Novel substrates for kinases involved in the biosynthesis of inositol pyrophosphates and their enhancement of atpase activity of a kinase

Article abstract of DOI:10.3390/molecules26123601

IP6K and PPIP5K are two kinases involved in the synthesis of inositol pyrophosphates. Synthetic analogs or mimics are necessary to understand the substrate specificity of these enzymes and to find molecules that can alter inositol pyrophosphate synthesis. In this context, we synthesized four scyllo‐inositol polyphosphates—scyllo‐IP5, scyllo‐IP6, scyllo‐IP7 and Bz‐scyllo‐IP5—from myo-inositol and studied their activity as substrates for mouse IP6K1 and the catalytic domain of VIP1, the budding yeast variant of PPIP5K. We incubated these scyllo‐inositol polyphosphates with these kinases and ATP as the phosphate donor. We tracked enzyme activity by measuring the amount of radiolabeled scyllo‐inositol pyrophosphate product formed and the amount of ATP consumed. All scyllo‐inositol polyphosphates are substrates for both the kinases but they are weaker than the corresponding myo‐inositol phosphate. Our study reveals the importance of axial-hydroxyl/phosphate for IP6K1 substrate recognition. We found that all these derivatives enhance the ATPase activity of VIP1. We found very weak ligand‐induced ATPase activity for IP6K1. Benzoyl‐scyllo‐IP5 was the most potent ligand to induce IP6K1 ATPase activity despite being a weak substrate. This compound could have potential as a competitive inhibitor.

Full text of DOI:10.3390/molecules26123601

molecules
Article
Novel Substrates for Kinases Involved in the Biosynthesis of
Inositol Pyrophosphates and Their Enhancement of ATPase
Activity of a Kinase
Raja Mohanrao 1 , Ruth Manorama 2, Shubhra Ganguli 2,3, Mithun C. Madhusudhanan 1 , Rashna Bhandari 2,
*
and Kana M. Sureshan 1,
*
1
2
School of Chemistry, Indian Institute of Science Education and Research Thiruvananthapuram,
Kerala 695551, India; rajamohanrao1@gmail.com (R.M.); mithun716@iisertvm.ac.in (M.C.M.)
Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500039, India;
manorama@cdfd.org.in (R.M.); shubhraganguli@cdfd.org.in (S.G.)
3
Manipal Academy of Higher Education, Manipal 576104, India
*
Correspondence: rashna@cdfd.org.in (R.B.); kms@iisertvm.ac.in (K.M.S.)
Abstract: IP6K and PPIP5K are two kinases involved in the synthesis of inositol pyrophosphates.
Synthetic analogs or mimics are necessary to understand the substrate specificity of these enzymes
and to find molecules that can alter inositol pyrophosphate synthesis. In this context, we synthe-
sized four scyllo-inositol polyphosphates—scyllo-IP5, scyllo-IP6, scyllo-IP7 and Bz-scyllo-IP5—from
myo-inositol and studied their activity as substrates for mouse IP6K1 and the catalytic domain of
VIP1, the budding yeast variant of PPIP5K. We incubated these scyllo-inositol polyphosphates with
these kinases and ATP as the phosphate donor. We tracked enzyme activity by measuring the amount
of radiolabeled scyllo-inositol pyrophosphate product formed and the amount of ATP consumed. All
scyllo-inositol polyphosphates are substrates for both the kinases but they are weaker than the corre-
sponding myo-inositol phosphate. Our study reveals the importance of axial-hydroxyl/phosphate
for IP6K1 substrate recognition. We found that all these derivatives enhance the ATPase activity of
VIP1. We found very weak ligand-induced ATPase activity for IP6K1. Benzoyl-scyllo-IP5 was the
most potent ligand to induce IP6K1 ATPase activity despite being a weak substrate. This compound
could have potential as a competitive inhibitor.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Mohanrao, R.;
Manorama, R.; Ganguli, S.;
Madhusudhanan, M.C.; Bhandari, R.;
Sureshan, K.M. Novel Substrates for
Kinases Involved in the Biosynthesis
of Inositol Pyrophosphates and Their
Enhancement of ATPase Activity of a
Kinase. Molecules 2021, 26, 3601.
https://doi.org/10.3390/
molecules26123601
Keywords: kinase; inositol pyrophosphate; IP6K1; VIP1 KD; ATPase
Academic Editors: Brullo Chiara,
Marie Migaud and Gerd Wagner
Received: 6 May 2021
Accepted: 7 June 2021
Published: 11 June 2021
1. Introduction
Phosphoinositols play a variety of roles in cellular signaling [
polyphosphates (PPIPns) are a class of highly phosphorylated myo-inositol derivatives that
have one or two pyrophosphate moieties and occur only in eukaryotes [ ]. PPIPns present
in mammals include 5-PP-IP4, 5-PP-IP5, 1-PP-IP5 and 1,5-[PP]2-IP4, and their cellular levels
are regulated by (i) IP6Ks, kinases which pyrophosphorylate the 5-position of myo-inositol,
(ii) PPIP5Ks, kinases which pyrophosphorylate the 1-position and (iii) DIPPs, phosphatases
which dephosphorylate the pyrophosphate to phosphate (Figure 1A) [6–8]. PPIPns play
vital roles in apoptosis, cellular signaling, vesicular trafficking, metabolic homeostasis,
cytoskeletal dynamics, exocytosis, insulin signaling, DNA damage repair, telomere main-
tenance, stress response, etc. [
are yet to be ascertained [23 25
the structure-activity relationships (SAR) and mechanism of their actions, it is necessary
to synthesize these molecules and their analogs [ 22 28 30]. Recently, Jessen et al. re-
ported elegant approaches to synthesize various PPIPn analogs [31 33]. These analogs
served as tools to unravel various biological roles played by inositol pyrophosphates [34].
1,2]. Diphospho-inositol
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3–5
Copyright:
© 2021 by the authors.
4,
9–
24]. Clear mechanisms of their wide-ranging functions
–27]. For biochemical and genetic exploration to unravel
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
,
2, , –
–
Potter et al. [35] and Fiedler et al. [29
,36
,
37] independently synthesized PPIPn analogs
Molecules 2021, 26, 3601. https://doi.org/10.3390/molecules26123601
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3601
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having non-hydrolyzable pyrophosphate surrogates and established, through biochemical
assays, that this modification does not prevent target recognition and action of metabo-
lizing enzymes. Both of these studies have also established that the recognizing enzymes
can tolerate 2-O-substitution with a hydrophobic group. An extension of this study not
only validated the previously reported [38] ligand induced ATPase activity of one of the
kinases, PPIP5K2, but also unraveled the mechanism for the same [39]. Here we report the
syntheses and biochemical studies of four novel substrate analogs of kinases involved in
PPIPn biosynthesis, and ATPase activities of both these kinases.
Figure 1. (A) Biosynthesis of inositol pyrophosphates in mammalian cells. (B) Proposed ligands to probe kinase activity.
As mentioned earlier, IP6Ks and PPIP5Ks are involved in the biosynthesis of PPIPns.
IP5, IP6 and 1-PP-IP5 are the natural substrates for IP6Ks and get phosphorylated to
5-PP-IP4, 5-PP-IP5 and 1,5-[PP]2-IP4, respectively (Figure 1A). Similarly, IP6 and 5-PP-IP5
are the natural substrates for PPIP5Ks and get phosphorylated to 1-PP-IP5 and 1,5-[PP]2-IP4,
respectively (Figure 1A) [40
function and substrate-specificity of these enzymes using various analogs as synthetic
tools [30 35 36 39 44]. An important strategy to make analogs of IPns is to alter the inositol
–43]. There is great interest in understanding the mechanism,
,
,
,
,
core. We chose scyllo-inositol, the C2-epimer of myo-inositol as the central core to synthe-
size PPIPn analogs. Scyllo-inositol phosphates constitute the second naturally abundant
inositol phosphates [45
organisms and in the environment [48
–
47] and many scyllo-inositol phosphates have been found in living
49]. Utilizing these scyllo-inositol phosphates as
,
substrates for the PPIPn synthesizing kinases, would provide valuable information re-
garding enzyme function. In this context, we planned to synthesize scyllo-IP5, scyllo-IP6
and scyllo-IP7 (Figure 1B) to test them as substrates for Vip1 (budding yeast S. cerevisiae
ortholog of PPIP5K) and mouse IP6K1. While the first two are mimics of IP5 and IP6

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respectively, scyllo-IP7 is a mimic of both 1-PP-IP5 and 5-PP-IP5. We also planned to
synthesize Bz-scyllo-IP5, as 2-O-benzyl/2-O-benzoyl substituted ligands are known to
activate PPIP5K2 [39].
2. Results and Discussion
2.1. Synthesis of Scyllo-Inositol Phosphates
Scyllo-inositol derivative
ing the reported procedure [45]. Acidic hydrolysis of
scyllo-inositol ( ), which, on phosphitylation with N, N-dibenzyl diisopropylphospho-
ramidite in the presence of 1H-tetrazole followed by oxidation with m-CPBA at
78 ◦C,
gave hexaphosphorylated scyllo-inositol derivative (Scheme 1). Hydrogenolysis of
yielded scyllo-IP6 ( ). Benzoylation of followed by acidic hydrolysis of the ester
benzoyl-scyllo-inositol ( ). Phosphorylation of
pentakisphosphate ( ), which, on hydrogenolysis, yielded the benzoyl-scyllo-IP5 (
pound on treatment with 1 N LiOH gave scyllo-IP5 ( ), which was purified by passing
through Dowex H+ resin.
1
was synthesized from myo-inositol in four steps by follow-
1
with 1 N HCl in ethanol yielded
2
−
3
3
4
1
5
gave
6
6
resulted in the fully protected scyllo-inositol
). Com-
7
8
8
9
Scheme 1. Synthesis of scyllo-IP6 (4), Bz-scyllo-IP5 (8) and scyllo-IP5 (9).
Next, we turned our attention to synthesize scyllo-IP7 (Scheme 2). Attempts to remove
the benzoyl group selectively from compound 7 failed. This prompted us to pursue another
approach for the synthesis of scyllo-IP7 (20) starting from myo-inositol derivative 10 [50].
Selective O-alkylation of the equatorial hydroxyl group using p-methoxybenzyl chloride
gave compound 11, which, on triflylation, gave the triflate 12. Nucleophilic displace-
ment of the trifllate with acetate gave scyllo-inositol derivative 13, which, on saponi-
fication, yielded the pentol 14. Phosphitylation of pentol 14 followed by in situ oxi-
dation resulted in the formation of fully protected pentakisphosphate 15. Oxidative
removal of the PMB group afforded compound 16, whose structure was unambigu-
ously confirmed by single crystal XRD analysis (see Figure 7 in Section 3.2.15., Crystal
Data for 16). Compound 16 was then phosphitylated with the unsymmetrical phosphorami-
date, 2-cyanoethyl-N,N-diisopropyl benzylphosphoramidte (see Scheme 3 in Section 3.2.16.),
followed by oxidation to get differently protected hexakisphosphate derivative 17. Depro-
tection of the cyanoethyl group gave the phosphodiester 18. Phosphorylation of 18 using
dibenzylphosphoryl chloride [30
,51] yielded fully protected scyllo-IP7 derivative 19, which
was used without purification for hydrogenolysis to get the scyllo-IP7 (20).

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Scheme 2. Synthesis of scyllo-IP7 (20).
2.2. Analysis of Scyllo-Inositol Phosphates as Substrates for IP6K and PPIP5K
We investigated the ability of these scyllo-inositol phosphates to act as substrates
for mouse IP6K1 (UniProt entry Q6PD10) and S. cerevisiae VIP1 (UniProt entry Q06685)
in comparison with myo-IP5 and IP6. All these substrate analogs were incubated with
ATP and purified N-terminally 6xHis-tagged mouse IP6K1 or the kinase domain (amino
acid residues 1–535) of S. cerevisiae VIP1 fused N-terminally to GST (VIP1-KD). The IPn
substrates and their PPIPn products were resolved by polyacrylamide gel electrophoresis
(PAGE) and detected by staining with toluidine blue dye (Section 3.3). Upon the incubation
of myo-IP5 or myo-IP6 with IP6K1 or VIP1-KD, we detected one or more myo-PPIPn products
(Supplementary Figure S1). Our observations are in concordance with an earlier report
demonstrating multiple PP-IP products arising from the action of these IP kinases on myo-
IP5 or myo-IP6, many of which are uncharacterized [52]. All scyllo-IPns served as substrates
for both IP6K1 and VIP1-KD (Supplementary Figure S1). PAGE analysis of PP-IPs is not
suitable for the quantitative comparison of scyllo- vs. myo-IPs as substrates, owing to
the variation in staining of different IPns by toluidine blue dye. Therefore, to quantify
the PPIPn products arising from myo- and scyllo-IPn substrates, we resorted to the use
of radiolabeled ATP. Equimolar quantities of the different IPn substrates were incubated
with either enzyme in the presence of [γ
-32P]ATP; the enzymatic reactions were quenched
by adding perchloric acid and then neutralized with potassium carbonate and EDTA
(Section 3.3). The products were separated by strong anion exchange HPLC, and 1 mL
fractions were collected and counted using a liquid scintillation counter. The substrates and
the corresponding chromatograms showing the peaks due to phosphorylated products are
shown in Figure 2. Each HPLC peak represents the addition of a radiolabeled phosphate
onto the IPn substrate. Owing to the inability to use an ATP regeneration system along
with radiolabeled ATP (see Section 3.3), in many cases, we observed fewer products by
HPLC compared with PAGE analysis.

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Figure 2. Chromatograms of the phosphorylated PPIPn products (indicated by arrows) formed by the catalysis of IP6K1
(blue) and VIP1-KD (red) on various IPn substrates. The IPn substrate used in each case is indicated above the chromatogram.
The reaction products were resolved by strong anion exchange HPLC as described in Section 3.3. Retention times for
standard myo-IPs were marked using radiolabeled inositol phosphates extracted from budding yeast (Section 3.3 and
Supplementary Figure S2B). The traces represent one of four independent experiments. The well-characterized products
of myo-IP5 and myo-IP6 are indicated. The PPIPn products of scyllo-IPs are likely to be as follows: A. PP-scyllo-IP4;
B. PP-Bz-scyllo-IP4; C. PP-scyllo-IP5; D. [PP]2-scyllo-IP4/PPP-scyllo-IP5; E. [PP]2-scyllo-IP4/PPP-scyllo-IP5. The corresponding
PPIPn products observed by PAGE analysis are similarly annotated in Figure S1. See Figure S2B,C for chromatograms of
other myo-IPn and myo-PPIPn standards, and ATP, under the same conditions.
We have proposed probable structures for the scyllo-PPIPn products seen by gel elec-
trophoresis and HPLC (see legends for Figure 2 and Supplementary Figure S1). Our
proposed structures are based on the known catalytic properties of IP6K1 and VIP1 to-
wards myo-inositol substrates. IP6K1 catalyzes the addition of a
α- phosphate, and a -phosphate onto a -phosphate on C5 of myo-IP5 and myo-IP6 [19
whereas VIP1 acts to add a -phosphate on C1 of myo-IP6 and myo-IP7 [13 17]. As all
β-phosphate onto an
γ
β
,
53],
β
,
equatorial carbon atoms of scyllo-inositol are indistinguishable from each other, we propose
that the same scyllo-PPIPn products arise by the action of both enzymes. Therefore, IP6K1
and VIP1 probably act on scyllo-IP5, Bz-scyllo-IP5 and scyllo-IP6 to yield PP-scyllo-IP4, PP-Bz-
scyllo-IP4 and PP-scyllo-IP5 (i.e., scyllo-IP7), respectively (peaks A, B and C in Figure 2). The
product formed by the addition of two phosphates onto scyllo-IP6 (peak D), and one phos-
phate onto scyllo-IP7 (peak E), are likely to correspond to a scyllo-inositol with 8 phosphate
groups. This could be [PP]2-scyllo-IP4 or PPP-scyllo-IP5. The only unambiguous means
of identifying the structures of any of the scyllo-PPIPn products is via NMR. However, it
is not trivial to obtain sufficient quantities of purified, enzymatically synthesized PPIPn
products required for such an analysis. The masses of the products we have proposed are
based on the bands visualized by PAGE (separated by a single phosphate units), and the
resolution of 32P-labeled inositol phosphates by HPLC. Despite repeated attempts we were
unable to detect enzymatically synthesized scyllo-PPIPn products by mass spectrometry—
HPLC-purified radiolabeled products cannot be loaded onto a mass spectrometer due
to radiation safety protocols, and contaminating substances from polyacrylamide gels
interfered with our attempts to analyze gel-eluted scyllo-IPs. For the above reasons, the

Molecules 2021, 26, 3601
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proposed structures of the scyllo-PPIPn products detected by PAGE and HPLC analysis are
likely, but not certain.
Analysis of the conversion of the substrates to products indicates that all scyllo-IPns
tested are substrates for IP6K1, but they are relatively poorer substrates than the correspond-
ing myo-inositol phosphates (Figure 3A). For IP6K1, myo-IP5 shows the best conversion
rate and is approximately 1.5-fold the conversion rate of myo-IP6. This is in agreement
with the trend reported previously [53]. Interestingly, scyllo-IP5 and scyllo-IP6 are poor
substrates for IP6K1 compared with their myo-counterparts. This suggests that an axial
C2-hydroxyl/phosphate has an important role in active-site binding. This requirement
was however partially offset by the presence of a benzoyl group on scyllo-IP5. It has been
shown that IP6K1 can act on 5PP-myo-IP5 to generate a triphosphate containing species—
5PPP-myo-IP5 [19]. We observed that IP6K1 can also act on scyllo-IP7 to generate a product
(Figure 2, peak E), which possibly contains a triphosphate moiety.
Figure 3. Extent of conversion of the substrates to products by (
(B) VIP1-KD in presence of ATP (40 M) and IPn (100 M). PPIPn product formation was calculated as described in
Section 3.3. The graphs show individual data points and mean SEM from four independent experiments. Data
A) IP6K1 in presence of ATP (1 mM) and IPn (200 µM).
µ
µ
±
was analyzed using a two-tailed Mann–Whitney test to compare the PPIPn product of myo-IPn with its corresponding
scyllo-derivative. p values are indicated where significant.
In contrast to IP6K1, all scyllo-IPns acted as moderate substrates for VIP1 (Figure 3B).
myo-IP5 is the poorest substrate of all those tested. This is in agreement with the fact that
1-PP-IP4 has not been found in vivo and also with a previous report that myo-IP5 is not a
substrate for PPIP5Ks, mammalian orthologs of Vip1 [17]. myo-IP6 is the best substrate; the
approximately 3-fold difference between myo-IP5 and myo-IP6 phosphorylation by VIP1-KD
suggests that the 2-phopshate is crucial for active site binding/effective phosphotransfer.
All scyllo-IPns tested were weaker substrates for VIP1 compared with myo-IP6, although
the difference was not statistically significant. This suggests that the loss of the axial
C2-phosphate can be partially compensated by an equatorial phosphate at that position.
Interestingly, the presence of a benzoyl group on scyllo-IP5 did not interfere with its ability
to serve as a substrate for VIP1. It is to be noted that structurally similar 2-O-benzyl-myo-IP5
was reported to be an inhibitor of PPIP5K but an activator of its ATPase activity [39].
In order to get further insights about the IPn-kinase interactions, we conducted
molecular docking with the kinases and myo-IP6 and scyllo-IP6 as ligands using CCDC
GOLD suit (see Supplementary Materials and Supplementary Figure S3). Since exact
structures for ScVIP1 and IP6K1 are not available, the molecular docking experiments
were carried out at the active sites of analogous hPPIP5K2 and IP6KA kinase of Entamoeba
histolytica (PDB code: 3T9C, 4O4F) [17,54]. With both the enzymes, myo-IP6 showed better
fitness score than scyllo-IP6 and this is in agreement with the experimental observation
of higher turnover for the myo-IPns. Furthermore, total number of attractive interactions
between the enzyme and ligands was more for myo-IP6 in both the cases. While myo-IP6 had

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21 interactions with IP6K, the scyllo-isomer had only 17 interactions. Similarly, PPIP5K2
kinase domain had 28 interactions with the myo-IP6 and 26 interactions with the scylllo-IP6.
Interestingly, the axial C2 phosphate in myo-IP6 shows seven interactions with the IP6K
side-chains (K101, R119, S207, T106, R134), but the corresponding equatorial phosphate
in scyllo-IP6 shows four interactions (R119, K118, R134, T106). In the case of PPIP5K2, the
axial phosphate at C2 position shows seven interactions (K329, R262, K248, R281, Y250)
and equatorial phosphate makes five interactions (K329, K248, R262, R281) (Figure 4). This
difference in number and strength of such interactions can influence the substrate binding
and, thus, the kinase activity.
Figure 4. Molecular Docking results of myo and scyllo-IP6 on PPIP5K2 and EhIP6KA (GOLDScore); (
A
) myo-IP6 at the
) scyllo-IP6
active site of PPIP5K2; ( ) scyllo-IP6 at the active site of PPIP5K2; ( ) myo-IP6 at the active site of EhIP6KA; (D
B
C
at the active site of EhIP6KA. All interactions were visualized using PLIP while further image processing was carried out
using PyMOL [55].
The non-productive activation of PPIP5K2 by inositol phosphates leading to the
hydrolysis of ATP to ADP (ATPase activity) has been reported [38]. Later, biochemical
studies and mutagenesis studies, using several synthetic analogs, revealed the existence of
a surface-exposed second binding site within the kinase domain of PPIP5K2, which acts
as a capture site for the substrate, and that the natural substrate occupies this site only
transiently before being delivered to the catalytic site [39]. The ATPase activity was found
to be associated with ligand occupancy at the capture site. This study also found that
2-O-benzyl-myo-IP5 interacts weakly with the catalytic site but binds better at the capture
site of PPIP5K2, and consequently it is not a substrate of this kinase but a ligand that
induces its ATPase activity [39]. We were curious to investigate the ability of scyllo-inositol
phosphates to induce the ATPase activity of VIP1. A recent report describing a high-
throughput assay for IP6K1 activity suggested that measuring ADP production from ATP
is an easier alternative to HPLC-based measurement of 5-PP-IP5 produced from IP6 [56]. By
extension, PPIPn products of scyllo-IPs should also lead to concomitant ADP release from
ATP, which can be used to measure kinase activity against these substrates. Furthermore,

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many kinases are known to possess inherent ATPase activity [57
–63], and PPIP5K2 shows
substrate-stimulated ATP hydrolysis (38, 39). With these considerations, we studied
ADP production by both kinases, IP6K1 and VIP1-KD. We ruled out the presence of any
contaminating ATPase or phosphatase in the preparation of 6xHis-mIP6K1 or GST-VIP1-KD
purified from the E. coli expression system (see Section 3.4 and Supplementary Figure S4).
We used the ADP-GloTM kinase assay kit to monitor the conversion of ATP to ADP
by IP6K1 and VIP1-KD in the presence of their IP substrates (Section 3.4) [56]. ADP-GloTM
kinase assay is a luminescence detection assay method to measure the percentage of
ATP that is converted to ADP during a kinase reaction. VIP1-KD showed considerable
basal activity, converting close to 40% of the ATP supplied during the reaction to ADP
(Figure 5A), which is in agreement with its known inherent ATPase activity [38]. In contrast
to the stimulation of non-productive ATPase activity by IP5 and IP6 reported in the case of
PPIP5K2 [38,39], in our hands, both IP5 and IP6 showed only negligible ATPase activation
of VIP1 over the basal level (Figure 6A). This may be due to differences in the behavior
of VIP1 and PPIP5K2. It may also be a reflection of the significantly lower levels of ATP
used in our assay—at high concentrations of ATP (5–10 mM), the presence of IP5 and IP6
leads to a 2- to 4-fold elevation in the release of Pi from ATP by PPIP5K2 (38). Under
physiological ATP concentrations, IP5 or IP6 may stimulate ATPase activity of the VIP1-KD;
substrate-independent ATPase activity of VIP1-KD may also increase at physiological
[ATP]. The 4–6% of ATP consumed in the presence of IP6 (Figure 6A) is sufficient for
the conversion of approximately 2% of IP6 to 1-PP-IP5, in agreement with the extent of
product formation shown in Figure 3B. All the scyllo-IPns tested showed up to two-fold
enhancement in ATPase activity of VIP1 (Figure 5A). Interestingly, scyllo-IP5 and scyllo-IP6
enhance ATPase activity four times more than their corresponding myo-IPns (Figure 6A).
This property of scyllo-IP5 and scyllo-IP6 to induce non-productive ATPase activity while
being moderate substrates, suggests that these ligands may bind better at the capture
site than the catalytic site of VIP1. Bz-scyllo-IP5 and scyllo-IP7 are also potent inducers of
VIP1 ATPase activity; while the ATPase activity of the former is in agreement with the
previously reported effect of 2-O-benzyl substituted myo-IP5 enhancing non-productive
ATPase activity of PPIP5K2 [39], in case of the latter, the absence of an axial-phosphate
group may weaken its interaction with the active site and thus increase its residence time
at the capture site, stimulating futile ATPase activity.
Figure 5. The effects of the myo- and scyllo-inositol phosphates upon ATPase activity of (
in presence of ATP (40 µM) and IPn (100 M). ( ) IP6K1 in presence of ATP (1 mM) and IPn (200 µM).
The graphs show individual data points and mean SEM from three independent experiments.
A) VIP1-KD
µ
B
±
Data was analyzed using one-way ANOVA to compare each condition with ATPase activity in the
absence of substrate. * p ≤ 0.05; ns, not significant, p > 0.05.

Molecules 2021, 26, 3601
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Figure 6. Percentage of ADP generated in the kinase reactions for (
A
) VIP1-KD and (
B
) IP6K1, after
subtraction of basal ADP generated in the absence of substrate, for the data shown in Figure 5. The
graphs show individual data points and mean SEM from three independent experiments. Data was
±
analyzed using one-way ANOVA to compare ATPase activity in the presence of each scyllo-IPn with
ATPase activity in the presence of either myo-IP5 or myo-IP6 (which were not significantly different
from each other). ** p ≤ 0.01; * p ≤ 0.05; ns, not significant, p > 0.05.
IP6K1 showed a very low basal activity, suggesting that it is not an inherent ATPase
(Figure 5B). The myo- and scyllo-inositol phosphates increased ADP production marginally
(Figure 6B), in keeping with the transfer of the gamma phosphate from ATP to generate
the corresponding inositol pyrophosphate (Figure 3A). An intriguing exception to this
was Bz-scyllo-IP5, which yielded a low amount of inositol pyrophosphate product, but
significantly stimulated ATPase activity in IP6K1. Further studies will be required to
establish non-productive ATPase activity in IP6K1.
In conclusion, we synthesized a series of scyllo-inositol phosphates as substrates for the
two kinases involved in diphosphoinositol polyphosphate synthesis. This study revealed
the tolerance of these enzymes for the myo- to scyllo- isomers. The relative potentials of
these compounds as substrates in comparison with natural myo-inositol based substrates
gave details and structural factors influencing binding of substrate to the enzymes. This
study established that the absence of an axial hydroxyl/phosphate at the C2 position of an
inositol phosphate reduces its potential as a substrate for IP6K1. In contrast, VIP1 appeared
to accommodate the absence of an axial-2-phosphate more easily compared with IP6K1.
All the scyllo-inositol derivatives tested are efficient in enhancing the ATPase activity of
VIP1, which may be due to the preference of these compounds for the capture site on VIP1.
Design of novel inhibitors based on these SAR studies and studies to identify the structures
and biological effects of the products of these unnatural substrates are underway.
3. Materials and Methods
3.1. General
All chemicals and solvents were purchased from commercial suppliers and used
directly. Solvents were dried using standard laboratory procedures and stored under
anhydrous conditions. Reactions were monitored by thin layer chromatography (TLC)
using Merck pre-coated silica plates (60 F254) (Darmstadt, Germany). TLC plates were
visualized under ultraviolet light at 254 nm and also by charring using cerium ammonium
molybdate solution (235 mL of distilled water, 12 g of ammonium molybdate, 0.5 g of
cerium sulfate and 15 mL of concentrated sulphuric acid). Column chromatography was
1
performed on silica gel (200–400 mesh). H, 19F, 13C and 31P-NMR spectra were recorded
using 500, 470, 125 and 202.4 MHz NMR spectrometers (Bruker, Mannheim, Germany),
respectively. Proton chemical shifts (δ) are relative to tetramethylsilane (TMS, δ = 0.0) as in-
ternal standard and expressed in parts per million (ppm). Spin multiplicities were given as
s (singlet), d (doublet), t (triplet), (qt) quartet, dd (doublet of doublet), dt (doublet of triplet),

Molecules 2021, 26, 3601
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ddd (doublet of doublet of doublet) and m (multiplet). Coupling constants (J) are given
in Hertz (Hz). The assignment of protons and carbons were done using two-dimensional
spectroscopic techniques COSY, HMQC and HMBC. IR spectra were recorded using IR
Prestige-21 (Shimadzu) spectrometer. Melting points were determined by using SMP-30
melting point apparatus (Stuart Scientific, Staffordshire, UK). Elemental analyses were done
on Elementar, vario MICRO cube elemental analyzer (Elementar Americas, Ronkonkoma,
NY, USA). Ion-exchange chromatography was performed on Biologic DuoFlow (Bio-Rad,
Hercules, CA, USA) by using Q-Sepharose as Fast Flow resin, 2 M triethylammonium bicar-
bonate (TEAB) buffer and MilliQ water were used as gradient solvents. Phosphate analysis
was done using Briggs and Ames phosphate assays. X-ray intensity data measurements of
freshly grown crystals were carried out at 298K on a Bruker-KAPPA APEX II CCD (Bruker,
Mannheim, Germany)diffractometer with graphite monochromatized (MoK = 0.71073 Å)
radiation. The X-ray generator was operated at 50 kV and 30 mA. Data were collected
with scan width of 0.3◦ at different settings of
ϕ
(0◦, 90◦ and 180◦), keeping the sample
to detector distance fixed at 40 mm. The X-ray data collection was monitored by SMART
program (Bruker, 2003) [64]. All the data were corrected for Lorentzian, polarization and
absorption effects using SAINT and SADABS programs (Bruker, 2003). SHELX-97 was used
for structure solution and full matrix least-squares refinement on F2 [65]. All the hydrogen
atoms were placed in geometrically idealized position and refined in the riding model
approximation with C-H = 0.95 Å, and with Uiso (H) set to 1.2Ueq (C). Molecular diagrams
were generated using ORTEP. Geometrical calculations were performed using SHELXTL
(Bruker, 2003) and PLATON. Statistical analysis was performed using GraphPad Prism 5
(GraphPad Software, San Diego, CA, USA). Results are represented as mean ± SEM. The
differences between multiple groups were analyzed by one-way ANOVA, using Tukey’s
multiple comparison test. p ≤ 0.05 is treated as statistically significant.
3.2. Synthesis
3.2.1. Synthesis of Scyllo-Inositol (2)
To a solution of compound 1 [45] (1 g, 2.33 mmol) in ethanol (15 mL), 1 N HCl solution
(5 mL) was added and refluxed for 1h. After completion of the reaction, the reaction mass
was filtered and the resultant solid was washed with DCM, and dried under reduced
1
pressure, to get compound
2
(0.3 g, 72%) as a white solid. H-NMR of compound
2
was
identical with the reported data [46].
3.2.2. Synthesis of 1,2,3,4,5,6-Hexakis-O-(dibenzyloxyphosphoryl)-Scyllo-Inositol (3)
Dibenzyl N,N-diisopropylphosphoramidite (2.3 g, 6.66 mmol) was added to a mixture
of scyllo-inositol ( ) (100 mg, 0.55 mmol) and 1H-tetrazole (0.46 g, 6.66 mmol) in acetonitrile
(10 mL). The mixture was stirred for 15 h at room temperature. After 15 h, temperature
of the reaction mixture was cooled down to
78 ◦C, then m-CPBA (1.3 g, 8.3 mmol) was
2
−
added and the reaction mixture was slowly warmed to room temperature. The reaction
mass was extracted with ethyl acetate and washed successfully with sat. aq. Na2SO3, sat.
aq. NaHCO3, water and brine. The organic layer was dried over anhydrous Na2SO4 and
the solvent was evaporated under reduced pressure. The crude product thus obtained was
purified by column chromatography using ethyl acetate and petroleum ether (1:1, v/v) as
1
eluent, to get compound 3 (380 mg, 0.22 mmol, 40%) as sticky liquid. H and 31P-NMR of
compound 3 were identical with the reported data [46].
3.2.3. Synthesis of Scyllo-Inositol Hexakisphosphate (4)
To a solution of 1,2,3,4,5,6-hexakis-O-(dibenzyloxyphosphoryl)-scyllo-inositol (3) (320 mg,
0.18 mmol) in a mixture of MeOH/H2O (2:1, v/v, 10 mL), 10% Pd/C (100 mg) was added
at room temperature and stirred under hydrogen atmosphere (using balloon) for 10 h.
After completion of the reaction, the reaction mass was filtered through a PTFE filter and
the filtrate was concentrated under reduced pressure. Then, the residue was purified by
ion-exchange chromatography on Q-sepharose Fast Flow resin eluting with a gradient of

Molecules 2021, 26, 3601
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aqueous TEAB (0 to 1.0 M) to obtain the triethylammonium salt of scyllo-inositol hexak-
1
isphosphate (
4
, 104 mg, 0.16 mmol, 89%) as a white foamy solid. H and 31P-NMR of the
compound were identical with the reported data [46].
3.2.4. Synthesis of 6-O-Benzoyl-2,4-Di-O-(p-Methoxybenzyl)-1,3,5-O-Methylidyne-
Scyllo-Inositol (5)
◦
To a solution of compound
1
(1 g, 2.33 mmol) in dry pyridine (20 mL) at 0 C, benzoyl
chloride (0.4 mL, 3.48 mmol) and DMAP (50 mg, 0.41 mmol) were added and stirred for
4–5 h. After completion of the reaction, water was added to quench the excess benzoyl chlo-
ride. The crude reaction mass was extracted with ethyl acetate and then washed thoroughly
with saturated NaHCO3 solution, water and brine. The organic layer was dried over anhy-
drous Na2SO4 and the solvent was evaporated under reduced pressure. The crude product
thus obtained was purified by column chromatography using ethyl acetate and petroleum
◦
ether (2:8, v/v) as eluent, to get compound
5
(1.2 g, 96%) as a white solid. The m. p. = 92 C;
RF = 0.7 (ethyl acetate/petroleum ether, 3:7, v/v); 1H-NMR (500 MHz, CDCl3)
δ: 3.69 (s, 6H,
2 x OCH3), 4.31 (brs, 2H), 4.37 (d, J = 10.4 Hz, 2H, ArCH2), 4.48 (d, J = 10.4 Hz, 2H, ArCH2),
4.56 (brs, 3H), 5.52 (s, 1H), 5.57 (brs, 1H), 6.59 (d, J = 8.2 Hz, 4H, ArH), 6.96–7.0 (m, 6H, ArH),
7.33 (t, J = 7.4 Hz, 1H, ArH), 7.74 (d, J = 7.7 Hz, 2H, ArH). 13C-NMR (CDCl3, 125 MHz)
δ: 55.2, 66.6, 67.9, 68.4, 71.6 (ArCH2), 72.7, 103.1, 113.6, 128.0, 128.5, 129.5, 129.6, 129.62,
130.2, 130.3, 132.8, 159.1, 166.1. Elemental analysis Calcd for C30H30O9: C, 67.41; H, 5.66.
Found C, 67.23; H, 5.75.
3.2.5. Synthesis of 1-O-Benzoyl-Scyllo-Inositol (6)
To a solution of compound
5 (1.0 g, 1.87 mmol) in ethanol (15 mL), 1 N HCl (5 mL)
was added and refluxed for 1h. After completion of the reaction, the reaction mass was
filtered and the obtained solid was washed with DCM and dried under reduced pressure
1
to get compound
6
as a white solid (0.42 g, 79%). H-NMR of compound
6
was identical
with the reported data [46].
3.2.6. Synthesis of 6-O-Benzoyl-1,2,3,4,5-Pentakis-O-(Dibenzyloxyphosphoryl)-
Scyllo-Inositol (7)
Dibenzyl N,N-diisopropylphosphoramidite (0.49 g, 1.41 mmol) was added to a mixture
of 1-O-benzoy-scyllo-inositol (6) (40 mg, 0.14 mmol) and 1H-tetrazole (99 mg, 1.41 mmol)
in dry acetonitrile (10 mL). The mixture was stirred for 15 h at room temperature. After
◦
15 h, temperature of the reaction mixture was cooled down to
−
78 C, then m-CPBA
(60%, 0.33 g, 2.1 mmol) was added and the reaction mixture was slowly warmed to room
temperature. The reaction mass was extracted with ethyl acetate and washed successfully
with sat. aq. Na2SO3, sat. aq. NaHCO3, water and brine. The organic layer was dried
over anhydrous Na2SO4 and the solvent was evaporated under reduced pressure. The
crude product thus obtained was purified by column chromatography using ethyl acetate
and petroleum ether (1:1, v/v) as eluent, to get compound
7 (0.2 g, 0.13 mmol, 93%) as
a sticky liquid; 1H-NMR (500 MHz, CDCl3)
δ: 4.46 (dd, J = 9.3 Hz, 11.7 Hz, 2H, ArCH2),
4.67 (d, J = 7.4 Hz, 1H, ArCHA), 4.70 (d, J = 7.4 Hz, 1H, ArCHB), 4.74 (d, J = 8.1 Hz, 1H,
ArCHA), 4.76 (d, J = 8.1 Hz, 1H, ArCHB), 5.01–4.77 (m, 19H, ArCH2 & H-2, H-3, H-4, H-5,
H-6), 5.69 (t, J = 7.6 Hz, 1H, H-1), 6.85 (m, 4H, ArH), 6.98 (m, 4H, ArH), 7.05–7.23 (m, 42H,
ArH), 7.23 (t, J = 7.9 Hz, 1H, ArH), 7.42–7.38 (td, J = 1.2 Hz, 7.5 Hz, 1H, ArH), 7.98 (dd,
J = 1.2 Hz, 8.3 Hz, 1H, ArH). 13C-NMR (CDCl3, 125 MHz)
δ: 69.4, 69.5, 69.6, 69.7, 69.9, 69.92,
69.97, 70.0, 70.04, 72.1, 75.9 (31P coupled), 76.3 (31P coupled), 127.7, 127.9, 128.1, 128.15,
128.2, 128.28, 128.31, 128.35, 128.4, 128.42, 128.44, 129.3, 130.2, 133.2, 135.3, 135.37, 135.6,
135.66, 135.7, 165.2. 31P-NMR (202.4 MHz, CDCl3)
analysis Calcd for C83H81O22P5: C, 62.88; H, 5.15. Found C, 62.76; H, 5.32.
δ:
−
1.77 (2P),
−
1.84 (3P). Elemental
3.2.7. Synthesis of 6-O-Benzoyl-Scyllo-Inositol-1,2,3,4,5-Pentakisphosphate (8)
To a solution of 6-O-benzoyl-1,2,3,4,5-pentakis-O-(dibenzyloxyphosphoryl)-scyllo-inositol (7)
(160 mg, 0.1 mmol) in a mixture of MeOH/H2O (2:1, v/v, 15 mL), 10% Pd/C (50 mg)

Molecules 2021, 26, 3601
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was added at room temperature and stirred under hydrogen atmosphere (using bal-
loon) for 10 h. After completion of the reaction, the reaction mass was filtered through a
PTFE filter and the filtrate was concentrated under reduced pressure. Then the residue
was purified by ion-exchange chromatography on Q-sepharose Fast Flow resin eluting
with a gradient of aqueous TEAB (0 to 1.0 M) to obtain the triethylammonium salt of
1-O-benzoyl-scyllo-inositol-2,3,4,5,6-pentakisphosphate (8) (50 mg, 0.073 mmol, 73%) as
a white foam; 1H-NMR (500 MHz, CDCl3)
δ: 4.44–4.36 (m, 3H, H-3,H-4, H-5), 4.51
(qt, J = 9.3 Hz, 2H, H-2, H-6), 5.53 (t, J = 9.6 Hz, 1H, H-1), 7.4 (t, J = 7.4 Hz, 2H, ArH),
7.53 (t, J = 7.5 Hz, 1H, ArH), 7.95 (t, J = 7.9 Hz, 2H, ArH). 13C-NMR (CDCl3, 125 MHz)
δ: 71.8 (C-1), 75.3 (C-2, C-6), 76.3 (C-4), 76.4 (C-3, C-5), 128.5, 128.9, 129.8, 133.9, 167.7.
31P-NMR (202.4 MHz, CDCl3) δ: 2.12 (1P), 1.91 (2P), 1.61 (2P). Elemental analysis Calcd for
C13H21O22P5: C, 22.82; H, 3.09. Found C, 22.75; H, 3.33.
3.2.8. Synthesis of Scyllo-Inositol-1,2,3,4,5-Pentakisphosphate (9)
The 1 N LiOH (5 mL) was added to 6-O-benzoyl-scyllo-inositol-1,2,3,4,5-pentakisphosphate (8)
◦
(50 mg, 0.073 mmol) and heated at 80 C for 3 h. After completion of the reaction the
reaction mass was passed through Dowex 50WX8-100 (H+ form) and eluted with water.
The filtrate was concentrated under reduced pressure to obtain compound
9 (34 mg,
1
0.058 mmol, 80%) as a sticky solid. H and 31P-NMR of the compound were identical with
the reported data [46].
3.2.9. Synthesis of 1-O-p-Methoxybenzyl-3,4,5,6-Tetra-O-Benzoyl-Myo-Inositol (11)
To a solution of 1,4,5,6-tetra-O-benzoyl-myo-inositol (10) [50] (7 g, 11.7 mmol) in dry
acetonitrile (150 mL), dibutyltinoxide (3.2 g, 12.87 mmol) and tetrabutylammonium bro-
mide (6.49 g, 17.6 mmol) were added at room temperature and the mixture was refluxed for
3–4 h. The reaction mass was then brought to room temperature, p-methoxybenzylchloride
◦
(2.76 g, 17.6 mmol) was added and heated at 60 C for 12 h. After completion of the
reaction, Et3N (1.18 g, 23.4 mmol) was added and refluxed for 1 h. Then the reaction mass
was extracted with ethyl acetate, washed successively with sat. aq. NaHCO3 solution,
water and brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was
evaporated under reduced pressure. The crude product thus obtained was purified by
column chromatography using a mixture of ethyl acetate and petroleum ether (3:7, v/v) as
eluent, to get compound 11 (7.04 g, 9.82 mmol, 84%) as a white solid. The m. p. = 188 ◦C;
Rf = 0.6 (ethyl acetate/petroleum ether, 3:7, v/v); 1H-NMR (500 MHz, CDCl3)
δ: 2.73 (s, 1H,
2-OH), 3.67 (s, 3H, OCH3), 3.82 (dd, J = 2.2 Hz, 9.75 Hz, 1H, H-1), 4.40 (d, J = 11.8 HZ, 1H,
CH2), 4.58 (m, 2H, ArCH2 & H-2), 5.23 (dd, J = 1.9 Hz, 10.3 Hz, 1H, H-3), 5.70 (t, J = 10 Hz,
1H, H-5), 5.98 (t, J = 9.9 Hz, 1H, H-6), 6.23 (t, J = 10.2 Hz, 1H, H-4), 6.59 (d, J = 6.6 Hz, 2H,
ArH of PMB), 7.0 (d, J = 8.5 Hz, 2H, ArH of PMB), 7.21 (m, 3H), 7.34–7.29 (m, 7H), 7.47–7.43
(m, 2H), 7.70 (t, J = 7.6 Hz, 4H), 7.82 (d, J = 7.3 Hz, 2H), 7.93 (d, J = 7.3 Hz, 2H). 13C-NMR
(CDCl3, 125 MHz) δ: 55.2, 67.4 (C-2), 69.8 (C-4), 71.2 (C-5), 71.3 (C-6), 71.9 (ArCH2), 72.1
(C-3), 76.3 (C-1), 113.9, 128.2, 128.3, 128.5, 128.7, 128.8, 129.0, 129.2, 129.5, 129.6, 129.72,
129.76, 129.8, 129.9, 133.0, 133.1, 133.2, 133.5, 159.6, 165.4, 165.5, 165.8, 166.0. Elemental
analysis Calcd for C42H36O11: C, 70.38; H, 5.06. Found C, 70.49; H, 4.82.
3.2.10. Synthesis of 1-O-p-Methoxybenzyl-3,4,5,6-Tetra-O-Benzoyl-2-O-
Trifluoromethanesulfonyl-Myo-Inositol (12)
To a solution of 1-O-p-methoxybenzyl-3,4,5,6-tetra-O-benzoyl-myo-inositol (11) (7.0 g,
9.76 mmol) in a mixture of dry DC◦M (50 mL) and dry pyridine (20 mL), triflic anhydride
(5.51 g, 19.5 mmol) was added at 0 C. After completion of the reaction, reaction mass was
extracted with DCM and the organic layer was washed successfully with sat. aq. NaHCO3,
water and brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was
evaporated under reduced pressure. The crude product thus obtained was purified by
column chromatography using a mixture of ethyl acetate and petroleum ether (2:8, v/v) as
eluent, to get compound 12 (5.38 g, 6.34 mmol, 65%) as a white solid; m. p. = 126 ◦C; 19
F

Molecules 2021, 26, 3601
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1
NMR (470 MHz, CDCl3) δ: −76.0. H-NMR (500 MHz, CDCl3) δ: 3.68 (s, 3H, OCH3), 3.97
(dd, J = 1.9 Hz, 10.2 Hz, 1H, H-1), 4.34 (d, J = 12.2 Hz, 1H, ArCHA), 4.70 (d, J = 12.2 Hz, 1H,
ArCHB), 5.44 (dd, J = 2.1 Hz, 10.5 Hz, 1H, H-3), 5.59 (brs, 1H, H-2), 5.72 (t, J = 10.1 Hz, 1H,
H-5), 5.89 (t, J = 10.1 Hz, 1H, H-6), 6.09 (t, J = 10.27 Hz, 1H, H-4), 6.58 (d, J = 8.5 Hz, 2H,
ArH of PMB), 7.0 (d, J = 8.5 Hz, 2H, ArH of PMB), 7.17–7.21 (m, 1H), 7.29–7.35 (m, 8H), 7.47
(m, 3H), 7.73 (t, J = 6.9 Hz, 4H), 7.78 (d, J = 7.3 Hz, 2H), 7.92 (d, J = 7.3 Hz, 2H). 13C-NMR
(CDCl3, 125 MHz) δ: 55.0, 55.2, 69.2 (C-3), 69.3 (C-4), 70.1 (C-6), 70.6 (C-5), 72.0 (ArCH2),
72.9 (C-1), 82.0 (C-2), 113.9, 127.8, 128.0, 128.3, 128.4, 128.5, 128.6, 129.1, 129.7, 129.8, 129.9,
130.0, 130.1, 133.3, 133.36, 133.4, 134.0, 159.7, 164.9, 165.1, 165.6, 165.7. Elemental analysis
Calcd. for C43H35F3O13S: C, 60.85; H, 4.16. Found C, 60.68; H, 4.11.
3.2.11. Synthesis of 2-O-Acetyl-1-O-p-Methoxybenzyl-3,4,5,6-Tetra-O-Benzoyl-
Scyllo-Inositol (13)
To a solution of 1-O-p-methoxybenzyl-3,4,5,6-tetra-O-benzoyl-2-O-trifluoromethanesulfonyl-
myo-inositol (12) (5 g, 5.89 mmol) in N,N-dimethylacetamide (30 mL), potassium acetate
◦
(2.89 g, 29.5 mmol) was added and heated at 70 C for 2 h. After completion of the reaction,
the reaction mass was extracted with ethyl acetate and thoroughly washed with water
and brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was
evaporated under reduced pressure. The crude product thus obtained was purified by
column chromatography using a mixture of ethyl acetate and petroleum ether (2:8, v/v)
as eluent, to get compound 13 (4.25 g, 5.59 mmol, 95%) as a white solid; m. p. = 254 ◦C;
1H-NMR (500 MHz, CDCl3)
δ: 1.83 (s, 3H, OAc), 3.64 (s, 3H, OCH3), 3.99 (t, J = 9.2 Hz, 1H,
H-1), 4.47 (d, J = 11.8 Hz, 1H, ArCHA), 4.52 (d, J = 11.7 Hz, 1H, ArCHB), 5.55 (t, J = 9.7 Hz,
1H, H-6), 5.65 (t, J = 9.7 Hz, 1H, H-5), 5.82–5.71 (m, 3H, H-2, H-3, H-4), 6.59 (d, J = 8.5 Hz,
2H, ArH of PMB), 6.96 (d, J = 8.5 Hz, 2H, ArH of PMB), 7.33–7.27 (m, 7H), 7.46–7.42 (m,
2H), 7.70 (d, J = 7.5 Hz, 4H), 7.82 (t, J = 6.6 Hz, 4H). 13C-NMR (CDCl3, 125 MHz)
δ: 20.6,
55.2, 70.6 (C-2 or C-3 or C-4 or C-5), 70.7 (C-3 or C-4 or C-5 or C-2), 71.0 (C-4 or C-5 or C-2
or C-3), 71.9 (C-6), 72.2 (C-5 or C-2 or C-3 or C-4), 74.5 (ArCH2), 77.2 (C-1), 113.7, 128.2,
128.3, 128.4, 128.5, 128.6, 128.7, 129.1, 129.2, 129.7, 129.8, 129.9, 133.2, 133.3, 133.5, 159.3,
165.2, 165.3, 165.5, 165.6, 169.4. Elemental analysis Calcd. for C44H38O12: C, 69.65; H, 5.05.
Found C, 69.59; H, 4.83.
3.2.12. Synthesis of 1-O-p-Methoxybenzyl-Scyllo-Inositol (14)
To a solution of 2-O-acetyl-1-O-p-methoxybenzyl-3,4,5,6-tetra-O-benzoyl-scyllo-inositol
(4.0 g, 5.27 mmol) in methanol (50 mL), sodium methoxide (57 mg, 1.05 mmol) was added
and refluxed for 4 h. After completion of the reaction, Dowex® 50WX8 H+ (1 g) was added
to the reaction mass and stirred at room temperature for 10 min. Then, the mixture was
filtered and the resin was washed with methanol. The combined filtrate was evaporated
under reduced pressure to get the title compound 14 as a white solid (1.55 g, 5.16 mmol,
◦
1
98%). Charred at 220 C without melting; H-NMR (500 MHz, CDCl3)
4H), 3.12–3.17 (m, 2H), 3.73 (s, 3H, OCH3), 4.69 (s, 2H, ArCH2), 4.81 (brs, 5H, OH), 6.86 (d,
J = 8.5 Hz, 2H, ArH), 7.33 (d, J = 8.5 Hz, 2H, ArH). 13C-NMR (CDCl3, 125 MHz)
: 55.5,
δ: 2.94–3.04 (m,
δ
73.7, 74.4, 74.5, 74.8, 83.3, 113.7, 129.6, 132.2, 158.8. Elemental analysis Calcd for C14H20O7:
C, 55.99; H, 6.71. Found C, 55.82; H, 6.65.
3.2.13. Synthesis of 6-O-p-Methoxybenzyl-1,2,3,4,5-Pentakis-O-(Dibenzyloxyphosphoryl)-
Scyllo-Inositol (15)
Dibenzyl N,N-diisopropylphosphoramidite (5.76 g, 16.7 mmol) was added to a solu-
tion of 1-O-p-methoxybenzyl-scyllo-inositol (14) (0.5 g, 1.67 mmol) and 1H-tetrazole (1.17 g,
16.7 mmol) in dry acetonitrile (30 mL). The mixture was stirred for 15 h at room tempera-
ture. After 15 h, the reaction mixture was cooled down to
−
78 ◦C, then m-CPBA (3.92 g,
25.05 mmol) was added and the reaction mixture was slowly warmed to room temperature.
The reaction mass was extracted with ethyl acetate and washed successively with sat. aq.
Na2SO3, sat. aq. NaHCO3, water and brine. The organic layer was dried over anhydrous
Na2SO4 and the solvent was evaporated under reduced pressure. The crude product thus

Molecules 2021, 26, 3601
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obtained was purified by column chromatography using a mixture of ethyl acetate and
petroleum ether (1:1, v/v) as eluent, to get compound 15 (2.4 g, 15.03 mmol, 90%) as a
1
sticky liquid. H-NMR (500 MHz, CDCl3)
δ
: 3.56 (s, 3H, OCH3), 4.06 (t, J = 4.5 Hz, 1H), 4.4
(brs, 2H), 4.80–4.99 (m, 25H), 6.56 (d, J = 8.5 Hz, 2H), 7.1–7.2 (m, 52H). 13C-NMR (CDCl3,
125 MHz)
: 55.1, 69.6, 69.7, 69.8, 69.9, 72.5, 76.9 (31P coupled), 77.3 (31P coupled), 79.3 (31P
coupled), 113.6, 128.0, 128.1, 128.3, 128.4, 128.5, 129.3, 129.5, 135.57, 135.63, 135.65, 135.68,
135.7, 135.71, 135.73, 159.1. 31P-NMR (202.4 MHz, CDCl3)
1.9, 2.0. Elemental analysis
Calcd for C84H85O22P5: C, 63.00; H, 5.35. Found C, 62.82; H, 5.61.
δ
δ
:
−
−
3.2.14. Synthesis of 1,2,3,4,5-Pentakis-O-(Dibenzyloxyphosphoryl)-Scyllo-Inositol (16)
To a solution of 6-O-p-methoxybenzyl-1,2,3,4,5-pentakis-O-(dibenzyloxyphosphoryl)-
scyllo-inositol (15) (1 g, 0.62 mmol) in anhydrous 1,2-dichloroethane (20 mL), DDQ (170.0 mg,
0.75 mmol) was added and heated at 60 ◦C for 3–4 h. After completion of the reaction, the
reaction mass was concentrated under reduced pressure and the crude residue was purified
by column chromatography using a mixture of ethyl acetate and petroleum ether (1:1, v/v)
as eluent to get compound 16 (670 mg, 0.45 mmol, 73%) as a white solid. The m. p. = 112 ◦C;
1H-NMR (500 MHz, CDCl3)
δ: 3.98 (td, J = 2.5 Hz, 8.2 Hz, 1H, H-1), 4.65 (qt, J = 8.1 Hz,
2H, H-2 & H-6), 4.65 (m, 1H, H-4), 4.73 (qt, J = 7.8 Hz, 2H, H-3 & H-5), 4.91–5.13 (m,
20H), 5.29 (d, J = 2.6 Hz, 1H, OH-1), 7.15–7.29 (m, 50H). 13C-NMR (CDCl3, 125 MHz)
δ: 69.64, 69.68, 69.72, 69.77, 69.85, 69.89, 69.96, 70.01, 70.05, 70.09, 72.4 (C-1), 75.4 (C-4, 31
P
coupled), 75.9 (C-3 & C-5, 31P coupled), 79.1 (C-2 & C-6, 31P coupled), 127.9, 128.03, 128.07,
128.1, 128.3, 128.33, 128.38, 128.4, 128.47, 128.5, 135.5, 135.62, 135.69, 135.71, 135.76, 135.8,
135.86. 31P-NMR (202.4 MHz, CDCl3)
δ:
−
1.55,
−
1.50,
−
0.79. Elemental analysis Calcd for
C76H77O21P5: C, 61.62; H, 5.24. Found C, 61.85; H, 4.87.
3.2.15. Crystal Data for 16
CCDC No. 1946543 (Figure 7) Molecular formula = C76H77O21P5, Formula weight = 1481.23,
colorless blocks, 0.25 0.20 0.15 mm, Triclinic, space group P-1, 12.9915(9), b = 16.3817(11),
c = 19.4914(14) Å, V = 3759.2(5) Å3, Z = 2, T = 293(2) K, 2
F(000) = 1552,
(mm−1) = 0.194, 55445 reflections measured, 13192 unique reflections
×
×
◦
max = 50.00 , D calc (g cm−3) = 1.309,
θ
µ
(Rint = 0.0907), multi-scan absorption correction, Tmin = 0.9530, Tmax = 0.9714, number of
parameters = 847, number of restraints = 396, GoF = 0.986, R1 = 0.0884, wR2 = 0.2343, R
indices based on 13192 reflections with I > 2s(I). ∆ρmax = 0.555, ∆ρmin = −0.358 (eÅ−3).
Figure 7. ORTEP diagram of compound 16 at 30% probability level.

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3.2.16. Synthesis of 2-Cyanoethyl-N,N-Diisopropylbenzylphosphoramidite
Diisopropylethylamine (0.87 mL, 5.0 mmol) was added to a mixture of
chloro-2-cyanoethyl-N,N-diisopropylphosphoramidite (1.1 mL, 5.0 mmol) and benzyl
alcohol (0.52 mL, 5.0 mmol) at room temperature and stirred for 1 h (Scheme 3). The reac-
tion mixture was extracted with DCM, washed with sat. aq. NaHCO3 and the organic layer
was dried over anhydrous Na2SO4. The organic layer was concentrated under reduced
1
pressure and used without any further purification. H and 31P-NMR of the compound
(1.4 g, 4.54 mmol, 91%) were identical with the reported data [66].
Scheme 3. Synthesis of 2-Cyanoethyl-N,N-Diisopropylbenzylphosphoramidite.
3.2.17. Synthesis of 6-((Benzyloxy)(2-Cyanoethoxy)Phosphoryl)-1,2,3,4,5-Pentakis-O-
(Dibenzyloxyphosphoryl)-Scyllo-Inositol (17)
To a solution of 1,2,3,4,5-pentakis-O-(dibenzyloxyphosphoryl)-scyllo-inositol (16) (0.2 g,
0.135 mmol) in dry DCM (10 mL), 2-cyanoethyl N,N-diisopropylbenzylphosphoramidite
(0.41 g, 1.35 mmol) and 1H-tetrazole (0.094 g, 1.35 mmol) were added. The mixture was
stirred for 4 h at room temperature and then m-CPBA (0.32 g, 2.03 mmol) was added at
−
78 ◦C and the reaction mixture was slowly warmed to room temperature. The reaction
mass was then extracted with ethyl acetate and washed successively with sat. aq. Na2SO3,
sat. aq. NaHCO3, water and brine. The organic layer was dried over anhydrous Na2SO4
and the solvent was evaporated under reduced pressure. The crude product thus obtained
was purified through column chromatography using a mixture of acetone and petroleum
ether (3:7, v/v) as eluent, to get compound 17 (160 mg, 0.09 mmol, 67%) as a sticky liquid;
1H-NMR (500 MHz, CDCl3)
7.12–7.20 (m, 55H). 13C-NMR (CDCl3, 125 MHz)
76.4, 116.6, 128.0, 128.11, 128.19, 128.3, 128.5, 128.52, 128.6, 128.66, 128.7, 128.8, 135.2, 135.3,
135.52, 135.58. 31P-NMR (202.4 MHz, CDCl3)
1.98, 2.78. Elemental analysis Calcd for
δ: 2.29 (t, J = 6.4 Hz, 2H), 3.93 (m, 2H), 4.87–5.08 (m, 28H),
δ: 62.4, 62.5, 69.9, 70.0, 70.1, 70.3, 70.4, 76.3,
δ:
−
−
C86H87NO24P6: C, 60.60; H, 5.14; N, 0.82. Found C, 60.53; H, 5.27; N. 0.88.
3.2.18. Synthesis of 6-O-Benzyloxyphosphoryl-1,2,3,4,5-Pentakis-O-
(Dibenzyloxyphosphoryl)-Scyllo-Inositol (18)
To a solution of 6-((benzyloxy)(2-cyanoethoxy)phosphoryl)-1,2,3,4,5-pentakis-O-
(dibenzyloxyphosphoryl)-scyllo-inositol (17) (0.1 g, 0.059 mmol) in dry acetonitrile (1.0 mL),
DBU (35 µL, 0.24 mmol) wasaddedfollowedbytheadditionofbis(trimethylsilyl)trifluoroacetamide
(0.5 mL) under nitrogen atmosphere. After 1 h, the reaction mixture was filtered through
Dowex-H+ resin. The filtrate was concentrated under reduced pressure and the crude reac-
tion mass was purified by column chromatography using methanol and dichloromethane
(1:9, v/v) as eluent to afford compound 18 (88 mg, 0.053 mmol, 90%) as a sticky solid;
1H-NMR [500 MHz, CDCl3:CD3OD (3:1)]
δ: 1.61–1.70 (m, 5H), 2.48–2.50 (m, 2H), 3.31–3.37
(m, 4H), 3.75 (m, 1H), 4.77–4.88 (m, 24H), 4.93 (m, 2H), 5.17 (m, 2H), 6.99–7.13 (m, 55H).
13C-NMR (CDCl3:CD3OD (3:1), 125 MHz)
δ: 23.1, 26.5, 28.1, 29.7, 31.8, 36.1, 36.7, 45.3, 49.9,
67.6, 67.7, 69.8, 69.9, 69.98, 70.0, 70.2, 70.3, 70.36, 70.4, 74.7 (31P coupled), 76.8 (31P coupled),
77.0 (31P coupled), 77.5 (31P coupled), 112.4, 114.6, 117.0, 119.2, 127.3, 127.4, 127.9, 127.98,
128.0, 128.1, 128.3, 128.4, 128.47, 134.9, 135.0, 135.07, 135.2, 135.22, 135.26, 137.5, 159.1,
159.4, 159.7, 159.8, 160.0, 177.9. 31P-NMR (202.4 MHz, CDCl3)
Elemental analysis Calcd for C83H84NO24P6: C, 60.37; H, 5.13. Found C, 60.31; H, 5.19.
δ:
−
1.92,
−
2.55,
−
3.03,
−
3.0.

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3.2.19. Synthesis of 6-O-Diphospho-Scyllo-Inositol-1,2,3,4,5-Pentakisphosphate (Scyllo-IP7) (20
To a solution of 6-O-benzyloxyphosphoryl-1,2,3,4,5-pentakis-O-(dibenzyloxyphosphoryl)-
scyllo-inositol (18) (50 mg, 0.03 mmol) and trimethylamine (9 L, 0.06 mmol) in anhydrous
)
µ
DCM, dibenzylphosphoryl chloride (0.18 mL, 10% in toluene, 0.06 mmol) was added at
◦
0 C. After 2 h, the solvents were removed by applying vacuum, and the residue was
completely dried at 10−2 mbar. The residue (19) was dissolved in t-BuOH/H2O (6:1, v/v,
14 mL) to which 10% Pd/C (100 mg) was added and stirred under hydrogen atmosphere
(balloon pressure) for 10 h. The catalyst was removed by filtration through a PTFE syringe
filter and the filtrate was concentrated under reduced pressure. The obtained residue was
purified by ion-exchange chromatography on Q-sepharose Fast Flow resin eluting with a
gradient of triethylammonium bicarbonate (TEAB) buffer (0 to 2.0 M) and water to obtain
the triethylammonium salt of 20 (10 mg, 44%) as a white solid. 31P-NMR (202.4 MHz, D2O)
δ
: 1.99 (5P),
−
6.1 (1P),
−
11.0 (1P); HRMS (ESI) mass calculated for C6H19O27P7 [M
−
H]+,
738.8199, found 738.5057.
3.3. Generation and Detection of PPIPn Products
The 6xHis-tagged mouse IP6K1 (mIP6K1) and GST-tagged kinase domain of S. cere-
visiae Vip1 (amino acid residues 1–535) (GST-VIP1-KD) were expressed and purified as
described earlier [67]. Assays to detect synthesis of PPIPn product were set up in reaction
buffer (20 mM Tris-Cl pH 7.4, 60 mM NaCl, 6 mM MgCl2, 1 mM DTT) in a 25
volume for PAGE analysis and a 50 L reaction volume for HPLC analysis. For PAGE
analysis, the reactions were supplemented with 1 mM Mg2+-ATP, an ATP regeneration
system (6 mM phosphocreatine and 25 U/mL creatine phosphokinase), ~1.2 M purified
M IPn substrate (for
µL reaction
µ
µ
enzyme, and either 200
µ
M IPn substrate (f◦or 6xHis-IP6K1) or 100
µ
GST-VIP1 KD), and were incubated at 37 C for 18 h. The reactions were mixed with
0.1% orange G containing loading dye and were resolved on a 35.8 % polyacrylamide
gel, overnight at 500 V in 1X TBE (Tris/borate/EDTA), as described earlier [52]. The gels
were stained with 0.1% Toluidine blue (w/v) in 20% methanol and 2% glycerol and were
destained in 20% methanol and 2% glycerol. The gels were scanned on a desktop scanner
(Laserjet M1136 MFP).
For HPLC analysis, the reactions were supplemented with 1 mM Mg2+-ATP and
200 µM IPn substrate for 6xHis-mIP6K1, and 40 µ µM IPn substrate
M Mg2+-ATP and 100
for GST-VIP1-KD. The concentration of ATP used in the assay was chosen on the basis
of the reported affinities of IP6K1 and VIP1-kinase domain for ATP (Km value for ATP
for mIP6K1 is 1.1 mM [68]; Km value for ATP has not been measured for S. cerevisiae
VIP1, but the kinase domain of human PPIP5K2 has a Km of 20–40 µM for ATP [38]. All
reactions contained 2.5
~1.2 µM purified enzyme, and were incubated at 37 C for 18 h. A negative control was set
µ
Ci [
γ
-32P]ATP (Regional ◦Centre Hyderabad, BRIT, India) and
up by incubating myo-IP6 in the presence of ~1
40
M Mg2+-ATP. The reactions were terminated by the addition of 40
followed by 100 L of 0.6 M perchloric acid, followed by 33 L neutralization solution
µM BSA, along with 2.5
µCi [
γ
-32P] ATP and
µ
µL of 6 mM EDTA,
µ
µ
(1 M potassium carbonate and 5 mM EDTA). The inositol phosphates were resolved by
HPLC (515 HPLC pumps, Waters) on a SAX Partisphere column (4.6 mm diameter and
125 mm length column, Whatman) using a gradient of buffer A (1 mM EDTA) and buffer
B (1 mM EDTA and 1.3 M (NH4)2HPO4, pH 3.8) as follows: 0–5 min, 0% B; 5–10 min,
0–20% B; 10–70 min, 20–100% B; 70–80 min, 100% B (see Supplementary Figure S2A for the
elution gradient profile). One-milliliter fractions were mixed with 4 mL scintillation cock-
tail (Ultima-Flo AP, Perkin Elmer) and counted for 1 min in a liquid scintillation analyzer
(Tri-Carb 2910 TR, Perkin Elmer). To identify the retention times for standard myo-IPs, we
utilized radiolabelled inositol phosphates extracted from yeast strains harboring deletions
of enzymes involved in inositol phosphate synthesis, as described earlier [69]. S. cerevisiae
strain BY4741 wild type (WT), ipk1∆, kcs1∆ and vip1∆ were grown in synthetic medium
containing yeast nitrogen base with ammonium sulphate and without inositol (MP Biomed-
icals), 2% dextrose (Sigma-Aldrich), 76 mg/mL uracil (HiMedia Laboratories), synthetic

Molecules 2021, 26, 3601
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complete supplement mixture (SCSM; Formedium) without inositol, and supplemented
with 5 µCi/mL of myo-2-[3H] inositol (15–20 Ci/mmol; American Radiolabeled Chemicals).
The yeast was inoculated at OD600 0.001 and grown to OD600 ~1.0 at 30 ◦C, harvested by
centrifugation (2000× g, 3 min), lysed in 350
0.2 mg/mL IP6, 2 mM EDTA) by bead beating using glass beads (Sigma-Aldrich). The
soluble cell extracts were harvested by centrifugation (21,000 g for 5 min) and neutralized
µL chilled lysis buffer (1 M perchloric acid,
×
with 1 M potassium carbonate/5 mM EDTA solution. The extracted inositol phosphates
were resolved using the column and gradient described above. One-milliliter fractions
were mixed with 3 mL scintillation cocktail and counted for 5 min, as described above.
In this way, we were able to identify the retention time for myo-IP5, myo-IP6, myo-PP-IP4,
myo-[PP]2-IP3, myo-PP-IP5 and myo-[PP]2-IP4, based on the IP profile described earlier for
these strains [70] (Supplementary Figure S2B). The retention time of [γ
-32P]ATP, deter-
mined from the negative control reaction (see Supplementary Figure S2C), was found to be
~20 min. Therefore, to avoid any carryover of ATP, fractions collected from 35–70 min of the
elution gradient were monitored to identify the PPIPn product. Minor variations in column
back-pressure during consecutive HPLC runs led to inter-assay variations in retention
time. However, the column was calibrated for each experimental set by making a note of
the retention time of 5PP-myoIP4, the product of myo-IP5 formed in the presence of IP6K1,
and of 1PP-myo-IP5 and 5PP-myo-IP5, the products of myo-IP6 formed in the presence of
VIP1-KD and IP6K1, respectively, as indicated in Figure 2. The conversion of IPn substrate
(10 nmoles and 5 nmoles respectively for IP6K1 and VIP1-KD reactions) to PPIPn product
(expressed in pmoles) was calculated based on the specific activity of ATP. All product
peaks that were at least 5-fold above the baseline were counted. Data shown reflect the
mean
±
S.E.M. from four independent assays, and were analyzed using GraphPad PRISM.
3.4. Detection and Quantification of ATP Consumed and ADP Generated during the
Kinase/ATPase Reaction
To monitor ATP consumption by purified recombinant 6 His-mIP6K1 and GST-
×
VIP1-KD, we used the ATPlite luminescence detection system (Perkin Elmer) as per the
manufacturer’s protocol. To rule out the possible co-purification of contaminating ATPases
or phosphatases from the E. coli expression system, we used the generic ATPase inhibitor
sodium orthovanadate (2 mM), or a mixture of phosphatase inhibitors (Phosphatase In-
hibitor Cocktail 3, Sigma-Aldrich P0044, used at the recommended concentration). The
reactions were set up with ~0.4
buffer (20 mM Tris-Cl pH 7.4, 60 mM NaCl, 6 mM MgCl2, and 1 mM DTT) in a final volume
of 25 L. Bz-scyllo-IP5 (200 M for IP6K1 and 100 M for VIP1-KD) was used to stimulate
µM 6xHis-mIP6K1 or ~0.2 µM GST-VIP1-KD in 1X reaction
µ
µ
µ
the ATPase activity of the enzymes. The ATPase or phosphatase inhibitor, or a vehicle
control, was added to the reaction for 15 min on ice, prior to the addition of ATP. The
◦
reactions were initiated by the addition of 40 µ
M Mg2+-ATP, incubated for 1 h at 37 C and
suitably diluted for measurement of ATP. A standard curve was plotted with increasing
amount of ATP and the luminescence values were extrapolated to obtain the amount of
ATP remaining after the reaction was terminated. Data shown reflect the mean
from two independent experiments and were analyzed using GraphPad PRISM.
±
range
The kinase/ATPase reactions for 6xHis-mIP6K1 and GST-VIP1-KD were set up in
reaction buffer in a 10 L or 12.5 L reaction volume, respectively. The reactions were
supplemented with 1 mM ATP and 200 M IPn substrate for 6xHis-mIP6K1, and 40
ATP and 100 M IPn substrate for GST-VIP1-KD, and ~1.2 M purified enzyme. The final
µ
µ
µ
µM
µ
µ
amount of ATP in the reaction was 10 nmoles and 500 pmoles, respectively, for IP6K1
and Vip1. Reactions were carried out for 18 h at 37 ◦C, cooled to 25 ◦C, and the amount
of ADP generated was assayed using the ADP-GloTM kinase assay kit (Promega, Cat.
# V6930), according to the manufacturer’s protocol. The ADP-GloTM kinase assay is a
luminescence-based ADP detection method to measure the amount of ADP generated in
the kinase reaction, and also reflects non-productive ATPase activity. After completion
of the kinase/ATPase reaction, an equal volume of ADP-GloTM reagent was added and
incubated at 25 ◦C for 30 min to terminate the reaction and to deplete the remaining ATP.

Molecules 2021, 26, 3601
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The reaction mixture was transferred to a solid white 96-well plate, and double the volume
of kinase detection reagent was added under dark, and incubated for 1 h to generate ATP
from ADP, and measure the newly synthesized ATP using a luciferase/luciferin reaction.
Luminescence was measured using a Multimode plate reader (EnSpire, Perkin Elmer,
Waltham, MA, USA). The luminescence values were correlated with the percentage of ADP
generated in the reaction by using an ATP-to-ADP standard curve. Appropriate volumes of
ATP and ADP stock solutions were mixed to generate an ATP-to-ADP conversion standard
curve. Separate standard curves were generated for IP6K1 and VIP1-KD—for IP6K1, 1 mM
ATP and ADP solutions were mixed to obtain a range of ADP from 0% to 10% of ATP;
and for Vip1, 40
0% to 40% of ATP. In every sample used for the standard curve, we included 1X reaction
buffer and 100 M or 200 M IP6 respectively for VIP1-KD or IP6K1 assays, to account for
µM ATP and ADP solutions were mixed to obtain a range of ADP from
µ
µ
any effects of buffer components or IPns on the luminescence reading. Standard curves
were plotted and data was interpolated from three independent assays each for IP6K1 and
VIP1-KD. Data shown reflect the mean
analyzed using GraphPad PRISM.
±
S.E.M. from three independent assays, and were
As shown in Figure 5, we observed significant basal ATPase activity without the
addition of IPn substrate, especially in the case of VIP1-KD. The graphs shown in Figure 6
clearly depict the percentage ADP generated in the presence of IPn substrate, after sub-
tracting the amount of ADP generated in the absence of substrate.
Supplementary Materials: The following are available online, Section S1. PAGE analysis of PPIPn
products; Section S2. Calibration of SAX HPLC for resolution of IPs and PPIPs; Section S3. Molecular
Docking; Section S4. Effect of inhibitors on ATPase activity of IP kinases; Section S5. Spectral data.
References [52,69–75] are cited in the Supplementary Materials.
Author Contributions: Conceptualization, K.M.S. and R.B.; synthesis and characterization com-
pounds, R.M. (Raja Mohanrao); biochemical assays, R.M. (Ruth Manorama) and S.G.; molecular
docking, M.C.M.; preparation of the manuscript, R.M. (Raja Mohanrao), R.M. (Ruth Manorama),
S.G., M.C.M., R.B. and K.M.S. All authors have read and agreed to the published version of
the manuscript.
Funding: K.M.S. thanks the Department of Science and Technology, Govt. of India for a Swarna-
Jayanti Fellowship. R.B. acknowledges support from the Science and Engineering Research Board,
Department of Science and Technology, Govt. of India (CRG/20l9/002597); Department of Biotech-
nology, Govt. of India (BT/PR29960/BRB/10/1762/2019); and CDFD core funds.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the Supplementary Materials.
Acknowledgments: R.B. thanks members of the Laboratory of Cell Signalling for their valuable feedback.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Some of the samples are available in small quantities. This may be available
for some time.
References
1.
Irvine, R.F.; Schell, M.J. Back in the water: The return of the inositol phosphates. Nat. Rev. Mol. Cell Biol. 2001, 2, 327–338.
[CrossRef] [PubMed]
2.
Best, M.D.; Zhang, H.; Prestwich, G.D. Inositol polyphosphates, diphosphoinositol polyphosphates and phosphatidylinositol
polyphosphate lipids: Structure, synthesis, and development of probes for studying biological activity. Nat. Prod. Rep. 2010, 27,
1403–1430. [CrossRef]
3.
4.
Wilson, M.; Livermore, T.M.; Saiardi, A. Inositol pyrophosphates: Between signalling and metabolism. Biochem. J. 2013, 452,
369–379. [CrossRef]
Bennett, M.; Onnebo, S.M.N.; Azevedo, C.; Saiardi, A. Inositol pyrophosphates: Metabolism and signaling. Cell. Mol. Life Sci.
2006, 63, 552–564. [CrossRef] [PubMed]

Molecules 2021, 26, 3601
19 of 21
5.
6.
7.
8.
9.
Shah, A.; Ganguli, S.; Sen, J.; Bhandari, R. Inositol Pyrophosphates: Energetic, Omnipresent and Versatile Signalling Molecules. J.
Indian Inst. Sci. 2017, 97, 23–40. [CrossRef]
Barker, C.J.; Illies, C.; Gaboardi, G.C.; Berggren, P.-O. Inositol pyrophosphates: Structure, enzymology and function. Cell. Mol.
Life Sci. 2009, 66, 3851–3871. [CrossRef]
Shears, S.B.; Weaver, J.D.; Wang, H. Structural insight into inositol pyrophosphate turnover. Adv. Biol. Regul. 2013, 53, 19–27.
[CrossRef] [PubMed]
Wang, H.; Gu, C.; Rolfes, R.J.; Jessen, H.J.; Shears, S.B. Structural and biochemical characterization of Siw14: A protein-tyrosine
phosphatase fold that metabolizes inositol pyrophosphates. J. Biol. Chem. 2018, 293, 6905–6914. [CrossRef]
Auesukaree, C.; Tochio, H.; Shirakawa, M.; Kaneko, Y.; Harashima, S. Plc1p, Arg82p, and Kcs1p, Enzymes Involved in Inositol
Pyrophosphate Synthesis, Are Essential for Phosphate Regulation and Polyphosphate Accumulation in Saccharomyces cerevisiae.
J. Biol. Chem. 2005, 280, 25127–25133. [CrossRef]
10. Azevedo, C.; Szijgyarto, Z.; Saiardi, A. The signaling role of inositol hexakisphosphate kinases (IP6Ks). Adv. Enzym. Regul. 2011
,
51, 74–82. [CrossRef] [PubMed]
11. Bhandari, R.; Chakraborty, A.; Snyder, S.H. Inositol Pyrophosphate Pyrotechnics. Cell Metab. 2007, 5, 321–323. [CrossRef]
[PubMed]
12. Burton, A.; Hu, X.; Saiardi, A. Are inositol pyrophosphates signalling molecules? J. Cell. Physiol. 2009, 220, 8–15. [CrossRef]
13. Lin, H.; Fridy, P.C.; Ribeiro, A.A.; Choi, J.H.; Barma, D.K.; Vogel, G.; Falck, J.R.; Shears, S.B.; York, J.D.; Mayr, G.W. Structural
analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate
kinases are 1/3-kinases. J. Biol. Chem. 2009, 284, 1863–1872. [CrossRef]
14. Majerus, P.W. A discrete signaling function for an inositol pyrophosphate. Sci. STKE 2007, 2007, pe72. [PubMed]
15. Mulugu, S.; Bai, W.; Fridy, P.; Bastidas, R.J.; Otto, J.C.; Dollins, D.E.; Haystead, T.A.; Ribeiro, A.A.; York, J.D. A Conserved Family
of Enzymes That Phosphorylate Inositol Hexakisphosphate. Science 2007, 316, 106–109. [CrossRef] [PubMed]
16. Onnebo, S.M.N.; Saiardi, A. Inositol Pyrophosphates Get the Vip1 Treatment. Cell 2007, 129, 647–649. [CrossRef]
17. Wang, H.; Falck, J.R.; Hall, T.M.T.; Shears, S.B. Structural basis for an inositol pyrophosphate kinase surmounting phosphate
crowding. Nat. Chem. Biol. 2011, 8, 111–116. [CrossRef] [PubMed]
18. Wundenberg, T.; Mayr, G.W. Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates),
regulators of cell homeostasis. Biol. Chem. 2012, 393, 979–998. [CrossRef]
19. Saiardi, A. Cell Signalling by Inositol Pyrophosphates. Prokaryotic Cytoskelet. 2012, 59, 413–443. [CrossRef]
20. Tusi, M.M.; York, J.D. Roles of inositol phosphates and inositol pyrophosphates in development, cell signaling and nuclear
processes. Adv. Enzyme Regul. 2010, 50, 324–337.
21. Thota, S.G.; Bhandari, R. The emerging roles of inositol pyrophosphates in eukaryotic cell physiology. J. Biosci. 2015, 40, 593–605.
[CrossRef]
22. Brown, N.W., Jr.; Marmelstein, A.M.; Fiedler, D. Chemical tools for interrogating inositol pyrophosphate structure and function.
Chem. Soc. Rev. 2016, 45, 6311–6326. [CrossRef] [PubMed]
23. Lee, Y.-S.; Huang, K.; Quiocho, F.A.; O’Shea, E.K. Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an
inositol pyrophosphate. Nat. Chem. Biol. 2008, 4, 25–32. [CrossRef] [PubMed]
24. Yin, M.-X.; Catimel, B.; Gregory, M.; Condron, M.; Kapp, E.; Holmes, A.B.; Burgess, A.W. Synthesis of an inositol hexakisphosphate
(IP6) affinity probe to study the interactome from a colon cancer cell line. Integr. Biol. 2016, 8, 309–318. [CrossRef] [PubMed]
25. Lee, Y.-S.; Mulugu, S.; York, J.D.; O’Shea, E.K. Regulation of a Cyclin-CDK-CDK Inhibitor Complex by Inositol Pyrophosphates.
Science 2007, 316, 109–112. [CrossRef]
26. Chakraborty, A.; Koldobskiy, M.A.; Bello, N.T.; Maxwell, M.; Potter, J.J.; Juluri, K.R.; Maag, D.; Kim, S.; Huang, A.S.;
Dailey, M.J.; et al. Inositol pyrophosphates inhibit Akt signaling, thereby regulating insulin sensitivity and weight gain. Cell 2010
,
143, 897–910. [CrossRef] [PubMed]
27. Luo, H.B.R.; Huang, Y.E.; Chen, J.M.C.; Saiardi, A.; Iijima, M.; Ye, K.Q.; Huang, Y.F.; Nagata, E.; Devreotes, P.; Snyder, S.H. Inositol
pyrophosphates mediate chemotaxis in dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions. Cell 2003
,
114, 559. [CrossRef]
28. Albert, C.; Safrany, S.; Bembenek, E.M.; Reddy, K.M.; Falck, J.R.; Bröcker, M.; Shears, B.S.; Mayr, W.G. Biological variability in
the structures of diphosphoinositol polyphosphates in Dictyostelium discoideum and mammalian cells. Biochem. J. 1997, 327,
553–560. [CrossRef]
29. Wu, M.; Dul, B.E.; Trevisan, A.J.; Fiedler, D. Synthesis and characterization of non-hydrolysable diphosphoinositol polyphosphate
messengers. Chem. Sci. 2012, 4, 405–410. [CrossRef] [PubMed]
30. Zhang, H.; Thompson, J.; Prestwich, G.D. A Scalable Synthesis of the IP7 Isomer, 5-PP-Ins(1,2,3,4,6)P5. Org. Lett. 2009, 11,
1551–1554. [CrossRef]
31. Capolicchio, S.; Thakor, D.T.; Linden, A.; Jessen, H.J. Synthesis of Unsymmetric Diphospho-Inositol Polyphosphates. Angew.
Chem. Int. Ed. 2013, 52, 6912–6916. [CrossRef]
32. Capolicchio, S.; Wang, H.; Thakor, D.T.; Shears, S.B.; Jessen, H.J. Synthesis of Densely Phosphorylated Bis-1,5-Diphospho-
myo-Inositol Tetrakisphosphate and its Enantiomer by Bidirectional P-Anhydride Formation. Angew. Chem. Int. Ed. 2014, 53,
9508–9511. [CrossRef]

Molecules 2021, 26, 3601
20 of 21
33. Pavlovic, I.; Thakor, D.T.; Bigler, L.; Wilson, M.; Laha, D.; Schaaf, G.; Saiardi, A.; Jessen, H.J. Prometabolites of 5-Diphospho-myo-
inositol Pentakisphosphate. Angew. Chem. Int. Ed. 2015, 54, 9622–9626. [CrossRef]
34. Pavlovic, I.; Thakor, D.T.; Vargas, J.R.; McKinlay, C.J.; Hauke, S.; Anstaett, P.; Camuña, R.C.; Bigler, L.; Gasser, G.; Schultz, C.; et al.
Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo. Nat.
Commun. 2016, 7, 10622. [CrossRef]
35. Riley, A.M.; Wang, H.; Weaver, J.D.; Shears, S.B.; Potter, B.V.L. First synthetic analogues of diphosphoinositol polyphosphates:
Interaction with PP-InsP5 kinase. Chem. Commun. 2012, 48, 11292–11294. [CrossRef]
36. Wu, M.; Chong, L.S.; Capolicchio, S.; Jessen, H.J.; Resnick, A.C.; Fiedler, D. Elucidating Diphosphoinositol Polyphosphate
Function with Nonhydrolyzable Analogues. Angew. Chem. Int. Ed. 2014, 53, 7192–7197. [CrossRef] [PubMed]
37. Wu, M.; Chong, L.S.; Perlman, D.H.; Resnick, A.C.; Fiedler, D. Inositol polyphosphates intersect with signaling and metabolic
networks via two distinct mechanisms. Proc. Natl. Acad. Sci. USA 2016, 113, E6757–E6765. [CrossRef] [PubMed]
38. Weaver, J.D.; Wang, H.; Shears, S.B. The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against
the consequences of a deteriorating cellular bioenergetic environment. Biosci. Rep. 2013, 33, e00022. [CrossRef] [PubMed]
39. Wang, H.; Godage, H.Y.; Riley, A.M.; Weaver, J.D.; Shears, S.B.; Potter, B.V.L. Synthetic Inositol Phosphate Analogs Reveal that
PPIP5K2 Has a Surface-Mounted Substrate Capture Site that Is a Target for Drug Discovery. Chem. Biol. 2014, 21, 689–699.
[CrossRef]
40. Shears, S.B.; Ali, N.; Craxton, A.; Bembenek, M.E. Synthesis and Metabolism of Bis-diphosphoinositol Tetrakisphosphate in Vitro
and in Vivo. J. Biol. Chem. 1995, 270, 10489–10497. [CrossRef] [PubMed]
41. Craxton, A.; Caffrey, J.J.; Burkhart, W.; Safrany, S.; Shears, B.S. Molecular cloning and expression of a rat hepatic multiple inositol
polyphosphate phosphatase. Biochem. J. 1997, 328, 75–81. [CrossRef]
42. Kilari, R.S.; Weaver, J.D.; Shears, S.B.; Safrany, S. Understanding inositol pyrophosphate metabolism and function: Kinetic
characterization of the DIPPs. FEBS Lett. 2013, 587, 3464–3670. [CrossRef] [PubMed]
43. Wundenberg, T.; Grabinski, N.; Lin, H.; Mayr, G.W. Discovery of InsP6-kinases as InsP6-dephosphorylating enzymes provides a
new mechanism of cytosolic InsP6 degradation driven by the cellular ATP/ADP ratio. Biochem. J. 2014, 462, 173–184. [CrossRef]
[PubMed]
44. Dollins, D.E.; Bai, W.; Fridy, P.C.; Otto, J.C.; Neubauer, J.L.; Gattis, S.G.; Mehta, K.P.M.; York, J.D. Vip1 is a kinase and
pyrophosphatase switch that regulates inositol diphosphate signaling. Proc. Natl. Acad. Sci. USA 2020, 117, 9356–9364. [CrossRef]
[PubMed]
45. Riley, A.M.; Trusselle, M.; Kuad, P.; Borkovec, M.; Cho, J.; Choi, J.H.; Qian, X.; Shears, S.B.; Spiess, B.; Potter, B.V.L. scyllo -Inositol
Pentakisphosphate as an Analogue of myo -Inositol 1,3,4,5,6-Pentakisphosphate: Chemical Synthesis, Physicochemistry and
Biological Applications. ChemBioChem 2006, 7, 1114–1122. [CrossRef]
46. Chung, S.-K.; Kwon, Y.-U.; Chang, Y.-T.; Sohn, K.-H.; Shin, J.-H.; Park, K.-H.; Hong, B.-J.; Chung, I.-H. Synthesis of all possible
regioisomers of scyllo-Inositol phosphate. Bioorganic Med. Chem. 1999, 7, 2577–2589. [CrossRef]
47. Kwon, Y.-U.; Im, J.; Choi, G.; Kim, Y.-S.; Choi, K.Y.; Chung, S.-K. Synthesis of three enantiomeric pairs of scyllo-inositol phosphate
and molecular interactions between all possible regioisomers of scyllo-inositol phosphate and inositol 1,4,5-trisphosphate 3-kinase.
Bioorganic Med. Chem. Lett. 2003, 13, 2981–2984. [CrossRef]
48. Thomas, M.P.; Mills, S.J.; Potter, B.V.L. The “Other” inositols and their phosphates: Synthesis, biology, and medicine (with recent
advances in myo-inositol chemistry). Angew. Chem. Int. 2016, 55, 1614–1650. [CrossRef]
49. Turner, B.L.; Cheesman, A.W.; Godage, H.Y.; Riley, A.M.; Potter, B.V.L. Determination of neo- and d-chiro-Inositol Hexakisphos-
phate in Soils by Solution 31P NMR Spectroscopy. Environ. Sci. Technol. 2012, 46, 4994–5002. [CrossRef]
50. Sureshan, K.M.; Kiyosawa, Y.; Han, F.; Hyodo, S.; Uno, Y.; Watanabe, Y. Resolution of synthetically useful myo-inositol derivatives
using the chiral auxiliary O-acetylmandelic acid. Tetrahedron Asymmetry 2005, 16, 231–241. [CrossRef]
51. Babouri, R.; Traore, L.; Bekro, Y.-A.; Matveeva, V.I.; Sadykova, Y.M.; Voronina, J.K.; Burilov, A.R.; Ayad, T.; Volle, J.-N.;
Virieux, D.; et al. Golden Face of Phosphine: Cascade Reaction to Bridgehead Methanophosphocines by Intramolecular Double
Hydroarylation. Org. Lett. 2018, 21, 45–49. [CrossRef] [PubMed]
52. Losito, O.; Szijgyarto, Z.; Resnick, A.C.; Saiardi, A. Inositol Pyrophosphates and Their Unique Metabolic Complexity: Analysis by
Gel Electrophoresis. PLoS ONE 2009, 4, e5580. [CrossRef]
53. Draškovicˇ, P.; Saiardi, A.; Bhandari, R.; Burton, A.; Ilc, G.; Kovacˇevicˇ, M.; Snyder, S.H.; Podobnik, M. Inositol Hexakisphosphate
Kinase Products Contain Diphosphate and Triphosphate Groups. Chem. Biol. 2008, 15, 274–286. [CrossRef] [PubMed]
54. Wang, H.; Derose, E.F.; London, R.E.; Shears, S.B. IP6K Structure and the Molecular Determinants of Catalytic Specificity in an
Inositol Phosphate Kinase Family. Nat. Commun. 2014, 5, 4178. [CrossRef]
55. The PyMOL Molecular Graphics System, version 2.0 Schrodinger, LLC. Available online: https://pymol.org/2/support.html?
#citing (accessed on 9 June 2021).
56. Wormald, M.; Liao, G.; Kimos, M.; Barrow, J.; Wei, H. Development of a homogenous high-throughput assay for inositol
hexakisphosphate kinase 1 activity. PLoS ONE 2017, 12, e0188852. [CrossRef] [PubMed]
57. Paudel, H.; Carlson, G. The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity. J. Biol.
Chem. 1991, 266, 16524–16529. [CrossRef]
58. Armstrong, R.N.; Kondo, H.; Kaiser, E.T. Cyclic AMP-dependent ATPase activity of bovine heart protein kinase. Proc. Natl. Acad.
Sci. USA 1979, 76, 722–725. [CrossRef]

Molecules 2021, 26, 3601
21 of 21
59. Ward, N.E.; O’Brian, C.A. The intrinsic ATPase activity of protein kinase C is catalyzed at the active site of the enzyme. Biochemistry
1992, 31, 5905–5911. [CrossRef]
60. Ahmad, S.; Hughes, M.A.; Johnson, G.L.; Scott, J.E. Development and Validation of a High-Throughput Intrinsic ATPase Activity
Assay for the Discovery of MEKK2 Inhibitors. J. Biomol. Screen. 2013, 18, 388–399. [CrossRef] [PubMed]
61. Fox, T.; Fitzgibbon, M.J.; Fleming, M.A.; Hsiao, H.-M.; Brummel, C.L.; Su, M.S.-S. Kinetic mechanism and ATP-binding site
reactivity of p38gamma MAP kinase. FEBS Lett. 1999, 461, 323–328. [CrossRef]
62. Ferreira-Cerca, S.; Sagar, V.; Schaefer, T.; Diop, M.; Wesseling, A.-M.; Lu, H.; Chai, E.; Hurt, E.; LaRonde-LeBlanc, N. ATPase-
dependent role of the atypical kinase Rio2 on the evolving pre-40S ribosomal subunit. Nat. Struct. Mol. Biol. 2012, 19, 1316–1323.
[CrossRef]
63. Trajtenberg, F.; Graña, M.; Ruétalo, N.; Botti, H.; Buschiazzo, A. Structural and Enzymatic Insights into the ATP Binding and
Autophosphorylation Mechanism of a Sensor Histidine Kinase. J. Biol. Chem. 2010, 285, 24892–24903. [CrossRef] [PubMed]
64. Bruker. SADABS (Version 2.05), SMART (Version 5.631), SAINT (Version 6.45) and SHELXTL (Version 6.14); Bruker AXS Inc.:
Madison, WI, USA, 2003.
65. Sheldrick, G.M. Crystal structure refinement with SHELXL. Acta Cryst. 2015, 71, 3–8.
66. Wang, X.J.; Xu, B.; Mullins, A.B.; Neiler, F.K.; Etzkorn, F.A. Conformationally locked isostere of phosphoSer cis-pro inhibits pin1
−
23-fold better than phosphoSer−trans-pro isostere. J. Am. Chem. Soc. 2004, 126, 15533–15542. [CrossRef]
67. Bhandari, R.; Saiardi, A.; Ahmadibeni, Y.; Snowman, A.M.; Resnick, A.C.; Kristiansen, T.Z.; Molina, H.; Pandey, A.; Werner, J.K.;
Juluri, K.R.; et al. Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event. Proc. Natl. Acad. Sci. USA
2007, 104, 15305–15310. [CrossRef]
68. Saiardi, A.; Erdjument-Bromage, H.; Snowman, A.M.; Tempst, P.; Snyder, S.H. Synthesis of diphosphoinositolpentakisphosphate
by a newly identified family of higher inositol polyphosphate kinases. Curr. Biol. 1999, 9, 1323–1326. [CrossRef]
69. Azevedo, C.; Saiardi, A. Extraction and analysis of soluble inositol polyphosphates from yeast. Nat. Protoc. 2006, 1, 2416–2422.
[CrossRef] [PubMed]
70. Onnebo, S.M.N.; Saiardi, A. Inositol pyrophosphates modulate hydrogen peroxide signalling. Biochem. J. 2009, 423, 109–118.
[CrossRef]
71. Jones, G.; Willett, P.; Glen, R.C.; Leach, A.R.; Taylor, R. Development and validation of a genetic algorithm for flexible docking. J.
Mol. Biol. 1997, 267, 727–748. [CrossRef]
72. Dennington, R.; Keith, T.A.; Millam, J.M. GaussView, Version 6.1; Semichem Inc.: Shawnee Mission, KS, USA, 2016.
73. Avogadro: An Open-Source Molecular Builder and Visualization Tool. Version 1.2.0. Available online: http://avogadro.cc/
(accessed on 11 June 2021).
74. Riley, A.M.; Unterlass, J.; Konieczny, V.; Taylor, C.W.; Helleday, T.; Potter, B.V.L. A synthetic diphosphoinositol phosphate
analogue of inositol trisphosphate. MedChemComm 2018, 9, 1105–1113. [CrossRef]
75. Salentin, S.; Schreiber, S.; Haupt, V.J.; Adasme, M.F.; Schroeder, M. PLIP: Fully automated protein–ligand interaction profiler.
Nucleic Acids Res. 2015, 43, W443–W447. [CrossRef] [PubMed]