Highly potent and selective ectonucleotide pyrophosphatase/ phosphodiesterase i inhibitors based on an adenosine 5′-(α or γ)-Thio-(α,β- or β,γ)-methylenetriphosphate scaffold

Article abstract of DOI:10.1021/jm500196c

Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure-activity relat

Full text of DOI:10.1021/jm500196c

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Article
Highly potent and selective ecto-nucleotide pyrophosphatase/
phosphodiesterase I inhibitors based on an adenosine 5’-
# or #) thio, (#,#- or #,#)-methylene triphosphate scaffold
(
Yael Nadel, Joanna Lecka, Yocheved Gilad, Gal Ben-David, Daniel Förster, Georg Reiser, Sarah
Kenigsberg, Jean Camden, Gary A Weisman, Hanoch Senderowitz, Jean Sévigny, and Bilha Fischer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500196c • Publication Date (Web): 20 May 2014
Downloaded from http://pubs.acs.org on May 30, 2014
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Highly potent and selective ectoꢀnucleotide pyrophosphatase/phosphodiesterase I
inhibitors based on an adenosine 5’ꢀ (α or γ) thio, (α,βꢀ or β,γ)ꢀmethylene
triphosphate scaffold
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a§
b,c§
a
a
d
Yael Nadel , Joanna Lecka , Yocheved Gliad , Gal BenꢀDavid , Daniel Förster ,
d
a
e
e
Georg Reiser , Sarah Kenigsberg, Jean Camden , Gary A. Weisman ,
a
b,c
a
Hanoch Senderowitz *, Jean Sévigny, * and Bilha Fischer *
a
Department of Chemistry, BarꢀIlan University, RamatꢀGan 52900 Israel,
b
Département de microbiologieꢀinfectiologie et d’immunologie, Faculté de Médecine,
Université Laval, Québec, QC, Canada.
c
Centre de recherche du CHU de Québec, Québec, QC, Canada.
d
Otto von Guericke University, Leipziger Str. 44, 39120, Magdeburg, Germany
e
Biochemistry Department, 540E Bond Life Sciences Center, University of Missouri
Columbia, Columbia, Missouri 65211
*§
These authors contributed equally to this work
Address correspondence to either coꢀsenior author:
Bilha Fischer
*
Department of Chemistry, BarꢀIlan University
RamatꢀGan 52900, Israel
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Fax: 972ꢀ3ꢀ6353907; tel.: 972ꢀ3ꢀ5318303
eꢀmail: bfischer@mail.biu.ac.il
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Jean Sévigny
Centre de Recherche du CHU de Québec
2705 Laurier Blvd., Room T1ꢀ39
Québec, QC, Canada G1V 4G2
Fax: 418ꢀ654ꢀ2765; tel.: 418ꢀ654ꢀ2772
Email: Jean.Sevigny@crchul.ulaval.ca
Hanoch Senderowitz
Department of Chemistry, BarꢀIlan University
RamatꢀGan 52900, Israel
Fax: 972ꢀ3ꢀ7384588; tel.: 972ꢀ3ꢀ7384588
eꢀmail: hsenderowitz@gmail.com
2
+
2+
Abbreviations: [Ca ] , intracellular Ca concentration; CDI, carbonyldimidazole; NPP,
i
ectoꢀnucleotide
pyrophosphatase/phosphodiesterase;
NTPDase,
ectoꢀnucleoside
triphosphate diphosphohydrolase; ESI, electron spray ionization; HRMSꢀMALDI, highꢀ
resolution mass spectrometry matrixꢀassisted laser desorption ionization; P2R, P2
receptor; NTP, nucleoside–5’ꢀtriphosphate; pNPꢀTMP, thymidine 5'ꢀmonophosphate pꢀ
nitrophenyl ester; RT, room temperature; SD, standard deviation; TEAA,
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triethylammonium acetate; TEAB, triethylammonium bicarbonate; CPPD, calcium
pyrophosphate dihydrate; GFP, green fluorescent protein; hERG, human Etherꢀàꢀgoꢀgoꢀ
Related Gene; PDB, protein database.
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Abstract
Aberrant nucleotide pyrophosphatase/phosphodiesteraseꢀ1 (NPP1) activity is associated
with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1
inhibitors as therapeutic agents, and the scarceness of their structureꢀactivity relationship,
encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATPꢀαꢀthioꢀ
β,γꢀCH (1), ATPꢀαꢀthioꢀβ,γꢀCCl (2), ATPꢀαꢀCH ꢀγꢀthio (3), and 8ꢀSHꢀATP (4), and
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established their resistance to hydrolysis by NPP1,ꢀ3 and NTPDase1,ꢀ2,ꢀ3 and ꢀ8 (<5%
hydrolysis). Analogues 1ꢀ3 at 100 ꢀM inhibited pNPꢀTMP hydrolysis by NPP1 and
NPP3 by > 90% and 23ꢀ43%, respectively, and only slightly affected (0ꢀ40%) hydrolysis
of ATP by NTPDase1,ꢀ2,ꢀ3,ꢀ8. Analogue 3 is the most potent NPP1 inhibitor currently
known, K 20 nM and IC 0.39 ꢀM. Analogue 2a is a selective NPP1 inhibitor with K
i
50
i
6
85 nM and IC50 0.57 ꢀM. Analogues 1ꢀ3 were found mostly to be nonꢀagonists of
P2Y /P2Y /P2Y receptors. Docking analogues 1ꢀ3 into NPP1 model suggested that
1
2
11
activity correlates with the number of Hꢀbonds with binding site residues. In conclusion,
we propose analogues 3 and 2a as highly promising NPP1 inhibitors.
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INTRODUCTION
Two major families of ectonucleotidases, namely the ectoꢀnucleoside triphosphate
diphosphohydrolases (NTPDases) family and the ectoꢀnucleotide pyrophosphatase/phosꢀ
phodiesterase (NPPs) family terminate nucleotide signaling through the hydrolysis of
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1
, 2
nucleotide agonists of the P2X and P2Y receptors.
The NTPDase family consists of
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eight Ca /Mg ꢀdependent ectonucleotidases (NTPDase1ꢀ8) which hydrolyze
extracellular nucleosideꢀ5'ꢀtriphosphate and nucleosideꢀ5'ꢀdiphosphate, to nucleoside 5'ꢀ
1
3, 4
monophosphate. The NPP family consists of seven ectoꢀenzymes
which exist as
5
membrane glycoproteins with an extracellular divalentꢀion containing active site, yet, are
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also found extracellularly as secreted enzymes. NPP1ꢀ3, the only nucleotideꢀhydrolyzing
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members of this group, have a wide tissue distribution, and are capable of hydrolyzing
phosphodiester and pyrophosphate bonds of nucleotides. NPPs are more specific for
phosphodiester vs. phosphomonoester substrates. NPP1 and NPP3 both hydrolyze
7
extracellular nucleoside di/triphosphates derivatives. NPP1 can catalyze the hydrolysis
3
of ATP to either ADP and Pi or AMP and PPi. NPP2 has a much lower ATPase activity
than NPP1/3.
NPP1ꢀ3 can control P2 and P1 receptor signaling because their enzymatic action
results in the hydrolysis of signaling nucleotides and the generation of new messengers
6
like AMP, adenosine or pyrophosphate.
NPP1 is expressed in different tissues. It was reported to exist in bone
(osteoblasts) and cartilage (chondrocytes) and has a role in regulating mineralization
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processes. Extracellular pyrophosphate (ePPi), the product of ATP hydrolysis by NPP1,
is a likely source of inorganic phosphate to sustain hydroxyapatite (HA) formation when
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hydrolyzed by phosphatases, and is also a potent inhibitor preventing apatite mineral
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deposition and growth. The main hydrolyzing agent of PP within chondrocytes is tissue
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non specific alkaline phosphatase (TNAP). TNAP is a highly similar ectoenzyme to
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NPP1 containing a similar catalytic site and hydrolyzing its substrates in a similar
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mechanism. Although TNAP's main substrate within chondrocytes is PP , TNAP can
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also act as a phosphodiesterase and hydrolyze nucleoside–5’ꢀtriphosphates (NTPs).
Although PPi is required to prevent ectopic mineralization, its overproduction leads to
deposition of the pathological mineral calcium pyrophosphate dihydrate (CPPD), most
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often in the articular cartilage.
This condition, known as chondrocalcinosis,
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frequently accompanies ageꢀrelated osteoarthritis.
1
3
A role for NPP1 in insulin receptor signaling has also been proposed. Defective
insulinꢀstimulated insulin receptor autophosphorylation in type 2 diabetes patients was
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3,14ꢀ16
found to result from an overexpression of NPP1.
In addition, NPP1 was found in
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human astrocytic brain tumors and was correlated with tumor gradation.
The activity of NPP1 is endogenously regulated by AMP (i.e., product
8
inhibition). Yet, nonꢀendogenous NPP inhibitors have been scarcely reported. Recent
reports suggested that quinazolinꢀ4ꢀpiperidineꢀ4ꢀmethyl sulfamide is a NPP1 inhibitor
1
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lacking binding affinity for hERG, and that 1,3,4ꢀoxadiazole (thiadiazole)ꢀ2 (3H)ꢀ
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thiones are nonꢀcompetitive human NPP1 inhibitors with K values of 360 micromolar.
i
Previously, we reported that diꢀ2'ꢀdeoxyadenosineꢀα,βꢀδ,εꢀdimethyleneꢀpentaphosphonate
2
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(Figure 1A), is an NPP1 inhibitor with an IC of 13 ꢁM and a K of 9 ꢁM. In addition,
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we recently reported that adenosine 5’ꢀαꢀboraneꢀβ,γꢀmethylene triphosphate (isomer A)
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2
(
Figure 1B) is a selective inhibitor of NPP1 with a K of 0.5 ꢁM.
i
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Here, we continue our search for highly potent and selective NPP1 inhibitors.
Specifically, we describe the synthesis of a series of new ATP analogues bearing both
phosphonate and thiophosphate modifications, the evaluation of their activities as NPP1
inhibitors, and their selectivity for NPP1 vs. other ectonucleotidases and representative
adenine nucleotide binding receptors, P2Y1,2,11 receptors. Furthermore, we performed
docking simulations of the new analogues (Figure 2) to the catalytic site of a model of
human NPP1 and analyzed the resulting poses to deduce the interaction pattern with
binding site residues and the origin of NPP1 inhibitory activities.
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RESULTS
Design and synthesis of potential NPP1 inhibitors
NPPs hydrolyze P ꢀP phosphorodiester as well as P ꢀP phosphoroester bonds. In an
α
β
β
γ
attempt to confer hydrolytic stability and possibly inhibitory activity of nucleotide ligands
at NPP1, we have designed compounds 1 and 2 in which the P phosphate moiety was
α
modified to a thiophosphate group and a methylene or dichloromethylne group,
respectively, was introduced between P and P .
β
γ
In compound 3 we replaced the nucleotide bridging P ꢀP oxygen atom with a CH group
α
β
2
and modified the Pγ phosphate moiety to thiophosphate. The catalytic binding site of
2+
23
NPP1 was suggested to contain two Zn ions, although other reports suggested also
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activation by Mg and Ca ꢀions.
Hence, thiophosphate groups were designed to
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chelate the tentative Zn ions. For this reason, we further replaced H8 of the adenine
base in ATP by a thiol group, analogue 4.
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Synthesis of ATPꢀαꢀthioꢀβ,γꢀCX analogues 1ꢀ2
2
Adenosine 5'ꢀtriphosphateꢀαꢀthioꢀβ,γꢀCX analogues 1 and 2 were prepared from 2',3'ꢀ
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methoxymethylideneꢀadenosine, compound 5 (Scheme 1), to ensure a selective reaction
of adenosine at 5'ꢀOH. The 5'ꢀOH of nucleoside 5 was activated with 2ꢀchloroꢀ4Hꢀ1,3,2ꢀ
benzodioxaphosphorinꢀ4ꢀone to give compound 6 which was condensed with
bis(tributylammonium) methylenediphosphonate or dichloromethylenediphosphonate to
form the cyclic triphosphate intermediates, compound 7 (Scheme 1). Treatment of
compound 7 with powdered sulfur yielded, after hydrolysis with triethylammonium
bicarbonate (TEAB) buffer, analogues 1 or 2 in moderate yields (9ꢀ17%).
1
31
The identity and purity of the products were established by H and P NMR, ESI
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HR mass spectrometry, and HPLC in two solvent systems. The P NMR spectra of
analogues 1 and 2 showed a typical Pα signal as a doublet at about 43 ppm (J = 34 Hz).
1
The H NMR spectrum of analogue 1 showed methylene hydrogen atoms as a triplet at
about 2.3 ppm (J = 20 Hz). Due to the chiral center at Pα, analogues 1 and 2 were each
1
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obtained as a pair of diastereoisomers in a 1:1 ratio. In both H and P NMR spectra,
there was a slight difference between the chemical shifts of the diastereoisomers of
analogues 1 and 2. For instance, diastereoisomers 1a,b (Figure 3), yielded two sets of
signals for H8, at 8.67 and 8.62 ppm. These isomers were well separated by reverseꢀ
phase HPLC with about 6ꢀ10 min difference in their retention times. The first and second
eluting isomers were designated as isomers A and B, respectively.
Assignment of the absolute configurations of 1a,b and 2a,b diastereoisomers
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We employed H NMR spectroscopy to elucidate the absolute configuration at Pα of 1a,b
and 2a,b diastereoisomers. A difference in the chemical shift of H8 was observed
between the two diastereoisomers of ATPꢀβ,γꢀmethyleneꢀαꢀS. The signal of H8 of isomer
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A in H NMR spectrum was more shielded than that of the H8 of isomer B possibly due
to the effect of the proximal negatively charged Pα group of thiophosphate (8.62 vs 8.67
ppm). Pα is much further away from H8 in isomer B than in isomer A (Figure 3). Thus,
the Sp configuration may be attributed to isomer A, analogue 1a, and the Rp
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configuration to isomer B, analogue 1b.
Synthesis of Pγꢀthioꢀα,βꢀCH ATP, analogue 3, and 8ꢀSHꢀATP, analogue 4
2
Adenosine 5'ꢀtriphosphateꢀα,βꢀCH ꢀγꢀthio, analogue 3, was prepared in several steps from
2
protected adenosine, compound 5, (Scheme 2). 5'ꢀOH of 5 was activated with tosyl
chloride and a catalytic amount of dimethylamino pyridine. Adenosine 2',3'ꢀmethoxy
methylideneꢀ5'ꢀtosyl, compound 8, was phosphorylated with methylene diphosphonate
28
bis(tributylammonium) salt to form 5'ꢀadenosine diphosphonate compound 9. Next, the
protecting group was removed with 10% HCl and then with 10% NH OH. Activation of
4
compound 10 with carbonyl diimidazole (CDI) for 4ꢀ5
h
resulted in
phosphoroimidazolide intermediate, the yield of which was ca. 50% by NMR.
Subsequently, MeOH was added to decompose the excess CDI, followed by addition of
+
+
thiophosphate (Bu NH) /(Oct NH) salt and ZnCl . The latter was added to protect the
3
3
2
thiophosphate sulfur atom from reaction with phosphorimidazolide. Finally, compound
1 was obtained in 29% yield after LC separation.
1
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The P NMR spectrum of analogue 3 showed a typical Pγ signal as a doublet at about 39
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ppm (J = 32 Hz). The H NMR spectrum of analogue 3 showed methylene hydrogen
atoms as a triplet at about 2.5 ppm (J = 20 Hz).
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Analogue 4, was obtained in two steps from 8ꢀBrꢀadenosine (Scheme 3). 8ꢀMercaptoꢀ
adenosine, obtained in a quantitative yield from 8ꢀBrꢀadenosine upon treatment with 10
equivalents of NaSH in wet DMF at 100 °C overnight, was 5'ꢀtriphosphorylated first by
addition of POCl in the presence of proton sponge in TMP for 3 h and then, by the
3
addition of pyrophosphate in DMF for 2 h at ꢀ15°C, to give analogue nucleotide 4 at a
6
0% yield.
Analogues 1ꢀ3 are not substrates of NTPDase1, ꢀ2, ꢀ3, ꢀ8, NPP1, ꢀ3, or TNAP
Experiments were conducted with protein extracts from COSꢀ7 cells transfected
separately with an expression vector encoding each ectonucleotidase i.e., NPP1, NPP3
and NTPDase1, ꢀ2, ꢀ3, and ꢀ8. The protein extracts of nonꢀtransfected COSꢀ7 cells
exhibited less than 5% NTPDase or NPP activity compared with COSꢀ7 cells transfected
with NTPDases or NPPs, thus allowing the analysis of each ectonucleotidase in its native
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membrane bound form.
Analogues 1ꢀ3 (100 µM, n=3) were stable to hydrolysis by NTPDase1, ꢀ2, ꢀ3 and ꢀ8 when
compared to ATP (4.4ꢀ5.5% hydrolysis over 1 h, Table 1). Analogues 2 and 3 (100 µM)
also were neither catabolized by NPP1 nor by NPP3. Analogue 1a (100 µM) was fully
stable to NPP1 hydrolysis and was hardly hydrolysed by NPP3 over 1h (~1%) compared
to the physiological substrate ATP. Analogue 1b was weakly hydrolyzed by both NPP1
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and NPP3 (~4%). Analogue 4 was hydrolyzed by NPP1 and NPP3 at 32ꢀ54% over 1 h
and therefore, was not studied further as an inhibitor of these enzymes.
In addition, the metabolic stability of the promising candidates, 2a and 3, was further
proven by their resistance to enzymatic hydrolysis by TNAP. Compound 2a was fully
stable to TNAP hydrolysis during 1 h vs. 100% hydrolysis of ATP, and 3 was negligibly
hydrolysed (2.5%) (data not shown).
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Analogues 1ꢀ3 are selective inhibitors of NPP1
The effect of analogues 1ꢀ3 on NPP and NTPDase activities and their selectivity were
tested using a synthetic substrate (pNPꢀTMP) as well as a natural substrate (ATP),
respectively. Analogues 1ꢀ3 at (100 ꢁM, n=3) effectively inhibited pNPꢀTMP (100 ꢁM)
hydrolysis by NPP1 by over 90% (Figure 4A). The hydrolysis of the physiological
substrate ATP by NPP1 was inhibited more potently by analogues 2a and 3 vs. 1a/b and
2
b (Figure 4B). Similar inhibition was observed when osteocarcinoma cells (HTB85
cells, also known as SaOS 2) were used as a native source of NPP1 (Figure 4C).
Analogues 1ꢀ3, at 100 ꢁM, were NPP1selective inhibitors, since they inhibited NPP3
activity by only 23ꢀ43% (Figure 4A, B).
Analogues 1a, 1b, 2a and 2b did not interfere with the hydrolysis of ATP by human
NTPDase1, ꢀ2, ꢀ3 and ꢀ8 (Table 2). Analogue 3 at 100 µM inhibited human NTPDase1
and ꢀ3 by 60% and 40%, respectively (Table 2). Both compounds 2a and 3 at 100 ꢁM
showed low inhibition of TNAP, 17% and 8% respectively.
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We have estimated IC (a parameter that shows the ability of a molecule to inhibit an
5
0
enzyme under specific conditions and substrate concentration), and inhibition constant Ki
(a kinetic parameter that represents an absolute value for each tested inhibitor) towards
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NPP1. The kinetic parameters indicate that analogue 3 is the most potent inhibitor of
NPP1 with K value of 20 nM (Table 3, Figure 5). Analogue 2a was also a good NPP1
i
inhibitor exhibiting a K value of 685 nM. Under these experimental conditions the IC s
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of both analogues were also the lowest being 390 nM and 600 nM for 3 and 2a,
respectively (Table 3). Using the methods of Dixon (Figure 5A and Table 3) and Cornishꢀ
Bowden (Figure 5B and Table 3) that estimate dissociation constant for EIS complex
(
K ’), we determined that the inhibitors 1bꢀ3 presented in Table 3, showed mixed type
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inhibition, predominantly competitive.
Evaluation of analogues 1ꢀ3 as agonists of P2Y , P2Y , and P2Y receptors
1
2
11
Adenine nucleotides are not only substrates of ectonucleotidases but are also potent
33
agonists at P2Y , P2Y , and P2Y Gꢀproteinꢀcoupled receptors. These receptors are
1
2
11
expressed in many tissues and are involved in many diseases. Of relevance to the current
study is the expression of P2Y receptors in bone tissues. P2Y receptors are expressed in
1
30
osteoclasts and osteoblasts, P2Y receptors are expressed in osteobloasts where they
2
3
4
block bone formation and the P2Y11 receptors are expressed in human osteoclast
35
cultures. This expression pattern may lead to selectivity problems for inhibitors if they
act as P2YR agonists.
With this in mind, we evaluated the activity of NPP1 inhibitors, analogues 1ꢀ3, at
P2Y1,2,11 receptors. GFP (green fluorescent protein) constructs of human P2Y , P2Y and
1
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P2Y receptors were expressed in 1321N1 astrocytoma cells, which lack endogenous
1
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6
expression of P2X and P2Y receptors. These cells were then incubated with various
2
+
concentrations of analogues 1ꢀ3. The Ca responses to analogues 1ꢀ3 were compared
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with ATP. Analogues 1ꢀ3 were weak agonists (EC50 > 10 ꢁM) of the P2Y receptor
1
(Table 4). Analogues 1a and 2a were not agonists of the P2Y ꢀreceptor. In contrast,
1
1
analogues 1b and 3 were 7ꢀfold more potent than the endogenous P2Y ꢀ receptor ligand,
1
1
ATP (EC 6.7 ꢁM) (Figure 6). In addition, analogue 2b was a P2Y ꢀ receptor agonist
50
11
(2ꢀfold more active than ATP). Analogues 2a and 3 (100 ꢁM) had no activity, as agonists
at P2Y R. These analogues did not inhibit the typical response to UTP observed for the
2
P2Y R in 1321N1 cells (data not shown).
2
Docking simulations at human NPP1
Docking simulations were used in order to provide insight into the inhibitory activities of
the analogues studied in this work. We have previously reported a model of human NPP1
22
based on the recently solved structure of mouse NPP1 and demonstrated that docking
simulations using Glide were able to successfully reproduce the crystallographic pose of
AMP in this structure with a RMSD of 0.73 Å and to provide plausible binding mode for
ATP in which it is stabilized within the binding site through an array of aromatic,
hydrophobic, and Hꢀbond interactions with binding site residues. These previous docking
simulations suggest that Glide is a suitable docking tool for this system.
Analogues 1ꢀ3 were docked into the NPP1 site and their representative poses were
analyzed. These were obtained by first clustering all poses obtained by the docking
simulations within the binding site and then by selecting the pose which is closest to the
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center of the largest cluster. The representative poses of all five analogues (1ꢀ3) were
found to adopt ATPꢀlike conformations, suggesting that they could compete with ATP for
binding site interactions (Figure 7, Table S1). Furthermore, similar to ATP, all five
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analogues coordinate Zn1 through their P oxygen atom (although other chelation
α
2
patterns were also observed). An excellent correlation (R = 0.95; Figure 8a) was found
between Boltzmann averaged Glide scores of the largest cluster and between
experimental ꢂꢂG values (calculated from K values at the experimental temperature of
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3
7°C using analogue 3 as a reference) for all five analogues considered in this work.
Furthermore, this correlation was largely maintained when only the representative poses
2
from each cluster were considered (R = 0.8; Figure 8b). In addition, a very good
2
correlation (R = 0.95; Figure 8c) was found between the analogues' activities and the
hydrogen bond term which forms part of Glide's scoring function. A detailed analysis of
the interaction patterns of the representative poses of all analogues with binding site
residues is provided in Table S1.
DISCUSSION
A series of ATP analogues modified at the P /P /P positions by bridging methylene and
α
β
γ
thiophosphate moieties (analogues 1ꢀ3) or by including a 8ꢀSH group (analogue 4) was
designed and synthesized to identify potent and selective NPP1 inhibitors. Analogue 4
was hydrolyzed by NPP1 and NPP3 at about 50% the rate of ATP (Table 1), and
therefore it could not serve as a good NPP inhibitor. Of the remaining compounds, ATPꢀ
α,βꢀCH ꢀγꢀS, 3, was found to be an extremely potent NPP1 inhibitor (K 20 nM, Figure 5,
2
i
Table 3) followed by ATPꢀαꢀSꢀβ,γꢀCCl , 2a (K 685 nM, Figure 5, Table 3), while the
2
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other three analogues were less active (1b, K 1.3 ꢀM; 1a, K 4.5 ꢀM; 2b, K 15 ꢀM,
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Table 3).
In order to provide structural insight into these results as well as tools for the design of
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yet more potent NPP1 inhibitors, analogues 1ꢀ3 were docked into the binding site of our
22
previously reported NPP1 model. Similar to other nonꢀhydrolysable ATP derivatives
22
studied by us in the past, the representative poses of all five analogues (1a, b, 2a, b and
3
) adopt ATPꢀlike conformations, suggesting that they could effectively compete with
ATP for binding site interactions (Figure 7, Table S1). Furthermore, similar to ATP, all
simulated analogues coordinate the Zn1 through their P oxygen atom (although other
α
chelation patterns were also observed). While zinc is known to be a "thiophilic" ion, our
docking results demonstrate preference for zinc chelation through the nonꢀbridging
oxygen atom of the phosphate chain rather than through the sulfur atom. Yet, we argue
that this nonꢀtypical chelation pattern may be the consequence of binding site constraints.
With this in mind we have searched the PDB for protein complexes where a ligand
containing both oxygen and sulfur atoms chelates a zinc ion. Twenty four such
complexes were found out of which seven demonstrated OꢀZn chelation. Interestingly,
out of this list of ligands, the two ligands most similar to analogues 1ꢀ3, (ATPꢀγꢀS, PDB
code 3ZEU and a phosphorothioate derivative, PDB code 1KRP) show zinc chelation via
2+
oxygen. In the present study, the potential role of the binding site in controlling Zn ꢀ
chelation pattern is nicely demonstrated for the most active compound, analogue 3. As
the data in Table S1 clearly indicate, zinc chelation via the P sulfur atom, rather than via
γ
Pα oxygen atom, results in loss of important interactions with binding site residues
including hydrogen bonds with Thr256, Lys295, and Tyr340 and an important salt bridge
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with Lys255. These findings suggest that the role of the sulfur atom may not be to
enhance binding by providing better chelation to the zinc ion but rather by affecting the
degree of ionization of the terminal phosphate. Indeed the pK of thiophosphate is 5.5
a
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whereas that of phosphate is 6.5.
The docking results were analyzed using a Boltzmann averaging of glide scores over the
2
largest cluster of each analogue and resulted in an excellent correlation (R = 0.95; Figure
8
a) with experimental ꢂꢂG values calculated from the kinetic parameters (K s). This
i
correlation was largely maintained when considering only a single (representative)
2
structure pose for each cluster (R = 0.8; Figure 8b). The representative poses of all
ligands considered in this work are given in Figure 7 and an analysis of their interaction
patterns with binding site residues as well as key Glide scores (total score, Hꢀbond score
and electrostatic score) are given in Table S1. In particular, the hydrogen bond
2
component of the scoring function shows an excellent correlation (R = 0.95) with the
experimental data (Figure 8c). This is reflected in the hydrogen bonding and salt bridge
patterns of the representative poses of the different ligands (Figure 7 and Table S1). Thus,
while the most active compound 3 is hydrogen bonded to five bindingꢀsite residues
(Thr256x2, Asn277, Lys295, and Tyr340), and forms a salt bridge with Lys255, the less
active analogues have fewer Hꢀbonds (2a: two Hꢀbonds with Asn277 and Tyr340; 1b:
two Hꢀbonds with Asn277 and Lys295; 1a: one Hꢀbond with Asn277) while maintaining
the salt bridge with Lys255, and the least active analogue has only two Hꢀbonds and no
salt bridge (2b: two Hꢀbonds with Asn277 and Lys295). In addition, analogue 3 features
the most favorable electrostatic energy component of the Glide score (ꢀ65.7 kcal/mol)
although no clear correlation is observed between this score and the binding affinities
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(
Table 4 and S1). Presumably, for 1b vs. 2b the introduction of the bulkier chlorine atoms
shifts the position of the 2b analogue within the binding site leading to the loss of the
important electrostatic interaction with Lys255.
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Analogues 2a (the second most active compound) and 2b (the least active compound)
differ only in the chirality at P (S and R, respectively). While this reversed chirality is
α
reflected in the more favorable Hꢀbond and salt bridge pattern of the more active
compound (see above), it also leads to different exposures of polar surface areas to the
solvent. As the data in Figure 9 indicate, analogue 2a exposes a more polar "face" to the
solvent than analogue 2b. This in turn may lead to more favorable ligandꢀsolvent
interactions for this analogue and consequently to a stronger protein binding.
A detailed analysis of the representative poses of the five analogues does not provide a
clear interpretation as to the role of the two chlorine atoms since they are not involved in
specific interactions with binding site residues. These atoms were originally introduced
into the ATP skeleton in order to reduce the pK of the terminal oxygen of the
a
phosphonate moiety bringing it closer to that of phosphate (the pK of phosphonate,
a
3
8, 39
dichlorophosphonate and phosphate are 8.4, 6.7ꢀ7.0, and 6.5, respectively),
making it
a more potent inhibitor. This was indeed the case for analogues 1a and 2a (4.5 and 0.685
µM, respectively, Table 3) but not for analogues 1b and 2b (1.3 and 15.2 µM,
respectively, Table 3).
Analogues 1ꢀ3 at 100 ꢀM inhibited selectively NPP1 vs. NPP3 (Figure 4A), inhibiting
the former by 90ꢀ100%, and the latter by only 23ꢀ43%, using pNPꢀTMP as the substrate.
Analogues 2a and 3 at 100 ꢀM inhibited about 90% of ATP hydrolysis by NPP1 (Figure
4B). The results were confirmed by assays with osteocarcinoma cells (Figure 4C).
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Analogues 2a and 3 exhibited Ki and IC values of 685 nM and 600 nM, and 20 nM and
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90 nM, respectively. IC values that do not parallel Ki values may perhaps result from
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conditions that may influence the type of observed inhibition (see Fig. 7D, F and Table
3).
2
2
As previously reported, the preferred binding of all analogues to NPP1 rather than to
NPP3, could be readily explained by comparing the binding sites of the two proteins. In
NPP1, a unique arrangement of Lys residues (seven in total) which is absent in NPP3 (a
single binding site Lys residue), results in a highly positive electrostatic potential which
renders this site more suitable for binding negatively charged nucleotides.
Analogues 1ꢀ2 at 100 ꢀM had a minor inhibitory effect on NTPDase1, ꢀ2, ꢀ3, ꢀ8 activity
(0ꢀ25% inhibition) (Table 2). In contrast, analogue 3 inhibited NTPDase1 by 60% and
NTPDase3 by 40%. In addition, the two most promising candidates 2a and 3 had a
limited effect on TNAP activity showing minor inhibition (8ꢀ17%).
As adenine nucleotides are also the endogenous agonists of P2Y1,2,11 receptors, we tested
40, 41
analogues 1ꢀ3 at these receptors involved in numerous physiological activities.
Compounds 1a, b did not activate the P2Y receptor, consistent with earlier reports that a
1
methylene modification tends to reduce agonist potency at P2Y receptors as compared to
4
2ꢀ44
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ATP.
Both ATP (pEC = 4.77) and ATPꢀγꢀS (pEC 5.52) activate P2Y receptor
50
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11
30
with ATPꢀγꢀS being the most potent agonist of the two. Consistent with this, ATPꢀα,βꢀ
CH ꢀγꢀthio, 3, was found here to be ~7ꢀfold more potent than ATP at this receptor. This
2
finding is also in agreement with our previously reported model of hP2Y ꢀR and its
1
1
preference for ATPꢀγꢀS over ATP binding as a result of the tighter fit of the larger P ꢀS
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moiety. Adenosine 5'ꢀtriphosphateꢀαꢀthioꢀβ,γꢀCH isomer B, 1b, and adenosine 5'ꢀ
2
triphosphateꢀαꢀthioꢀβ,γꢀCCl isomer B, 2b, showed ~7ꢀ and ~2ꢀfold, respectively, higher
2
potency than ATP at the P2Y11 receptor. The respective isomers A, 1a and 2a were
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inactive at the P2Y receptor.
1
1
4
7
This is in accordance with our previous findings reporting that the P2Y receptor
1
1
prefers isomer B of Pαꢀsubstituted ATP derivatives over isomers A. Recently we found
4
8
that adenosine 5'ꢀtriphosphate 2ꢀpropylthioꢀαꢀthioꢀβ,γꢀCCl , isomers A and B, showed
2
~3ꢀ and ~4 fold, respectively, higher potency than ATP at the P2Y11 receptor. In
accordance with our findings presented here, the αꢀthioꢀβ,γꢀCCl ꢀsubstituted ATP
2
derivatives were very weak agonists at the P2Y receptor or not active at the P2Y
2
1
,
receptor. The nucleotides used in the present study lack the 2ꢀpropylthio modification of
48
potent P2Y R agonists, but have the αꢀthio β,γꢀCCl – substitution. Taken together,
11
2
these results suggest that the αꢀthio β,γꢀCCl – substitution generally increases the affinity
2
and selectivity of ATP derivatives at the P2Y receptor.
1
1
Siteꢀdirected mutagenesis and computational analysis identified some amino acid
residues responsible for the ligand recognition of the P2Y and P2Y receptor, as
1
11
2
7, 46, 49, 50
reported before.
However, only little is known about the molecular structure of
the binding pocket of all the P2Y receptors. Even though they belong to the same
phylogenetic family, P2Y receptor subtypes show significant differences in amino acid
51, 52
sequences.
P2Y , P2Y , and P2Y receptors differ in their preference for the natural
1 2 11
5
3
agonists. Therefore, different EC values are expected for the nucleotide derivatives
50
tested here (Table 4).
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The newly identified NPP1 inhibitors were found to be highly potent, as compared to
currently available inhibitors. Specifically, analogues 2a and 3 were 525ꢀfold and 18,000ꢀ
fold, respectively, more potent human NPP1 inhibitors than the recently reported [4ꢀ(tꢀ
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butyldimethylsilyloxy)ꢀphenyl]ꢀ1,3,4ꢀoxadiazoleꢀ2 (3H)ꢀthione. Likewise, analogue 3
was found to be 405ꢀfold and 25ꢀfold more potent than our previously reported NPP1
21
inhibitors ꢀ diꢀ2'ꢀdeoxyadenosineꢀα,βꢀδ,εꢀdimethyleneꢀpentaphosphonate (Figure 1)
4
4
and adenosineꢀ5'ꢀOꢀ(αꢀboranotriphosphate)ꢀβ,γꢀCH (K 0.5 ꢁM; Figure 2), respectively.
2
i
The greater activity of analogue 3 relative to adenosineꢀ5'ꢀOꢀ(αꢀboranotriphosphate)ꢀβ,γꢀ
2+
CH could possibly be attributed to its ability to coordinate the M (Zn1) ion through its
2
Pα nonꢀbridging oxygen atom. Based on docking results, such coordination is not possible
for the borano compound since in this compound, the nonꢀchelating borano group at Pα
2
2
points towards the Zn1 ion.
The kinetic data presented in this work coupled with the structural insight into the origin
of the analogues’ activities available from the docking simulations suggest that analogues
3 and 2a, together with the NPP1 and NPP3 models, are good starting points for the
design of efficacious and selective NPP1 inhibitors. Nevertheless, being ATPꢀbased,
these analogues are not classical "drugꢀlike" compounds. Yet, related compounds such as
thiazoleꢀ4ꢀcarboxamide adenine dinucleotide and Denufosol have found their way into
54, 55
clinical trials.
Developing these compounds into drugs may require proꢀdrug
5
5
approaches, appropriate formulations and/or administration modes other than oral.
However, even if these compounds will not be eventually developed into drugs they are
still likely to serve as important mechanistic tools for the study of the complex process of
mineralization.
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Experimental
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General. All commercial reagents were used without further purification, unless
otherwise noted. All airꢀ and moisture sensitive reactions were conducted in flameꢀdried,
nitrogenꢀflushed, twoꢀneck flasks sealed with rubber septa, and the reagents were
introduced with a syringe. Progress of reactions was monitored by TLC using precoated
Merck silica gel plates (60Fꢀ253). Reactants and products were visualized using UV
light. Compounds were characterized by NMR using a Bruker ACꢀ200, DPXꢀ300, or
1
DMXꢀ600 spectrometer. H NMR spectra were recorded at 200, 300, or 600 MHz.
31
Nucleotides were characterized also by P NMR in D O using 85% H PO as an external
2
3
4
reference on Bruker ACꢀ200 and DMXꢀ600 spectrometers. Highꢀresolution mass spectra
were recorded on an AutoSpecꢀE FISION VG mass spectrometer. Nucleotides were
analyzed using electron spray ionization (ESI) on a QꢀTOF microinstrument (Waters).
Primary purification of the nucleotides was achieved on a LC (Isco UAꢀ6) system using a
°
column of Sephadex DEAEꢀA25, swollen in 1 M NaHCO at 8 C for 24 h. The resin was
3
washed with deionized water before use. LC separation was monitored by UV detection
at 280 nm. Final purification of the nucleotides was achieved on a HPLC (MerckꢀHitachi)
system using a semipreparative reverse phase column [Gemini 5u Cꢀ18 110A, 250 mm X
1
0 mm, 5 ꢁm (Phenomenex, Torrance, CA)]. The purity of the nucleotides was evaluated
on an analytical reverseꢀphase HPLC column system [Gemini 5u Cꢀ18 110A, 150 mm X
3
.60 mm, 5 ꢁm (Phenomenex)] in twoꢀsolvent systems with either solvent system I or II.
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6
Solvent system I consisted of (A) 100 mM triethylammonium acetate (TEAA) (pH 7) and
(
B) CH CN. Solvent system II consisted of (A) 46 mM PBS (pH 7.4) and (B) CH CN.
3
3
The details of the solvent system gradients used for the separation of each product are
provided below. The products, obtained as triethylammonium salts, were generally ≥95%
pure. All reactants for moistureꢀsensitive reactions were dried overnight in a vacuum
0
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6
oven. 2',3'ꢀOꢀMethoxymethylidene adenosine, compound 5, 5’ꢀtosylꢀ adenosine,
5
6
56
56
compund 8, α,βꢀmethyleneꢀADP, compound 9, and 8ꢀSHꢀATP, compound 4, were
prepared as previously described.
Adenosine 5'ꢀPαꢀthioꢀβ,γꢀ(methylene)ꢀtriphosphate (analogues 1a,b). A solution of 2ꢀ
chloroꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ4ꢀone (98 mg, 0.48 mmol) in anhydrous DMF
(0.75 mL), was added via syringe to a solution of 2',3'ꢀorthoformate protected adenosine
(compound 5) (100 mg, 0.32 mmol) and anhydrous pyridine (250 ꢁL) in 0.5 mL of
◦
anhydrous DMF at 0 C under nitrogen. After stirring at RT for 1 h, tributylamine (500
ꢁ
L) was added, followed by
a
solution of bisꢀ(tetrabutylammonium)
methylenediphosphonate (68 mg, 0.38 mmol) in anhydrous DMF (0.5 mL). The reaction
◦
mixture was stirred at RT for 2 h then sulfur (21 mg, 0.64 mmol) was added at 0 C. The
solution color changed to orange and afterwards to brown. After stirring at room
temperature for 1.5 h, the mixture was dripped to a cold 1 M TEAB solution (10 mL)
until pH~7 was attained. The resulting mixture was stirred at room temperature for 30
min. During that time the color of the solution changed to yellow. The solution was
extracted (2 X 10 mL) with ether. The aqueous phase was freezeꢀdried twice. The product
was then deprotected by addition of 10% HCl until pH 2.3 was obtained and the mixture
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was stirred for 3 h. Afterwards 24% NH OH was added to give pH~9 and the mixture
4
was stirred for another 45 min and freezeꢀdried overnight.
The crude residue was separated on a DEAEꢀSephadex A25 column with a linear
gradient of ammonium bicarbonate (from 0.1 M to 0.4 M ammonium bicarbonate. Total
gradient volume ꢀ 600 mL). The relevant fraction was freezeꢀdried for four times to
afford 20 mg (9% yield) of adenosine 5'ꢀPαꢀthioꢀβ,γꢀ(methylene)triphosphate ammonium
salt. Final seperation of analogues 1a, b (two diastereoisomers) was carried out by HPLC
10
11
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13
14
15
16
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18
19
20
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22
23
24
25
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29
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on a semipreparative reverseꢀphase column with a TEAA/CH CN gradient from 96.6:3.4
3
to 95:5 over 22 min at a flow rate of 4.5 mL/min: retention time (t ) (isomer 1a) = 15.8
R
1
min, t (isomer 1b) = 21.4 min. Isomer 1a: H NMR (D O, 200 MHz) δ 8.62 (s, Hꢀ8,
R
2
1H), 8.27 (s, Hꢀ2, 1H), 6.15 (d, J = 6.0 Hz, Hꢀ1', 1H), 5.00 (m, H2', 1H), 4.60 (m, Hꢀ3',
31
1
H), 4.42 (m, Hꢀ4', 1H), 4.28 (m, Hꢀ5' , 2H), 2.28 (t, J = 20.0 Hz, CH , 2H) ppm;
P
2
NMR (D O, 81 MHz) δ 42.9 (d, J = 33.5 Hz, PαꢀS, 1P), 13.0 (br m, Pγ and Pβ, 2P) ppm;
2
TLC (2:7:11 NH OH/H O/2ꢀpropanol) R = 0.22. The following purity data were
4
2
f
obtained on an analytical column: t = 6.48 min (96% purity) using solvent system I with
R
a TEAA/CH CN isocratic elution 95:5 over 10 min at a flow rate of 1 mL/min; t = 2.94
3
R
min (95% purity) using solvent system II with a PBS/CH CN isocratic elution 98:2 over
3
1
8
min at a flow rate of 1 mL/min. Isomer 1b: H NMR (D O, 200 MHz) δ 8.67 (s, Hꢀ8,
2
1H), 8.25 (s, Hꢀ2, 1H), 6.15 (d, J = 6.0 Hz, Hꢀ1', 1H), 5.00 (m, H2', 1H), 4.59 (m, Hꢀ3',
31
1
H), 4.41 (m, Hꢀ4', 1H), 4.28 (m, Hꢀ5' , 2H), 2.31 (t, J = 21.0 Hz, CH , 2H) ppm;
P
2
NMR (D O, 81 MHz) δ 43.2 (d, J = 32.4 Hz, PαꢀS, 1P), 13.2 (br s, Pγ, 1P), 11.9 (br d, Pβ,
2
ꢀ
J = 32.4 Hz, 1P) ppm; HRMS ESI (negative) m/z: calculated for C H N O P S
1
1
17
5
11 3
5
19.9864, found 519.9853, low resolution mass was measured for both isomers
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andHRMS was measured for one of the isomers; TLC (2:7:11 NH OH/H O/2ꢀpropanol)
4
2
R = 0.22. The following purity data were obtained on an analytical column: t = 9.12 min
f
R
(
97% purity) using solvent system I with a TEAA/CH CN isocratic elution 95:5 over 15
3
0
1
2
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4
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0
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9
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2
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4
5
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9
0
min at a flow rate of 1 mL/min; t = 4.17 min (97% purity) using solvent system II with a
R
PBS/CH CN isocratic elution 98:2 over 10 min at a flow rate of 1 mL/min.
3
Adenosine 5'ꢀPαꢀthioꢀβ,γꢀ(dichloromethylene)triphosphate (analogues 2a, b)
A solution of 2ꢀchloroꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ4ꢀone (117 mg, 0.58 mmol) in
anhydrous DMF (1 mL), was added via syringe to a solution of 2',3'ꢀorthoformate
protected adenosine (compound 5) (100 mg, 0.32 mmol) and anhydrous pyridine (260
◦
ꢁ
L) in 1.5 mL of anhydrous DMF at 0 C under nitrogen. After stirring at RT for 1 h,
tributylamine (626 ꢁL) was added, followed by a solution of bisꢀ(tetrabutylammonium)
dichloromethylendiphosphonate (127 mg, 0.42 mmol) in anhydrous DMF (1 mL). The
reaction mixture was stirred at RT for 2 h then sulfur (25 mg, 0.77 mmol) was added at 0
◦
C. The solution color changed to orange and then to brown while stirring at RT for 1.5 h.
The mixture was dripped to a cold 1 M TEAB solution (10 mL) until pH~7 was attained.
The resulting mixture was stirred at RT for 30 min. During that time the color of the
solution changed to yellow. The solution was extracted with ether (2 X 10 mL). The
aqueous phase was freezeꢀdried twice. The product was then deprotected by addition of
10% HCl until pH 2.3 was obtained and the mixture was stirred for 3 h. Afterwards 24%
NH OH was added to give pH~9 and the mixture was stirred for another 45 min and
4
freezeꢀdried overnight.
The crude residue was separated on a DEAEꢀSephadex A25 column with a linear
gradient of ammonium bicarbonate (from 0.1 M to 0.4 M ammonium bicarbonate. Total
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gradient volume ꢀ 600 mL). The relevant fraction was freezeꢀdried for four times to
afford 43 mg (17% yield) of adenosine 5'ꢀPαꢀthioꢀβ,γꢀ(dichloromethylene)triphosphate
ammonium salt. Final separation of analogues 2a,b (two diastereoisomers) was carried
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out by HPLC on a semipreparative reverseꢀphase column with a TEAA/CH CN gradient
3
from 96.5:3.5 to 95.5:4.5 over 31 min at a flow rate of 4.5 mL/min: t (isomer 2a) = 20.3
R
1
min, t (isomer 2b) = 30.6 min. Isomer 2a: H NMR (D O, 200 MHz) δ 8.72 (s, Hꢀ8,
R
2
1
H), 8.29 (s, Hꢀ2, 1H), 6.16 (d, J= 6.0 Hz, Hꢀ1', 1H), (H2' and Hꢀ3' signals are hidden by
the water signal at 4.78), 4.63 (m, Hꢀ4', 1H), 4.35 (m, Hꢀ5' , 2H), 3.35 (t, Et N , 24H),
3
31
2
.05 (s, AcOH , H), 1.42 (d, Et N , 36H) c; P NMR (D O, 81 MHz) δ 44.0 (d, J = 35.4
3
2
Hz, PαꢀS, 1P), 8.0 (d, J = 19.3 Hz, Pγ, 1P), ꢀ1.0 (dd, J = 19.3 Hz, J = 35.4 Hz, Pβ, 1P)
ꢀ
ppm; HRMS ESI (negative) m/z: C H Cl N O P S calculated 587.9084, found
11
15
2
5
11 3
5
87.9073, low resolution mass was measured for both isomers and HRMS was measured
for one of the isomers; TLC (2:7:11 NH OH/H O/2ꢀpropanol) Rf = 0.35. The following
4
2
purity data were obtained on an analytical column: t = 9.5 min (99% purity) using
R
solvent system I with a TEAA/CH CN isocratic elution 96:4 over 15 min at a flow rate of
3
1 mL/min; t = 3.35 min (99% purity) using solvent system II with a PBS/CH CN
R
3
1
isocratic elution 98:2 over 8 min at a flow rate of 1 mL/min. Isomer 2b: H NMR (D O,
2
2
00 MHz) δ 8.65 (s, Hꢀ8, 1H), 8.29 (s, Hꢀ2, 1H), 6.16 (d, J= 6.2 Hz, Hꢀ1', 1H), (H2'
signal is hidden by the water signal at 4.78), 4.64 (m, Hꢀ3', 1H), 4.45 (m, Hꢀ4', 1H), 4.38
31
(
m, Hꢀ5' , 2H), 3.15 (t, Et N, 24H), 2.05 (s, CH CO H, 3H), 1.38 (d, Et N, 36H) ppm;
3 3 2 3
P NMR (D O, 81 MHz) δ 43.9 (d, J = 35.5 Hz, PαꢀS, 1P), 8.00 (d, J = 19.1 Hz, Pγ, 1P), ꢀ
2
0
.9 (dd, J = 19.1 Hz, J = 35.5 Hz, Pβ, 1P) ppm; TLC (2:7:11 NH OH/H O/2ꢀpropanol) R
4 2 f
=
0.36. The following purity data were obtained on an analytical column: t = 10.0 min
R
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(
98% purity) using solvent system I with a TEAA/CH CN isocratic elution 95:5 over 15
3
min at a flow rate of 1 mL/min; t = 4.54 min (98% purity) using solvent system II with a
R
PBS/CH CN isocratic elution 98:2 over 10 min at a flow rate of 1 mL/min.
3
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Adenosine 5' ꢀα,βꢀmethyleneꢀγꢀthioꢀtriphosphate (analogue 3). α,βꢀMethyleneꢀ
+
+
+
+
ADP(Bu NH) /(Oct NH) and thiophosphate(Bu NH) /(Oct NH) salts were prepared by
3
3
3
3
applying the corresponding phosphate deriviatives through a column of activated Dowex
+
5
0WXꢀ8 (200 mesh, H form). The eluate was collected in an iceꢀcooled flask containing
tributylamine, triꢀnꢀoctylammine (1 eq) and EtOH. The resulting solution was freezeꢀ
+
+
dried
to
yield
α,βꢀmethyleneꢀADP(Bu NH) /(Oct NH)
and
3
3
+
+
thiophosphate(Bu NH) /(Oct NH) salts, each as a viscous oil.
3
3
Adenosineꢀ5'ꢀ α,βꢀmethylene diphosphate tributylammonium and triꢀnꢀoctylammonium
salt (75 mg, 0.17 mmol) were suspended in anhydrous DMF (2 mL), 1,1'ꢀ
carbonyldiimidazole (260 mg, 1.64 mmol) was added at RT and the mixture was stirred
for 5 h. Next, dry MeOH (66 ꢁL) was added and the reaction was stirred for 8 min,
followed by addition of anhydrous ZnCl (220 mg, 1.60 mmol) and thiophosphate
2
tributylammonium and triꢀnꢀoctylammonium salt (170 mg, 0.98 mmol) in dry DMF (1
mL) were added. After 3 h, the reaction was added to EDTA solution (580 mg, 1.5 mmol
in 20 mL of water), brought to pH~7 with triethylammonium bicarbonate and the solution
was freezeꢀdried overnight. The residue was separated on a DEAEꢀSephadex A25
column with an isocratic elution of ammonium bicarbonate (from 0 to 0.4 M ammonium
bicarbonate. Total gradient volume ꢀ 600 mL.). The solution was freezeꢀdried for four
times to afford adenosineꢀ5'ꢀtriphosphateꢀα,βꢀmethyleneꢀγꢀthiophosphate ammonium salt
(30 mg, 29% yield). Final purification of analogue 3 was carried out by HPLC on a
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semipreparative reverseꢀphase column, using isocratic elution of 4% CH CN, 96% TEAA
3
1
over 15 min at a flow rate of 4.5 mL/min: t = 10.25 min; H NMR (D O, 200 MHz) δ:
R
2
8
.58 (s, Hꢀ8, 1H), 8.26 (s, Hꢀ2, 1H), 6.09 (d, J= 5.8 Hz, Hꢀ1', 1H), 4.90 (m, H2', 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
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4
.56 (m, Hꢀ3', 1H), 4.36 (m, Hꢀ4', 1H), 4.20 (m, Hꢀ5' , 2H), 3.20 (t, Et N , 24H), 2.48 (t,
3
3
1
J=20.00 Hz, CH , 2H), 1.30 (d, Et N, 36H) ppm; P NMR (D O, 81 MHz) δ: 39.0 (d, J
2
3
2
=
32 Hz, PγꢀS, 1P), 18.2 (d, J = 9 Hz, Pα, 1P), 6.9 (dd, J = 9 Hz, J = 32 Hz, Pβ, 1P) ppm;
2
ꢀ
HRMS ESI (negative) m/z: C H N O P S calculated 519.9853, found 519.9820; TLC
11
17
5
11 3
(2:7:11 NH OH/H O/2ꢀpropanol) R = 0.30. The following purity data were obtained on
4
2
f
an analytical column: t = 5.23 min (90% purity) using solvent system I with a
R
TEAA/CH CN isocratic elution 96:4 over 10 min at a flow rate of 1 mL/min; t = 6.72
3
R
min (93% purity) using solvent system II with a PBS/CH CN isocratic elution 97:3 over
3
1
0 min at a flow rate of 1 mL/min.
Plasmids
The plasmids used in this study have all been described in published reports: human
57
58
NTPDase1 (GenBank accession no. U87967) , human NTPDase2 (NM_203368) ,
5
9
60
human NTPDase3 (AF033830) , human NTPDase8 (AY330313) , human NPP1
6
1
62
(NM_006208) and human NPP3 (NM_005021) .
Cell culture and transfection
Ectonucleotidases were expressed by transiently transfecting COSꢀ7 cells in 10 cm plates
63
by use of Lipofectamine (Invitrogen, Burlington, ON, Canada), as previously described.
Briefly, 80–90% confluent cells were incubated for 5 h at 37°C in Dulbecco’s modified
Eagle’s medium nutriment mix Fꢀ12 (DMEM/Fꢀ12) in the absence of fetal bovine serum
(FBS) with 6 ꢀg of plasmid DNA and 23 ꢀL Lipofectamine reagent. The reaction was
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5
5
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5
5
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6
stopped by the addition of an equal volume of DMEM/Fꢀ12 containing 20% FBS and the
cells were harvested 33–72 h later.
Preparation of membrane fraction
0
1
2
3
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7
8
9
0
1
2
3
4
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0
1
2
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4
5
6
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8
9
0
For the preparation of protein extracts, transfected cells were washed three times with
Trisꢀsaline buffer at 3°C, collected by scraping in the harvesting buffer (in mM, 95 NaCl,
0
.1 phenylmethylsulphonyl fluoride (PMSF) and 35 Tris at pH 7.5), and washed twice by
00 × g centrifugation for 10 min at 3°C. Cells were resuspended in the harvesting buffer
3
ꢀ1
containing 10 mgꢃmL aprotinin and sonicated. Nuclei and cellular debris were discarded
by centrifugation at 300 × g for 10 min at 3°C and the supernatant (crude protein extract)
was aliquoted and stored at –80°C until used for activity assays. Protein concentration
was estimated by the Bradford microplate assay using bovine serum albumin (BSA) as a
64
standard of reference.
NPPs and TNAP stability assay
Evaluation of the hydrolysis percentage (compared to ATP) of analogues 1ꢀ4 by human
NPP1,3 and of 2a and 3 by TNAP was conducted at 37°C in incubation buffer 0.573 mL
containing: 1 mM CaCl , 130 mM NaCl, 5 mM KCl, and 50 mM Tris (pH 8.5 for NPPs
2
and pH 9 for TNAP) with or without lysates of cells expressing NPP1,3 or TNAP.
Analogues 1ꢀ4 were used at a final concentration of 100 ꢁM. Recombinant human
NPP1,3 or TNAP cell lysates were added to the above mixture and preincubated at 37°C
for 3 min. The reaction was initiated by addition of the analogues or ATP (control). The
reaction was stopped after 1 h by addition of 0.375 mL iceꢀcold 1 M perchloric acid. The
samples were centrifuged for 1 min at 13 000 g. Supernatants were neutralized with 2 M
KOH (0.136 mL) at 4°C and centrifuged for 1 min at 13000 g. Each mixture sample was
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Page 29 of 58
Journal of Medicinal Chemistry
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filtered and freezeꢀdried once for storage overnight. The samples were dissolved in HPLC
water (0.2 mL) and filtered again. An aliquot of 20 ꢁL was separated by reverseꢀphase
HPLC to evaluate the nucleotide content of each reaction sample.
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Separation and quantification of nucleotides by HPLC
An aliquot of 20 ꢁL of the reaction products (described above) was used for nucleotide
analysis on an analytical reverseꢀphase HPLC column [Gemini 5u Cꢀ18 110A, 150 mm X
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.60 mm, 5 ꢁm (Phenomenex)]. Analogues 1ꢀ4 and their hydrolysis products were
separated with a mobile phase consisting of 100 mM TEAA (99ꢀ90%, pH 7.0), and 1ꢀ
0% CH CN, at a flow rate of 1 mL/min for 20 min. Separated nucleotides were detected
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by UV absorption at 260 nm, identified, and quantified by their peak integration and by
comparison of the retention times with those of appropriate standards.
Inhibition of NTPDase (EC 3.6.1.5) ꢀ activity assays
1
Activity was measured as described previously in 0.2 mL of TrisꢀRinger buffer (in mM),
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3
20 NaCl, 5 KCl, 2.5 CaCl , 1.2 MgSO , 25 NaHCO , 5 glucose, 80 Tris, pH 7.3) at
2 4 3
7°C with or without analogues 1ꢀ3 (final concentration 100 µM), and with or without
100 µM ATP as a substrate. The analogues were added alone when tested as potential
substrate, and with ATP when tested for their effect on nucleotide hydrolysis. NTPDases
protein extracts were added to the incubation mixture and preꢀincubated at 37°C for 3
min. The reaction was initiated by the addition of substrate (ATP and/or analogues 1ꢀ3)
and stopped after 15 min with 50 µL of malachite green reagent. The released inorganic
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phosphate (Pi) was measured at 630 nm according to Baykov et al.
Inhibition of NPP and TNAP (EC 3.1.4.1; EC 3.6.1.9) ꢀ activity assays
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