Identification and Directed Development of Non-Organic Catalysts with Apparent Pan-Enzymatic Mimicry into Nanozymes for Efficient Prodrug Conversion

Article abstract of DOI:10.1002/anie.201812668

Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near-exclusively performing redox reactions. We present an unexpected discovery of non-proteinaceous enzymes based on metals, metal oxides, 1D/2D-materials, and non-metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan-glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human metabolites and privileged prodrugs in the field of enzyme-prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti-inflammatory and antibacterial agents, as well as “nanozyme prodrug therapy” to mediate antibacterial measures.

Full text of DOI:10.1002/anie.201812668

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Accepted Article
Title: Identification and directed development of non-organic catalysts
with apparent pan-enzymatic mimicry into nanozymes for efficient
prodrug conversion
Authors: Raoul Walther, Anna Winther, Anne Sofie Fruergaard, Wouter
van den Akker, Lise Sørensen, Signe Maria Nielsen, Morten
Jarlstad Olesen, Yitao Dai, Henrik Jeppesen, Paolo Lamagni,
Aleksandr Savateev, Søren Pedersen, Camilla Frich, Cecile
Vigier-Carrière, Nina Lock, Mandeep Singh, Vipul Bansal,
Rikke Meyer, and Alexander N. Zelikin
This manuscript has been accepted after peer review and appears as an
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using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
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content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201812668
Angew. Chem. 10.1002/ange.201812668
Link to VoR: http://dx.doi.org/10.1002/anie.201812668
http://dx.doi.org/10.1002/ange.201812668

10.1002/anie.201812668
Angewandte Chemie International Edition
COMMUNICATION
Identification and directed development of non-organic catalysts
with apparent pan-enzymatic mimicry into nanozymes for
efficient prodrug conversion
Raoul Walther,a # Anna K. Winther,a # Anne Sofie Fruergaard,a Wouter van den Akker,a Lise Sørensen,a
Signe Maria Nielsen,a,b Morten T. Jarlstad Olesen,a,b Yitao Dai,b Henrik S. Jeppesen,b Paolo Lamagni,b
Aleksandr Savateev,c Søren Lykke Pedersen,a Camilla Kaas Frich,a Cécile Vigier-Carrière,a Nina
Lock,a,b Mandeep Singh,d Vipul Bansal,d Rikke L. Meyer,b Alexander N. Zelikina,b
*
Abstract: Nanozymes, nanoparticles that mimic the natural activity of
enzymes, are intriguing academically and are important in the context
of Origin of Life. However, current nanozymes offer mimicry to a
narrow range of mammalian enzymes, near-exclusively performing
redox reactions. In this work, we present an unexpected discovery of
non-proteinaceous enzymes based on metals, metal oxides, 1D/2D-
materials, and non-metallic nanomaterials. Specific novelty of our
findings lies in the identification of nanozymes with apparent mimicry
of diverse mammalian enzymes, including unique pan-glycosidases.
Further novelty lies in the identification of the substrate scope for the
lead candidates, specifically in the context of bioconversion of
glucuronides, that is, human metabolites and privileged prodrugs in
the field of enzyme-prodrug therapies. Lastly, nanozymes are
employed for conversion of glucuronide prodrugs into marketed anti-
inflammatory and antibacterial agents, as well as “nanozyme prodrug
therapy” to mediate antibacterial measures.
dimension) is considered highly important in the context of Origin
[3]
of Life.
However, successful in their own right, current
nanozymes and mineral surfaces offer mimicry to a very narrow
range of mammalian enzymes, almost exclusively performing
redox reactions.[4] Compared to the magnitude of enzymes in the
human body, this range is only humble at the very best.
In this work, we present a highly unexpected identification of
nanozymes with apparent mimicry of diverse mammalian
enzymes performed under physiological conditions, including
non-proteinaceous pan-glycosidases. This was accomplished via
a screen of ca. 100 enzyme-mimic candidate materials against ca.
20 fluoro/chromogenic substrates. Specific novelty lies in that we
identified non-proteinaceous enzymes based on metals, metal
oxides, 1D/2D-materials, and non-metallic nanomaterials
(detailed in Supporting Information, Figures S10-S31). Further
novelty lies in that we identified the substrate scope for the lead
nanozymes, specifically in the context of bioconversion of
glucuronides, that is, natural metabolites and privileged prodrugs
in the field of enzyme-prodrug therapies.[5] Lead nanozymes
performed efficient conversion of glucuronide prodrugs to
produce anti-inflammatory and antibacterial agents. To our
knowledge, we report the first example of pan-enzymatic mimicry
evidenced in non-proteinaceous materials, as well as the first
example of nanozymes as mimics of glucuronidase for enzyme-
prodrug therapy.
Nanoparticles that mimic the natural activity of enzymes, termed
nanozymes, comprise a unique set of materials at the interface
[1]
between inorganic and bio-organic chemistry.
Engineering
mammalian enzyme-like behavior into non-proteinaceous
molecules is intriguing academically and is poised to open up
immense opportunities in biotechnology and biomedicine.
Specifically, advantages of nanozymes over the natural enzymes
include stability, scalability, chemical diversity, and performance
in non-aqueous solvents.[2] From
enzymatic activity of inorganic materials (regardless of their
a different perspective,
[6]
With our broader interest in enzyme prodrug therapies (EPT),
the first screen of nanomaterials for potential enzymatic activity
performed in this work aimed to identify mimics of glucuronidase.
Glucuronides hold a privileged position in EPT[5] and are also
natural metabolites that are formed during phase II metabolism in
the human liver. Enzyme activity was evaluated with the use of a
turn-on fluorescent probe, resorufin--D-glucuronide (Figure 1A).
Over a 24 h incubation time, inorganic enzyme-mimics afforded
conversion of the substrate to yield fluorescent product in content
as high as 1 µM (Figure 1B). The catalytic activity of the particles
was readily registered by fluorescence imaging which showed
significant increase in the fluorescence of substrate solutions in
the presence of nanozymes (Figure 1C). Strikingly, enzyme
mimics were identified within each class of nanomaterials tested
and leads included metals, metal oxides, 1D/2D materials and
non-metallic nanoparticles.
[a]
[b]
R. Walther, A.K. Winther, W. van den Akker, A.S. Fruergaard, L.
Sørensen, S.M. Nielsen, M.T. Jarlstad Olesen, S.L. Pedersen, C.K.
Frich, C. Vigier-Carrièr,
Department of Chemistry, Aarhus University, Aarhus, Denmark
E-mail: zelikin@chem.au.dk
Y. Dai, H.S. Jeppesen, P. Lamagni, Dr. N. Lock, Dr. R.L. Meyer, Dr.
A.N. Zelikin
iNano Interdisciplinary Nanoscience Centre, Aarhus University,
Aarhus, Denmark
Dr. Aleksandr Savateev
Max-Planck Institute of Colloids and Interfaces, Potsdam, Germany
M. Singh, Prof. Vipul Bansal
RMIT University, Melbourne, Australia
These authors contributed equally
[c]
[d]
#
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. High throughput screen of nanozymes identifying nanoparticles with activity of β-Glucuronidase and pan-enzymatic activity. (A)
Conversion of weakly fluorescent resorufin β-D-glucuronide into the corresponding fluorescent product, resorufin, allowing for a fluorescence based readout
of nanozyme activity; (B) Glucuronidase mimicry screen for nanozymes based on metals, metal oxides and other nanomaterials using resorufin β-D-
glucuronide as an enzyme-specific fluorogenic probe; (C) imaging of catalytic output for selected nanozymes using resorufin β-D -glucuronide (Glu) and - β-
D -galactoside (Gal); (D) “Heat map” representation of the results of the screen for nanozyme activity (Cu, Fe/Ni, Cu/Zn, NiO, g-C3N4) using enzyme-
specific substrates corresponding to diverse mammalian enzymes. NP = 4-nitrophenol, AMC = 7-amido-4-methylcoumarin; MU = 4-methylumbelliferone.
“Positive” activity is defined herein as the one with statistically significant enhancement of the catalytic substrate conversion over non-catalyzed reaction
(p≤0.05). Statistical evaluation was performed using one-way ANOVA with Dunnett’s multiple comparison test or unpaired t-tests with or without Welch’s
correction.
Optimization of the catalytic output of the nanozymes (performed
using commercial copper nanoparticles) was performed using the
computer-assisted design of experiments. Solvent pH and
temperature had a non-negligible influence on the catalytic output
of the nanozyme particles. However, the highest level of influence
was revealed by the choice of the buffer, phosphate buffer being
a significantly better medium than HEPES for catalysis, and the
presence of physiological concentration of sodium chloride
(Figure S1). This observation indicates that nanozyme activity, at
least for copper nanozymes, is strongly affected by the ion
adsorption to the metal surface. [7] With regards to the kinetics of
(bio)conversion of resorufin--D-glucuronide, nanozyme based
on commercially available copper nanoparticles expectedly
revealed Kcat values that were significantly lower than for the
natural catalyst, while Km values were surprisingly similar to β-
glucuronidase (-Glu) (Table 1, raw data presented in Figures S6-
S9). We note that while contribution of the metal ions released
from the particles in the overall catalytic performance [8] cannot be
fully ruled out, we found that ion-mediated catalysis was
significantly lower, e.g. for copper ions compared to the copper
nanozyme (Figure S2); this consideration is altogether irrelevant
for non-metallic nanozymes such as graphitic carbon nitride (g-
C3N4).
Table 1. Enzyme and copper-based nanozyme kinetics for
bioconversion of resorufin--D-glucuronide. Km – Michaelis
constant, Kcat – catalytic constant.
K
cat/Km
entry
β-Glu
Cu NP
K
m [µM]
Kcat [s-1]
2.7 ± 0.2
[mM-1 s-1]
25 ± 2
108 ± 2
41 ± 24
0.05 ± 0.02
1.2 ± 0.2
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Figure 2. (A) Substrate scope of the copper nanozyme as a β-glucuronidase mimic is closely related to the quality of the leaving group in the substrate. HPLC
characterization of conversion for glucuronide prodrugs of estradiol (aliphatic, estradiol 17-(-D-glucuronide) and phenolic, estradiol 3-(-D-glucuronide)), 4-
nitrophenol, and salicylic acid acyl glucuronide (from left to right, increasing quality of the leaving group). Experimental conditions: 37 °C, phosphate buffered
saline pH 7.4; prodrug concentration of 0.1 g L-1 and nanozyme concentration of 5 g L-1. Conversion time was 24 h (all substrates) or 1 h (salicylic acid acyl-
glucuronide); (B) HPLC quantitative analysis of nanozyme-catalysed hydrolysis of salicylic acid acyl glucuronide at a concentration of 0.14 g L-1 in phosphate
buffered saline pH 7.4 optionally supplemented with fetal bovine serum (FBS) (3.5% and 10% v/v% FBS) over 1 h incubation at 37 °C. Statistical significance is
calculated relative to the negative control (non-catalyzed decomposition of SAG) using a one-way ANOVA test followed by a Dunnett’s multiple comparisons test.
We next performed a screen of selected nanozymes for mimicry
against 20 enzymes (esterases, phosphatases, peptidases,
cytochromes, glycosidases, etc), in each case using commercially
available chromogenic or fluorogenic probes (Figure 1D). The
threshold activity used to distinguish between positive and
negative mimicry was set using statistical significance of the
“leaving group”, not the enzyme affinity ligand in the reporter
molecule, defines the substrate scope.
We further scrutinized the substrate scope for nanozymes
focusing on glucuronides. To this end, we performed
glucuronidase mimicry tests using prodrugs with varied quality of
the leaving group,[11] increasing from aliphatic alcohol to phenol
(pKa > 10) to 4-nitrophenol (pKa < 10) and carboxylic acid (Figure
2A). The former two prodrugs were glucuronides of estradiol,
while the latter was a salicylic acid acyl glucuronide (SAG). Using
HPLC to monitor prodrug conversion in phosphate buffered saline
over 24 h, we observed that the copper nanozyme afforded
negligible conversion of the estradiol glucuronides. In contrast,
conversion of 4-nitrophenyl glucuronide was confirmed by HPLC.
Finally, conversion of the acyl glucuronide was pronounced even
within 1 h of observation (accompanied by broadening of peaks
which we attribute to the well-documented acyl migration
phenomenon[12]). These experiments confirm that the substrate
scope for Cu nanozymes with regards to their glucuronidase
mimicry is closely related to the quality of the leaving group in the
prodrug. We also found that conversion of SAG was enhanced by
the presence of sodium chloride, as was the case for the
catalytic output compared to
a
non-catalyzed substrate
decomposition. The striking observation from this map is that for
each enzyme considered in this work (except phosphatase, for
which nanozymes have been reported previously[9]), there was at
least one nanomaterial with an apparent enzymatic activity
evidenced by the conversion of the enzyme-specific substrate into
its corresponding product. Furthermore, nanozymes revealed
apparent pan-glycosidase activity in this screen, a feature hardly
observed in nature.
Results in Figure 1D are surprising and illustrate that the same
nanozyme readily accepts multiple, diverse enzyme-specific
substrates for conversion. The substrates used in Figure 1D are
employed as enzyme-specific probes in the overall majority of
studies on enzymes, nanozymes, and other types of enzyme
10]
mimics.[1,
However, it appears that these substrates alone
cannot provide sufficient evidence to postulate specific enzyme
mimicry. It appears that nanozymes do not obey the substrate
specificity of the natural enzymes and therefore enzyme mimicry
most likely does not draw origin from the specific substrate
recognition. To validate this conclusion, we performed inhibition
studies and observed that glucuronic acid expectedly inhibits
activity of the glucuronidase enzyme (via product-mediated
competitive enzyme inhibition) but has no such effect on the
copper nanozyme, Figure S3. Instead, we observe that all
substrates used in Figure 1D are engineered to contain an
enzyme-specific affinity ligand attached to a “good leaving group”
(4-nitrophenol, 4-methylumbelliferone, resorufin, fluorescein) thus
constructing the weakest bond in the molecule. In contrast to the
proteinaceous enzymes, for nanozymes it appears that the
fluorogenic
substrates,
suggesting
that
ion-mediated
enhancement of nanozyme activity is specific to the nanoparticle
rather than to the substrate (Figure S4). To our knowledge, results
in Figure 2A present the first example of the use of nanozymes
applied to convert the privileged glucuronide prodrugs into
corresponding therapeutic (salicylic acid).
Nanozymes used in the experiments presented above were
based on commercial copper nanoparticles and proved to be
active as glycosidases in aqueous buffered solutions but failed to
exhibit catalytic activity in complex biological media. Specifically,
these nanozymes were inactive in the presence of serum proteins
(data not shown). Such activity is indispensable with the view of
practical applications of the nanozymes in biomedicine. To
achieve this, we performed nanoengineering of copper-based
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[13]
[14]
nanozymes using several literature protocols.
Poly(vinyl
While conversion of acyl glucuronides is encouraging, the
pyrrolidone)-stabilized Cu nanocubes were particularly active as
artificial glycosidases, Figure 2B. These nanozymes successfully
mediated catalytic decomposition of SAG in phosphate buffered
saline and in the presence of increasing amounts of serum,
reaching the serum content as typically used in cell culture
medium. These results illustrate a successful engineering of
nanozymes to optimize their activity as artificial glycosidases.
latter are unstable and are prone to spontaneous
decomposition.[12] We aimed to broaden the scope of nanozymes
to non-acyl derivatives with inferior quality of the leaving group but
with a higher stability in aqueous solutions. To achieve this, we
used 4-hydroxybenzyl alcohol (PHBA) as a self-immolative linker
between the glucuronic acid and the drug (Figure 3). This linker is
typically used to enhance the accessibility of the scissile bond to
the enzyme and is already found in marketed drugs (Brentuximab
vedotin).[15] We envisioned that the phenolic nature of the leaving
group coupled with an irreversible reaction character (Figure 3A)
would make the prodrugs engineered around this linkage fit into
the desired substrate scope for nanozymes. HPLC monitoring
was used to validate the stability of the glucuronide prodrugs and
their conversion by the nanoengineered Cu nanozymes, Figure
3B. Prodrugs revealed minor if any spontaneous decomposition
over 24 h of incubation in absence of nanozyme (which is pivotal
for applications in EPT). In contrast, nanoengineered Cu
nanozymes as used in Figure 2B successfully converted
synthesized glucuronides into the corresponding antibiotics – thus
fully validating the proposed prodrug design. The carboxybenzyl
carbamate analogues (lacking glucuronide group and the
glycosidic linkage) showed no release of antibiotic in presence of
the nanozyme (Figure S5) providing evidence that hydrolysis of
the glucuronide prodrugs occurs at the anomeric position rather
than the benzylic. In this regard, copper nanozymes are true
mimics of the natural enzyme, glucuronidase.
Next, we applied the knowledge on the nanozyme substrate
scope to perform de novo engineering of glucuronide prodrugs.
Specifically, we used commercially successful antibiotics,
ciprofloxacin and moxifloxacin, and synthesized corresponding
prodrugs that would fall within the substrate scope for nanozymes.
We then performed the zone of bacterial growth inhibition (ZOI)
assay as a cell culture test for the engineered catalytic
nanoparticles and the glucuronide prodrugs as a nanozyme-
mediated antibacterial measure (Figure 3C). Nanoengineered
copper nanozymes, despite prior evidence of antibacterial activity
of copper, revealed no ZOI and bacterial growth was non-inhibited.
As expected, prodrugs on their own at the tested concentration
(10minimal inhibitory concentration of the corresponding drug,
ciprofloxacin) also had no antibacterial effect. In contrast, a
combination of the nanozyme and the prodrug achieved a
pronounced antibacterial effect and afforded a significant ZOI of
bacterial growth. We believe that this is the first example of
nanozyme-mediated conversion of glucuronide prodrugs applied
as “nanozyme-prodrug-therapy” to mediate antibacterial
measures. It is important to note that prodrug design using the
PHBA linker is modular: keeping the scissile bond between
glucuronic acid and proximal end of the linker the same, it is
possible to attach to the distal end virtually any drug with an amine
or a hydroxyl functionality (thus being the overall majority of drugs
on the market). [5]
Figure 3. Antibacterial prodrugs and nanoengineered copper nanozymes afford
strong antibacterial effect (zone of inhibition). (A) Proposed mechanism of drug
release from the glucuronide prodrugs containing 4-hydroxybenzyl alcohol self
immolative linker and ciprofloxacin/moxifloxacin as antibiotics, and the chemical
structure of carboxybenzyl carbamate analogue that showed no release of
antibiotics in the presence of the nanozymes. (B) HPLC traces illustrating
release of antibiotics from the synthesized prodrugs through β-glucuronidase –
like activity of the copper nanozyme at 100 M prodrug concentration and 5 g
L-1 Cu-nanozyme concentration in PBS, pH 7.4 after 24 h incubation; controls
were performed under the same conditions in absence of the nanozyme, (C)
“zone of inhibition” experiment revealing that nanozyme and the ciprofloxacin
prodrug individually have no antibacterial effect against E.Coli. but together
produce a strong zone of bacterial growth. Experimental conditions: 0.9 mg L-1
/1.5 µM ciprofloxacin glucuronide (corresponding to the 10x minimum inhibitory
concentration of the corresponding drug against E.Coli) in tryptic soy broth, 24
h growth phase. For full details, see supporting information.
Taken together, this work presents the identification of apparent
pan-glycosidase activity in nanozymes, the realization of the
substrate scope for nanoparticle-based biocatalysts, and the
development of “nanozyme prodrug therapy” as an antibacterial
treatment. These achievements became possible through a
combination of successful selection of the nanozymes with a
rational design of prodrugs. Current nanozymes, already highly
powerful, provide mimicry to a narrow range of enzymes, mostly
associated with the redox reactions. Our work goes well beyond
the state of art, presents unique nanozymes and guidelines for
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Acknowledgements.
ANZ acknowledges funding from the European Research Council
Consolidator Grant (ERC-2013-CoG 617336 BTVI) and Aarhus
University Research Foundation (grant no. AUFF-E-201 6-FLS-9-
32). NL acknowledges the Villum Foundation Young Investigator
Programme (VKR 023449) and the Danish National Research
Foundation (Carbon Dioxide Activation Center, DNRF 118). VB
acknowledges the Australian Research Council for a Future
Fellowship (FT140101285), funding support through a Discovery
Grant (DP170103477), and support from the RMIT Microscopy
and Microanalysis Facility (RMMF).
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Keywords: nanozyme • glucuronide • enzyme-prodrug therapy •
prodrug • enzyme mimicry
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Entry for the Table of Contents
COMMUNICATION
R. Walther, A.K. Winther, A.S.
Fruegaard, W. van den Akker,
L.Sørensen, S. M. Nielsen, M.T. Jarlstad
Olesen, Y. Dai, H.S. Jeppesen, P.
Lamagni, A. Savateev, S.L. Pedersen,
C.K. Frich, C. Vigier-Carrière, N. Lock,
M. Singh, V Bansal, R.L. Meyer, A.N.
Zelikin *
Unexpected apparent pan-enzymatic activity is discovered of non-
proteinaceous materials. Identification of the substrate scope for these
nanozymes led to the development of efficient mimics for β-glucuronidase that
convert glucuronide prodrugs for localized synthesis of anti-inflammatory and anti-
bacterial drugs.
Page No. – Page No.
Title
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