Small molecule colorimetric probes for specific detection of human arylamine N-acetyltransferase 1, a potential breast cancer biomarker

Full text of DOI:10.1021/ja909165u

Published on Web 02/19/2010
Small Molecule Colorimetric Probes for Specific Detection of Human
Arylamine N-Acetyltransferase 1, a Potential Breast Cancer Biomarker
Nicola Laurieri,†,‡ Matthew H. J. Crawford,†,‡ Akane Kawamura,‡ Isaac M. Westwood,‡
James Robinson,‡ Ai M. Fletcher,† Stephen G. Davies,† Edith Sim,‡ and Angela J. Russell*,†,‡
Department of Chemistry, Chemistry Research Laboratory, UniVersity of Oxford, Mansfield Road, Oxford,
OX1 3TA, U.K., and Department of Pharmacology, UniVersity of Oxford, Mansfield Road, Oxford, OX1 3QT, U.K.
Received October 28, 2009; E-mail: angela.russell@chem.ox.ac.uk
Probes to enable the specific detection of biomacromolecules
have been widely used in cellular biology and pathology. Selective
detection of native proteins using antibodies or recombinant proteins
using affinity tags is now well established.1 Small molecule probes2
to selectively label native3 or engineered4 proteins have been
reported, but the majority involve covalent tagging of the target.
We describe here a specific, noncovalent sensor to detect a potential
protein biomarker in its native form without the need for a tag or
an antibody.
Arylamine N-acetyltransferases (NATs) are found in a range of
species and catalyze the transfer of an acetyl group from acetyl
coenzyme A (AcCoA) to arylamines.5 Human NATs were originally
identified as drug metabolizing enzymes,5a but more recently human
NAT1 (hNAT1) has been implicated in cancer6 and, along with its
homologue mouse Nat2 (mNat2), in development.7 Human NAT1
(hNAT1) is one of the 10 most highly overexpressed genes in
estrogen-receptor-positive (ER+ve) breast tumors, in contrast with
ER-ve tumors, and a strong association exists between hNAT1
overexpression and breast tumor grade.8 This suggests that hNAT1
plays a role in breast cancer progression and is a candidate
biomarker in ER+ve breast cancer.8 To identify hNAT1 inhibitors
to probe its endogenous role,9 we screened 5000 compounds against
5 different NATs.10 Among the specific hNAT1 inhibitors identified
in the screen was naphthoquinone 1, which was observed to exhibit
some striking properties, described herein (Figure 1).
nM). Moreover, a solution of 1 was observed to undergo a
distinctive color change, observable by eye, from red to blue in
the presence of either hNAT1 or mNat2 but not the other isozymes.
This is consistent with the established functional similarity between
hNAT1 and mNat2: they have been shown to share a substrate
specificity profile9b and biological properties.7 The observed
differences between substrate and inhibitor specificity profiles in
this isozyme family exist even though all the isozymes share over
70% identity in the amino acid sequence (Supplementary Table
1).5b,9b,11
When an acid-base titration was carried out, the color change
was shown to be a pH-dependent event with a pKa of ∼9.512 (Figure
1). Thus, we hypothesized that 1 was undergoing a deprotonation
within the actiVe site of hNAT1 or mNat2 upon selectiVe binding,
but no deprotonation was occurring with other NAT isoforms.13
To probe the scope of the proposed recognition event between
1 and hNAT1 and mNat2, a series of analogues of 1 were next
prepared Via a two-step protocol from dichloronaphthoquinone 2.
Intermediates 3 and 4 were first prepared from 2 in 72% and 54%
yields, respectively. Optimal conditions for the second substitution
were determined to be treatment of 3 or 4 with the requisite aniline
and cerium chloride14 to afford 1 and 5-8 in 10-88% yield (Table
1). Analysis of the colorimetric properties of 1 and 5-8 and their
inhibitory activities against hNAT1 and mNat2 were next under-
taken. All the compounds showed, to varying degrees, inhibitory
activity against the enzymes (Figure 2, Table 2), and many of the
compounds again showed a distinctive color change upon basifi-
cation or in the presence of mNat2.13
Table 1. Preparation and Assessment of Compounds 1, 5-8 As
Inhibitors of mNat2a
yield
(%)
IC50
(µM)
entry
substrate
R1
R2
product
Figure 1. Top right: 1 (400 µM) in aq. Tris ·HCl (pH 8.0); 20%
trichloroacetic acid (TCA); 4 M aq. NaOH; or mNat2 in aq. Tris ·HCl (pH
8.0). Bottom left: Visible spectra of 1 (10 µM) in aq. Tris ·HCl (pH 8.0,
1
2
3
4
5
3
3
3
3
4
Ph
Ph
Ph
Ph
Ph
1
5
6
7
8
63
10
60
53
88
1.86
0.88
5.86
0.99
3.12
4-BrC6H4-
4-tBuC6H4-
3,5-Me2C6H3-
Ph
red line, λmax ) 489 nm), 1 in aq. NaOH solution (dark blue line, λmax
)
561 nm), and 1 with 2 equiv of mNat2 in aq. Tris ·HCl (pH 8.0, light blue
line, λmax ) 610 nm). Bottom right: pH titration curve for 1 (pKa ∼9.5).
2-thienyl
a mNat2 activity was determined as hydrolysis of AcCoA in the
presence of para-amino benzoic acid (pABA).
1 was shown to be a selective inhibitor of hNAT1 and mNat2,
with IC50 ) 1.65 and 1.86 µM, respectively, but showed no
inhibition against the other human and mouse NAT enzymes. 1
was also shown to be a competitive inhibitor of mNat2 (Ki ) 520
Compound 7 was identified as showing moderately improved
potency (IC50 0.99 µM vs mNat2), inhibition of hNAT1 in breast
cancer cell lysates (90% inhibition at 5 µM), and a markedly
increased absorption at 498 and 610 nm (Figure 2), and subsequent
studies therefore focused on 7.
† Department of Chemistry.
‡ Department of Pharmacology.
9
3238 J. AM. CHEM. SOC. 2010, 132, 3238–3239
10.1021/ja909165u  2010 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Left panel: Screening of 1, 5-8 (5 µM) against pure mNat2
(dark blue columns), hNAT1 in Escherichia coli cell lysate (blue columns),
and hNAT1 in breast cancer cell lysate (ZR-75-1 cells, 1 and 7 only, light
blue columns). NAT activity was determined as acetylation of pABA in
the presence of AcCoA. Right panel: Compounds 1, 5-8 (2 mM) in
Tris·HCl buffer (pH 8.0, top) or aq. NaOH (bottom).
Figure 4. Assessment of the number of binding sites and Kd value for 7
with mNat2 by Michaelis-Menten curve (left) and Scatchard plot (right).
In summary, we have identified a family of reagents which bind
selectively to human NAT1 and its murine homologue mouse Nat2
with a concomitant color change driven by a proton transfer event.
As the expression of hNAT1 is related to tumor type, this property
may be subsequently exploited for diagnosis.
Table 2. Color Change of 1, 5-8 (10 µM) with aq. NaOH or
mNat2
Compound
1
5
6
7
8
Acknowledgment. We thank Research Councils UK for a
fellowship (A.J.R.), Cancer Research UK for a studentship (N.L.),
and Siu Po Lee for mNat2 purification.
λmax (nm) in buffer
∆λmax with NaOH (nm)
∆λmax with mNat2 (nm)
489
+72
+121
487
+49
+123
484
0
0
498
+74
+127
490
+69
+125
Supporting Information Available: All experimental procedures;
compound characterization data; complete ref 6a. This material is
available free of charge via the Internet at http://pubs.acs.org.
As an alternative explanation, the susceptibility of the naphtho-
quinone function toward reduction led us to investigate whether
any reducing agents may give rise to the color change of 7,
especially DTT which is present in the recombinant protein solution
at 34 µM. Treatment of 7 with DTT at 34 µM gave no reduction
and did not affect its color, but at high concentration (100 mM)
DTT led to a decolorization of a solution of 7, which was reversed
upon exposure to air.15 Other reducing agents such as NAD(P)H
gave no visible change, and only treatment with base gave the
characteristic red shift in the visible spectrum of 7.
To assess specificity more broadly, 7 was tested for inhibitory
activity and colorimetric properties with a range of other mammalian
and bacterial NAT enzymes (Table 3). Although partial crossre-
activity was observed with mNat1, the color change was only
observed with mNat2 and hNAT1 (Figure 3).
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Figure 3. Visible spectra of 7 (10 µM) with different NATs (20 µM).
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