substituted (19) derivatives
were already identified in the screening and inhibited Fhit activity
by more than 95% at 10 µM.
Scheme 1. A) Structural motive found recurring in the identified Fhit inhibitors.
B) Structure of the two clusters of Fhit inhibitors found in the screen. C), D)
Synthesis scheme for cluster I (C) and cluster II (D) using a one-pot protocol
with microwave irradiation. E) Synthesized derivatives and yield of synthesis.
Derivatives of cluster II were synthesized from 1,3-indanedione,
5,5-dimethyl-3-amino-cyclohex-2-enone and the respective
aldehydes in a 1:1:1 ratio. p-TsOH was added and the mixture
was reacted in a microwave at 150 °C for 10 min in H2O. The
reaction was quenched with 10% NaOH and the compounds
were purified by silica gel column chromatography and reversed-
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phase medium pressure column chromatography. The synthesis
of compounds 14 and 15 was described previously.[25, 26] To the
best of our knowledge, the other cluster II compounds have not
been synthesized before. In total, 18 compounds were
synthesized. The respective yields are depicted in Scheme 1E.
Next, we tested the inhibitory potential of the synthesized
compounds using the in vitro FRET assay described above.
Four compounds, 12, 13, 14 and 15, were found to inhibit Fhit
more potently than lead compound 2. Interestingly, all of these
compounds derive from cluster II and all the derivatives of this
structural motive are better inhibitors than the respective
derivatives of cluster I with the same R3 group.
amounts of substrate 1 were applied to pre-incubated mixtures
of inhibitor 15 and Fhit. A higher turnover was achieved with
rising concentrations of 1 (Figure S7B). This suggests that 15
acts as a competitive inhibitor and that the substrate is able to
compete with the binding of 15 and displace it.
Next, all compounds were tested for their cytotoxic properties in
HEK 293T and lung cancer H1299 cell lines, that are often used
in Fhit research.[14, 27–29] Therefore a colorimetric assay was
employed which determines the metabolic activity of cells, and
thus cell viability, as a function of the reduction of the tetrazolium
dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide
(MTT) into insoluble formazan by NAD(P)H-dependent
oxidoreductases.[30] The results for the halogenated cluster II
compounds 13, 14 and 15 are displayed in Figure 3A and 3B
(the MTT results for all compounds are displayed in Figure S8).
All three compounds show similar cytotoxicity. Interestingly, HEK
293T cells are more sensitive towards the Fhit inhibitors than
H1299 cells. The viability of the former is already affected in the
10 µM range, while the latter still exhibit more than 50% viability
even when 100 µM of compound are applied. Overall, the
compounds show a moderate to no cytotoxicity, depending on
the cell line and are therefore ideally suited for further in vivo
studies.
The halogenated and alkylated structures have
a higher
inhibitory potential than the pyridinyl- and thiophenyl-substituted
derivatives. The Br-substituted compound 15 is the most potent
inhibitor with an IC50 value of 49 ± 7 nM, followed by Cl (14,
51 ± 32 nM), Et (12, 125 ± 44 nM) and F (13, 154 ± 55 nM). The
effect of the halogenated and alkylated compounds might be
due to the altered electronic properties of the aromatic systems.
The IC50 values of all synthesized compounds are shown in
Table 1 (the respective dose-response curves are displayed in
Figure S4).
Since the synthesized compounds have largely conjugated π-
systems, an additional negative control was performed in which
the influence of the potential inhibitors on the FRET assay was
studied when no Fhit was applied. We found that all inhibitors
showed no effect on the fluorescent read-out (see Figure S5).
Furthermore, as depicted by the results of the structure-activity
relationship, slight changes of compound structure that do not
alter the π-system may alter the inhibitory potential significantly
(e.g., see IC50 values of e.g. compound 2 and 9). Furthermore,
several other compounds with largely conjugated π-systems
were screened and did not show any inhibition of Fhit (see
Figure S6). This shows that the depicted system is highly
sensitive to slight structural changes of the small molecule
excluding that just any extended aromatic system is an inhibitor.
Figure 3. MTT assay of halogenated compounds of cluster II derivatives (13,
14, 15) in HEK 293T (A) and H1299 (B) cells. Lower concentrations than the
ones displayed did not alter the relative cell viability.
Table 1. IC50 values of all synthesized derivatives.
Compounds
Cluster I
Compound
Cluster II
IC50 [nM]
IC50 [nM]
In conclusion, we developed a high-throughput assay using
FRET-Ap3A probe 1 to identify a main structural motive as
potential small molecule inhibitor of the activity of the tumor
suppressor Fhit. Based on this, we synthesized 18 compounds,
the synthesis of several thereof previously unknown, and
identified five of these, 2, 12, 13, 14 and 15, that inhibited Fhit
with IC50 values of < 200 nM. The best inhibitor (15) had an IC50
value of 49 ± 7 nM and was bound by Fhit in a reversible manner.
The compounds were further tested for their cytotoxic properties
in HEK 293T and H1299 cells, two commonly studied cell lines
in Fhit research. The inhibitors showed moderate to no
cytotoxicity, whereby HEK 293T cells seem to be more affected
than H1299. Interestingly, HEK 293T cells constitutively express
Fhit,[27] while H1299 are Fhit negative.[31]
3
4
589 ± 160
388 ± 26
11
12
13
14
15
16
17
18
19
275 ± 1
125 ± 44
154 ± 55
51 ± 32
5
762 ± 105
2
199 ± 10
6
379 ± 54
49 ± 7
7
6,682 ± 1,948
2,225 ± 721
16,840 ± 3,160
925 ± 198
4,245 ± 459
1,654 ± 241
5,204 ± 261
499 ± 187
8
9
10
Compound 15 was further tested on its effect on Fhit when the
natural substrate Ap3A was used instead of probe 1. As
analyzed by HPLC, Fhit was competitively inhibited by 15 when
the Fhit:inhibitor ratio was 1:1000 and higher, a value that
corresponds well to the conducted FRET assay (Figure S7A).
To further examine details about the enzymatic inhibition of Fhit,
another FRET-based assay was conducted in which increasing
Overall we identified several highly effective Fhit inhibitors that
will be useful to study the enzymatic activity of Fhit in more detail
under various conditions. However, in human cells seven
members of the histidine triad (HIT) protein-family are described,
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Funding by the DFG and support by the Konstanz Research
School Chemical Biology is gratefully acknowledged. We thank
S. Müller, Screening Center of the University of Konstanz, for
technical assistance. S. M. H. also acknowledges the
Studienstiftung des deutschen Volkes and the Zukunftskolleg of
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Keywords: Fhit • Inhibitors • Ap3A • FRET • Nucleotides
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Small molecule inhibitors have been
identified and developed by high-
throughput screening and organic
synthesis that suppress enzymatic
hydrolysis of diadenosine triphosphate
(Ap3A) into adenosine monophosphate
(AMP) and adenosine diphosphate
(ADP) by the human tumor suppressor
Fhit.
Sandra Lange, Stephan M. Hacker,
Philipp Schmid, Martin Scheffner and
Andreas Marx*
Page No. – Page No.
Small molecule inhibitors of the
tumor suppressor Fhit
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