Identification of a selective inhibitor of murine intestinal alkaline phosphatase (ML260) by concurrent ultra-high throughput screening against human and mouse isozymes

Article abstract of DOI:10.1016/j.bmcl.2013.12.043

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified - one tissue-nonspecific (TNAP) and three tissue-specific - named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.

Full text of DOI:10.1016/j.bmcl.2013.12.043

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1000–1004
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a selective inhibitor of murine intestinal alkaline
phosphatase (ML260) by concurrent ultra-high throughput screening
against human and mouse isozymes
Robert J. Ardecky a, , Ekaterina V. Bobkova a, , Tina Kiffer-Moreira a, , Brock Brown a, Santhi Ganji a,
Jiwen Zou a, Ian Pass a, Sonoko Narisawa a, Flávia Godoy Iano a, Craig Rosenstein a, Anton Cheltsov a,
Justin Rascon a, Michael Hedrick a, Carlton Gasior a, Anita Forster a, Shenghua Shi a, Russell Dahl a,
Stefan Vasile b, Ying Su a, Eduard Sergienko a, Thomas D. Y. Chung a, Jonathan Kaunitz b,
Marc F. Hoylaerts c, Anthony B. Pinkerton a, ,à, José Luis Millán a, ,à
⇑
⇑
a Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, United States
b UCLA School of Medicine, Los Angeles, CA 90095, United States
c Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are
capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In
humans, four AP isozymes have been identified—one tissue-nonspecific (TNAP) and three tissue-spe-
cific—named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP)
and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic
implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity
can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endo-
toxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate
secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series
of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High
throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with spec-
ificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.
Received 4 November 2013
Revised 6 December 2013
Accepted 10 December 2013
Available online 19 December 2013
Keywords:
Alkaline phosphatase
Inhibitors
Enzymes
Intestinal alkaline phosphatase
Inflammatory bowel disease
Ó 2013 Elsevier Ltd. All rights reserved.
Alkaline phosphatases (APs) are well-studied enzymes known
for their ability to dephosphorylate a wide spectrum of substrates.1
In humans, four AP isozymes have been identified, classified on
their predominant tissue localization: intestinal (IAP), placental
(PLAP) and germ cell (GCAP) APs. The expression of the fourth iso-
zyme, designated as tissue-nonspecific alkaline phosphatase
(TNAP), is ubiquitous, at high levels in the developing neural tube,
kidney, liver and mineralizing tissues such as cartilage and bone.
Its function is intimately associated with mineralization of skeletal
and dental tissues, as deficiency in TNAP function in humans and
mice leads to a heritable form of rickets/osteomalacia known as
hypophosphatasia.1 Mice also have four active AP genes: Alpl
(encoding TNAP), the Akp5 gene encoding embryonic AP (EAP)
and two genes expressed in the gut, Akp3 and Akp6, encoding a
duodenal specific IAP (dIAP) and a globally expressed IAP (gIAP),
respectively.1
Recent work using Akp3 knockout mice indicates that dIAP facil-
itates fat absorption,2,3 maintains gut barrier function4–6 and af-
fects the composition of the gut microbiota.7 Many studies in the
literature also relate human IAP with diarrhea-predominant dis-
eases, such as inflammatory bowel disease (IBD) or pathogenic
infections. Wada et al. reported that infection with Aeromonas
sobria hemolysin causes diarrhea; IAP, by binding hemolysin
appears to be involved with its pathogenesis.8 In IBD, genetic and
environmental factors along with chronic deregulation of the host
immune system response to gut flora appear to play key roles in its
pathogenesis.9–11
Exogenous purified IAP may be useful therapeutically for these
conditions. IAP may detoxify bacterial products such as lipopoly-
saccharide (LPS), reducing excessive intestinal inflammation.12
For example, the naso-duodenal delivery of calf IAP to ulcerative
colitis (UC) patients improved clinical and serological measures.13
⇑
Corresponding authors. Tel.: +1 858 795 5320 (A.B.P.), tel.: +1 858 795 3130
(J.L.M.).
E-mail addresses: apinkerton@sanfordburnham.org (A.B. Pinkerton), millan@
sanfordburnham.org (J.L. Millán).
These authors contributed equally to this work.
These authors share senior authorship.
à
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.043

R. J. Ardecky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1000–1004
1001
Figure 1. Screening hit.
Figure 3. General structure for dihydrobenzo[d]oxazoles.
Table 2 (continued)
Entry
R1
R2
H
n
IC50 (lM)
muIAP
TNAP
>100
PLAP
O
O
26
2
44
>100
Figure 2. Overall SAR strategy.
27
28
29
30
31
33
34
35
36
37
38
39
40
41
42
3-Trifluoromethylbenzyl
Benzyl
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
70
81
84
88
89
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Table 1
Summary of characteristic of initial hit CID24790981
3,4-Dimethylphenyl
4-Chlorobenzyl
4-Methylbenzyl
4-Fluorobenzyl
2,5-Di-Me-phenyl
3-MeO-benzyl
2-MeO-benzyl
2-F-phenyl
4-OH-benzyl
4-CF3-benzyl
3-F-benzyl
2-Cl-benzyl
Entry
IC50 (lM)
dIAP
1.82
huIAP
>100
TNAP
>100
PLAP
>100
1A
Table 2
3-Cl-benzyl
SAR elucidation of dihydrobenzo[d]oxazole series (n = chain length)
O
Entry
R1
R2
n
IC50 (lM)
43
H
0
>100
>100
>100
O
3,4-Di-OMe-phenylethyl
2-MeO-benzyl
4-HO-phenethyl
4-MeO-phenethyl
2,4-Di-MeO-phenyl
3-F-benzyl
muIAP
TNAP
PLAP
44
45
46
47
48
49
50
H
H
H
H
H
H
H
3
3
3
3
3
3
3
1.5
2
4.2
7.4
6.3
13
96
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1
2
3
4
5
6
7
3-OMe-benzyl
2,4-di-OMe-benzyl
4-OMe-benzyl
3-CN-phenyl
4-CN-phenyl
H
H
H
H
H
H
H
0
0
0
0
0
0
0
40
89
98
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2,5-Di-Me-phenyl
3,4-Di-OMe-phenyl
2,4-Di-MeO-benzyl
37
O
O
51
H
3
45
>100
>100
8
H
0
>100
>100
>100
O
3,4-Di-MeO-benzyl
2-Cl-phenyl
2-Me-benzyl
3,4-Di-OMe-phenyl
O
2,5-Di-Me-phenyl
3-OMe-benzyl
4-OMe-benzyl
4-CN-phenyl
52
53
54
55
H
H
H
H
3
3
3
3
78
>100
>100
1.5
>100
>100
>100
>100
>100
>100
>100
>100
9 ML260
H
H
H
H
H
H
1
1
1
1
1
1
0.54
0.73
2.1
35
74
>100
81
>100
>100
>100
>100
>100
>100
>100
>100
10
11
12
13
14
4.7
3-CN-phenyl
2,4-Di-OMe-benzyl
>100
>100
O
More recently, we showed that endogenous IAP likely protects the
host from IBD, since oral supplementation of IAP ameliorates clin-
ical signs and symptoms of IBD in two mouse models of chronic
colitis6 and prevents metabolic syndrome in Akp3À/À mice.14 De-
spite the ability of IAP enzyme to detoxify LPS, how IAP affects
intestinal inflammation has not been fully elucidated. Knowledge
of this mechanism would thus be a key factor for the development
of a successful therapy for the treatment of IBD patients. More
importantly, immunomodulatory therapy of IBD patients is associ-
ated with severe side effects.15
In the present study, we describe a multi-pronged screening ap-
proach that enabled the identification of dIAP inhibitors. SAR ef-
forts based on parallel testing of analogs against different AP
isozymes generated a potent inhibitor of the murine dIAP with
IC50 = 540 nM, at least 65-fold more selective against human IAP
than TNAP, and >185-fold more selective than PLAP. Furthermore,
the inhibitor proved to be selective against the Akp3 encoded dIAP
but not the Akp5- or Akp6-encoded EAP and gIAP isozymes. These
15
H
1
>100
>100
>100
O
O
16
H
1
>100
>100
>100
O
17
18
19
20
2,5-Di-Me-phenyl
H
H
H
H
2
2
2
2
0.4
0.53
2
>100
37
>100
>100
>100
>100
>100
>100
3,4-Di-OMe-phenyl
3-CN-benzyl
4-OMe-benzyl
2.3
O
21
22
H
H
2
2
2.6
7.7
>100
>100
>100
>100
O
3,4-Di-hydroxybenzyl
O
23
24
H
H
2
2
12
>100
>100
>100
>100
O
3-F-benzyl
O
66
19
25
H
2
>100
>100
O

1002
R. J. Ardecky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1000–1004
O
S
O
O
O
S
O
NH2
O
S
O
O
TFA, DCM
Cl
O
O
O
HO
O
O
N
H
N
H
O
O
N
H
O
DCM, ET3N
O
N
H
N
H
1
2
3
EDC, HOBT, NMM,
DMF
O
S
O
NH2
H
N
O
N
H
O
O
R1
N
H
R1
4
5
Scheme 1. Synthesis of 5, reagents and conditions: (a) dichloromethane, triethylamine, (70–88% yield); (b) trifluoroacetic acid, dichloromethane, 0 °C warm to rt (100%
yield); (c) EDC, HOBT, NMM, DMF, (40–55%).
compounds are likely to be useful tools in probing the functional
roles of human and mouse IAPs during the bacterial endotoxins
detoxifying process, absorption of fatty acids and bicarbonate
secretion.
contain either a 2,5-di-methylphenyl, 3,4-di-methoxyphenyl,
4-methoxybenzyl, 3-cyanobenzyl or methylene-di-oxybenzyl
substituents (entries 17–21). Most of these compounds display lit-
tle activity against TNAP or PLAP with IC50 values >100 lM. Meth-
Identification of isozyme-specific inhibitors was part of a
platform-based approach where the entire NIH’s small molecule
collection (MLSMR) was interrogated against dIAP and hIAP iso-
zymes in parallel, while assessment of selectivity against TNAP
and PLAP isozymes was based on the results of prior screening
campaigns.16,17 This parallel screening strategy, using the same
CDP StarÒ luminescent assay format, not only afforded a direct
comparison between several high-throughput screens, but also
allowed an efficient elimination of the artifacts.
ylation of the nitrogen atom of the dihydrobenzo[d]oxazole
heterocycle completely eliminates the activity (entry 34). We next
focused our attention on increasing the chain length by one carbon
atom, where n = 3. Generally these compounds are less active than
comparable compounds in the n = 2 series of molecules. (entries
50–61). The series of compounds where n = 0 produced very disap-
pointing results as all compounds are inactive with IC50 values
ranging from 40 to >100 lM. (entries 1–8 and 16).
Finally, we investigated a series of compounds where n = 1. We
synthesized 8 analogs with the most potent analog containing the
2,5-di-methylphenyl group, entry 9. Consideration of the potency
and in vitro selectivity data presented herein we nominated com-
pound 9 as our probe molecule ML260 as it was the most potent
1536-Well high throughput screens of MLMSR library compris-
ing 330,480 compounds against dIAP and hIAP isozymes were
conducted at 10 lM compound concentration, as described in
PubChem (AID 2544). Ultimately only one compound, hit
CID24790981 (Fig. 1), was selective against TNAP and PLAP.
CID24790981 has an IC50 = 1.82 lM in the dIAP assay and displays
excellent selectivity against TNAP and PLAP (Table 1).
The general SAR strategy we pursued around this scaffold from
the screening hit is depicted in Figure 2. We focused on changing
the nature and number of the R1 substituents attached to the phe-
nyl ring highlighted in yellow and we investigated changes in the
chain length, increasing and decreasing the carbon chain length
(n = 0, 1, 2, or 3) highlighted in red. Finally, we investigated if it
is possible to replace the hydrogen atom at R2 by alkyl groups
highlighted in green.
We developed an efficient synthesis for our lead series of
molecules that was straightforward and followed the general
methods outlined in Scheme 1. Treatment of the commercially
available sulfonyl chloride 1 with the tert-butyl 2-aminoacetate
afforded the (sulfonamido)acetic acid 2. Removal of the Boc-pro-
tecting group of compound 2 with trifluoracetic acid afforded the
free acid 3 in excellent yields. Coupling of acid 3 with various
ML260
100
dIAP
80
TNAP
60
40
20
PLAP
0
amines
4
produced the desired dihydrobenzo[d]oxazole
compounds 5 directly.
-2
-1
0
1
2
The results of our efforts are summarized in Table 2 below. Ini-
tially we focused our SAR on the R1 group in Figure 3, where n = 2.
Generally, mono substituents, either electron donating or electron
withdrawing, are inactive (entries 27–42). Interestingly, we found
the position of substituents on the phenyl ring was critical for
activity. For example entry 17 and entry 34 both contain a di-
methyl substituent on the phenyl ring, and this scaffold greatly
prefers the 2,5 substitution pattern of entry 17 over the 3,4 substi-
tution pattern of entry 34. We found the most active compounds
Log [ML260], µM
Figure 4. Potency and selectivity of ML260. The potency of the inhibitor against
dIAP (o), PLAP (+) and TNAP (x) was assessed by 16pt dose–response testing in
1536-well format in duplicate in 1Â Assay Buffer, containing 100 mM DEA, pH 9.8,
0.02 mM ZnCl2, 1 mM MgCl2, and 1:250, 1:1000 and 1:2000 diluted enzymes,
respectively, in a total volume of 4
of the CDP-star substrate to the final concentrations of 200
reaction, and 250 M for the PLAP and TNAP reactions, with the luminescence
intensity measured after 30 min incubation at room temperature.
l
L/well. The reactions were started by addition
lM
for the dIAP
l

R. J. Ardecky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1000–1004
1003
dIAP inhibitor in this series while having excellent selectivity
against both PLAP and TNAP. The multiple dose response titrations
of ML260 against dIAP, TNAP and PLAP are shown in Figure 4.
When the relative IAP selectivity was further tested using
p-nitrophenyl phosphate as a substrate the Akp3-encoded dIAP
was inhibited selectively over murine EAP and gIAP isozymes as
dIAP
EAP
gIAP
hIAP
well as the hIAP isozyme with an IC50 equivalent to 3.8
(Fig. 5). Further analysis, using pNPP as a substrate revealed that
ML260 inhibits dIAP competitively with an apparent Ki = 3.2
(Fig. 6). For this reason, a concentration of 10 M was selected to
lM
lM
l
inhibit dIAP activity expressed by transfected Cos-1 cells. Figure 7
shows that the dIAP activity expressed in these cells could be fully
inhibited with ML260 at 10 lM.
In conclusion, we have discovered the first selective murine IAP
inhibitor, ML260, which represents a tool compound to further
explore the functional role of dIAP in a variety of disease states,
in particular as a gut mucosal defense factor18 and as a mediator
in fatty acid absorption. Current efforts to examine the effects of
IAP inhibition in these models are underway.19
Log[ML260] (mM)
Figure 5. Selectivity of intestinal isozyme inhibition by ML260. Dose-dependency
of inhibition of dIAP (IC50 = 3.8 M), versus inhibition of EAP (IC50 = 21.1 M), gIAP
(IC50 = 21 M) and hIAP (IC50 = 27.6 M), as indicated.
l
l
l
l
Acknowledgments
0.20
0.15
0.10
0.05
No inhibitor
0.5 mM
1 mM
3 mM
4 mM
This work was supported by NIH grants X01-MH077602-01 and
R01 DE12889 to J.L.M. and an NIH Molecular Libraries grant
(U54HG005033-03) to the Conrad Prebys Center for Chemical
Genomics at the Sanford Burnham Medical Research Institute,
one of the comprehensive centers of the NIH Molecular Libraries
Probe Production Centers Network (MLPCN).
6 mM
Supplementary data
-1.0 -0.5 0.0
0.5
1.0
1.5
2.0
1/[pNPP] (mM)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
12.043.
Figure 6. Mechanism of dIAP inhibition by ML260. Double reciprocal plots of
residual dIAP activity versus the concentration of pNPP, constructed for each of the
indicated ML260 concentrations.
Figure 7. CHO cells expressing dIAP protein, stained with ELF97 in the absence (A) or presence of 10
respectively. Bar = 50 m.
lM ML260 (C). (B) and (D) are phase contrast views of (A) and (C)
l

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R. J. Ardecky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1000–1004
8. Wada, A.; Wang, A. P.; Isomoto, H.; Satomi, Y.; Takao, T.; Takahashi, A.; Awata,
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