We describe a rapid electrophoresis-based method for profiling of carbonic anhydrase inhibitors. In addition to the pharmacophore moiety intended for reversible interaction with a target enzyme, a fluorescent template with a built-in azide group for photoaffinity labeling is also included as a part of the inhibitor design. Following incubation and irradiation, gel electrophoresis with visualization under UV allows assessment of the efficiency of cross-linking. The relative efficiency of cross-linking of various probes can be regarded as a reflection of their inhibition potencies, an assumption supported by the trend in their IC50/K i values. The method has the advantage of being applicable to impure enzyme preparations and also can be used to screen several inhibitors including their promiscuity in parallel in a short time as has been currently demonstrated with HCA II.
We describe a rapid electrophoresis-based method for profiling of carbonic anhydrase inhibitors. In addition to the pharmacophore moiety intended for reversible interaction with a target enzyme, a fluorescent template with a built-in azide group for photoaffinity labeling is also included as a part of the inhibitor design. Following incubation and irradiation, gel electrophoresis with visualization under UV allows assessment of the efficiency of cross-linking. The relative efficiency of cross-linking of various probes can be regarded as a reflection of their inhibition potencies, an assumption supported by the trend in their IC50/K i values. The method has the advantage of being applicable to impure enzyme preparations and also can be used to screen several inhibitors including their promiscuity in parallel in a short time as has been currently demonstrated with HCA II.
Identification of target
receptors/enzymes whose over- or underexpression
is often correlated with a disease plays a primary role in the drug
discovery process.[1] Once the target is
identified, the classical screening can be performed for small-molecule
libraries against a purified form of these receptors or enzymes using
in vitro assays.[2] However, this approach
usually provides little information about target selectivity and interaction
with potential off-targets in a complex proteomic mixture present
in the system (Figure ). It would be more convenient if the assay can be carried out in
whole cell lysates, which not only provides information about the
affinity of the small molecule substrates toward the protein but also
reveals any off-target interactions. In this respect, affinity-guided
targeting of a protein of interest in a mixture has emerged as a viable
technique.[3] In the 1960s,[4] affinity-guided probes have a long history since their
inception. The technology was recently applied for the identification
of irreversible inhibitors by Bogyo and co-workers[5] using radio-labeled probes and a subsequent electrophoresis-based
assay. This rapid screening approach resulted in the identification
of a cathepsin B-selective inhibitor.[6]
Figure 1
Classical
method of potential inhibitor screening of a mixture of proteins
A, B, C, and D.
Classical
method of potential inhibitor screening of a mixture of proteins
A, B, C, and D.More recently, Li et
al.[7] used activity-based
protein profiling (ABPP) for competitive profiling to screen libraries
of carbamates as irreversible inhibitors of uncharacterized serine
hydrolases. Subsequently, Cravatt et al.[8] have successfully applied the completive affinity-based technique
for screening of reversible inhibitors using a competitive fluorescent
rhodamine-based assay (Figure ). The advantage of this method is that promiscuous inhibitors
lacking
specificity can be readily rejected without any further processing.
The method, though ingenious and novel, can be technically challenging
as it requires previous knowledge of the kinetics of binding.
Figure 2
Cravatt’s
strategy for screening of reversible inhibitors.
Cravatt’s
strategy for screening of reversible inhibitors.Strategy for inhibitor screening described in this paper.A comparatively straightforward method would be
to have a fluorescent
photo-cross-linker part (acting as a reporter) included in the inhibitor
design by attaching it to a possible pharmacophore. In that case,
the reversible enzyme–inhibitor complex can be permanently
bonded to the target enzyme via photoirradiation. Subsequent gel electrophoresis
of the photo-cross-linking experiment followed by visualization, for
example, under UV and using Coomassie blue, should indicate the efficacy
of the inhibitor. However, one issue could be the combined size of
the fluorophore and the photo-cross-linker, which may weaken the binding.
For this not to happen, one needs to use a photoaffinity group-embedded
fluorescent label of smaller steric size as compared to the frequently
used ones like rhodamine/fluorescein (Figure ). To have a minimal effect of the reporter
moiety on the pharmacophore activity, this has been kept apart by
a linker of optimized length (reported earlier[9]) with three methylene groups. Keeping this in mind, we have designed
and synthesized a series of naphthalimide–aryl sulfonamide
hybrids (1–11) and have shown that
it is possible to compare their relative inhibition potencies by their
relative cross-linking efficiencies. The method is validated by doing
the cross-linking experiment against a control compound (vide infra)
as well as by comparison of IC50/Ki values. The design, synthesis, and screening of potential
inhibitors are described in this paper in detail.
Figure 3
Strategy for inhibitor screening described in this paper.
Results and Discussion
Our starting point was the recently described[9] linker-based azidonaphthalimide template, which serves
three functions simultaneously. It has the in-built photo-cross-linker
in the form of an azide, a less sterically bulky naphthalimide moiety
as a fluorescence template, and a variable linker ending up in the
carboxylic acid functionality. This 3-in-1 template has been shown
to considerably improve the utility of template-based affinity probes.
It could be easily connected to the selectivity hands that are potential
reversible binders of humancarbonic anhydrase II (HCA II)[10] or penicillin-binding proteins (PBPs).[11] We were able to detect the presence of these
enzymes in the presence of other proteins as well as in cell lysates.To expand the scope of this 3-in-1 template, we attached various
zinc binding motifs such as sulfonamide and its derivatives, carboxamide,
terpyridine, and hydroxamate to produce the inhibitor molecules (structures 1–11 as shown in Figure ). Our design was also inspired by the report
by Supuran et al.[12] of excellent inhibition
shown by a naphthalimide–sulfonamide hybrid (12) (Ki in a low nanomolar level). As a
positive control for our photo-cross-linking studies, we synthesized
the azido version (13) of Supuran’s molecule.
Figure 4
Desired
inhibitors.
Desired
inhibitors.Synthesis of the target
inhibitors started with the GABA-attached
azido naphthalimide as depicted in Scheme . Sulfonamides 1–5 and 7–9 and carboxamide 6 were synthesized by esterification with the corresponding
bromoacylated derivatives. Hydroxamate 11 was prepared
via nucleophilic displacement of the ethyl ester with free hydroxylamine.[13] For the synthesis of terpyridine derivative 10, the 4-methyl phenyl terpyridine was first brominated,[14] which was then esterified with the GABA-naphthalimidecarboxylic acid. The control compound 13 was prepared
via direct imide formation from azidonaphthalic anhydride and aminomethyl
benzene 4-sulfonamide. All the compounds were isolated by simple crystallization
from hexane ethyl acetate and were fully characterized by NMR and
HRMS analysis (included in the Supporting Information).
Scheme 1
Synthesis of the Potential Inhibitors
With the target compounds in hand, we then proceeded to
investigate
their photo-cross-linking ability for HCA II. The enzyme (at a fixed
concentration) was incubated for 15 min with each compound (20 μM)
separately and then photoirradiated (30 min). The irradiated mixture
was then subjected to gel electrophoresis under denaturing conditions.
The gel was first visualized under UV and then stained with Coommassie
blue. In both cases, the results were documented and then analyzed
by Image J software.[15] The gel pictures
and the relative efficiency of cross-linking are shown in Figure .
Figure 5
(A) SDS PAGE of purified
HCA II cross-linked with the final compounds.
C stands for control (only protein) and M stands for MW marker. Each
compound (20 μM) has been used in each respective lane. (B)
Image J analysis of the gel pictures to compare the relative efficiencies
of cross-linking. L1-L18 denotes the corresponding lane numbers in
(A). (C) Top: Relative
cross-linking efficiencies of the control compound 13 and the two analogous compounds 2 and 3. Bottom: Image J analysis of the gel picture at the top.
(A) SDS PAGE of purified
HCA II cross-linked with the final compounds.
C stands for control (only protein) and M stands for MW marker. Each
compound (20 μM) has been used in each respective lane. (B)
Image J analysis of the gel pictures to compare the relative efficiencies
of cross-linking. L1-L18 denotes the corresponding lane numbers in
(A). (C) Top: Relative
cross-linking efficiencies of the control compound 13 and the two analogous compounds 2 and 3. Bottom: Image J analysis of the gel picture at the top.Analysis of the gel pictures along with an overall
image analysis
clearly shows that all the 4-substituted benzene sulfonamides (1–3) showed the highest and also similar
(considering the error range of ±5%) cross-linking efficiencies
when compared with the control compound 13 (Figure C). Interestingly,
the 3-aminosulfonamide
(4) behaves similarly to the 4-susbtituted derivatives.
Except for the benzoyl sulfonamide (8), which showed
low but perceptible cross-linking efficiency, all other compounds
including the 2-aminosulfonamide (5) showed insignificant
cross-linking.To check the selectivity and any off-targets,
compounds 3 and 4 were incubated with the
lysate of Escherechia coli cells overexpressed
with HCA II.[16] Sulfonamide 3 was found to be selective,
while sulfonamide 4 was found to cross-link with other
proteins to a significant extent (comparison of lanes 2 and 3 in Figure ).
Figure 6
Cross-linking of compounds 3 and 4 with
cell lysate. Lane 1: molecular weight marker. Lane 2: compound 4. Lane 3: compound 3. The gel picture was taken
under UV and then stained with Coomassie blue. The amount of compound
used in each lane is 20 μM.
Cross-linking of compounds 3 and 4 with
cell lysate. Lane 1: molecular weight marker. Lane 2: compound 4. Lane 3: compound 3. The gel picture was taken
under UV and then stained with Coomassie blue. The amount of compound
used in each lane is 20 μM.To validate the results based on cross-linking, we determined
the
IC50 values of some of the compounds with strong and weak
photo-cross-linking activity and compared these with those of the
reference compound 13. The IC50 values (shown
along with the curves in Figure ) also follow the same order as that observed from
the gel-based assay. The Ki values for
compound 3 showing the highest cross-linking efficiency
were compared with those of compounds 5 and 6 with insignificant cross-linking. The trend remains similar. Incidentally,
the reference compound 13 having a similar cross-linking
efficiency to that observed for 3 also has a nanomolar Ki value (table in Figure ).
Figure 7
Determination of IC50 and Ki values of some key compounds.
Determination of IC50 and Ki values of some key compounds.In conclusion, the present method offers a rapid
way of initial
screening of potential inhibitors, and based on the efficiency of
photo-cross-linking, compounds can be selected for proceeding further,
and weakly cross-linked or promiscuous inhibitors showing lack of
selectivity can be discarded. To check whether the fluorescent photoreactive
template works for other enzymes, derivatives with an appropriate
selectivity functionality were made to target the penicillin-binding
protein (PBP), metallo-β-lactamases like NDMs, and the fatty
acid dehydratase enzymes HadAB and HadBC. In all cases, we could detect
successful cross-linking demonstrating that the template has little
effect on the binding efficiency of the selectivity hand. While the
details of successful cross-linking with PBP and NDMs have already
been published,[9,17] the cross-linking results with
other enzymes will be reported after more elaborative studies.
Experimental
Section
General Procedure
All the reactions under an inert
atmosphere were conducted with oven-dried glassware with anhydrous
solvents dried using standard methods and purified by distillation
prior to use. All common reagents were of commercial grade unless
otherwise specified. Thin-layer chromatography (TLC) was performed
on aluminum-backed plates coated with Silica gel 60. A locally available
ultraviolet light chamber was used as the TLC spot indicator. All
new compounds were characterized using 1H nuclear magnetic
resonance (NMR) and 13C NMR spectroscopies. The NMR spectra
were recorded using Bruker 400 MHz and 600 MHz spectrometers. Proton
and carbon spectra were referenced internally to solvent signals using
values of δ = 2.50 for proton and δ = 39.52 for carbon
(middle peak) in DMSO-d6, of δ =
7.26 for proton and δ = 77.16 for carbon (middle peak) in chloroform-d, and of δ = 2.05 for proton and δ = 206.26
and 29.84 for carbon (middle peak) in acetone-d6. The following abbreviations have been used for NMR peak
assignments: s = singlet, bs = broad singlet, d = doublet, t = triplet,
p = pentet, m = multiplet, and dd = double of doublet. All biochemical
experiments have been done as described in our previous papers.[18,17]
Preparation of GABA-Carboxylic Acid (14)
The
procedure for preparation of the GABA-carboxylic acid derivative
is same as that reported earlier.[9] To a
solution of 4-azido-1,8-naphthalic anhydride (0.05 g, 0.2 mmol) in
dry ethanol (7 mL), DMAP (0.002 g, 0.02 mmol) was added and stirred
for 10 min. 4-Aminobutyric acid (GABA) (0.272 mmol) was added and
the mixture was refluxed for 12 h. After cooling, the precipitated
yellow solids were separated from the solution, washed with cold ethanol,
and air-dried to furnish the azidonaphthalimideGABA-carboxylic acid.
The compound was reprecipitated from the DCM–hexane mixture
and washed with hexane to get the carboxylic acid as a yellow solid.
General Method for Preparation of the Bromoacetyl Derivative
of Sulfonamides (15–19 and 21–23) and Carboxamide (20)
To a solution of sulfanilamide/carboxamide (0.35 g, 2.03
mmol) in dry THF (10 mL), K2CO3 (0.561 g, 4.06
mmol) was added and stirred for 30 min. It was cooled to 0 °C,
and bromoacetyl chloride (0.2 mL, 2.44 mmol) was added dropwise to
the reaction mixture and was stirred for 30 min at 0 °C. Water
was added, and the mixture was extracted with EtOAc (50 mL ×
2), washed with brine, dried over Na2SO4, and
concentrated in vacuo to get the product as a white crystalline solid.
The spectral data and other details are mentioned below.
Synthesis, NMR, and HRMS data
reported earlier.[14]
General Method for Preparation
of Sulfonamides 1–9
To a
solution of azidonaphthalimideGABA-carboxylic acids (0.15 mmol) in dry DMF (5 mL) under N2, anhydrous K2CO3 (0.18 mmol) was added and
stirred for 30 min at room temperature. A solution of bromoacetyl
derivatives of sulfonamide/carboxamide (0.18 mmol) in dry DMF (2 mL)
was added, and stirring was continued for 10 h at room temperature.
The reaction was quenched by adding water (30 mL), and the aqueous
layer was extracted with EtOAc (30 mL × 2). The combined organic
layers were washed with brine, aqueous NaHCO3, and water,
dried over anhydrous Na2SO4, and concentrated
in vacuo. The yellowish brown gummy product was first precipitated
from the acetone–hexane mixture to get the yellow precipitate,
which was washed with hexane 2–3 times to furnish the target
materials as yellow solids. The spectral and other details are mentioned
below
Synthesis of 2-(5-(4-([2,2′:6′,2″-Terpyridin]-4′-yl)phenyl)-4-oxopentyl)-6-azido-1H-benzo[de]isoquinoline-1,3(2H)-dione (10)
The GABA-carboxylic acid derivative
of 4-azido-1,8-naphthalimide (14) (0.11 g, 0.35 mmol)
was dissolved in methanol, and NaHCO3 (29 mg, 0.35 mmol)
was dissolved in a minimum amount of water. To the solution of methanol,
a NaHCO3 solution was added. The mixture was stirred for
2 h, and the compound was lyophilized for 2 h to obtain the sodium
salt of 14 as a yellow solid. To this solid in dry DMF,
compound 24 (0.12 g, 0.35 mmol) was added. The reaction
mixture was stirred for 12 h under a N2 atmosphere at room
temperature. The organic compound was extracted with ethyl acetate
and washed with water to remove DMF. The yellow compound was further
precipitated from the ethyl acetate–hexane layer. Yellow gummy
solid; 1H NMR (400 MHz, chloroform-d)
δ 8.75–8.58 (m,7H), 8.55 (d, J = 8.0,
1H), 8.40 (d, J = 8.4, 1H), 7.87 (d, J = 6.0, 4H), 7.75–7.67 (m, 1H), 7.48–7.41 (m, 3H),
7.35 (t, J = 6.2, 2H), 5.16 (s, 2H), 4.26 (t, J = 7.0, 2H), 2.54 (t, J = 7.4, 2H), 2.15
(p, J = 7.1, 2H). 13C NMR (125 MHz, chloroform-d) δ 172.9, 164.2, 163.7, 156.3, 156.1, 149.9, 149.3,
143.6, 138.4, 137.0, 132.4, 131.9, 129.3, 129.0, 128.8, 127.6, 127.0,
124.5, 124.0, 122.7, 121.5, 119.0, 114.8, 66.0, 39.7, 32.1, 23.6.
IR (KBr, cm–1): 3404, 2931, 2123, 1708, 1352, 1272,
1111, 784, 726, 612. HRMS: Calcd for C38H27N7O4H (M + H)+ 646.2203, found 646.2201.
Synthesis of 4-(6-Azido-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N-hydroxybutanamide (11)
To a solution of compound 14 (0.1 g, 0.31 mmol) in dichloromethane at 0 °C, ethyl
chloroformate (0.04 g, 0.37 mmol) and N-methylmorpholine
(0.041 g, 0.40 mmol) were added, and the mixture was stirred for 10
min. In another reaction vessel, an alcoholic solution of hydroxylamine
was prepared by stirring hydroxylamine hydrochloride (0.043 g, 0.62
mmol) with potassium hydroxide (0.035 g, 0.62 mmol) in methanol for
15 min followed by filtration of the solution. The freshly prepared
hydroxylamine in methanol was added to the previous reaction mixture
in dichloromethane and stirred for 15 min. The solvent was removed
under reduced pressure to obtain compound 11. Yellow
gummy solid; 1H NMR (400 MHz, methanol-d4) δ 8.58 (d, J = 7.3, 1H), 8.55
(d, J = 8.0, 1H), 8.49 (d, J = 8.5,
1H), 7.80 (t, J = 7.8, 1H), 7.64 (d, J = 8.0, 1H), 4.18 (t, J = 7.0, 2H), 2.20 (t, J = 7.6, 2H), 2.02 (p, J = 7.7, 2H). 13C (125 MHz, methanol-d4) δ
172.2, 165.5, 165.1, 145.3, 133.1, 133.1, 130.4, 130.0, 128.1, 125.6,
123.7, 119.8, 116.3, 40.7, 31.5, 25.4. IR (KBr, cm–1): 3309, 2946, 2123, 1730, 1272, 1119, 733, 624. HRMS: Calcd for
C16H13N5O4Na (M + Na)+ 362.0865, found 362.0868.
Synthesis of Compound 13
To a solution
of 4-azido-1,8-naphthalic anhydride (0.1 g, 0.4 mmol) in dry ethanol
(7 mL), DMAP (0.010 g, 0.08 mmol) was added and stirred for 10 min.
Homosulfamine hydrochloride (0.098 g, 0.44 mmol) was added to the
reaction mixture and refluxed for 12 h. After cooling, the precipitated
yellow solids were separated from the solution, washed with cold ethanol,
and were air-dried to furnish compound 13, and it was
characterized without any further purification. Yellow solid; mp >195
°C. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 7.3 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H),
7.84 (t, J = 7.9 Hz, 1H), 7.78–7.68 (m, 1H),
7.53 (d, J = 8.2 Hz, 1H), 7.31 (s, 1H), 5.28 (s,
1H). 13C NMR (100 MHz, DMSO-d6) δ 163.3, 162.8, 143.2, 142.8, 141.3, 131.9, 131.8, 128.6,
128.4, 127.8, 127.3, 125.8, 123.5, 122.0, 117.9, 116.0, 42.7, 40.14.
HRMS: Calcd for C19H13N5O4SH (M + H)+ 408.0766, found 408.0764.
Authors: Partha Sarathi Addy; Baisakhee Saha; N D Pradeep Singh; Amit K Das; Jacob T Bush; Clarisse Lejeune; Christopher J Schofield; Amit Basak Journal: Chem Commun (Camb) Date: 2013-01-30 Impact factor: 6.222
Figure 1
Figure 2
Figure 3
Figure 4
Scheme 1
Figure 5
Figure 6
Figure 7