Supramolecular Interaction of Molecular Cage and β-Galactosidase: Application in Enzymatic Inhibition, Drug Delivery and Antimicrobial Activity

Article abstract of DOI:10.1002/cbic.202100008

Enzyme inhibitors play a crucial role in diagnosis of a wide spectrum of diseases related to bacterial infections. We report here the effect of a water-soluble self-assembled Pd^II₈ molecular cage towards β-galactosidase enzyme activity. The molecular cage is composed of a tetrapyridyl donor (L) and cis-[(en)Pd(NO₃)₂] (en=ethane-1,2-diamine) acceptor and it has a hydrophobic internal cavity. We have observed that the acceptor moiety mainly possesses the ability to inactivate the β-galactosidase enzyme activity. Kinetic investigation revealed the mixed mode of inhibition. This inhibition strategy was extended to control the growth of methicillin-resistant Staphylococcus aureus. The internalization of the Pd(II) cage inside the bacteria was confirmed when bacterial solutions were incubated with curcumin loaded cage. The intrinsic green fluorescence of curcumin made the bacteria glow when put under an optical microscope. Furthermore, this curcumin loaded molecular cage shows an enhanced antibacterial activity. Thus, Pd^II₈ molecular cage is quite attractive due to its dual role as enzyme inhibitor and drug carrier.

Full text of DOI:10.1002/cbic.202100008

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Supramolecular Interaction of Molecular Cage and β-
Galactosidase: Application in Enzymatic Inhibition, Drug
Delivery and Antimicrobial Activity
[a]
[b]
[a]
[a]
Avijit Mondal, Imtiyaz Ahmad Bhat, Subbaraj Karunakaran, and Mrinmoy De*
Enzyme inhibitors play a crucial role in diagnosis of a wide
spectrum of diseases related to bacterial infections. We report
here the effect of a water-soluble self-assembled Pd 8 molecular
to control the growth of methicillin-resistant Staphylococcus
aureus. The internalization of the Pd(II) cage inside the bacteria
was confirmed when bacterial solutions were incubated with
curcumin loaded cage. The intrinsic green fluorescence of
curcumin made the bacteria glow when put under an optical
II
cage towards β-galactosidase enzyme activity. The molecular
cage is composed of a tetrapyridyl donor (L) and cis-[(en)
Pd(NO ) ] (en=ethane-1,2-diamine) acceptor and it has a
microscope. Furthermore, this curcumin loaded molecular cage
3
2
II
8
hydrophobic internal cavity. We have observed that the accept-
or moiety mainly possesses the ability to inactivate the β-
galactosidase enzyme activity. Kinetic investigation revealed the
mixed mode of inhibition. This inhibition strategy was extended
shows an enhanced antibacterial activity. Thus, Pd molecular
cage is quite attractive due to its dual role as enzyme inhibitor
and drug carrier.
Introduction
supramolecular coordination architecture with protein, we have
chosen β-galactosidase (β-Gal) as a model protein and a water-
1
6+
Biomolecules are natural targets for the development of many
soluble [Pd L ]
(1) molecular cage as metallasupramolecular
8
4
[
1]
inorganic drugs. Several small molecules, nanomaterials and
architecture.
[2]
macromolecules have been developed to target various
biomolecules. Metal based supramolecular coordination archi-
tectures have been exploited extensively in catalysis, sensing,
host-guest study, cavity induced unusual organic transforma-
β-Gal is a therapeutically relevant enzyme which catalyses
the hydrolysis of β-D-galactoside to galactose and alcohol,
which are the key sources to produce energy. It is a vital
enzyme for the human body; the deficiency of which can cause
[
3]
[10]
tions, antimicrobial activity etc. Similarly, their biomolecular
interaction also opened up new avenues for biomedical
galactosialidosis or Morquio B syndrome. β-Gal is an essential
enzyme for various microorganisms as well. Enhanced prolifer-
ation rate of various pathogenic bacteria like methicillin-
resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli)
rely on galacto-oligosaccharides which is formed through
[
4]
research which include, sensing of biomolecules, recognition
[5]
[6]
of proteins, recognition of nucleotide base, anticancer
[7]
[8]
therapy, drug delivery etc. Among them, recognition of
protein is key to control a range of cellular processes, such as
cellular signal transduction, protein antigen/antibody interac-
[11]
several enzymatic reactions catalysed by β-Gal.
Although
there is a little difference exists between human β-Gal and
bacterial β-Gal in their oligomerization state and domain
organization, but the mode of action is nearly same. The loop
region of β-domain 2 (residues 482–491) in human β-Gal
execute the same role as the complementation loop of E. coli β-
Gal. Moreover, the active site of human β-Gal is easily
approachable from the bulk solvent whereas the active site of
[
9]
tion and DNA transcription.
However, the effect of
supramolecular coordination assemblies on protein is not well
studied. Kamiya et al. have shown the interaction of saccharide
coated M L molecular spheres with protein concanavalin-A
12 24
[
5]
and peanut agglutinin. The polysaccharides present on the
outer surface of the sphere help to recognize the protein
surface of concanavalin A. Though M L sphere can recognize
[
12]
E. coli β-Gal is not easily accessible from the bulk solvent.
1
2 24
protein surface but the mode of interaction of the metallacage
with protein and their kinetic behaviour have not been
explored so far. To get insight about the interaction of
Hence, developing β-Gal inhibitor from E. coli benefits to tune
the activity for various relevant application. In literature various
small molecules have been reported as β-Gal inhibitor, such as
L-ribose, D-galactose, D-galactonolactone etc. Not only small
molecules but also several nanomaterials have been developed
to alter the enzymatic activity of β-Gal. Surface engineered gold
[13]
[
a] A. Mondal, S. Karunakaran, Dr. M. De
Department of Organic Chemistry, Indian Institute of Science
Bangalore 560012 (India)
[14]
[15]
[16]
nanoparticles, MoS nanosheets, ZnO nanoparticles and
2
[17]
E-mail: md@iisc.ac.in
b] I. A. Bhat
positively charged graphene oxide (GO) have been used to
tune the activity of β-Gal. By controlling the activity of β-Gal,
bacterial growth can also be controlled as reported by Kotov
[
Department of Inorganic and Physical Chemistry
Indian Institute of Science, Bangalore 560012 (India)
[16]
et al. However, to the best of our knowledge, metal based
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbic.202100008
supramolecular coordination architectures have not yet been
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[
18]
1
explored as β-Gal enzyme inhibitor. Compared to existing
systems, metallasupramolecular architecture-based inhibitors
possibly hold many other advantages. Not only we can tune the
surface property at the molecular level through the judicious
selection of metals/ligands but also their high positive charges,
large surface area, and high water solubility provide an
attractive means to control the activity of enzyme.
ure 1). The molecular cage 1 was characterized by H NMR,
ESI-MS spectrometry to reconfirm the structure and purity
[18]
before the biological study. Four ligands (L) are present along
the four walls of the square-barrel and are connected together
by eight acceptor units present along the edges to form the
overall square barrel structure. The cage possesses a hydro-
3
phobic internal cavity with dimensions of 12.2×12.2×15.3 Å .
We report here our study on the interaction of a water
Also, the presence of the eight cis-[(en)Pd(NO ) ] groups in cage
3
2
1
6+
soluble metallasupramolecular [Pd L ]
Figure 1). The cage 1 was constructed employing a sym-
cage (1) with β-Gal
1 makes it highly water soluble and cationic (with +16 charge)
in nature. Due to its cylindrical and barrel-shaped structure, the
molecular cage 1 possesses windows similar to its cavity size
which makes it suitable for the easy ingress and egress of the
hydrophobic drug molecules. In biological systems, barrel-
shaped molecules such as β- barrel proteins are known for
diffusing the small molecules and ions across cell
8
4
[18]
(
metrical tetrapyridyl donor (L) with cis-[(en)Pd(NO ) ] (en=
3
2
ethane-1,2-diamine) acceptor. To our surprise, we have ob-
served that the acceptor moiety is mainly responsible for
enzyme inhibition. We have studied the mode of interaction of
1
with β-Gal through kinetic measurements. Also, inspired by
[16]
[19]
the work of Kotov et al., we have extended our strategy to
control the bacterial growth. The study revealed that the
molecular cage 1 has higher antibacterial activity compared to
the precursor building block cis-[(en)Pd(NO ) ] against methicil-
membranes. In this regard, various barrel-shaped molecules
having large intrinsic cavities have been reported in literature
[20]
with potential applications in biology.
3
2
lin-resistant Staphylococcus aureus (MRSA) bacteria. We ex-
pected that the cage 1 would penetrate the cell more efficiently
and inhibit β-Gal in the cytosolic environment. In order to
support the internalization of the cage 1 inside the bacteria cell,
we have used curcumin as a trapping reagent. Curcumin is
insoluble in water, but the hydrophobic cavity of Pd L cage (1)
β-Gal activity assay and inhibition kinetics
β-Gal is a large tetrameric protein which has overall negatively
[14]
charged surface.
The active site of the β-Gal contains
Glutamic acids residues which are mainly responsible for
enzyme catalysis. The most possible mode of inhibition is due
to the binding of the cage to β-Gal through electrostatic
interaction followed by coordination Pd complex with amino
acid residues at active site. As the active site of the β-Gal has
negatively charged residues and the cage is overall positively
charged, so there is also a possibility of blocking the active site
of β-Gal by cage. Not only into the active site but also cage can
bind to the surface of the enzyme and thereby altering its
8
4
offers to encapsulate curcumin to form the water-soluble
[18]
inclusion complex (1�curcumin). Thus, the inclusion complex
1
�curcumin can be used for targeting the enzyme and as a
drug carrier simultaneously.
Results and Discussion
II
Synthesis and characterization of Pd 8 molecular cage
enzymatic
activity
as
reported
by
using
other
[
21]
macromolecules. To support the above hypothesis, activity
assays were performed to test the ease of inhibition by cage 1
using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a chro-
mogenic substrate (Figure 2a). The experiment was carried out
by pre-incubating 0.5 nM β-Gal with variable concentrations of
the cage 1 (ranging from 0 to 310 nM) in 5 mM sodium
phosphate buffer (pH 7.4) for 30 min. The enzymatic activity
was monitored from the rate of hydrolysis of ONPG. The activity
of β-Gal in the absence of 1 was taken as control. The activities
in the presence of the cage 1 were normalized after subtracting
the blank (without enzyme). The enzymatic activity was reduced
to ~10% when the concentration of 1 reached 310 nM
First, we synthesised the water-soluble tetrafacial Pd L coordi-
8
4
nation cage 1 by self-assembly of the symmetric tetrapyridyl
donor L with a 90° acceptor M [where M=cis-(en)Pd(NO ) ;
3
2
en=ethane-1,2-diamine] following the reported procedure (Fig-
(Figure 2b). But in case of control experiment with cis-[(en)
Pd(NO ) ] complex shows a similar extent of enzymatic activity
3
2
inhibition (since one cage is formed by 8 [cis-(en)Pd(NO ) ]
3
2
components) (Figure 2d). The IC50 value for the cage 1 is
5
9
3.8 nM (Figure 2c) whereas the IC50 value for the Pd acceptor is
25 nM (figure 2d). This suggest that cage is better inhibitor
than only Pd-acceptor possibly due to the higher local
concentration. We have carried out the same experiment in
0
.01% triton X-100 to verify the possibility of aggregation
1
6+
Figure 1. Synthesis of [Pd
8
L
4
]
molecular cage (1) and the inhibitory
induced inhibition (supporting information, Figure S1). The
experimental data shows nearly closer value of IC50 in PBS
interaction with β-Gal enzyme.
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of the enzyme. When the value of α=1, inhibition is non-
competitive. In this kind of inhibition, the inhibitor does not
affect the binding of substrate to the enzyme, instead, it binds
with the enzyme substrate complex and free enzyme with equal
affinity. A value of α!1 indicates uncompetitive inhibition. In
uncompetitive inhibition, inhibitor binds to the enzyme-sub-
strate complex exclusively. By using the above expression, the
curve fitting analysis gave α value ranging from 1.34 to 2.44
which could be due to the mixed mode of inhibition
(supporting information, Figure S2). The mode of inhibition can
also be assessed by using the Lineweaver-Burk plot (Figure 3b).
From the curve fitting analysis, we observed the value of K to
i
be 95.34 nM. Compared to other nanomaterial based inhibitors
[16]
(
e.g. 0.72 μM for ZnO nanopyramid ) and conventional
inhibitors (e.g. 1 μM for phenylethyl thio-β-D-galactoside
[
24]
[24]
[24]
(
0
PETG),
24 mM for D-galactose,
0.24 mM for L-ribose,
[24]
.7 mM for D-galactonolactone) cage 1 exhibits very high
enzymatic inhibition without any effect on secondary structure
Supporting Information, Figure S3).
(
Antibacterial activity against MRSA
Figure 2. (a) Scheme of ONPG hydrolysis with β-Gal in 5 mM sodium
phosphate buffer (pH 7.4) using ONPG as a substrate. (b) Normalized activity
of β-Gal (0.5 nM) as a function of different concentrations of cage 1. (c) IC50
for cage 1 and hill slope. (d) Normalized activity of β-Gal (0.5 nM) as a
[
11]
As the β-Gal is highly responsible for bacterial growth,
cytosolic inhibition of its activity can control the growth of
various microorganisms. Applying this hypothesis, earlier small
function of different concentrations of cis-[(en)Pd(NO
cis-[(en)Pd(NO ] complex and hill slope.
3 2
) ] complex. (e) IC50 for
3 2
)
[25]
molecule such as ginkgolic acid (C15:1)
and ZnO
[16]
nanopyramids were used for antibacterial activity. Consider-
ing those reports, the cage 1 or the cis-[(en)Pd(NO ) ] complex
3
2
(53.8 nM) and in 0.01% triton X-100 (51.8 nM), which excludes
the possibility of aggregation induced inhibition. The close
value of IC50 of free complex and the cage suggests that the
cage 1 in the presence of β-Gal was disassembled and each
acceptor unit present in the cage played a key role to inhibit
the activity of β-Gal. Even though it was well established that
the active site of β-Gal (His-418) is highly responsible for metal
ion binding and hence has a direct effect on enzymatic
[22]
activity, however there are no metal complex based inhibitor
reported so far.
Understanding the mode of enzyme inhibition helps to
determine the nature of the interaction with cage 1. The
enzyme velocity data as a function of substrate (S) concen-
tration at different fixed concentrations of 1 was used to
calculate the inhibition constant (K). The data were fitted
i
according to nonlinear regression using Graph-Pad Prism 5
software. The equation used to determine the velocity (V) of an
enzymatic reaction in the presence of an inhibitor (I) is:
V
½Sꢀ
þ ð1 þ Þ
max
V ¼
�
ꢀ
I
½Iꢀ
½
Sꢀ 1 þ
aKi
Ki
The calculated value of α from the above equation is
important to determine whether the mode of interaction is
[23]
competitive, non-competitive, or uncompetitive. When the
value of α @1, it indicates competitive inhibition. In compet-
itive inhibition, the inhibitor binds exclusively to the active site
Figure 3. (a) Enzyme velocity as a function of substrate concentration at
various fixed concentration of cage 1. 2.5 nM β-gal concentration was
maintained throughout the experiment. (b) Lineweaver-Burk plot.
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might be expected to show antibacterial effects. We have
carried out the growth kinetics of MRSA bacteria in presence of
various concentration of cage 1 and it shows MIC at 0.1 mM
concentration (Figure 4a). Similarly, we have also explored the
effect in case of gram-negative bacteria. For that purpose, we
have considered P. aeruginousa (PA) and we have observed
almost equally effective antimicrobial activity (Figure 4b). In
order to test the bacteria killing efficacy of cage 1 and Pd
complex, we have checked the colony forming ability of MRSA
bacteria in agar plate. MRSA bacteria having OD 0.01 were
incubated at 37°C for 5–6 hours with different concentrations
cytosol, it inhibits the β-Gal and thus prevents the bacterial
growth. This is confirmed by checking the amount of active β-
Gal remained inside the bacteria after treating with cage 1 and
cis-[(en)Pd(NO ) ] complex (Supporting Information, Figure S5).
3
2
This was done by using treated bacteria cell lysates. It was
observed that bacteria treated with cage 1 possesses very less
amount of active β-Gal in cell lysates compared to the bacteria
treated with cis-[(en)Pd(NO ) ] complex. This confirms the better
3
2
internalization of cage molecule compare to the acceptor
building blocks. The better internalization ability of 1 through
the bacterial cell membrane followed by disintegration by
enzymes also can be used as a carrier of various water insoluble
drug molecules. In order to further support the better internal-
ization of the cage molecule and carrier of hydrophobic drugs
we have used curcumin as a trapping agent. Curcumin, the core
ingredient of turmeric, is known to have a broad range of
pharmacological properties and is a popular antimicrobial,
antidiabetic, anti-inflammatory, anticancer, and antioxidant
of cage 1 (ranging from 0.1 mM to 0.25 mM) and cis-[(en)
Pd(NO ) ] complex (ranging from 0.8 mM to 2 mM; 8 times
3
2
compared to cage 1 concentration). The bacterial solution
without the cage 1 or Pd acceptor complex was taken as
control. After incubation, bacterial solutions were diluted
1
00 times and 10 μL of the solution streaked on agar plate to
check the bactericidal efficiency of cage 1 and cis-[(en)Pd(NO ) ]
3
2
[26]
complex (Figure 4). There is a significant reduction in colony
formation in the presence of cage 1 (Figure 4c); however, the
cis-[(en)Pd(NO ) ] complex failed to show any significant
agent. Also, the intrinsic green fluorescence of curcumin was
[27]
used as an optical biomarker. Though curcumin has a wide
range of applications, but its poor solubility in water limits its
3
2
[28]
reduction in colony formation even at 2 mM concentration
Figure 4d). The ligand (L) also did not show any antibacterial
bioavailability and pharmacological potential. We have shown
(
previously that the hydrophobic interior of the cage 1 can
[18]
effect (Supporting Information, Figure S4).
efficiently encapsulate curcumin and we found that, 15 μM
cage 1 can encapsulate 16.3 μM of curcumin (Supporting
Information). Following the similar method, the internalization
of 1 within the bacterial cell was confirmed when the bacterial
solution was incubated with curcumin loaded cage (1�curcu-
Based on the above observation, we can conclude that,
even though the cis-[(en)Pd(NO ) ] complex is responsible for β-
3
2
Gal enzyme inhibition, it is not suitable for bacterial cell wall
and plasma membrane penetration. Whereas the bacteria killing
efficiency of cage 1 suggests that the self-assembled cage is
suitable for cell internalization, possibly due to the presence of
amine bound cationic acceptor on the corners (with +16
charge). Once the cage 1 is internalized inside the bacteria
min). The intrinsic green fluorescence of curcumin made the
bacteria fluoresce when analysed under the optical microscope
(Figure 5a).
However, the bacteria treated only with curcumin did not
show any fluorescence (Figure 5b). This indicates that the cage
1
is internalized in bacterial cells followed by its disintegration
inside the bacteria to release the curcumin in cytosol. As the
sparingly water-soluble curcumin has antibacterial activity, we
have also tested the effect of the curcumin loaded cage system
(
1�curcumin) on the growth of MRSA bacteria. As speculated,
the antibacterial efficacy of 1�curcumin system was found to
exhibit higher compared to the only cage 1 molecule. While
checking the antibacterial efficacy of 1�curcumin, we have
found that 1�curcumin shows higher antibacterial activity
compared to cage 1 alone. Though there were a few colonies
noted at 0.25 mM cage concentration (Figure 4c), but no
bacterial colony formation was observed at this concentration
for the curcumin loaded cage (1�curcumin) (Figure 5c). In a
control experiment only with curcumin, an uncountable
number of colonies were formed (Figure 5d) (curcumin concen-
tration was varied from 0.16 mM to 0.27 mM). This observation
further established that the poor solubility and hence less
bioavailability of curcumin is inefficient for therapeutic applica-
tions. But cage 1 forms inclusion complex (1�curcumin) which
enhances the effective solubility and efficiently internalized by
bacteria cells. After internalization, the cage was disintegrated
inside the cell and released the curcumin drug that led to
higher antibacterial effect. In our previous report we have
demonstrated that the toxicity of 1�curcumin as well as cage 1
Figure 4. Bacterial growth curve in presence different concentration of cage
1
(a) MRSA (b) PA. Colony-forming ability of MRSA after treating with:
c) different concentrations of cage 1; (d) different concentrations of cis-[(en)
Pd(NO ] complex.
(
3 2
)
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Experimental Section
Materials and methods: β-Galactosidase (from Escherichia coli), o-
nitrophenyl-β-D-galactopyranoside (ONPG) and curcumin were
purchased from Sigma. Bruker 400 MHz NMR instrument was used
to record NMR spectra. Agilent 6538 Ultra-High Definition (UHD)
Accurate Mass Q-TOF spectrometer was used for ESI-MS experi-
ments. Activity assays were done by using Thermo Scientific
Varioskan Flash Multimode Reader (ultraviolet-visible measure-
ment). JASCO, J-815 CD spectrometer was used to measure circular
dichroism spectra. Bacterial optical density was measured by
Eppendorf Bio Spectrometer UV-vis spectrometer. Bacterial imaging
was performed by an Olympus IX73 microscope.
Synthesis and characterization of Pd L cage 1: Pd L molecular
8
4
8 4
cage
1 was synthesized according to our earlier reported
[18]
procedure. Briefly, in a glass vial ligand L (21.4 mg, 0.05 mmol)
and cis-[(en)Pd(NO ) ] (30 mg, 0.1 mmol) were taken and 1.5 mL of
3
2
Millipore water was added to it. The suspension was sonicated for
proper mixing and then heated at 60°C with constant stirring for
1
2 h. The faint bluish solution formed was centrifuged to get the
clear supernatant solution. Methanol was slowly diffused into the
solution to obtain shining single crystals of 1 after 15 days. Isolated
À 1
yield: 28.5 mg, 55%. IR: υ (cm )=3101, 1599, 1495, 1314, 1215,
1
1
059, 824, 593. H NMR (400 MHz, D O): δ=8.51 (d, 32H), 7.85 (s,
2
Figure 5. Fluorescence imaging of MRSA bacteria incubated with (a) curcu-
�
curcumin) and (b) only curcumin. (c) Colony-forming
8H), 7.46 (d, 32H), 7.24 (s, 8H) and 3.18(s, 32H). ESI-MS (m/z)=
umin loaded cage (1
À
+3 +4
À
3
ability of MRSA after treating with different concentrations of curcumin
1268.0125 [1–3NO ] , 936.3440 [1–4NO ] and 603.9027 [1–
3
À
+6
loaded cage (1�curcumin) (top concentration is for cage and bottom
6NO ]
.
3
concentration is for loaded curcumin) and (d) Control and different
concentrations of only curcumin.
Activity assay: All the experiments were carried out in sodium
phosphate buffer (pH 7.45) at 25°C. For the enzyme inhibition
studies, 0.5 nM of β-galactosidase was incubated with varying
concentration of the molecular cage 1 (concentrations ranging
from 0 to 310 nM) and [cis-(en)Pd(NO ) ] complex (concentrations
[18]
is very low based on cellular toxicity assay using HeLa cells.
3
2
Hence development of this kind of cage system is always
attractive for drug loading purpose. By changing the building
block of cage system, the internal cavity size can be tuned to
encapsulate various hydrophobic drug of comparable sizes.
Although the cage system is very attractive for drug loading, it
has few limitations as well. A small internal cavity cannot
encapsulate the drugs of bigger sizes. Moreover, the presence
of a specific enzyme is one of the criteria to release the drug
inside the bacteria.
ranging from 0 to 2.48 μM) for 30 minute in 96-well plate. After
0 minutes of incubation, the enzymatic hydrolysis reaction was
3
initiated by adding 4.41 mM ONPG stock solution (final concen-
tration 441 μM). The enzyme activity was monitored by estimating
the formation of o-nitrophenolate anion from ONPG over a period
of 3 hour at λ=405 nm with a microplate reader (Thermo Scientific
Varioskan Flash Multimode Reader (ultraviolet-visible measure-
ment)). The assays were performed in triplicates. In parallel same
experiments were carried out taking every other component except
the enzyme and these blank values were subtracted from the
experimental time points. All the concentration mentioned here are
the final concentrations. The tetrapyridyl ligand is insoluble in water
so it is not possible to carry out the control study with the ligand.
Conclusion
Kinetic study: To elucidate the mode of binding, and to calculate
the inhibition constant (K) β-galactosidase (2.5 nM) was incubated
i
In summary, we have explored the biomolecular interaction of a
with various concentration of molecular cage 1 (0 nM, 7.8 nM,
15.7 nM and 23.5 nM). For each concentration of molecular cage 1
initial velocity was measured at various concentration of ONPG. The
initial velocity was determined in all cases by the linear fitting of o-
nitrophenolate anion (hydrolysis by-product) formation over the
time intervals at λ=405 nm. The experiments were performed in
triplicates. All fittings were performed using GraphPad Prism 5,
where the mixed-model inhibition equation (which is a general
Pd L supramolecular coordination cage 1 with β-Gal enzyme.
8
4
We observed that in presence of the β-Gal enzyme the cage 1
disintegrates and the cis-[(en)Pd(NO ) ] building block of the
3
2
cage is mainly responsible for mixed mode enzyme inhibition.
This is the first report of a metal complex based β-Gal inhibition.
Also, we observed the cage 1 can be internalized inside the
bacteria more efficiently compared to the acceptor building
unit and exhibits higher antibacterial activity against MRSA
bacteria. Moreover, the hydrophobic interior of the water-
soluble cage helps to encapsulate hydrophobic antibacterial
drug curcumin and can offer as a potential drug carrier.
Combining the cellular internalization, enzymatic inhibition and
hydrophobic drug encapsulation, curcumin loaded cage 1
exhibits better antibacterial activity compared to the cage
alone.
velocity equation) was used to calculate the inhibition constant K.
i
Mixed-model inhibition includes competitive, uncompetitive, and
noncompetitive inhibition as special cases. The control plot gives
the Km value of ONPG 728 μM. The reciprocals of enzymatic
velocity enzyme velocity and substrate concentration plot is known
as Lineweaver-Burk plot which helps to determine the mode of
inhibition.
Antimicrobial assays: The antibacterial activity of the molecular
cage 1, [cis-(en)Pd(NO ) ] complex, inclusion complex 1�curcumin
3
2
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was tested against the growth of Methicillin Resistant Staph-
ylococcus aureus (MRSA, USA300). The freeze-dried bacterial stock
was revived on nutrient agar plates. Primary culture was then
prepared by taking few colonies of bacteria from agar plate and
culturing it in Luria broth media (LB, HiMedia – 20 g/L) overnight
Keywords: cage compounds
antibacterial activity · self-assembly · supramolecular chemistry
·
enzymatic inhibition
·
[
[
1] T. K. Ritter, C. H. Wong, Angew. Chem. Int. Ed. 2001, 40, 3508–3533;
Angew. Chem. 2001, 113, 3616–3641.
2] A. S. Abd-El-Aziz, C. Agatemor, N. Etkin, Biomaterials 2017, 118, 27–50.
3
7°C for 10–12 h. For secondary culture 100 μL of primary culture
was sub-cultured in 10 mL of fresh LB until it reaches to the mid-
log phase (OD6
~0.3). The final optical density of the bacterial
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Acknowledgements
The authors would like to thank BRNS (37(2)/14/07/2017-BRNS)
and SERB (CVD/2020/000855) for financial support. The authors
would also like to thank Prof. Partha Sarathi Mukherjee from
Inorganic and Physical Chemistry Department, IISc, Bangalore for
providing the cage materials for this study.
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Conflict of Interest
Manuscript received: January 7, 2021
Revised manuscript received: March 31, 2021
Accepted manuscript online: April 4, 2021
Version of record online: ■■■, ■■■■
The authors declare no conflict of interest.
ChemBioChem 2021, 22, 1–7
www.chembiochem.org
6
© 2021 Wiley-VCH GmbH
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These are not the final page numbers!

FULL PAPERS
A. Mondal, I. A. Bhat, S. Karunakar-
an, Dr. M. De*
1
– 7
Supramolecular Interaction of
Molecular Cage and β-Galactosi-
dase: Application in Enzymatic In-
hibition, Drug Delivery and Anti-
microbial Activity
8
The effect of a water-soluble PdII
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hydrophobic curcumin inside the
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aureus.
molecular cage towards β-galactosi-
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cage disintegrates in the presence of
the enzyme and inhibits the enzyme
by mixed mode of inhibition. This in-