Entropically driven Polymeric Enzyme Inhibitors by End-Group directed Conjugation

Article abstract of DOI:10.1002/chem.201800168

A new generic concept for polymeric enzyme inhibitors is presented using the example of poly(2-methyl-2-oxazoline) (PMOx) terminated with an iminodiacetate (IDA) function. These polymers are shown to be non-competitive inhibitors for horseradish peroxidase (HRP). Mechanistic investigations revealed that the polymer is directed to the protein by its end group and collapses at the surface in an entropy-driven process as shown by isothermal titration calorimetry. The dissociation constant of the complex was determined as the inhibition constant K_i using HRP kinetic activity measurements. Additional experiments suggest that the polymer does not form a diffusion layer around the protein, but might inhibit by inducing minor conformational changes in the protein. This kind of inhibitor offers new avenues towards designing bioactive compounds.

Full text of DOI:10.1002/chem.201800168

A Journal of
Accepted Article
Title: Entropically driven Polymeric Enzyme Inhibitors by End-Group
directed Conjugation
Authors: Montasser Hijazi, Christian Krumm, Sueleyman Cinar, Loana
Arns, Wasim Alachraf, Wolf Hiller, Wolfgang Schrader,
Roland Winter, and Joerg Tiller
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
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
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201800168
Link to VoR: http://dx.doi.org/10.1002/chem.201800168
Supported by

Chemistry - A European Journal
10.1002/chem.201800168
COMMUNICATION
Entropically driven Polymeric Enzyme Inhibitors
by End-Group directed Conjugation
[
a]
[a]
[b]
[c]
[d]
[c]
[b]
M. Hijazi , C. Krumm , S. Cinar , W.Alachraf , Wolf Hiller , W. Schrader , R. Winter , and J.C.
[
a]
Tiller*
Abstract: A new generic concept for polymeric enzyme inhibitors is
presented using the example of poly(2-methyl-2-oxazoline) (PMOx)
terminated with an iminodiacetate (IDA) function. These polymers
are shown to be non-competitive inhibitors for horseradish
peroxidase (HRP). Mechanistic investigations revealed that the
polymer is directed to the protein by its end group and collapses at
the surface in an entropy-driven process as shown by isothermal
titration calorimetry. The dissociation constant of the complex was
the polymer end group can be used to couple inhibitors to
macromolecules. Human matrix metalloproteinases (MMPs)
such as collagenase are inhibited by telechelic poly(2-
oxazolines) with N,N-dimethyldodecylammonium (DDA) as end
[
9]
groups, which were used for application in dental adhesives.
Furthermore, the bacterial topoisomerase II and IV inhibitor
ciprofloxacin and the transpeptidase inhibitor penicillin were
attached to poly(2-oxazolines). This changed the activity profiles
of the antibiotics and increased the hydrolysis stability against
i
found to be the inhibition constant K determined by HRP activity
[
10]
kinetic measurements. Additional experiments suggest that the
polymer does not form a diffusion layer around the protein, but might
inhibit by inducing minor conformational changes in the protein. This
kind of inhibitor offers new avenues towards designing bioactive
compounds.
penicillinase degradation in cases of the penicillin conjugates.
[
11]
One example for the third class are chitinase inhibitors that
are oligo- or polysaccharides with specific binding to the active
[
12]
site of the enzyme.
Polyacrylic acid derivatives are able to
inhibit the activity of luminal enzymes, e.g. trypsin and
carboxypeptidase. The polymers bind to the protein matrix
2
+
2+
stabilizing ions such as Ca in case of trypsin and Zn in case
of carboxypeptidase, using the specific metal complexing
Enzyme-inhibitors are of great importance in fields such as
medicine and agriculture. Their specific interactions with single
enzymes or enzyme classes are the reason for their application
as pharmaceuticals or pesticides. Although much is known
about low molecular weight enzyme inhibitors, particularly their
[13] [14]
character of their polymer backbone.
In this study, we follow a new concept of polymeric enzyme
inhibitors, where a hydrophilic polymer is driven onto the surface
of an enzyme by a specific end group that is not an inhibitor by
itself. The polymer is then changing the polarity of the surface of
the enzyme and/or blocking the active site, resulting in enzyme
inhibition (Schema 1).
[
1] [1b]
mode of action
, there is little known about polymeric
enzyme inhibitors. The latter can be classified by inhibitor
[
2]
releasing systems, e.g., for drug delivery , inhibitors covalently
[
3]
attached to polymers , and macromolecules that act due to their
[
4]
specific backbone structure .
The first class is used in a myriad of variations leading to highly
[5]
improved efficiency in many cases. Only a few examples are
known from the second class. For instance, the conjugation of
the anionic mucoadhesive polycarbophils such as poly(acrylic
acid) with different serine protease inhibitors is able to adhere to
the mucosa. The enables the use of less drug and to direct it to
[6] [7]
the infected tissue.
The conjugation of the Bowman-Birk
inhibitor (BBI) to the mucoadhesive polymer sodium
carboxymethylcellulose (Na-CMC) result in a polymer that
protects embedded insulin from enzymatically degradation by
[8]
serine proteases. Besides modifying the polymer backbone,
[
[
a]
b]
M. Hijazi, C. Krumm, Prof.Dr. J.C. Tiller
Department of Bio- and Chemical Engineering, TU Dortmund
TU Dortmund
Emil-Figge-Straße 66, 44227 Dortmund, Germany
E-mail: joerg.tiller@tu-dortmund.de
S. Cinar, L. Arns, Prof.Dr. R. Winter
Department of Chemistry and Chemical Biology, Physical Chemistry
Technical University of Dortmund, Dortmund, Germany
Otto-Hahn-Str. 4a D-44227 Dortmund, Germany
W. Alachraf, Prof. Dr. W. Schrader
Max-Planck Institut für Kohlenforschung
Kaiser Wilhelm Platz 1, D-45470 Mülheim an der Ruhr, Germany.
Prof. W. Hiller
Schema 1. Schematic representation of the proposed generic concept for
enzyme inhibition based on specifically binding anchor groups at inert
hydrophilic polymers.
[
[
c]
d]
Department of Chemistry and Chemical Biology
Technical University of Dortmund, Dortmund, Germany
Otto-Hahn-Str. 4a D-44227 Dortmund, Germany
This offers the opportunity to create new enzyme inhibitors
using functions that have not yet been considered as inhibitors.
In order to prove the concept of this approach, poly(2-
oxazoline) was chosen as hydrophilic and biocompatible
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201800168
COMMUNICATION
[
15]
polymer that has only weak interactions with proteins.
2, 2’-
the resulting polymers are fully functionalized with IDA groups
(Figure S4, Supplements).
Imino diacetate (IDA) was chosen as specific end group that has
the potential of binding to bivalent metals that are often found in
protein scaffolds. The enzyme horse radish peroxidase (HRP)
was chosen as model enzyme, because it contains two calcium
ions in the protein structure and is not inhibited by IDA (Schema
The interactions between HRP and IDA-PMOx33-IDA, CH
3
-
[16]
PMOx31-IDA, and CH -PMOx31-OH, respectively, were
3
investigated by isothermal titration calorimetry (ITC) adding the
concentrated polymer solution to the HRP solution. Titration in
phosphate buffer showed that addition of IDA-PMOx33-IDA to
HRP in phosphate buffer results in a strong exothermic signal
seemingly indicating a binding at a 1:1 molar ratio (Figure S5A,
Supplements). However, a similar titration curve is found when
adding the polymer solution to buffer without HRP (Figure S5B,
Supplements). Thus, we presume that this signal is due to
dissolution of polymer aggregates and overlaps any
polymer/enzyme interactions. Thus, the experiments were
repeated in water (see Fig. 3). Here, the polymer added to water
gives a weak exothermic signal, which can be attributed to the
dilution of the polymer solution (Figure S7, Supplements).
1).
The synthesis of the macromolecules was realized by cationic
ring opening polymerization of 2-methyl-2-oxazoline followed by
terminating the reaction with IDA dimethyl ester (IDA-2Me).
Cleaving the ester groups does then lead to the targeted
structure (Schema 1 and Figure 1, structure A). A series of
single and double side terminated polymers, respectively, with
different molecular weights was prepared this way. In addition to
H-NMR spectroscopy, the structural identity of the polymers
was investigated on the example of partially hydrolyzed IDA-
PMOx33-IDA by electrospray ionization mass spectrometry (ESI-
1
MS).
.
Figure 2. Calorimetric titration isotherm of the binding interaction between
PMOx derivatives and horseradish peroxidase (HRP) in water at 25°C. Each
peak corresponds to the repeated injection of 6 µl of polymer solution into the
reaction cell (V cell = 1.34 ml) in a low concentrated substance solution of 0.03
Figure 1. Electrospray ionization mass spectrum of IDA-PMOx33-IDA after
alkaline hydrolysis at 50 °C for 16 h in the presence of 0.025 M NaOH solution.
-
1
mmol·L HRP. (The heats of dilution of the polymers were determined in a
separate experiment and subtracted from this data). The upper part shows the
-
1
ITC peaks plotted as power (µcal·S ) against time (min). The lower part
shows the resulting integrated heats of the binding process. Left is the titration
As seen in Figure 1, three major generations of polymer masses
can be identified. All of them are carrying two positive charges.
The distance between the absolute masses is in all cases △M+2
curve of 6 mM CH
3
-PMOx31-IDA injected into the calorimeter cell containing
-PMOx31-OH injected into
0.03 mM of HRP. Right is the titration curve of CH
3
-1
=
42.52 g·mol , which is exactly one half of the molecular weight
the calorimeter cell containing 0.03 mM of HRP.
of the repeating unit. The masses of the main fraction (56% of all
signals) correspond to polymer chains of which the ester end
groups are fully converted to carboxylic groups (Figure 1,
structure A). The second polymer generation represents polymer
chains (35 mol%), which have only one ester group remaining
3
The ITC curve of the addition of CH -PMOx33-IDA to HRP
indicates an endothermic reaction (Figure 2A). This might be
caused by aggregation of polymer and enzyme, where the
entropy-driven release of polymer- and enzyme-bound water
(
Figure 1, structure B). The third generation (8 %) represents
[17]
polymer chains, which still contain two methyl groups (Figure 1,
structure C). Determining the overall amount of converted ester
groups by the ESI-MS data, results in 86 mol%, which is close to
molecules causes the endothermic effect.
Presuming the
formation of a 1:1 complex, the dissociation constant was
determined to 0.25 mM. In order to see if the interaction
between polymer and enzyme is caused by the IDA end group,
a PMOx with an OH end group was added to HRP, showing
practically no thermal response (Figure 2B). Thus, the IDA end
group on the PMOx is directing the polymer to the enzyme
1
the value of 83% calculated from H NMR analysis (Figure S3,
Supplements). Thus, the hydrolysis is not changing the PMOx
structure. After extended hydrolysis under the same conditions
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201800168
COMMUNICATION
surface. Interestingly, the titration with the double side IDA
terminated PMOx (IDA-PMOx33-IDA) shows practically the same
ITC curve (Figure S6, supplements) with the same dissociation
constant, indicating that one macromolecule might not be able to
bind at two different binding sites, simultaneously. Literature-
known entropic enzyme inhibitors are often hydrophobic
substances, e.g., the anesthetic propofol, that react via
hydrophobic interactions with the protein, affording a strong
conformational change, which leads to release of water
inhibition increases with greater molecular weight of the
polymers, which is unexpected, because the affinity of the IDA
group to the enzyme surface should be lower with larger coils
attached to IDA. The activity is fully diminished at
concentration of 7.5 to 15 mM IDA terminated polymer. The
most active polymer CH -PMOx31-DDA is inhibiting HRP by
more than 99% at 7.5 mM. No significant inhibition is observed
in the same experiment using CH -PMOx31-OH as control
polymer. The same is found after the treatment of CH -PMOx31
a
3
3
3
-
[18] [19]
molecules
. A similar effect of the here presented IDA
OH with NaOH (0.025 M, 59°C, 24h), at this concentration.
These results show that only the conjugation of IDA and PMOx
results in an enzyme inhibitor. This is consistent with the ITC
measurements shown above.
conjugated PMOx is unlikely, because the polymer is highly
hydrophilic. Circular dichroism (CD) measurements of HRP in
the presence of IDA-PMOx33-IDA revealed only a slight change
in protein conformation in the presence of the polymer that might
lead to inactivation, however, but the polymer does not afford
denaturation of the protein (Figure S8, Supplements). This
indicates that the entropic effect is most likely due to the surface
attachment of the PMOx and the related release of water
In order to obtain more insights in the nature of the inhibition, the
HRP activity was measured in the presence the inhibitor IDA-
PMOx33-IDA at different concentrations of hydrogen peroxide
(Figure 3c). The presence of the polymer lowers the maximum
[
20]
molecules.
DOSY-NMR experiments were performed on a mixture of CH
PMOX31-IDA and HRP in D O (Figure S13 and Figure S14,
To get further insights in the complex formation,
enzyme velocity Vmax, but not the Michaelis constant K
M
, which is
3
-
typical for a non-competitive enzyme inhibitor. The inhibition
2
constant K
equation 1.
.
i
was calculated to 1.74 mM ± 0.25 according to
Supplements). It was found that the polymer is indeed partially
forming aggregates with the enzyme.
The activity of HRP was measured by the oxidation of ABTS
with H O in the presence of varying amounts of different IDA
2 2
terminated PMOx (Figure 3).
Equation 1. The velocity equation for non-competitive inhibition.[20]
According to the proposed enzyme inhibition mechanism, this
value should be equal to the enzyme/polymer dissociation
constant of 0.26 mM determined by ITC, which is obviously not
the case. Presuming that the presence of phosphate ions
influences the binding of the inhibitor to the HRP surface, the
activity of the enzyme was measured in media with different
buffer concentration in the presence of IDA-PMOx33-IDA. As
seen in Figure 3d, the inhibition increases with lower salt
concentrations, proofing this presumption to be correct. The K
i
value of IDA-PMOx33-IDA in water was found to be 0.25 mM,
which perfectly corresponds to the dissociation constant of 0.26
mM measured by ITC. This shows that the formation of
polymer/HRP conjugate is solely responsible for the enzyme
inhibition.
Figure 3. a,b) Inhibition of horseradish peroxidase (HRP) induced by mono-
and bifunctional PMOx at varying concentrations (1.25, 2.5, 5, 7.5, 10, 12.5,
and 15 mM). c) Kinetics of the HRP inhibition by variation of the H O
2 2
concentration in the range of 0.37-3.3 mM. d) Inhibition of HRP induced by 1
mM IDA-PMOX33-IDA in different phosphate buffer concentrations. The
inhibition was measured with ABTS substrate (10 mM) at pH 5.0 for all
polymers. All measurements save the kinetics were performed in triplicate,
and the error bars are the standard deviation.
So far, we could establish that the IDA group directs PMOx to
the enzyme surface to inhibit its activity. To shed some light on
the possible mode of action, the HRP activity was measured in
2+
the presence of Ca ions and IDA-PMOx33-IDA (Figure S9,
Supplement). Interestingly, adding calcium chloride prior to the
polymer does diminish the inhibition from 80 to 28%. This
indicates that calcium ions are effective binding competitors for
the IDA group. In contrast, when adding calcium chloride to the
HRP solution after the polymer, the inhibition was not impaired.
This shows that the competitive binding of the calcium ions to
IDA does not take place once the enzyme/polymer complex is
formed. Therefore, we presume that the binding between
polymer and enzyme is primarily entropy-driven as already
As seen in Figure.3a and 3b the presence of the IDA-terminated
PMOx inhibits the activity of the HRP. The telechelic IDA-PMOx-
IDAs show a similar inhibition compared to the single side
terminated polymers, which confirms that only one IDA group
per polymer chain binds to the protein. Interestingly, the
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201800168
COMMUNICATION
indicated by the ITC data. Thus, whereas the IDA group only Acknowledgements
directs the PMOx via weak interactions to the surface of HRP,
subsequent stronger binding of the polymer is induced by
dehydration of the interacting interfaces.
According to its non-competitive character, the IDA terminated
PMOx does not attach directly to the heme group of the active
3
center. Thus, CH -PMOx-IDA attached to the enzyme surface
The authors of this paper would like to thank Thorsten Moll for
[17]
performing size exclusion chromatography and Dr. W. Hiller for
1
performing
H
NMR measurements. All polymers were
synthesized using CEM Discover microwaves, which were kindly
provided by CEM for undergraduate student education. DFG.
can either influence the protein conformation by changes of
surface polarity and/or by blocking the active site. CD spectra do
not show strong changes in protein conformation, which
supports the latter possibility. The fact that the polymer is
capable of fully inhibiting the enzyme is also an indication of
blocking the active site. Dynamic light scattering (DLS) data,
which show no formation of larger aggregates between HRP and
.
Keywords: Enzyme inhibitor, Poly(2-oxazoline), horse radish
peroxidase, functional polymer end groups
[
1] a) R. Copeland, Evaluation of Enzyme Inhibitors in Drug Discovery: A
3
CH -PMOx-IDA in water using the same concentration as
Guide for Medicinal Chemists and Pharmacologists, Second Edition
|, John Wiley & Sons, 2013, p; b) M. R. Arkin and J. A. Wells, Nat Rev Drug
Discov 2004, 3, 301-317.
applied for the ITC measurements, show that the IDA-terminated
polymers do not form a shell of many attaching polymers around
the protein (Figure S10, Supplement). This suggests specific
binding between protein and polymer at one binding site, which
could be near the active site. Nevertheless, these are only
indicators that are not excluding that a small conformational
change of the protein by changed polarity at the surface, found
[
2] T. Kimura, K. Sato, K. Sugimoto, R. Tao, T. Murakami, Y. Kurosaki and T.
Nakayama, Biological & Pharmaceutical Bulletin 1996, 19, 897-900.
3] R. Duncan, J. K. Coatsworth and S. Burtles, Human & Experimental
Toxicology 1998, 17, 93-104.
4] S. M. Ryan, G. Mantovani, X. Wang, D. M. Haddleton and D. J. Brayden,
Expert Opinion on Drug Delivery 2008, 5, 371-383.
5] P. Vivek Kumar, K. Saravanakumar, P. Nagaveni, A. Mohan Kumar and B.
[
[
[
Ramesh, International Journal of Research in Pharmaceutical Sciences 2016,
205-215%V 205.
[
21]
for liposome attaching PMOx, is responsible for the inhibition.
[
6] A. Bernkop-Schnurch, H. Zarti and G. F. Walker, Journal of Pharmaceutical
Sciences 2001, 90, 1907-1914.
7] A. Bernkop-Schnurch and C. E. Kast, Advanced Drug Delivery Reviews
2001, 52, 127-137.
8] M. K. Marschütz and A. Bernkop-Schnürch, Biomaterials 2000, 21, 1499-
507.
In order to broaden the validity of the shown concept, guaiacol
as second substrate was used and the inhibition of HRP in the
presence of IDA-PMOx33-IDA (1.25-7.5 mM) was investigated.
Similar results were obtained when using guaiacol as substrate
instead of ABTS (Figure S11, supplements). When fitting the
obtained kinetic data with the classic Michaelis-Menten-Model,
[
[
1
[9] C. P. Fik, S. Konieczny, D. H. Pashley, C. J. Waschinski, R. S. Ladisch, U.
Salz, T. Bock and J. C. Tiller, Macromolecular Bioscience 2014, 14, 1569-
1
579.
[
10] M. Schmidt, L. K. Bast, F. Lanfer, L. Richter, E. Hennes, R. Seymen, C.
kcat/K
M
(for guaiacol as substrate) changes from 6200 ± 900 to
Krumm and J. C. Tiller, Bioconjugate Chemistry 2017.
-1
-1
[11] M. Schmidt, L. K. Bast, F. Lanfer, L. Richter, E. Hennes, R. Seymen, C.
Krumm and J. C. Tiller, Bioconjugate Chemistry 2017, 28, 2440-2451.
[12] E. Cohen, Archives of Insect Biochemistry and Physiology 1993, 22, 245-
2100 ± 500 L·s ·mol ) when adding the polymeric inhibitor,
indicating non-competitive inhibition.
261.
In order to test if alkylated IDA group needs to be attached to the
polymer terminal for efficient HRP inhibition, the model
compound 2,2'-(propylazanediyl)diacetic acid (Pro-IDA) was
[
13] H. L. Lueßen, C. M. Lehr, C. O. Rentel, A. B. J. Noach, A. G. de Boer, J.
C. Verhoef and H. E. Junginger, Journal of Controlled Release 1994, 29, 329-
338.
[
14] G. Borchard, H. L. Lueβen, A. G. de Boer, J. C. Verhoef, C.-M. Lehr and
synthesized. This compound was mixed with the CH3-PMOx31
-
H. E. Junginger, Journal of Controlled Release 1996, 39, 131-138.
[15] P. Goddard, L. E. Hutchinson, J. Brown and L. J. Brookman, Journal of
Controlled Release 1989, 10, 5-16.
OH in 1:1 molar ratio and tested for HRP inhibition in the guiacol
test at the 7.5 mM. No significant inhibition could be observed
indicating that only the IDA-terminated PMOx is the inhibitor.
In closing, we have shown in this study that the combination of a
non-active polymer and a protein-binding group that is no
inhibitor can result in an effective enzyme inhibitor. The ITC data
suggest that weak interactions between the end group of the
polymer and the protein are sufficient to obtain a strong bond,
because the polymer/protein binding is driven by entropy. Thus,
this new inhibitor concept offers the chance to find new enzyme
inhibitors as pharmaceuticals, pesticides, or antibiotics by
combining so far not considered non-inhibiting but enzyme
binding functions with hydrophilic polymers. Such conjugates
might have different toxicology and immunology profiles
compared to currently used systems.
[
[
16] D. Tarn, M. Xue and J. I. Zink, Inorganic Chemistry 2013, 52, 2044-2049.
17] Q. Wei, T. Becherer, S. Angioletti-Uberti, J. Dzubiella, C. Wischke, A. T.
Neffe, A. Lendlein, M. Ballauff and R. Haag, Angewandte Chemie International
Edition 2014, 53, 8004-8031.
[
18] M. M. Lopez and D. Kosk-Kosicka, Biochemistry 1997, 36, 8864-8872.
[19] A. Miklavc and D. Kocjan, Journal of Chemical Information and Computer
Sciences 2004, 44, 422-426.
[
20] F. Zhao, X. Cheng, G. Liu and G. Zhang, The Journal of Physical
Chemistry B 2010, 114, 1271-1276.
[21] C. J. Waschinski, S. Barnert, A. Theobald, R. Schubert, F. Kleinschmidt, A.
Hoffmann, K. Saalwächter and J. C. Tiller, Biomacromolecules 2008, 9, 1764-
1771.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201800168
COMMUNICATION
COMMUNICATION
A new generic concept for polymeric
enzyme inhibitors is presented on the
example of poly(2-methyloxazoline)
terminated with a 2,2’-imino diacetate
function. These polymers are shown
to be non-competitive inhibitors for
M. Hijazi, C. Krumm, S. Cinar, L. Arns,
W. Alachraf, W. Hiller, W. Schrader, R.
Winter, J.C. Tiller*
Page 1. – Page 5
Title: Entropically driven Polymeric
Enzyme Inhibitors by End-Group
directed Conjugation.
horseradish
peroxidase
(HRP).
Mechanistic investigations revealed
that the polymer is directed to the
protein by its end group and collapses
at the surface in an entropy-driven
process inhibiting to enzyme this way.
This article is protected by copyright. All rights reserved.