Synthetic Inositol phosphate analogs reveal that PPIP5K2 has a surface-mounted substrate capture site that is a target for drug discovery

Article abstract of DOI:10.1016/j.chembiol.2014.03.009

Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) is one of the mammalian PPIP5K isoforms responsible for synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates; PP-InsPs), regulatory molecules that function at the interface of cell s

Full text of DOI:10.1016/j.chembiol.2014.03.009

Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
Article
Synthetic Inositol Phosphate Analogs Reveal
that PPIP5K2 Has a Surface-Mounted Substrate
Capture Site that Is a Target for Drug Discovery
Huanchen Wang,1,3 Himali Y. Godage,2,3 Andrew M. Riley,2 Jeremy D. Weaver,1 Stephen B. Shears,1,
*
and Barry V.L. Potter2,
*
1Inositol Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of
Health, Research Triangle Park, NC 27709, USA
2Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath, Somerset BA2 7AY, UK
3Co-first author
*Correspondence: shears@niehs.nih.gov (S.B.S.), b.v.l.potter@bath.ac.uk (B.V.L.P.)
http://dx.doi.org/10.1016/j.chembiol.2014.03.009
SUMMARY
PPIP5Ks synthesize the 1-diphosphate (Wang et al., 2012);
there are two isoforms in mammals (Thomas and Potter,
2014). Interest in this field has recently been heightened by
demonstrations that diphosphoinositol polyphosphates operate
at the interface of cell signaling and organismic homeostasis
(Choi et al., 2005; Szijgyarto et al., 2011; Shears, 2009; Illies
et al., 2007; Chakraborty et al., 2010; Pulloor et al., 2014).
Here, a dynamic balance between the activities of IP6Ks and
PPIP5Ks is of particular significance. For example, the syn-
thesis of 5-PP-InsP5 by IP6Ks inhibits the PtdIns(3,4,5)P3/
PDK1/AKT/mechanistic target of rapamycin (mTOR) cascade
(Chakraborty et al., 2010) that controls cell growth and meta-
bolism in response to changes in levels of nutrients, growth
factors, and bioenergetic status (Benjamin et al., 2011). This
Diphosphoinositol pentakisphosphate kinase
2
(PPIP5K2) is one of the mammalian PPIP5K isoforms
responsible for synthesis of diphosphoinositol poly-
phosphates (inositol pyrophosphates; PP-InsPs),
regulatory molecules that function at the interface
of cell signaling and organismic homeostasis. The
development of drugs that inhibit PPIP5K2 could
have both experimental and therapeutic applica-
tions. Here, we describe a synthetic strategy for
producing naturally occurring 5-PP-InsP4, as well
as several inositol polyphosphate analogs, and we
study their interactions with PPIP5K2 using inhibitory action of 5-PP-InsP5 is reversed through its further
phosphorylation by the PPIP5Ks (Gokhale et al., 2013). There
may be therapeutic value in inhibiting PPIP5K activity to elevate
biochemical and structural approaches. These ex-
periments uncover an additional ligand-binding site
on the surface of PPIP5K2, adjacent to the catalytic
pocket. This site facilitates substrate capture from
the bulk phase, prior to transfer into the catalytic
pocket. In addition to demonstrating a ‘‘catch-
and-pass’’ reaction mechanism in a small molecule
kinase, we demonstrate that binding of our analogs
5-PP-InsP5 levels and attenuate the mTOR pathway, which is
hyperactivated in 70% of human tumors, contributing to the
derangement of cell growth and metabolism that accompanies
cancer development and progression (Benjamin et al., 2011).
We recently published proof-of-principle of the latter idea by
demonstrating that AKT phosphorylation in myoblasts is in-
hibited when PPIP5K1 expression is ‘‘knocked-down’’ (Gokhale
to the substrate capture site inhibits PPIP5K2. This
work suggests that the substrate-binding site offers
new opportunities for targeted drug design.
et al., 2013). It is just such therapeutic motives that frequently
drive the development of drugs that can specifically target ki-
nases such as PPIP5Ks. Candidate molecules may be rationally
designed when information on protein structure is available. To
this end, we recently solved the structure of the N-terminal ki-
nase domain of PPIP5K2 (PPIP5K2KD) in complex with natural
substrate within the catalytic site (Wang et al., 2012). However,
INTRODUCTION
The process of signal transduction that governs many cellular the architecture of the active site exhibits substantial geometric
activities frequently relies upon evolutionarily conserved fam- and electrostatic constraints that raise challenges for the design
ilies of small, regulatory molecules. Among them are the of an effective yet specific inhibitor.
diphosphoinositol polyphosphates (inositol pyrophosphates:
In the current study, we set out to prepare substrate analogs
5-PP-InsP4, 1-PP-InsP5 [1-InsP7], 5-PP-InsP5 [5-InsP7], and that might modify PPIP5K2 activity. The synthesis of analogs
1,5-[PP]2-InsP4 [InsP8]; Figure 1), in which six to eight phos- of diphosphoinositol polyphosphates presents particular
phate groups are crammed around the six-carbon inositol technical challenges due to the reactive nature of the diphos-
ring. These high-energy molecules are synthesized by two phate group and the protected diphosphate intermediates
distinct classes of kinases, IP6Ks and PPIP5Ks. The IP6Ks (Best et al., 2010). The high negative charge density of these
add the 5-diphosphate group (Draskovic et al., 2008); mammals materials also presents purification problems (Capolicchio
express three IP6K isoforms (Thomas and Potter, 2014). The et al., 2013). Although several of the naturally occurring
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
Figure 1. Biosynthesis of Diphosphoinositol Phosphates
IP5K, inositol pentakisphosphate 2-kinase; IP6K, inositol hexakisphosphate 5-kinase; PPIP5K, diphosphoinositol pentakisphosphate 1-kinase.
diphosphoinositol polyphosphates have been synthesized Design and Synthesis of Inositol Phosphate and
(Albert et al., 1997; Best et al., 2010; Wu et al., 2013; Capolic- Diphosphoinositol Phosphate Analogs
chio et al., 2013), the preparation of useful analogs has only To increase insight into the origin of ligand-stimulated ATPase
recently been accomplished (Riley et al., 2012; Wu et al., activity of PPIP5K2KD, we explored the contributions of the
2013). In the latter studies, analogs of 5-PP-InsP4 and 5-PP- benzyl (Bn) and a-phosphonoacetyl (PA) groups in 2-O-Bn-
InsP5 were synthesized in which the diphosphate groups 5-PA-InsP4 (2). Our approach was to synthesize a series of
were replaced with metabolically stabilized phosphonoacetate analogs (Figure 2B) that exchanged Bn for either OH, mono-
(PA) or methylenebisphosphonate (PCP) groups. In the current phosphate, or alternative hydrophobes, whereas the PA group
study, we describe the synthesis of a series of diphosphoinosi- was replaced by either a monophosphate or a diphosphate.
tol polyphosphate analogs. We demonstrate how we used For analogs containing acyl groups at O-2, the appropriate
these reagents to gain insight into a previously described synthetic precursors are orthoesters of myo-inositol, because
(Weaver et al., 2013) substrate-stimulated ATPase activity of acid hydrolysis of these compounds gives rise to 2-O-acyl
PPIP5K2KD. These experiments also led us to uncover a esters with very high regioselectivity (Godage et al., 2006,
second ligand-binding site in PPIP5K2KD that performs an 2013). Thus, 2-O-benzoyl-InsP5 (4) and 2-O-butanoyl-InsP5
important aspect of the catalytic cycle by enhancing capture (5; Figures 2B and 3A) were synthesized from myo-inositol
of substrate from the bulk phase.
orthobenzoate (11) and myo-inositol orthobutanoate (12),
respectively. Acid hydrolysis of 11 and 12 (Godage et al.,
2013) gave pentaols 13 and 14, respectively, and subsequent
phosphorylation and deprotection provided 2-O-benzoyl-
InsP5 (4) and 2-O-butanoyl-InsP5 (5).
RESULTS AND DISCUSSION
Stimulation of the ATPase Activity of PPIP5K2KD by 5-
PA-InsP5 and 2-O-Bn-5-PA-InsP4
For analogs containing alkyl ethers or hydroxyl groups at
We recently reported that PPIP5K2KD exhibits an unusual, non- C-2, synthetic routes proceeding from the symmetrical
productive, substrate-stimulated ATPase activity (e.g., we butane-2,3-diacetal (BDA) protected diol 17 are expedient, as
observed
a 2- to 3-fold activation by 25 mM of either in our recent synthesis of 2-O-(2-aminoethyl)-InsP5 (6; Riley
Ins(1,3,4,5,6)P5 or InsP6; Figure 2A; Weaver et al., 2013). We et al., 2014). The remaining compounds 7–10 were also syn-
now report that 25 mM of either of two previously described thesized from diol 17 using a unified approach (Figures 2B
analogs of diphosphoinositol polyphosphates (Riley et al., and 3B). Thus, regioselective benzylation of the axial hydroxyl
2012) also stimulate ATP hydrolysis 5-fold by 5-O-a-phos- group of diol 17 gave 2-O-benzyl ether 18, and removal of
phonoacetyl-myo-inositol 1,2,3,4,6-pentakisphosphate (5-PA- BDA protecting groups gave pentaol 19. Phosphorylation of
InsP5 [1]), and 9-fold by 2-O-benzyl-5-O-a-phosphonoacetyl- 19 afforded the symmetrical pentakisphosphate 20 and
myo-inositol 1,3,4,6-tetrakisphosphate (2-O-Bn-5-PA-InsP4 [2]; cleavage of phosphate protection gave 2-O-Bn-InsP5 (7) in
Figures 2A and 2B). In view of the precise geometric and electro- 59% overall yield from 17.
static specificity constraints within the active site (Wang et al.,
For the synthesis of 2-O-Bn-5-PP-InsP4 (8) and 5-PP-InsP4 (9),
2012), we did not anticipate that it could accommodate the C-5 hydroxyl group of 18 was first phosphitylated with
2-O-Bn-5-PA-InsP4, which sports a bulky hydrophobic group. bis(cyanoethyl)(N,N-diisopropylamino)phosphine, followed by
It was therefore unexpected that 2-O-Bn-5-PA-InsP4 should be oxidation to provide the protected monophosphate 21. Removal
a more effective activator of ATPase activity than the two natural of BDA groups followed by phosphorylation afforded the fully
substrates, InsP6 (Figure 2A) and PP-InsP5 (Weaver et al., 2013). protected pentakisphosphate 23 in high yield (Figure 3B). We
We sought further information on this phenomenon.
constructed the diphosphate group by modifying a recently
2
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
Figure 2. The Effects of Inositol Phosphates and Analogs upon ATPase Activity of PPIP5K2KD
(A) The effects of the indicated inositol phosphates and analogs (25 mM) upon ATPase activity of PPIP5K2KD. Data represent means and SEs from three ex-
periments. Data for Ins(1,2,3,4,5)P5 (InsP5) and InsP6 are taken from Weaver et al., 2013.
(B) The structures of the reagents that were used.
described methodology (Capolicchio et al., 2013); compound 23 genolysis over palladium hydroxide, which removed the benzyl
was treated with DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) and esters from the phosphates; amines (either in DBU or in triethyl-
BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) to remove both amine; Riley et al., 2012) inhibit hydrogenolytic cleavage of the
cyanoethyl protecting groups on the 5-phosphate, followed by 2-O-benzyl ether. Note that 2-O-Bn-5-PP-InsP4 (8) is a synthetic
methanolysis of the temporary trimethylsilyl protection to give analog of an inositol pyrophosphate that retains the actual
the phosphate monoester at the C-5 position. Phosphitylation diphosphate group.
then afforded a mixed P(III)-P(V) intermediate, which was
oxidized to produce 24 (Figure 3B). We next prepared 2-O-Bn- yielded 5-PP-InsP4 (9), in 51% overall yield from 23 (Figure 3B).
5-PP-InsP4 (8) from crude 24 (which contained DBU), by hydro- This molecule is naturally occurring diphosphoinositol
Hydrogenolysis of purified 24 from which DBU was removed
a
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
Figure 3. Chemical Syntheses
(A) Syntheses of 2-O-Bz-InsP5 (4) and 2-O-But-InsP5 (5).
(B) Syntheses of 2-O-Bn-InsP5(7), 2-O-Bn-5-PP-InsP4 (8), 5-PP-InsP4 (9), and 2,5-di-O-Bn-InsP4 (10). Reagents and conditions: (a) TFA:H2O, 10:1; (b)
(BnO)2PN(iPr)2, 5-phenyl-1-H-tetrazole, DCM; (c) mCPBA, ꢀ40ꢁC to room temperature; (d) Pd(OH)2, H2, MeOH, H2O; (e) BnBr, NaH, DMF; (f) aq TFA (90%):DCM,
1:1; (g) (NCCH2CH2O)2PN(iPr)2, 5-phenyl-1-H-tetrazole, DCM; (h) tBuOOH, ꢀ40ꢁC to room temperature; (i) aq ammonia, 70ꢁC; (j) DBU, BSTFA; (k) MeOH, TFA; (l)
NaHCO3, Pd(OH)2, H2, tBuOH, H2O.
4
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
polyphosphate that features an unphosphorylated 2-OH group Inhibition of PPIP5K2KD Catalytic Activity by Inositol
(Figure 1); 5-PP-InsP4 has been implicated in telomere mainte- Phosphate Analogs
nance (York et al., 2005; Saiardi et al., 2005). Finally, for the syn- The six compounds that were selected for the dose-response
thesis of 2,5-di-O-Bn-InsP4 (10), diol 17 was benzylated followed curves in the assays of ATP hydrolysis (Figure 4A) were now
by cleavage of BDA groups. Subsequent phosphorylation and investigated for their effects upon diphosphoinositol poly-
deprotection of cyanoethyl phosphate esters gave (10) in 63% phosphate metabolism by PPIP5K2KD (Figure 4B). We used
overall yield from 17 (Figure 3B).
a high-throughput reverse-kinase assay that records ATP
generated from ADP during the dephosphorylation of 100 nM
1,5-[PP]2-InsP4 (Riley et al., 2012; Weaver et al., 2013). Each of
the tested analogs inhibited [PP]2-InsP4 metabolism by
Stimulation of the ATPase Activity of PPIP5K2KD by
Synthetic Inositol Phosphate Analogs
As discussed above (see also Figure 2A), 2-O-Bn-5-PA-InsP4 (2) PPIP5K2KD (Figure 4B). The two most potent of these analogs
strongly activated nonproductive ATPase activity of PPIP5K2KD
.
were 5-PA-InsP5 (1) and 2,5-di-O-Bn-InsP4 (10) (Figure 4B).
The degree of stimulation was dramatically reduced when the The inhibitory effect of 5-PA-InsP5 (1) was not in itself surprising
latter’s benzyl group was replaced with a hydroxyl group to becasue we have already demonstrated it to be a PPIP5K sub-
give 5-PA-InsP4 (3, the PA analog of 5-PP-InsP4; Riley et al., strate (Riley et al., 2012). However, the inhibition by 2,5-di-O-
2012; Figure 2). Furthermore, 5-PP-InsP4 (9) stimulated Bn-InsP4 (10) was more unexpected because due to its bulk,
ATPase activity at about half the rate shown by 2-O-Bn-5-PP- and its less negative charge compared to physiological sub-
InsP4 (8). Taken together, these data (Figure 2) lead to an unex- strates, we predict it to be a poor ligand for the active site of
pected conclusion that the ligand’s benzyl group makes an the highly specific PPIP5K2KD (Wang et al., 2012). Nevertheless,
important contribution to its association with PPIP5K2KD
.
its ability to inhibit reverse-kinase activity was directly confirmed
In 2-O-Bn-5-PP-InsP4 (8), the natural and more negatively by high-performance liquid chromatography (HPLC) analysis of
charged diphosphate (PP) group replaces the PA group at O-5 [PP]2-[3H]InsP4 dephosphorylation (Figures S1A–S1C available
in 2-O-Bn-5-PA-InsP4 (2). We found that 2-O-Bn-5-PP-InsP4 online). We confirmed that 2,5-di-O-Bn-InsP4 (10) also inhibited
(8) was slightly (20%) more effective at stimulating ATPase activ- PPIP5K2KD in the ‘‘forward,’’ kinase direction using InsP6, a
ity than was 2-O-Bn-5-PA-IP4 (2; Figure 2). Nevertheless, the PA physiologically relevant substrate (Figures S1D and S1E). A
group still contributes to efficacy more than a monophosphate Dixon plot demonstrated that inhibition by 2,5-di-O-Bn-InsP4
group because 2-O-Bn-InsP5 (7) was less effective than was (10) was competitive in nature (Ki = 240 nM; Figure 4C), arguing
2-O-Bn-5-PA-InsP4 (2; Figure 2).
that its actions are not allosteric in nature.
The rank order of potencies (as half-maximal inhibitory con-
Even in 2-O-Bn-InsP5 (7), the 2-O-benzyl ether has an
enhancing effect, roughly doubling the rate of ATP hydrolysis centration [IC50] values) with which this group of molecules in-
that is induced by InsP6, which has a phosphate group at C-2 hibited [PP]2-InsP4 dephosphorylation by PPIP5K2KD (Figure 4B)
(Figure 2). To investigate the effect of hydrophobes other than is different in two key respects from the rank-order of efficacy
benzyl at O-2, we compared 2-O-Bn-InsP5 (7) with 2-O-Bz- (EC50 values) for their separate stimulation of ATPase activity
InsP5 (4) and 2-O-But-InsP5 (5). The change from the 2-O-benzyl (Figures 4A and 4B). First, 2-O-Bn-InsP5 (7) is 4-fold less potent
ether to the structurally related 2-O-benzoyl ester led to only a than 2-O-Bn-5-PA-InsP4 (2) at stimulating ATPase activity (Fig-
minimal reduction in ATPase-stimulating activity, while substitu- ure 4A), whereas the two analogs are equally efficient at inhibit-
tion with a 2-O-butanoyl ester gave a sharp reduction in activity ing [PP]2-InsP4 metabolism (Figure 4B). Second, 5-PA-InsP5 (1),
(Figure 2). Furthermore, the analog with a positively charged the weakest activator of ATPase (Figure 4A), is the most potent
aminoethyl group at O-2 in (2-O-AminoEt-InsP5 [6]) did not stim- inhibitor of [PP]2-InsP4 dephosphorylation (Figure 4B). These
ulate ATPase activity (Figure 2).
are observations that suggest there is some uncoupling of
We next tested one further analog in which the diphosphate inositol phosphate turnover from the ATPase activity.
group in 2-O-Bn-5-PP-InsP4 (8) was replaced with a second
benzyl group (i.e., 2,5-di-O-Bn-InsP4; 10). This compound Structural Analysis Reveals that PPIP5K2KD Has Two
elicited the highest degree of ATPase activity among all of the Adjacent Ligand-Binding Sites
analogs that we have synthesized (Figure 2). This was initially a We have previously published an atomic-level description of
puzzling conclusion, as structural considerations indicate that both 5-PP-InsP5 and InsP6 substrates bound into the catalytic
2,5-di-O-Bn-InsP4 (10) would at best be a poor ligand for the pocket of PPIP5K2KD (Wang et al., 2012). In a separate study,
active site.
we (Riley et al., 2012) soaked 2 mM 5-PA-InsP5 (1) into crystals
Several of the compounds described above were selected of PPIP5K2KD, and reported that this ligand occupies the active
for detailed dose-response curves; we used the six compounds site in the same orientation as the natural substrate. At the time of
that yielded the greatest stimulation of ATPase activity, with that earlier study, we contoured the simulated annealing omit
the exception of 2-O-Bz-InsP5 (4), which in any case showed map at 2 s, and observed some additional but uninterpretable
similar efficacy to 2-O-Bn-InsP5 (7; Figure 4A). The most electron density at the entrance of the active site (data not
potent of this series of compounds was 2,5-di-O-Bn-InsP4 (10, shown). For the current study, we increased the soaking concen-
half-maximal effective concentration [EC50] = 340 nM; Figure 4A). tration of 5-PA-InsP5 (1) to 10 mM, and have now unequivocally
˚
In general, the rank order of the EC50 values for these detected an additional ligand-binding site, at 1.7 A resolution,
compounds approximated the rank order of their maximal that is located near the surface of PPIP5K2KD, at the entrance
effects, with the notable exception of 2-O-Bn-5-PP-InsP4 (8; to the catalytic center (Figures 5A and 5D; Table S1). This
Figure 4A).
surface-mounted ligand-binding site is too remote from ATP to
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
Figure 4. Effects of Inositol Phosphate Ana-
logs upon PPIP5K2KD Activities: ATPase,
1,5-[PP]2-InsP4 Dephosphorylation and
InsP6 Phosphorylation
(A and B) Dose-response curves are shown for the
indicated inositol phosphate analogs and their
stimulation of ATPase activities (A), and their
inhibition of ADP-driven dephosphorylation of
100 nM [PP]2-InsP4 (B). Error bars represent SEs
(n = 3). The EC50 and IC50 values for the molecules
tested are given in the key to the right of (A) and (B).
(C) A Dixon plot of the inhibition of InsP6 kinase
activity of PPIP5K2KD by 2,5-di-O-Bn-InsP4 (10);
the data are from a representative experiment (one
of three), performed in duplicate. Insets illustrate
the reaction being assayed.
5–10 mM of either 2-O-Bn-5-PA-InsP4
(2) or 2,5-di-O-Bn-InsP4 (10) into the
PPIP5K2KD crystals. X-ray analysis re-
vealed that both of these analogs were
exclusively associated with the second
ligand-binding site (Figures 5B, 5C, 5E,
and 5F; Table S1). Neither of these ana-
logs was found to occupy the catalytic
site. These data suggest that the stimula-
tion of ATPase activity by either natural
substrates or their analogs is associated
with their occupation of the second bind-
ing site, not the catalytic site. That expla-
nation is in turn consistent with other data
(Figures 4A and 4B, and see above) that
suggest ligand-stimulated ATPase activ-
ity is uncoupled from the inositol phos-
phate kinase activity.
The architecture of this second ligand-
binding site is represented by a deep
cleft, which is walled on one side by
K53, K54, and K103. The opposite face
is formed from R213, and a loop created
from residues E192 to H194 (Figure 5).
In this binding site, the shared groups in
5-PA-InsP5 (1) and 2-O-Bn-5-PA-InsP4
(2) exhibit almost identical conformations
(Figure 6A). As for 2,5-di-O-Bn-InsP4 (10)
this molecule is rotated ꢂ20ꢁ clockwise
(Figure 6B). For both 2-O-Bn-5-PA-InsP4
(2) and 2,5-di-O-Bn-InsP4 (10), their
phosphate groups at C-3 and C-4 would
clash with the positions that the C-4 and
permit ligand phosphorylation. It should be noted that both this C-3 phosphates of InsP6/5-PP-InsP5 normally occupy in the
site and the catalytic pocket cannot be occupied simultaneously active site (Figures S2A–S2C). This phenomenon presumably
due to steric clashing (Figures 5A and S2A). That is, these contributes to the potency of both 2-O-Bn-5-PA-InsP4 (2) and
particular structural data arise from a mixture of crystal com- 2,5-di-O-Bn-InsP4 (10) as inhibitors of inositol phosphate
plexes in which 5-PA-InsP5 (1) is separately bound to either of turnover by PPIP5K2KD (Figures 4B and 4C). These structural
the two sites.
The stimulation of ATPase activity of PPIP5K2KD by either 2-O- phosphate kinase activity without occupying the catalytic site.
Bn-5-PA-InsP4 (2) or 2,5-di-O-Bn-InsP4 (10) indicates that these
The association of 2-O-Bn-5-PA-InsP4 (2) and 2,5-di-O-Bn-
compounds also interact with PPIP5K2KD, so we soaked InsP4 (10) with the second binding site is facilitated by their
considerations explain how both compounds can inhibit inositol
6
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
Figure 5. X-Ray Analysis of Inositol Phosphate Analogs Bound to PPIP5K2KD Uncovers Two Ligand-Binding Sites
X-ray analysis was performed after we soaked crystals of PPIP5K2KD with either 10 mM of 5-PA-InsP5 (1), 5 mM of 2-O-Bn-5-PA-InsP4 (2), or 10 mM of 2,5-di-O-
Bn-InsP4 (10).
(A–C) Surface representations of ligand binding, with the analogs shown as stick-and-ball models. Atoms are blue for nitrogen; red for oxygen; orange for
phosphorus; and gray, cyan or green for carbon.
(D–F) The corresponding depictions of the protein as a ribbon diagram, with some key residues as stick models and numbered. For increased clarity, (D) only
shows the 5-PA-InsP5 (1) that binds to the capture site (see the text for the functional rationalization of this site). The carbons around the inositol ring are
numbered. AMPPNP is brown and magnesium atoms are magenta. Refined 2Fo-Fc maps are contoured at 1.0 s and are shown in gray mesh.
phosphate groups at C-3 and C-4 making multiple electrostatic bition was competitive (Figure 4C), further arguing against
interactions with K53, K54, and R213 (Figures 5, S2E, and S2F). an allosteric effect. Instead, the proximity to the active site
The terminal phosphonate of 2-O-Bn-5-PA-InsP4 (2) also forms of the second ligand-binding site (Figure 4) suggests that the
polar contacts with the backbone of K103 and the H194 side latter plays a role in catalysis of natural substrates, by facili-
chains (Figures 5 and S2E). Additionally, the 5-O-benzyl group tating their capture from the bulk phase. A precedent for
in 2,5-di-O-Bn-InsP4 (10) has van der Waals interactions with such a phenomenon has been reported for microbial anthrani-
the side chains of H101 and E192 (Figures 5 and S2F). The 2- late phosphoribosyltransferases (Castell et al., 2013; Marino
O-benzyl group in this analog is disordered, indicative of its et al., 2006); anthranilate substrate is first captured at a sur-
mobility. Nevertheless, our catalytic data (Figure 4) indicate face-mounted binding-site, before delivery to a proximal cata-
that this benzyl group makes a significant contribution to ligand lytic site (Castell et al., 2013). A similar substrate transfer is
potency.
feasible in PPIP5K2KD, and our structural data provide some
The data that we have obtained allow important conclusions atomic-level insight into such a phenomenon. For example,
to be drawn concerning the likely significance of the second we presume that delivery of natural substrate is efficient, so
binding site. For example, we can exclude the possibility that, that substrate only occupies the capture site transiently; this
as is the case with certain enzymes (Reed et al., 2010), the may explain why we could not detect substrate in the capture
role of the second, noncatalytic substrate-binding site is to site in our crystal structures. We found that such substrate
regulate enzyme activity by substrate inhibition; PPIP5K2KD transfer within PPIP5K2KD requires that the inositol ring be flip-
does not exhibit that property (Weaver et al., 2013). Other ped and rotated by ꢂ100ꢁ (Figures 6D and 6E). This motion is
enzymes use noncatalytic ligand-binding sites to promote presumably facilitated by the flexible amino-acid side chains
enzyme activation allosterically (Grant, 2012). That also seems that comprise the active site (Wang et al., 2012). Such confor-
unlikely to be the case for PPIP5K2KD, because our inositol mational dynamics can reinforce catalytic specificity (Hers-
phosphate analogs do not enhance kinase activity, but inhibit chlag, 1988), which is indeed a notable feature of PPIP5K2KD
it instead (Figure 4B; Riley et al., 2012). The nature of the inhi- (Wang et al., 2012).
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
Figure 6. Relative Orientations of Inositol
Phosphate Analogs in the Second Ligand-
Binding Site of PPIP5K2KD
Data for ligand orientation in the second binding
site were obtained as described in Figure 5.
Analogs are depicted as stick models. Atoms are
blue for nitrogen, red for oxygen, and orange for
phosphorus.
(A) Superimposition of 5-PA-InsP5 (1; carbons in
gray) and 2-O-Bn-5-PA-InsP4 (2; carbons in cyan).
(B and C) Superimposition of 2,5-di-O-Bn-InsP4
(10; carbons in green) and 2-O-Bn-5-PA-InsP4 (2;
carbons in cyan). Arrows in (B) emphasize spatial
separation, and (C) includes details of ligand in-
teractions with neighboring amino acid residues.
(D and E) illustrate that transfer of 5-PA-InsP5 (1)
between the two sites involves a lateral migration
ꢁ
˚
of 7 A, a ring flip, and a rotation of ꢂ100 . Atoms
are blue for nitrogen, red for oxygen, orange for
phosphorus, and gray for carbon. The inositol ring
is numbered.
2,5-di-O-Bn-InsP4 (10) (Figures 7B and
7C). These mutagenic data are consis-
tent with the idea that K54 and R213
participate in substrate capture.
The side chain of K103 is also suggested by our structural data
to interact with ligand that is bound to the capture site (Figures 5
Site-Directed Mutagenesis of the Substrate Capture
Site
Mutagenesis offers a valuable means of pursuing conclusions and S2). Nevertheless, a K103A mutant showed only a slight
drawn from structural analysis. The loop that is formed from res- reduction in 2-O-Bn-5-PA-InsP4 (2)-activated and 2,5-di-O-Bn-
idues A191 to H194 screens the catalytic site from ligands that InsP4 (10)-activated ATPase activity, compared to wild-type
are associated with the second binding site (Figures 5, 7A, and enzyme (Figures 7B and 7C). Likewise, the InsP6 kinase activity
S3). The influence of the carbonyl oxygen of A191 may be indi- of the K103A mutant (71 4 nmol/mg protein/min, n = 3) was
rect, through a hydrogen bond with a water molecule that in similar to that of wild-type enzyme (61 4 nmol/mg protein/min).
turn coordinates with an Mg2+ ion that interacts with the nucleo-
We also mutated E192. This residue is present in the loop
tide’s b-phosphate moiety (Figure 7A). The role of H194 seems between the two ligand-binding sites (Figure 7A). It is too distant
more direct; it can form a hydrogen bond with the oxygen from ATP and the catalytic site to influence either directly, and
atom of the nucleotide’s b-phosphate (Figures 7A and S3). This electrostatic repulsion would prevent it from interacting with
could stabilize the transition state, or have some other catalytic negatively charged groups on substrates located in the second
role. These interactions could be relevant to both kinase and binding site. Nevertheless, E192 is evolutionarily conserved in
ATPase activities of PPIP5K2KD. Consistent with these ideas, PPIP5Ks from mammals to yeast (not shown), suggestive of
an H194A mutant did not exhibit any detectable ATPase activity, functional significance. We investigated the significance of
either in the presence or absence of 2-O-Bn-5-PA-InsP4 (2) or E192 by preparing E192G and E192Q mutations that we posited
2,5-di-O-Bn-InsP4 (10), and its inositol phosphate kinase activity would eliminate electrostatic repulsion between the amino acid
was reduced 80-fold compared to that of the wild-type enzyme side chain and phosphorylated ligands, enhancing ligand bind-
(data not shown).
ing to the substrate capture site. We further hypothesized that
As discussed above, our structural and biochemical data led such an effect might reduce the efficiency of transfer of substrate
us to hypothesize that the stimulation of ATPase activity by to the catalytic site, which in turn would lead to a reduction in
either natural substrates or their analogs is associated with inositol phosphate kinase activity. Indeed, we found that these
their occupation of the substrate capture site, not the catalytic E192G and E192Q mutations reduced the rate of InsP6 phos-
site. We attempted to consolidate this idea by selecting for phorylation by 12- and 18-fold respectively, compared to wild-
mutation residues that might separate ATPase activity from type PIP5K2KD (Figure 7D). It was of further note that neither of
inositol phosphate kinase activity. For example, our X-ray these particular mutations altered the rate of nonproductive,
data (Figure 5) indicate that K54 and R213 interact with ligand ligand-stimulated ATPase activity elicited by either 2-O-Bn-
at the substrate capture site. However, previous mutagenic 5-PA-InsP4 (2), 2-O-Bn-5-PP-InsP4 (8), or 2,5-di-O-Bn-InsP4
work has shown that both K54 and R213 also contribute (10; Figure 7E), confirming the uncoupling of this aspect of
directly to inositol phosphate kinase activity (Wang et al., PPIP5K2KD activity from its kinase activity.
2012). Nevertheless, we did observe that K54A and R213A
We have shown that 2,5-di-O-Bn-InsP4 (10) binds to a sub-
mutants exhibited a substantially impaired degree of stimula- strate-capture site on the surface of PPIP5K2KD (Figure 5),
tion of ATPase activity by either 2-O-Bn-5-PA-InsP4 (2) or thereby inhibiting the enzyme’s catalytic activity (Figure 4). This
8
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
Figure 7. Impact on the Catalytic Properties of PPIP5K2KD Following Mutation of K54, K103, E192, and K213
(A) The spatial separation of AMPPNP from the PPIP5K2KD substrate capture site. Protein residues are shown as stick models. AMPPNP, and inositol phosphate
analogs 5-PA-InsP5 (1), 2-O-Bn-5-PA-InsP4 (2), and 2,5-di-O-Bn-InsP4 (10) and all are shown as stick models. Atoms are blue for nitrogen; red for oxygen; orange
for phosphorus; and gray, cyan, or green for carbon. Hydrogen bonds are shown as black dashed lines.
(B and C) Dose-response curves for the effects of 2-O-Bn-5-PA-InsP4 (2) and 2,5-di-O-Bn-InsP4 (10) upon the ATPase activities of wild-type PPIP5K2KD (closed
circles; data are taken from Figure 2A) and the following mutants: K54A mutant (closed squares), K103A (open circles), and R213A (open squares).
(D) The InsP6 kinase activities of wild-type, E192G, and E192Q mutants of PPIP5K2KD, determined with 5 mM substrate.
(E) The ATPase activities of wild-type and E192Q mutants of PPIP5K2KD obtained in the presence of the indicated analogs, at concentrations of 25 mM. Error bars
represent SEs from three experiments (error bars are not shown when they are smaller than the symbol).
particular ligand-binding site may be exploitable as a pharmaco-
In conclusion, our studies have uncovered a ‘‘catch-and-
logical target. We therefore examined the specificity of 2,5-di-O- pass’’ aspect to the catalytic cycle of PPIP5K2KD. Substrate
Bn-InsP4 (10) by investigating if it interacted with IP6K2, a (either InsP6 or 5-PP-InsP5) first associates with a ligand-
member of a different class of kinases that also phosphorylate binding site on the surface of the kinase. We propose that
InsP6 and synthesize diphosphoinositol polyphosphates (see this phenomenon enhances kinase activity by improving sub-
Figure 1). Because the affinity of IP6K2 for InsP6 (430 nM; Saiardi strate capture from the bulk phase; substrate is then delivered
et al., 2000) is very similar to that for InsP6 phosphorylation by into the catalytic pocket. We are not aware of any other exam-
PPIP5K2KD (390 nM; Weaver et al., 2013), an identical substrate ples of a substrate-capture mechanism in a small-molecule
concentration ([InsP6] = 500 nM) was used to assay both kinase. Indeed, the only example that we have found in the
enzymes. As shown in Figure 4C, 2,5-di-O-Bn-InsP4 (10) inhibits literature for a dedicated substrate capture site on any enzyme
PPIP5K2KD with an IC50 value of 1 mM (Figure 4C). This analog is that observed for certain microbial anthranilate phosphoribo-
was found to be a much weaker inhibitor of IP6K2 activity syl-transferases (Castell et al., 2013; Marino et al., 2006). Our
(IC50 = 63 mM; data not shown). Thus, 2,5-di-O-Bn-InsP4 (10) structural, biochemical, and mutagenic data have also led us
may be a useful lead molecule for future development of a to conclude that the stimulation of ATPase activity of PPIP5K
drug that can specifically inhibit PPIP5Ks and not IP6Ks, even by inositol phosphate analogs is associated with their occu-
though both groups of enzymes phosphorylate InsP6.
pation of the substrate capture site, not the catalytic site.
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Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
EXPERIMENTAL PROCEDURES
Moreover, the previously puzzling observation (Weaver et al.,
2013) that a degree of nonproductive ATP hydrolysis is also
stimulated by natural substrate, can now be rationalized as a
Protein Expression, Purification, Crystallization, and Structure
Determination
consequence of its interaction with the capture site. Further-
more, the fact that InsP6-stimulated ATPase activity is abol-
ished by an R213A mutation (Weaver et al., 2013) can now
be viewed as resulting from an impairment to substrate occu-
pation of the capture site.
The kinase domain of human PPIP5K2 (PPIP5K2KD; residues 1–366) and the
N-terminally truncated domain used for the crystallography studies (residues
41–366)weresubcloned,expressed,and purifiedasbefore. Thelatter wascrys-
tallized by hanging drop vapor diffusion against a well buffer of 12% (w/v) PEG
3350, 20 mM MgCl2, 0.1 M HEPES, pH 7.0, 1 mM AMP-PNP, and 2 mM CdCl2 at
4ꢁC. The crystals were then soaked with 5–10 mM compounds in a stabilizing
buffer containing 22% (w/v) PEG 3350, 10 mM MgCl2, 0.1 M sodium acetate,
pH 5.2 or 7.0, at 4ꢁC for 3 days. Cryosolvent was prepared by adding 33%
ethylene glycol into the soaking buffer. Diffraction data were collected using
APS beamlines 22-BM and 22-ID. All data were processed with the program
HKL2000. The structure was determined using rigid body and direct Fourier
synthesis, and refined with the equivalent and expanded test sets. The structure
was further manually rebuilt with COOT and refined with PHENIX and REFMAC
from the CCP4 package. Ligand topology and parameter files were prepared
using the PRODRG server. The molecular graphics representations were
Our atomic-level description of the substrate-capture site
on PPIP5K2KD indicates its structural determinants of specificity
differ substantially from those of the catalytic site. We have
further shown that synthesis of diphosphoinositol polyphos-
phates by PPIP5K2KD is inhibited by ligands that bind to the cap-
ture site but not the catalytic site. These findings in turn raise the
possibility that there may be cellular constituents that might
inhibit catalytic activity by binding to this site. Finally, the sub-
strate capture site offers a selective target for the purposes of
rational drug design, including screening in silico (Kitchen
et al., 2004), that is free from many of the usual specificity con-
straints within the catalytic site that typically complicate pharma-
cological targeting of small-molecule kinases. It may also be
possible to design a ligand that occupies both sites.
¨
prepared with the program PyMol (Schrodinger). The 2D ligand-protein interac-
tion diagrams were generated by LigPlot+.
Enzyme Assays
HPLC was used to assay [3H]InsP6 phosphorylation by human PPIP5K2KD or
human IP6K2 in 100 ml buffer containing 50 mM KCl, 20 mM HEPES pH 7.0,
7 mM MgSO4, 5 mM ATP, and 1 mM EDTA (for IP6K2, [MgSO4] was 12 mM
and [ATP] was 10 mM). The initial InsP6 concentration was either 500 nM,
70 nM, or 40 nM. Various concentrations of 2,5-di-O-Bn-InsP4 were present
as indicated. Radioactivity was assessed using an in-line Flo1 detector. The
ADP-driven dephosphorylation of 100 nM 1,5-[PP]2-InsP4 by PPIP5K2KD
was usually determined by a luminescence-based assay of ATP accumulation
(Riley et al., 2012; Weaver et al., 2013). The IC50 values were determined using
GraphPad Prism v6.02 (n R 3). In some experiments, 1,5-[PP]2-[3H]InsP4
dephosphorylation by PPIP5K2KD was assayed in 50 ml buffer containing
50 mM KCl, 20 mM HEPES pH 7.0, 7 mM MgSO4, 5 mM ADP, 1 mM EDTA.
Reactions were then quenched, neutralized, and analyzed with HPLC using
a Partisphere SAX column (Weaver et al., 2013); 1 ml fractions were collected
for liquid scintillation spectrometry. The ATPase activity of PPIP5K2KD
(10–270 mg/ml) was assayed from Pi release following incubation at 37ꢁC for
120–180 min in 20 ml reaction mixtures containing 20 mM Tris/HCl, pH 7.5,
10 mM ATP, 100 mM KCl, 0.1 mM EDTA, and 13 mM MgCl2.
SIGNIFICANCE
PPIP5K1 and PPIP5K2 are small-molecule kinases that
synthesize diphosphoinositol polyphosphates, which func-
tion at the interface of cell signaling and organismic homeo-
stasis. Synthetic chemical modulators of cell-signaling
enzymes such as the PPIP5Ks can provide valuable insight
into catalytic mechanisms, they decipher the biological
roles of the enzymes in situ, and they generate leads for
therapeutic drug development. Substrate analogs offer
one approach for the preparation of such chemical reagents.
However, the architecture of the active site of PPIP5Ks, as
revealed by recent X-ray analysis, has identified substantial
geometric and electrostatic constraints that limit the
options for designing a substrate analog that might be an
effective modulator. In the current study, we describe the
chemical synthesis of both the naturally occurring 5-
PP-InsP4 and a family of inositol polyphosphate analogs
that include molecules with hydrophobes at the 2- and/or
5-positions. Importantly, these molecules have led us to
characterize a second, less constrained substrate-binding
site on the surface of PPIP5K2, adjacent to the catalytic
pocket. We provide an atomic-level description of this sur-
face-mounted site and describe its role in the catalytic cycle
in capturing substrate from the bulk phase. With the assis-
tance of site-directed mutagenesis of this site, we show
that its occupation is associated with an unusual, ligand-
activated ATPase activity. This considerable amount of
information had not been accessible with our experiments
with natural substrates and represents a success for our
chemical biology approach. In addition to adding to
the repertoire of catalytic specializations of the PPIP5Ks,
our structural and functional characterization of this
ligand-binding site offers a promising target for drug devel-
opment; to this end, 2,5-di-O-Bn-InsP4 is a significant lead
compound.
Statistical Analysis
In the figures, error bars represent SEs from three experiments.
ACCESSION NUMBERS
The Protein Data Bank accession numbers for the protein/ligand structures
reported in this paper are 4NZM, 4NZN, and 4NZO.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
full experimental detail for the synthetic chemistry and NMR spectra, three fig-
ures, and one table and can be found with this article online at http://dx.doi.
org/10.1016/j.chembiol.2014.03.009.
AUTHOR CONTRIBUTIONS
H.W., H.Y.G., A.M.R., J.D.W., and S.B.S. carried out the experiments; S.B.S.
and B.V.L.P. designed and coordinated the research; and all authors provided
input during writing of the paper.
ACKNOWLEDGMENTS
We thank the Wellcome Trust (program grant 082837 to A.M.R. and B.V.L.P.)
for support. B.V.L.P. is a Wellcome Trust Senior Investigator (grant 101010).
10 Chemistry & Biology 21, 1–11, May 22, 2014 ª2014 Elsevier Ltd All rights reserved

Please cite this article in press as: Wang et al., Synthetic Inositol Phosphate Analogs Reveal that PPIP5K2 Has a Surface-Mounted Substrate Capture
Site that Is a Target for Drug Discovery, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.03.009
Chemistry & Biology
A PPIP5K Substrate Capture Site
This research was also supported by the Intramural Research Program of the
NIH/National Institute of Environmental Health Sciences (NIEHS). X-ray data
were collected at Southeast Regional Collaborative Access Team (SER-
CAT) 22-beamline at the Advanced Photon Source (APS), Argonne National
Laboratory. Supporting institutions may be found at http://www.ser-cat.org/
members.html. Use of the APS was supported by the US Department of En-
ergy, Office of Science, Office of Basic Energy Sciences, under contract no.
W-31-109-Eng-38. We also thank the NIEHS collaborative crystallography
group for assistance with data collection.
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