Synthesis and characterization of novel isoform-selective IP6K1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.126628

Inositol hexakisphosphate kinases (IP6Ks) have been increasingly studied as therapeutically interesting enzymes. IP6K isoform specific knock-outs have been used to successfully explore inositol pyrophosphate physiology and related pathologies. A pan-IP6K inhibitor, N2-(m-trifluorobenzyl)-N6-(p-nitrobenzyl) purine (TNP), has been used to confirm phenotypes observed in genetic knock-out experiments; however, it suffers by having modest potency and poor solubility making it difficult to handle for in vitro applications in the absence of DMSO. Moreover, TNP's pan-IP6K inhibitory profile does not inform which IP6K isoform is responsible for which phenotypes. In this report we describe a series of purine-based isoform specific IP6K1 inhibitors. The lead compound was identified after multiple rounds of SAR and has been found to selectively inhibit IP6K1 over IP6K2 or IP6K3 using biochemical and biophysical approaches. It also boasts increased solubility and IP6K1 potency over TNP. These new compounds are useful tools for additional assay development and exploration of IP6K1 specific biology.

Full text of DOI:10.1016/j.bmcl.2019.126628

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of novel isoform-selective IP6K1 inhibitors
⁎
Michael M. Wormalda, Glen Ernstb, Huijun Weia,b, James C. Barrowa,b,
a Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 855 North Wolfe Street, Baltimore, MD 21205, USA
b Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Inositol hexakisphosphate kinases (IP6Ks) have been increasingly studied as therapeutically interesting enzymes.
IP6K isoform specific knock-outs have been used to successfully explore inositol pyrophosphate physiology and
related pathologies. A pan-IP6K inhibitor, N2-(m-trifluorobenzyl)-N6-(p-nitrobenzyl) purine (TNP), has been
used to confirm phenotypes observed in genetic knock-out experiments; however, it suffers by having modest
potency and poor solubility making it difficult to handle for in vitro applications in the absence of DMSO.
Moreover, TNP’s pan-IP6K inhibitory profile does not inform which IP6K isoform is responsible for which
phenotypes. In this report we describe a series of purine-based isoform specific IP6K1 inhibitors. The lead
compound was identified after multiple rounds of SAR and has been found to selectively inhibit IP6K1 over
IP6K2 or IP6K3 using biochemical and biophysical approaches. It also boasts increased solubility and IP6K1
potency over TNP. These new compounds are useful tools for additional assay development and exploration of
IP6K1 specific biology.
Inositol hexakisphosphate kinase
Inositol pyrophosphate
Selective enzyme inhibitors
Structure activity relationships
Kinases
Inositol pyrophosphates (1PP-IP5, 5PP-IP5, 1,5PP-IP4, Fig. 1) are
becoming recognized as important signaling molecules that may help
regulate cellular metabolism and energy levels.1–3 The intracellular
pool of 5PP-IP5 is formed from inositol hexakisphosphate (IP6, phytic
acid) by a family of three inositol hexakisphosphate kinases (IP6K1,
IP6K2, and IP6K3). These kinases utilize ATP to form a high-energy
pyrophosphate bond when they convert IP6 to 5PP-IP5. 5PP-IP5 is
thought to integrate with a number of cellular processes including
signal transduction, vesicular trafficking, apoptosis, and metabolism.4
All three IP6K isoforms catalyze the same reaction; however, global
genetic knock out experiments have uncovered unique phenotypes as-
sociated with each isoform. IP6K1 knock out mice show metabolic
changes and resistance to diet-induced obesity and fatty liver.5,6 IP6K2
knock out mice are sensitive to carcinogen-induced tumor formation,7
and IP6K3 knock out mice are resistant to age-induced weight gain and
have an increased lifespan.8 IP6K1 (neuronal migration),9 IP6K2 (Pur-
kinje cell morphology and cerebellar synapse formation),10 and IP6K3
(synapse formation)11 have also been implicated with neuronal func-
tions. Each isoform has differential cell-type expression,8 sub-cellular
localization,12 and protein-protein interactions13–16 that allow for the
different phenotypes observed. Due to the importance of protein-pro-
tein interactions in maintaining normal cellular processes it is unclear if
phenotypes observed in knock-out studies are fully driven by
decreasing 5PP-IP5 production or if the loss of protein-protein interac-
tions and scaffolding have a significant effect.
Nearly
a decade ago N2-(m-trifluorobenzyl)-N6-(p-nitrobenzyl)
purine (TNP, compound 1, Table 1) was discovered as a pan-IP6K in-
hibitor.17 Since then TNP has been used in a number of studies to ex-
amine the effects of 5PP-IP5 in various contexts (see 4 for a compre-
hensive review). In particular, Ghoshal et al. demonstrated that mice
treated with TNP were resistant to high-fat diet induced weight gain
while remaining sensitive to glucose and insulin.18 While TNP has
proved itself a useful tool, it can certainly be improved – particularly in
terms of potency, solubility, and isoform selectivity in order to better
understand inositol pyrophosphate biology. TNP is reported to have low
micromolar potency against the IP6K family,17,19,20 however, it is
sparingly soluble in aqueous buffers and is thus difficult to work with in
vitro without precipitation and difficult to formulate for in vivo animal
studies. Finally, TNP does not preferentially inhibit one IP6K isoform
over the others. Therefore, it is not possible to use TNP to study the
effects of one IP6K isoform in a context where the others are also
present.
Puhl-Rubio and colleagues have identified a number of IP6K2 in-
hibitors after screening a library of kinase-focused compounds.20 Gu
et al. have also recently reported a series of flavonoids as inositol
pyrophosphate kinase inhibitors.21 However, we adopted a medicinal
⁎ Corresponding author at: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 855 North Wolfe Street, Baltimore,
MD 21205, USA.
E-mail address: james.barrow@libd.org (J.C. Barrow).
https://doi.org/10.1016/j.bmcl.2019.126628
Received 24 July 2019; Received in revised form 15 August 2019; Accepted 17 August 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:MichaelM.Wormald,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.126628

M.M. Wormald, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 2
Inhibitory activities of the second iteration of purine analogs with variation at
the N2-position.
Compound
R
IP6K1 pIC50
7
4.10
0.12
8
4.05
0.04
9
4.95
5.08
0.19
0.10
10
Fig. 1. Biosynthesis of inositol pyrophosphates.
11
4.03
0.09
Table 1
Inhibitory activities of the first iteration of purine analogs with variation at the
N6-position.
12
< 3.70
Data represented as the mean
SEM.
performance as described previously.23 Briefly, the assay is conducted
with physiologically relevant concentrations of ATP (1 mM) that is
consumed as the kinase reaction progresses over time. After a time, the
reaction is stopped, the remaining ATP is enzymatically depleted, and
the ADP produced is converted back to ATP with a recycling reaction.
This regenerated ATP reacts with luciferin/luciferase to produce a
bioluminescent signal proportional to IP6K activity. In this assay, TNP
performance is inconsistent with previously published work pre-
sumably due to the immense hydrophobic nature of the inhibitor
causing it to precipitate out of the assay. Nitrobenzyl replacement with
benzyl (4) or phenethyl (5) moieties caused a dramatic decrease in
potency compared to the published value for TNP. A methoxyethyl (6)
substitution produced a soluble compound capable of completely in-
hibiting IP6K1, albeit very weakly with a pIC50 of 3.93.
Compound
R
IP6K1 pIC50
< 3.70*
1, TNP
4
< 3.70
< 3.70
5
6
3.93
0.10
Data represented as the mean
SEM.
* Solubility issues preclude proper IC50 determination.
The N6-methoxyethyl substitution was initially retained for solubi-
lity while we attempted to regain potency at the 2-position (Table 2).
Replacing the mCF3-benzyl (6) with phenethyl (7) resulted in a modest
increase in potency. Removing electron density (8) from the phenyl ring
did not increase potency while adding electron density (9) increased
potency from pIC50 4.10 to 4.95. Large electron-rich aromatic rings
(indole, 10) at the 2-position further increase potency to pIC50 5.08
while large hydrophobic aromatic rings are less active (napthyl, 11). A
smaller imidazole group (12) similarly abolishes potency.
chemistry approach to identify IP6K1 specific inhibitors by system-
atically varying the N2 and N6 positions off of the purine core.
A simple two-step SNAr reaction depicted in Scheme 1 from 6-
chloro-2-fluoro purine allows for rapid exploration of the 6- and 2-po-
sitions on the purine core. Initial efforts were spent exploring the N6
position to determine if the nitrobenzyl moiety present on TNP was
essential for compound potency (Table 1). The nitrobenzyl group is
undesirable because it is capable of forming toxic metabolites in vivo
and reduces compound solubility.22 In this study, all compounds were
tested in the Promega ADP-Glo Max assay optimized for IP6K
Using compound 10 as a baseline, we set out to explore how es-
sential the hydrogen bond donors in the molecule are by alkylating the
NH groups with a methyl cap. We found that alkylating any of the
exocyclic NHs abolished potency and 1-methyltryptamine dramatically
reduced it (Supplementary Data Table 1). Likewise, purine N7 and N9
to carbon substitutions abolished potency. These data suggest that there
is an intricate hydrogen bond network in the IP6K1 active site.
We next explored the chemical space surrounding the tryptamine
(Table 3). Generally, substitutions at the 5 position on the indole are
not tolerated (13–16). Substitutions at the 2 and 6 positions increase
potency (17, 18). Increasing the linker length from 2 to 3 carbons (19)
dramatically decreases potency and constraining free rotation of the
tryptamine abolishes potency (data not shown).
Scheme 1. Preparation of N2- and N6-substituted purine analogs 2–25.
Reagents and reaction conditions: (a) R1-NH2, DIPEA, nBuOH, 110 °C (b) R2-
NH2, DIPEA, DMSO, 110 °C.
2

M.M. Wormald, et al.
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Table 3
Table 5
Inhibitory activities of the third iteration of purine analogs with exploration
Compound 24 thermally stabilizes IP6K1 to a greater degree than other purine
around an N2-tryptamine.
analogs.
Compound
ΔTm at 11.1 µM (⁰C)
ΔTmMax (⁰C)
24
5.5
3.6
2.4
1.8
6.6
4.7
4.3
3.3
0.6
0.6
0.9
0.6
17
10
1, TNP
Data represented as the mean
SEM.
Compound
R1
R2
R3
n
IP6K1 pIC50
is additive with 5-methyl-2-pyridone yielding for the first time a sub-
micromolar IP6K1 inhibitor with a pIC50 of 6.13.
13
14
15
16
17
18
19
H
OMe
OH
Cl
H
1
1
1
1
1
1
2
3.86
4.50
4.57
3.91
5.56
5.71
4.16
0.08
0.14
0.20
0.08
0.09
0.14
0.01
H
H
H
H
Compounds 1 (TNP), 10, 17, and 24 were selected for confirmation
of binding to IP6K1 in an orthogonal biophysical thermal melt assay. In
this assay purified IP6K1 was incubated over a temperature gradient in
the presence of different concentrations of purine analogs. Compounds
that bind tightly to IP6K1 will increase the protein melting temperature
to a greater degree. Here, we observe good correlation between IC50
values obtained in the biochemical assay and an increase in melting
temperature. As expected, compound 24 was able to increase the
melting temperature to a greater degree than the other compounds at
11.1 µM (Table 5). Moreover, compound 24 has a greater ΔTmMax than
the other compounds tested, including TNP, indicating greater stabili-
zation. Taken together with the biochemical data, these data suggest
that compound 24 inhibits IP6K1 both enzymatically as well as physi-
cally interacts with the enzyme in a meaningful way.
H
Me
H
H
H
OMe
H
Me
H
H
H
H
Data represented as the mean
SEM.
Table 4
Inhibitory activities of the fourth iteration of TNP analogs with nitro replace-
ments at N6.
In order to examine IP6K isoform selectivity we tested compound 24
against purified IP6K2 and IP6K3 in the ADP-Glo Max assay (Table 6).
We were gratified to find that compound 24 was ∼25 and 50 fold more
selective for IP6K1 over IP6K2 and IP6K3, respectfully, after converting
observed IC50 values into Ki values with the Cheng-Prusoff equation.24
These results suggest that there are distinct enough differences in the
IP6K family to allow for subtype specific inhibitors to be developed.
Compound 24 was broadly profiled for off-target kinase inhibition
in a diversity panel at Eurofins. This screen includes a number of ki-
nases from different families and signaling pathways (see
Supplementary Data Table 2). Compound 24 was tested at 10 µM
against 58 kinases and found to potently inhibit 4 kinases, 3 of which
are part of the RAF/MAPK pathway in addition to casein kinase γ. The
remaining 54 kinases were not significantly inhibited by compound 24.
Importantly, this panel included a number of metabolically important
enzymes including GSK3β, AKT, mTOR, and PI3K. 5PP-IP5 is thought to
interact intimately with the PI3K/AKT/GSK3β pathway.5 As such, it is
important that tools that manipulate 5PP-IP5 concentration do not in-
hibit these enzymes or the results may be confounded and difficult to
interpret. These profiling results suggest that, surprisingly, the purine-
based compound 24 is a relatively selective IP6K1 inhibitor against the
rest of the kinome.
Compound
R1
R2
H
R3
H
IP6K1 pIC50
20
3.82
0.00
21
22
H
H
H
H
5.00
4.33
0.11
0.17
23
H
H
5.76
0.14
24
25
H
OMe
H
6.13
5.52
0.08
0.07
Me
In summary, given the increasing body of evidence that IP6Ks and
inositol pyrophosphates play an important role in cellular processes and
pathologies, we became interested in developing IP6K specific in-
hibitors to serve as pharmacological compliments to the published ge-
netic knock out models. These chemical tools will help deconvolute
inositol pyrophosphate signaling and inform what each IP6K isoform is
contributing to a particular phenotype. Our approach to systematically
Data represented as the mean
SEM.
After achieving low micromolar potency with compounds 17 and 18
by optimizing the N2 position off of the purine core, we returned to the
N6 position to see if nitro replacements (relative to TNP) exist which
are more potent than methoxyethylamine (Table 4). Benzoxadiazole
(20), 4-cyanobenzyl (22), 4- and 5-methyl-2-pyridone (21, 23) deri-
vatives were synthesized and tested. Compound 23 increased potency
from pIC50 5.08 to 5.76 over the parent compound 10. This 5-methyl-2-
pyridone substitution was then combined with the more potent N2-6-
methoxytryptamine or N2-2-methyltryptamine derivatives to yield
compounds 24 and 25. N2-2-Methyltryptamine (25) is equipotent to
unsubstituted tryptamine (10); however, N2-6-methoxytryptamine (24)
Table 6
Selectivity of compound 24 against IP6K isoforms.
Compound 24
Selectivity (Ki/Ki IP6K1)
IC50 (µM)
Ki (µM)
IP6K1
IP6K2
IP6K3
0.75
20
0.20
4.88
9.62
–
24.4
48.1
15
3

M.M. Wormald, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 7
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Nitrobenzyl
Solubility
Removed
Present
51 µg/mL (PBS pH
7.4)
Mostly insoluble in 2%
DMSO
Potency
0.20 µM Ki
0.24 µM Ki (published
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IP6K selectivity
Microsome stability (t1/2
24.4 fold over K2
48.1 fold over K3
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Acknowledgements
The authors declare no competing financial interests. This research
was supported by National Institutes of Health grant U01 MH112658
and the Lieber Institute for Brain Development. M.M.W. acknowledges
support from National Institute of General Medical Sciences
T32GM135083.
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://
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