Switch-on fluorescent/FRET probes to study human histidine triad nucleotide binding protein 1 (hHint1), a novel target for opioid tolerance and neuropathic pain

Article abstract of DOI:10.1039/c7ob02472j

Histidine Triad Nucleotide Binding Protein 1 (Hint1) has emerged to be an important post-synaptic protein associated with a variety of central nervous system disorders such as pain, addiction, and schizophrenia. Recently, inhibition of histidine nucleotid

Full text of DOI:10.1039/c7ob02472j

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Switch-on Fluorescent/FRET Probes to Study Human Histidine
Triad Nucleotide Binding Protein 1 (hHint1), a Novel Target for
Opioid Tolerance and Neuropathic Pain
Rachit Shah,a Andrew Zhoua and Carston R. Wagnera
Received 00th January 20xx,
Accepted 00th January 20xx
Histidine Triad Nucleotide Binding Protein 1 (Hint1) has emerged to be an important post-synaptic protein associated with
a variety of central nervous system disorders such as pain, addiction, and schizophrenia. Recently, inhibition of histidine
nucleotide binding protein 1 (Hint1) with a small nucleoside inhibitor has shown promise as a new therapeutic strategy for
the treatment of neuropathic pain. Herein, we describe the first rationally designed small molecule switch-on probes with
dual fluorescence and FRET properties to study Hint1. Two non-natural fluorescent nucleosides with a fluorescent lifetime
of 20 and 25 ns were each coupled through a linker to the indole ring, i.e. probes 7 and 8. Both probes were found to be
water soluble and quenched intramolecularly via photoinduced electron transfer (PET) resulting in minimal background
fluorescence. Upon incubating with Hint1 compound 7 and 8 exhibited a 40- and 16- fold increase in the fluorescence
intensity compared to the control. Compounds 7 and 8 bind Hint1 with a dissociation constant of 0.121 ± 0.02 and 2.2 ±
0.36 μM, respectively. We demonstrate that probe 8 exhibits a switch-on FRET property with an active site tryptophan
residue (W123). We show the utility of probes in performing quantitative ligand displacement studies, as well as in
selective detection of Hint1 in the cell lysates. These probes should be useful for studying the dynamics of the active site,
as well as for the development of fluorescence lifetime based high throughput screening assay to identify novel inhibitors
for Hint1 in future.
DOI: 10.1039/x0xx00000x
www.rsc.org/
preclinical and clinically approved antiviral and anticancer
phosphoramidate proTides.6-9 In addition, Chou and Wagner et al.
Introduction
Histidine triad nucleotide binding protein 1 (Hint1) has emerged as
a key regulator of pain, opioid tolerance and addiction properties in
the central nervous system (CNS).1-3 Hint1 belongs to the histidine
triad (HIT) superfamily which are characterized by their conserved
have demonstrated that lysyl t-RNA synthetase generated lysyl-
AMP is a substrate for hHint1 in vitro. 10
Hint1 has been shown to play a role in modulating in N-methyl-D-
aspartate (NMDA) receptors activation.11 The interaction between
Hint1 and NMDA receptor has been proposed to mediate the co-
association of NMDAR with several G-protein coupled receptors
nucleotide binding motif, His-X-His-X-His-XX, where
X is a
hydrophobic residue. hHint1 exists as a homodimer and possesses
nucleoside phosphoramidase and acyl-AMP hydrolase activity, with
13
(GPCRs) in vivo, including the µ-opioid receptor (MOR).12,
a
preference for substrates with purine over pyrimidine
Consistent with this observation, Hint1-/- mice exhibit an increased
morphine analgesic response and sensitivity to amphetamine, while
reduced nicotine dependence has been observed in self-
administration studies.1-3 In addition, mutations or aberrant
expression of hHint1 is been associated with inherited peripheral
neuropathy, schizophrenia and bipolar disorders.14, 15 Nevertheless,
nucleosides.4, 5 The nucleoside phosphoramidase activity of hHint1
has been shown to be necessary for the activation of several
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the identity of the endogenous substrate that presumably is a) the probe should exhibit selective binding affinity for hHint1 and
participating in these signaling events has yet to be determined.
Based on the results of our substrate specificity analysis of Hint1,4
b) there should be a substantial difference in the fluorescence
property of the free and the bound probe. One of the characteristic
features of hHint1 is its nucleoside-binding motif, which exhibits a
preference for binding purine over pyrimidine nucleoside
monophosphates. Hence, we selected the fluorescent non-natural
nucleosides, 1, N6-ethenoadenosine (EtAd, exλmax 278, emλmax 410
nm) (7) and thG (exλmax 331, emλmax 450 nm), (8) to incorporate into
the design of our fluorescent probes (Supporting Figure S2). 21, 22
we
have
designed
several
competitive
inhibitors.16
Neuropharmacological studies with one of these inhibitors, 3 (TpGc,
Supporting Figure S1), revealed that it enhanced morphine
analgesia, blocked the development of tolerance and relieved
neuropathic pain in mice.17 We have also demonstrated that Hint1
regulates the function of MOR via its interaction with the NMDA
receptor.17 To facilitate interrogation of the molecular mechanisms The selection of EtAd and dGth was based on their high quantum
governing the cellular function of Hint1, a fluorescent probe that yields (Ø = 0.56 for EtAd and Ø = 0.34 for thG) and long fluorescent
switches on when bound to the protein would be a valuable lifetimes ( ~ 20-25 ns) in aqueous buffers. The guanosine mimic,
mechanistic tool. In addition, a fluorescent switch-on probe of the thG, has an additional advantage since it’s excitation and emission
Hint1 active site would aid in the development of high throughput maxima are at higher wavelengths compared to EtAd (Supporting
assays for the screening of potential Hint1 inhibitors.
Figure S2B) To address the second criteria, we decided to
incorporate an indolyl group as a fluorescent quencher. A wide
variety of chemical moieties can act as a quencher of fluorescence,
For example tryptophan is known to induce quenching of certain
dyes and fluorophores over relatively short distances via photon-
induced electron transfer (PET).23 Attachment of an indole side
chain using a water-soluble acyl-sulfamate linker to the fluorophore
nucleoside nearby resulting in quenching of the fluorescence
(Figure 1A). We therefore chose to prepare and investigate the
fluorescence properties of 5’-indole-3-propionic acid ribose 1, N6-
ethenoadenosine sulfamate (7) and 5’-indole-3-propionic acid
ribose-2-aminothieno[3,4-d]pyrimidin-4(3H)-one sulfamate (8)
(Figure 1C).
Currently, there are two classes of small molecule based switch-on
fluorescent probes: enzymatic activity-dependent probes and
enzymatic activity independent probes.18 In the later case,
generally, a solvatochromic fluorophore is attached to the ligand
specific to a target protein of interest. Upon binding to the target of
interest, the change in the polarity of the environment results in an
increase in the fluorescence intensity of the probe.19 Unfortunately,
these probes exhibit high background fluorescence and hence a low
ratio of signal to background fluorescence is observed. Herein we
report the first fluorescent reporter ligands for hHint1 with
switchable properties guided by the principle used in the design of
DNA or aptamer-based molecular beacons.20 Upon binding to
hHint1, these switchable probes undergo a conformational change Compound 7 has been recently reported as a sub-micromolar (Kd =
resulting in the quencher becoming less hybridized with the 0.23 μM) inhibitor of hHint1 and was prepared as previously
fluorophore and thus exhibiting and increased fluorescence described.16 The synthesis of compound
8 began with the
intensity. In the current work, we demonstrate the utility of such preparation of 2-Aminothieno[3,4-d]pyrimidine G mimic nucleoside
probes in performing hHint1-ligand dissociation studies and the (thG) as reported by Shin et. al21 with minor modifications (see
selective detection of hHint1 in vitro.
supplemental information for details). Next, we carried out the
acetonide protection of the 2´, 3´- hydroxyl of the ribose sugar of
thG, followed by sulfomylation of the 5’-hydroxyl with sulfamoyl
chloride to yield 6 (Scheme 1). Coupling of 6 with the activated
NHS-ester of an indole-3-propionic acid (22) in the presence of DBU
and subsequent removal of the acetonide group with aqueous TFA
generated 8 in 60 % overall yield. When compared to the parent
nucleosides, EtAd and thG, the quantum yields of compounds 7 and
Result and Discussion
Design and synthesis of intramolecularly quenched fluorescent
nucleotidomimetic probes for hHint1
We envisioned the following two important criteria for the
successful development of switch-on fluorescent probes for hHint1:
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A)
B)
Switch-off
Switch-on
Switch-off
Fluorescent
hHint1
Aqueous solution
Aqueous solution
hHint1
Minor population
Extended conformer
Major population
Stacked conformer
Minor population
Stacked conformer
Major population
Extended conformer
Fluorophore
Linker
Quencher
C)
Fluorophore
N
Quencher
O
N
N
N
NH
NH2
O
O
O
S
O
N
N
N
S
O
N
S
O
H
H
O
O
O
O
N
H
N
H
OH OH
OH OH
7
8
Figure 1. A) Concept of the designed intramolecularly quenched probes for hHint1 B) Upon incubation with hHint1, the probe
hybridizes with the complementary active site, resulting in the extended conformer and hence regain in the fluorescence C) Chemical
structures of the designed intramolecular quenched probes 7 and 8. Circled in red is a quencher (indole ring) and blue is the
fluorescentnucleobase
8 exhibited a 50- and 19- fold decrease, respectively (Table 1). The unimolecular rate constant (kq) for this process was calculated
Studies also revealed a 4- to 5- fold reduction in the fluorescence to be 1.7 x 108 per second for both 7 and 8. Thus, both probes
lifetime of 7 and 8 when compared to the parent nucleosides (Table undergo static and dynamic quenching.24
1, Supporting Figure S3). Based on the decrease in the fluorescence
Both probes exhibit switch-on fluorescence properties upon
intensity and lifetime of the fluorophore, we estimate that in an
incubation with hHint1 in an aqueous solution
aqueous solution, 89 % and 77% of the population of 7 and 8,
respectively, exists in a stacked conformation (Table 1). The
Since a stacked conformation for 7 and 8 would result in the
decrease in the average lifetime also indicates that both probes
quenching of the fluorescence, we reasoned that binding of the
continuously undergo end-to-end dynamic collisional quenching.
probes to hHint1 would lead to decreased end-to-end collision and
an increase in the observed fluorescence (Figure 1B). Such
phenomenon of fluorescence/FRET behavior has been previously
observed upon binding of the cofactor NADH to human 17-B-
dehydrogenase complex, but has not been exploited in the design
of an inhibitor for a target protein.25 As expected, an increase in
fluorescence intensity was observed upon incubation of these
probes in the presence of hHint1 (Figure 2A). Titration of these
probes with an increasing amount of hHint1 yielded a fluorescence
intensity curve consistent with active site binding in an extended
aReagents and conditions: i) perchloric acid and acetone 2 h, ii) NH SO Cl, DMA, 85%; iii) 22, DBU,
conformation (Figure 2B). Our observation is supported by X-ray co-
2
2
DMF 55%; iv) 80 % aq. TFA quant
crystal studies of 7 bound to hHint1.16
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Recently it was reported that different keto-amino tautomers of the The affinity of 7 is in agreement with the previously reported
exocyclic amine in the fluorescent thG nucleoside exist in aqueous dissociation constant of 0.23 μM obtained by isothermal titration
solution with each possessing
a
distinct spectral property.26 calorimetry (ITC).16 The dissociation constant of 8 was found to be
Mixtures of the tautomers was found to result in broad emission four-fold lower than the recently reported guanosine analog of 8 by
spectra in aqueous solutions with each tautomer either blue or red- Shah and Wagner et al.16 This result indicate that the replacement
shifted relatively along a narrow emission profile and characterized of nitrogen’s in the purine ring and the incorporation of a sulfur
by a high photostability. The emission profile of our bound probe atom at C8 position results in decreased binding affinity for hHint1.
resembles the red shifted tautomer indicating that binding of the
Utility of switch-on probes in hHint1-ligand binding analysis
protein is stabilizing a preferred nucleoside tautomer.
Since probes 7 and 8 occupy the active site of hHint1, monitoring
Upon inspection of the X-ray crystal structure of 7 bound to Hint1,16
their displacement by non-fluorescent ligands would allow us to
it can be estimated that the exocyclic amine in guanosine could
determine the dissociation constants of the ligands. As can be seen
participate in a hydrogen bonding interaction with backbone
in Figure 3, titration of the hHint1-7 or 8 complex with the inhibitors
carbonyl of His-42, which might result in the stabilization of one
Bio-AMS or TpGc yielded
a dose-dependent decrease in
tautomer in the bound state. The probes 7 and 8 exhibited a linear
increase in the intensity of the emission signal in the absence of
proteins, corresponding to EtAd and thG auto or background
fluorescence. (Supporting Figure S4). Fitting the observed data with
a one site-binding model provided dissociation constant for 7 of
0.121 ± 0.02 µM and 8, 2.2 ± 0.36 µM. (Figure 2)
fluorescence. IC50 values obtained from the plot of the % decrease
in fluorescence intensity vs. log dose response curve were then
converted using the Cheng-Prusoff equation to provide the
inhibition constant (Ki) values. The values for both the inhibitors
were found to be very similar to the previously reported
A)
Figure 2. A) Fluorescent spectra changes of
compound 7 and 8 (3 µM) upon addition of hHint1
(0.5ꢀ6 µM) (λex= 278 nm for 7 and 330 nm for 8) in
an aqueous assay buffer with slit width of 5 nm for
both excitation and emission. B) Specific binding of
probes to hHint1 (0.25 or 1 µM) observed with
increasing concentration of ligands. The total
Compound 7
Compound 8
400
300
200
100
0
200
160
120
80
40
0
increase in the fluorescence (λex= 278 nm for 7, λem=
350
400
450
500
400
450
500
550
Wavelength (nm)
Wavelength (nm)
410 slit 10 and 330 nm for 8, λem= 453 slit 5) was
subtracted from the background intensity and
plotted against the concentration of the respective
B)
compounds.
Data points
represent three
measurements including the standard deviations.
Table 1: Photophysical properties for the fluorescent
nucleoside analogues used in the current study
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make them suitable for the development of a high-throughput-
screening (HTS) assay in the identification of new nucleoside or
non-nucleoside inhibitors of hHint1. One of the significant
limitations that commonly arise in fluorescent-based high
throughput assays is the interference from the library compounds
that are fluorescent, resulting in false negative or positive hits.27
Typically, fluorescent compounds in the screening library have a
dissociation constant values of 0.32 and 3.65 μM for Bio-AMS and
TrpGc, respectively, via ITC.16 In similar fashion, the values of the
inhibition constants for the nucleoside monophosphates (NMPs) to
hHint1 ranged from 20-40 µM with
GMP≈AMP>CMP>UMP (Table 2).
a
rank order of
We next sought to evaluate whether the probes could be
used to determine the binding affinity of nucleoside
phosphoramidate substrates of hHint1. Instead of the catalytically
active enzyme, we employed the catalytically inactive mutant of
hHint1, H112A, in which the nucleophilic imidazole was replaced
with an alanine residue.10 As observed for the wild-type enzyme,
incubation of hHint1_H112A mutant with 8 resulted in an increase
in fluorescence intensity. Interestingly, the hHint1_H112A mutant
exhibited a moderate gain (~4 fold) in the binding affinity for 8
compared to the wild-type enzyme (Figure 3B, Table 2). When the
fluorescence lifetime of less than
5 ns. Hence, the use of
fluorescence lifetime as the readout parameters for HTS has been
shown to increase the robustness against artifacts and compound
29
related interferences.28,
Both our probes exhibit a remarkable
change in their fluorescence lifetime (4-5 fold) when unquenched or
bound to hHint1. This exceptionally long lifetime (20-25 ns) would
offer a robust assay with a high quality primary screening data.
A fluorescent switch-on FRET probe of the active site of hHint1
hHint1-8 complex was then titrated with variable concentrations of One of the key motivations in the development of 8 was the
the hHint1 substrate, TpAd,
dose-dependent decrease in realization that the maximum excitation wavelength of thG (exmax
fluorescence was observed corresponding to a dissociation constant 330-360 nm) overlaps with the fluorescence emission maxima
a
=
of 0.5 ± 0.01 M (Figure 3B, Table 2).
wavelength for tryptophan (emmax = 340 nm). The critical Föster
resonance distance (R0) between tryptophan and the fluorescent
nucleoside (thG) has been reported to be 22 Å.30 Inspection of the x-
ray crystal structure revealed the presence of a single tryptophan
residue (W123) located in the active site that is 13 Å away from the
nucleobase-binding pocket (S1) (Figure 4A).
Our fluorescent probes allow for
a
rapid mix and
measurement of fluorescence for a displacement assay for hHint1.
The assay circumvents high concentration of proteins required for
the recently reported ITC studies with hHint1 inhibitors. Compound
8 and its cognate fluorescent nucleoside displays properties that
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A)
KI= 0.32
± 0.03 ꢀM
KI= 1.04 ± 0.03 ꢀM
100
100
80
60
40
20
0
80
60
40
20
0
-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
Log Bio-AMS ( M)
-1.0 -0.5
0.0
0.5
1.0
1.5
2.0
ꢀ
Log TrpGc (ꢀM)
B)
Ki= 0.50 ± 0.03 ꢀM
100
80
60
40
20
0
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0
Log TrpAMP (ꢀM)
Figure 3. A) Competitive displacement studies (Left) Titration of Bio-AMS (0.025-12 μM) with hHint1 (0.25 μM) incubated with 0.4 μM of
compound 7. (Right) Titration of compound 3 (TrpGc, 1-40 μM) with hHint1 (1.0 μM) incubated with 12 μM M of compound 8. B) (Left)
Specific binding of compound 8 to hHint1_H112A (0.5) observed with increasing concentration of the ligand. The total increase in the
fluorescence (λex= 330 nm and λem= 453 nm, slit 5) was plotted against the concentration of the compound 8. Data points represent three
measurements including the standard deviations. (Right) Titration of Hint1 substrate (TrpAMP, 0.025-12 μM) with hHint1_H112A (0.25 μM)
incubated with 2.0 μM of compound 8. One site-binding model was used to fit the displacement curves. Data points represent three
measurements, including the standard deviations.
Table 2: hHint1-ligand binding constants calculated using fluorescence displacement studies with switch-on probes.
Ligand
Wild type hHint1
hHint1_H112A
IC50 (µM)
Ki (µM)
IC50 (µM)
Ki (µM)
BioꢀAMSa
3 (TrpGc)b
AMPb
1.77 ± 0.05
6.71 ± 0.04
141.9 ± 1.8
153.99 ± 2.0
246.82 ± 2.8
219.95 ± 2.4
ꢀ
0.32 ± 0.03
ꢀ
ꢀ
1.04 ± 0.03
22.1 ± 2.1
23.9 ± 1.9
38.3 ± 2.3
34.1 ± 2.4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
GMPb
ꢀ
ꢀ
UMPb
ꢀ
ꢀ
CMPb
ꢀ
ꢀ
2 (TrpAMP)b
1.94 ± 0.05
0.50 ± 0.03
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Such proximity provides an opportunity for the development of a compound
8
(Figure 5A). A similar pattern was observed for the
(Supporting Figure S6). The difference in the
fluorescence resonance energy transfer (FRET) based switch-on selectivity of probe
7
probe for hHint1. FRET was verified by measuring the fluorescence amount of unquenching also manifests the ability of the Hint
emission spectra of hHint1 alone and in the presence of compound isoforms to differentially bind acyl-NMP and their potential as in
8
(Figure 4A). Upon incubation with an excess amount of hHint1 vivo regulators. These results are in agreement with the decrease in
and excitation at 280 nm; the emission at 360 nm was decreased the Michaels-Menten constant observed for both hHint3 and echinT
with an increase in emission signal intensity at 450 nm. This when the substrate specificity studies of the isoforms and the
observation is in agreement with those typically seen with FRET hHint1 are compared. Interestingly, among these isoforms, only
pairing molecules. Since the structure of
8 incorporates an indole hHint1 and 2 contains a tryptophan, in the active site but not the
moiety, one might expect FRET from not only W123 but also from others. Consequently, we observed the FRET signal only in the
the intramolecular indole side chain. Distinguishing between the presence of hHint1 and 2 (Figure 5C). The ability of our probes to
two overlaps would be difficult. Hence, we synthesized an analog of light up upon binding to hHint1 provides a simple and easy means
9
, with a biotin instead of the indole side chain. Thermodynamic to evaluate the impact of the perturbations on the protein structure
binding analysis using ITC indicated that binds hHint1 with a on the acyl-NMP binding. For example, an increase in the binding
dissociation constant of 6.2 ± 0.34 μM (Figure S5 and Table S2). affinity was clearly evident with the catalytically inactive H112A
Upon incubation, with hHint1 compound
exhibited an increase in mutation in hHint1.31 The relative increase in the fluorescence
emission intensity at 450 nm. (Figure 4B). This result confirms FRET intensity of upon binding to hHint1 provides an insight into the
pairing of our probes and with the tryptophan in the active site first step in the formation of the ES complex. The ability of to
9
9
8
8
9
8
of hHint1. Our attempts to abolish FRET with a W123A mutation led differentiate between hHint1 and Hint2 despite their high sequence
to the loss of dimerization of hHint1 (data not shown). Mutations and structural similarity indicates the underlying differences in their
associated with a loss in dimerization of hHint1 have been reported ability to form an ES complex. Recently, numerous point mutations
to decrease its substrate specificity and Michaelis-Menten constant in hHint1 were identified from clinical patients and were described
for the substrates.31 Hence, we did not observed fluorescence to be a leading cause of peripheral neuropathy associated with
32
unquenching of compound
mutant (data not shown).
8
in the presence of hHint1_W123A neuromyotonia.14, In such scenario, our probes could serve as
valuable tools in evaluating the impact of these mutations on the
binding of potential substrates and products to hHint1.
Selectivity of switch-on fluorescence probes on binding to Hint1.
Next, we wanted to ask if compound 8 would display similar switch-
on properties in the presence of other adenylating or nucleoside
binding proteins. We chose an aminoacyl t-RNA synthetase (lysRs)
as a representative example and dihydrofolate reductase (DHFR) for
its ability to bind a nucleotide-based cofactor NADPH. We also used
an aminopeptidase protein trypsin in the study. We monitored the
The human genome encodes for three different isoforms of Hint
proteins: hHint1, 2 and 3, while prokaryotes such as Escherichia coli
encodes only
a single protein with phosphoramidase activity
(ecHint). Our kinetic and substrate specificity studies with Hint
enzymes have clearly shown differences among these isoforms.
Incubation of compound
8 with each hHint1 isoforms resulted in
increase in the fluorescence intensity of Compound
8 (λex = 330 nm,
differential amounts of fluorescence unquenching. Among them,
hHint1 showed the highest, while both hHint3 and echinT exhibited
the lowest amount of fluorescence intensity upon incubation with
λem = 450 nm) in the presence of both proteins. Incubation of these
proteins with Compound
8
yielded little or no increase in the
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fluorescence intensity (Figure 5A). Furthermore, incubation of 8 induction 1.5 h) resulted in a significant increase in the fluorescence
with the lysate of Escherichia coli (total protein 1 mg) yielded little intensity (Figure 5B). Together, these results demonstrate the
or no increase in the fluorescence intensity. However, incubation of ability of Hint1 to bind selectively and switch-on the fluorescence of
8
with E.coli lysate (total protein concentration 1 mg/ml) obtained
8.
from cells transformed with the hHint1 expression plasmid (IPTG
FRET
λex~ 280 nm
A)
600
500
400
300
200
100
0
W123
W123
13 Å
350
400
450
500
550
Wavelength (nm)
B)
Ex - 280 nm
FRET
Em - 350 nm
13 Å
Em - 450 nm
NH
W123
O
N
NH
NH2
O
O
S
H
O
N
N
S
O
H
O
O
HN
S
OH OH
9
Ex - 330 nm
Figure 4. A) (Left) X-ray crystal structure analysis of AMP bound to hHint1 (pdb: 3TW2) to measure the distance between W123 and the
nucleobase for FRET pairing (dotted line 13Å). (Right) FRET signal measurement upon incubating 8 μM of hHint1 alone (red) or in the
presence of
tryptophan, slit 5) of the protein was observed upon incubation with compound
without indole side chain) bound which is proposed to FRET pair (dotted line 13Å) with W123 in hHint1. (Right) FRET signal measurement
8
(2 μM) (blue, λex= 280 nm for
8
, slit 5) in an aqueous assay buffer. The decrease in the emission intensity (λem= 360 nm for
8
. B) Chemical structure of compound (analogue of 8
9
upon incubating hHint1 (1 μM) alone (red) or in the presence of increasing concentration of compound
slit 5) in an aqueous assay buffer.
9 (1.0-4.0 μM) (blue, λex= 280 nm,
A)
B)
C)
*
8 | J. Name., 2012, 00, 1-3
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Figure 5. Selectivity test of compound
8 with other proteins: Compound 8 (12 μM) was tested with A) 1 μM of Hint isoforms and three
other nucleoside/nucleotide binding proteins or B) E.coli lysate (1 mg/ml) without/with hHint1. C) Comparison of signal from FRET
pairing of compound 8 with tryptophan in the active site of Hint1 or 2 to other Hint proteins that lacks such tryptophan: Compound 8
(12 ꢁM) was tested with 1 ꢁM of Hint isoform proteins. The observed signal in Hint3ꢀ2 and echinT is resulting from the background
fluorescence of the unbound probe and hasn’t been normalized to that. Student t-test perform on the values indicate, **** p-values <
0.0001,
***
p-values
≥
0.0001
Conclusion:
measurement, the excitation wavelength for
8
was done at 280 nm,
and emission spectra were recorded from 320-520 nm.
In conclusion, we have developed a novel fluorescent/FRET probes
with switch on properties for selective detection of hHint1. The For hHint1-ligand binding studies hHint1 was incubated with
probes would serve a valuable tool for the future discovery of successive increase in the concentration of (0.05 -3 μM) or
7
8
inhibitors and substrates, as well as aid in studies of the (0.25-40 μM). The mixture was incubated for 30 s before the
conformation dynamics and molecular recognition of ligands by excitation and following with emission spectral intensity recording
hHint1.
at 410 nm and 450 nm. The increase in the fluorescence intensity
upon successive increase of the ligand concentration was
subtracted from the blank measurement. A blank was recorded in
the presence of just the ligand and no protein to yield a linear
curve. The resulting sigmoidal dose response curve was plotted in
the graph pad prism and fitted using one site-binding model to yield
the dissociation constant (Kd) values.
Acknowledgements
We thank Dr. Kurt J. Peterson at the fluorescence innovations and
Dr. Ji Li at the biophysical and spectroscopy facilities at University of
Minnesota for help with the fluorescence lifetime studies and
helpful discussions. We would also like to thank Dr. Courtney
Aldrich lab for providing NHS-ester of Biotin to synthesize
For hHint1-ligand displacement studies hHint1 was incubated with
compound
9.
either
7 or 8 at saturating concentrations. The mixture was
incubated for 60 s before the addition of the desired ligand for the
displacement of the fluorescence. The excitation and emission
intensity was recorded. The decrease in the fluorescence intensity
upon successive addition of the ligand was normalized to the blank
and the resulting dose response curve was plotted in the graph pad
prism. The dose response curve was fitted with one site-binding
model to yield the IC50 values. The values were then converted to
the inhibitory constant (Ki) values using Cheng-Prusoff equation.
Materials and Methods
Fluorescence
Spectroscopy: hHint1-Ligand binding
and
displacement studies
All fluorescence measurements were performed in an aqueous
assay buffer (20 mM Tris, 150 mM NaCl pH 7.4). All fluorescence
measurements were performed in a 1 cm four sided, 2 ml quartz
cuvette. The total sample of 600-μl solution including the protein
and ligand was used in the cuvettes to perform fluorescence
measurements. All the readings were recorded at room
temperature. The excitation and emission wavelengths and the slit
width are described in the legends of the respective figures. The
Fluorescence Spectroscopy: Quantum yield, Fluorescence lifetime
and Quenching studies
All the fluorescence and absorbance measurement was performed
in the assay buffer. All the readings were recorded at the room
temperature. The UV visible measurements were performed in the
cary eclipse UV spectrophotometer. 5 nm slit width was used for all
the measurements.
excitation wavelength for dGth and
spectral scan was recorded from 360-530 nm. The excitation
wavelength for EtAd and was 280 nm, and the emission spectral
8 was 330 nm and the emission
7
scan was recorded from 320-530 nm. In the case of FRET
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Quantum yields (ØF) for the dGth, nucleoside analogs
calculated using the following equation.
7
and
8
were control for the absence of an intermolecular quencher. and 0 is
the fluorescence lifetime in the presence and absence of the
quencher. The ratio of
/
gave the fraction of the population
0
ØF(x) = (As/Ax)(Fx/Fs)(nx/ns)2 ØF(s)
under dynamic quenching. The unimolecular rate constant (kq) for
the dynamic intramolecular quenching was calculated using
equation,
Where s is the standard, x is the nucleoside, A is the absorbance at
the excitation wavelength, F is the area under the emission curve, n
is the refractive index of the solvent and ØF is the quantum yield.
The standard compound used in the study was EtAd and its
reported quantum yield (0.56). The excitation wavelength of 280
(
/
0) = 1 + kq (
)
0
kq
=
1/ - 1/
0
Synthesis of the dGth nucleoside and other nucleoside acyl-
sulfamates:
nm for
the quantum yields.
7 8 was used in the calculation of
and 330 nm for dGth and
Time resolved fluorescence spectroscopy or fluorescence decay
curve measurements were recorded using time correlated single
photon count (TCSPC). Samples (1 μM ligands) were excited with
tunable dye laser range of 280-305 nm (instrument MatrixUV
The detail information on the synthesis of probes is described in the
supplemental information.
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