A fast, miniaturised in-vitro assay developed for quantification of lipase enzyme activity

Article abstract of DOI:10.1080/14756366.2019.1651312

The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive. High-throughput screening combined with novel computational tools can reduce these factors. Thus, we developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent to the in-silico library screening. While the in-silico screening yields in the identification of potential allosteric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lipase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay conditions were adjusted to CRL’s properties including pH, temperature and substrate specificity for two different substrates. The optimised assay conditions were validated and were used to characterise Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reproducible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated high-throughput screening workflow.

Full text of DOI:10.1080/14756366.2019.1651312

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
A fast, miniaturised in-vitro assay developed for
quantification of lipase enzyme activity
Ariane Menden, Davane Hall, Daniel Paris, Venkatarian Mathura, Fiona
Crawford, Michael Mullan, Stefan Crynen & Ghania Ait-Ghezala
To cite this article: Ariane Menden, Davane Hall, Daniel Paris, Venkatarian Mathura, Fiona
Crawford, Michael Mullan, Stefan Crynen & Ghania Ait-Ghezala (2019) A fast, miniaturised in-
vitro assay developed for quantification of lipase enzyme activity, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 1474-1480, DOI: 10.1080/14756366.2019.1651312
To link to this article: https://doi.org/10.1080/14756366.2019.1651312
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1474–1480
https://doi.org/10.1080/14756366.2019.1651312
RESEARCH PAPER
A fast, miniaturised in-vitro assay developed for quantification of lipase enzyme
activity
Ariane Mendena,b , Davane Halla , Daniel Parisa,b, Venkatarian Mathuraa , Fiona Crawforda,b
Michael Mullana,b , Stefan Crynena,b and Ghania Ait-Ghezalaa,b
,
b
aDepartment of Genomics, Roskamp Institute, Sarasota, FL, USA; ARC, Open University, Milton-Keynes, United Kingdom
ABSTRACT
ARTICLE HISTORY
Received 8 May 2019
Revised 25 July 2019
Accepted 26 July 2019
The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive.
High-throughput screening combined with novel computational tools can reduce these factors. Thus, we
developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent
to the in-silico library screening. While the in-silico screening yields in the identification of potential allo-
steric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lip-
ase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay
conditions were adjusted to CRL’s properties including pH, temperature and substrate specificity for two
different substrates. The optimised assay conditions were validated and were used to characterise
Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reprodu-
cible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated
high-throughput screening workflow.
KEYWORDS
Candida rugosa lipase;
hydrolase; enzyme activity
assay; lipase inhibitor;
Tropolone
1. Introduction
centre and the tunnel entrance and refolding itself into two
aa-helices11,12. One of the acyl groups on the glyceride can subse-
quently move into the accessible tunnel domain, which contains
several functional groups stabilising the hydrophobic acyl chain.
This positions the ester bond connecting the fatty acid and gly-
cerol in the catalytic centre, initiating the hydrolysis activity and
subsequently releasing a free fatty acid as well as diacylglyceride11.
Changes in the sequence by site-directed mutation of both the
loop and the tunnel domain can have effects on specificity and
activity of the enzyme7,13. In addition, it was discovered that CRL
isoforms’ activity is very sensitive to pH and temperature
changes4,14. While the application of immobilisation strategies as
well as the addition of hydrophobic organic solvents or emulsions
showed to enhance the enzyme’s activity by stabilising the
exposed hydrophobic areas in the open conformation15–18, certain
experimental conditions can even change the direction of the cata-
lytic reaction, meaning an ester bond formation instead of hydroly-
sis19. Although allosteric modulation is very effective and is a
biochemical method to alter enzymatic activity and specificity, it
has not been considered in the past due to the mentioned alterna-
tive procedures, complexity and cost-intensity of high-throughput
screening with numerous potential modulators. In modern
approaches, the identification of allosteric sites is simplified
through pre-screening of potential allosteric modulators by com-
putational analysis, which pre-sorts and reduces the pool of
Lipases or triacylglycerol acyl hydrolases (EC 3.1.1.3) catalyse the
hydrolysis of triacyl glycerides to release fatty acids and glycerol,
an established mechanism in yeast. They comprise a broad variety
of lipase structures with common structural traits1. Yeasts’ secre-
tion of lipases support their establishment in plants by hydrolys-
ing epicuticular waxes, which enables access to nutrition and
carbohydrates in the roots of plants as a survival mechanism2.
Candida rugosa is an especially potent species that secretes 5 iso-
forms of lipases with an identity of 77%. Despite their structural
similarity, the five isoforms of Candida rugosa lipase (CRL) covers a
broad specificity range in catalysing the breakdown of lipids into
fatty acids and glycerol3; which is also the reason why this subset
is widely used in the industry, for instance, in the production of
biodiesel and pharmaceuticals4,5. This broad specificity range ena-
bles Candida rugosa to hydrolyse fatty acids preferentially from
position 1 or 3 on the glyceride with an additional preference for
medium- and long-chain fatty acid, but no preference between
saturated and unsaturated fatty acids as well as cholesterol6–8
.
The varying specificity within the isoforms is caused by sequential
differences in a conserved loop and tunnel domain, which deter-
mines the activity and specificity of the respective isoforms7,9,10
.
The loop is composed of an a-helix in the closed conformation,
where it lays planar on the molecule’s surface covering the cata-
lytic centre (catalytic triad: Ser209-His449-Glu341, a water mol-
ecule, and the oxyanion hole) as well as the tunnel entrance
inhibiting catalytic activity11,12. For the transition in the open con-
formation, the respective loop has to travel 19 Å, positioning itself
screening component as well as complexity and cost-intensity20–22
.
However, in-silico based screening requires an X-ray-crystallog-
raphy structure of the respective enzyme as well as high-perform-
perpendicular to the enzyme’s surface exposing the catalytic ance software and hardware, an in-vitro screening assay, which can
CONTACT Ariane Menden
amenden@roskampinstitute.org
2040 Whitfield Avenue, Sarasota, FL, 34243, USA
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1475
verify the theoretically determined ligands and their influence on 2.4. Temperature and pH stability of enzyme activity assay
the enzyme’s activity.
Robustness of the assay conditions was tested by performing the
assay at a different temperature or pH conditions according to the
protocol in Section 2.2.
Therefore, we developed a rapid, miniaturised, high-throughput
assay starting from Dairaku et al.’s proposed method23, which can
be used as in-vitro identification tool in existing in-silico work-
flows elucidating the impact of in-silico determined ligands on the
enzyme’s activity. The assay is reproducible, reliable, and enables
flexible adjustments depending on the enzyme’s specificity and
enzyme class. For the assay’s development, Candida rugosa lipase
isoforms were selected due to the availability of their X-ray crystal-
lography structure (PDB: 1CRL, 1TRH) and their wide use in indus-
trial production processes.
2.4.1. Temperature
The standard curve was prepared in triplicates and several plates
incubated at different temperatures including enzyme plus sub-
strate and blank samples in triplicates for each temperature point
(4ꢀC; 15ꢀC; 25ꢀC; 28ꢀC; 37ꢀC; 45ꢀC). Each sample was corrected
for their individual blank at the respective temperature.
2.4.2. pH
2. Materials and methods
Sample containing the enzyme plus substrate and blank were also
run in duplicates for each pH. Reaction conditions were otherwise
maintained as described above in the assay protocol.
2.1. Materials
Candida rugosa lipase was received from Deerland (Kennesaw, GA,
USA). 4-Methylumbelliferyl palmitate (4-MUP) and Trigonelline
were obtained from Cayman Chemicals (Ann Arbour, MI, USA).
4-Methylumbelliferyl (4-MU), 4-Methylumbelliferyl butyrate (4-
MUB), dimethyl sulfoxide (DMSO), sodium phosphate dibasic hep-
tahydrate, sodium phosphate monobasic monohydrate, Tropolone
and Berberine were purchased from Sigma Aldrich (St. Louis, MO,
USA). b-Aescin was purchased from Santa Cruz Biotechnology
(Dallas, TX, USA). 85% o-phosphoric acid was received from Fisher
Scientific (Waltham, MA, USA). Sterile, black, mCLEAR, flat bottom,
96-well plates were obtained from VWR (Radnor, PA, USA).
2.5. Stability of CRL in water
CRL (1 mg/mL) was incubated in water for six days at room tem-
perature. Samples were collected at day 0, 1, 3, 5, and 6.
Subsequently, the collected samples were analysed on a stain-free
SDS gel and with the 4-MUB activity assay according to Section
2.2 to investigate changes of activity and quantity.
2.6. Inhibitor analysis
The in-silico determined inhibitor Tropolone was identified with
the CRL1 structure (PDB: 1CRL) and the compiled ligand screen
from the Zinc database (Zbc; Zbc Leads; Zbc Drugs; Zbc frags),
InterBioScreen database (natural compounds) and AnalytiCon
database (FRGx, MEGx) (supplementary method S1). Tropolone’s
impact on CRL was analysed by determining the IC50 with both
substrates (4-MUB and 4-MUP) according to the described proto-
col in Section 2.2. A 1 M Tropolone stock was prepared in DMSO
and diluted in phosphate buffer pH 7 to a final concentration
range of 2 mM to 10 mM in the reaction and compared to CRL
without inhibitor addition. In addition, Berberine’s, Trigonelline’s
and b-Aescin’s IC50 were analysed and compared to Tropolone.
Tested concentration ranges were based on published IC50’s and
required CRL’s concentration being reduced 10-fold.
2.2. Enzyme activity assay
Standards and samples were measured in triplicates in a black,
m-clear bottom 96-well plate. Dilutions and blanks were prepared
with 0.1 M phosphate buffer pH 7. The standard curve was pre-
pared with a 400 mM stock of 4-MU in DMSO and 0.1 M phosphate
buffer (pH 7.0) with a range of 7.813 mM to 500 mM (final:
1.95–62.5 mM). 50 mL buffer and 50 mL of standard solution was
added to respective standard wells, while 100 mL buffer were
added to the blank wells. 50 mL of CRL at 10 ng/mL (final concen-
tration: 2.5 ng/mL) for 4-MUB or 50 ng/mL CRL (final concentration
12.5 ng/mL) for 4-MUP and 50 mL of buffer or additive (enhancer,
inhibitor) were added to the respective sample wells. A 10 mM
stock of 4-MUB and 10 mM stock of 4-MUP were prepared in
DMSO and diluted in buffer (4-MUB) or buffer and 0.006% SDS
(w/V) incubated at 37 ꢀC (4-MUP) to a concentration of 0.25 mM.
50 mL of 4-MUB and 150 mL of 4-MUP were added to each well.
The 96-well plate was incubated at 37 ꢀC for 25 min. The reaction
was stopped with 50 mL of 10% o-phosphoric acid in MilliQ water
for 4-MUB and 10 mL for 4-MUP. The fluorescence was immediately
measured with the BioTek Cytation 3 system at k ¼ 326/472 with
a gain of 72.
2.7. Michaelis–Menten kinetics
Km and Vmax were determined by measuring eight different sub-
strate concentration at six-time points. Each concentration at
each time point had a separate blank. The plot of time vs. concen-
tration of fluorescent dye for each substrate concentration deter-
mined V0 (slope of linear graph) for each substrate concentration.
V0 values were subsequently plotted against the substrate concen-
tration based on the Michaelis–Menten plot to calculate Km and
Vmax
2.3. Validation of enzyme activity assay
½ ꢂ
Vmax
ꢁ
S
½ ꢂ
S
Blank and standards were diluted and prepared as described in
the enzyme activity section 2.2. Samples were run in triplicates
containing CRL in buffer, CRL with 200 mM 4-MU (final: 50 mM
4-MU) and CRL with 12.5 mM 4-MU (final: 3.125 mM 4-MU). The
experiment was executed over three consecutive days to deter-
mine intra- and inter-day CV for all three samples as well as intra-
and inter-day precision for both spiked reactions.
V0 ¼
(1)
Km
þ
Km ¼ Michaelis–Menten constant; Vmax ¼ maximal velocity;
V0 ¼ initial velocity; [S] ¼ substrate concentration.
In addition, the kinetics experiment was performed with two
different Tropolone concentrations to determine the type of
inhibitory activity imposed by Tropolone. All time points and

1476
A. MENDEN ET AL.
substrate concentration were maintained. Tropolone was added constant in both assays (250 mM), the concentration of the enzyme
prior to the experiment in a concentration of 100 mM or 400 mM was adjusted to account for SDS’s inhibitory effect on CRL (data
and Km and Vmax were compared.
not shown). Therefore, the 4-MUP assay required 10 ng/mL, while
the 4-MUB assay’s concentration needed to be reduced to 3.3 ng/
mL CRL (Supplementary Figure S3). Furthermore, it was deter-
mined that 10 mL and 50 mL of 10% o-H3PO4 was sufficient to
completely stop the reaction for 4-MUB and 4-MUP, respectively
(Supplementary Figure S1). Moreover, accuracy and precision
for each substrate were evaluated separately over three-day,
(Table 1), which confirmed reproducibility and comparability of
measurements within the assay and between days.
Statistical analyses were performed for determination of Km,
Vmax, and IC50 using Graphpad Prism 8 (San Diego, CA, USA).
3. Results
3.1. Enzyme activity assay development
Starting with Dairaku et al.’s proposed protocol for the human
lysosomal acid lipase23, we developed an enzyme activity assay,
which is applicable for two types of lipase specificities: short-chain
fatty acid (butyrate) and long-chain fatty acids (palmitate). The
selected substrates (4-Methylumbelliferryl butyrate (4-MUB) and
palmitate (4-MUP)) comprise these fatty acids connected to the
fluorescent dye 4-MU via an ester bond, respectively. 4-MU emits
fluorescence upon hydrolysis of the ester bond and excitation at
the corresponding wavelength. While 4-MUB is stable in phos-
phate buffer (pH 7.0), 4-MUP requires an additional stabilising
agent (SDS) for the hydrophobic longer-chain fatty acid palmitate
in aqueous solution (37 ꢀC). To translate the subsequent fluores-
cence emitted by 4-MU after CRL hydrolysis of the substrate into
concentration units, a seven-point standard curve of defined 4-MU
concentrations was included ranging from 1.563 to 62.5 mM
diluted in assay buffer. The curve (Figure 1(A)) is linear within the
defined range (Figure 1(B)) and was analysed with a linear regres-
sion fit. The standard curve allows the translation of relative fluor-
escent units into concentration (mM) within the Lower Limit of
Quantification (LLOQ) of 0.5 mM 4-MU and the Upper Limit of
Quantification of 62.5 mM.
3.2. Assay robustness
Although the assay was validated for both substrates, our data
shows superior aqueous solubility of 4-MUB as compared to
4-MUP requiring the use of SDS, which inhibits the enzyme’s activ-
ity (Supplementary Figure S3). Thus, we focussed on the substrate
4-MUB for further determination of validation parameters to avoid
interference with the enzyme’s activity. Subsequently, we investi-
gated the assay performance with 4-MUB at different tempera-
tures (Figure 2(A)) and pHs (Figure 2(B)).
Five different temperatures, 4, 15, 28, 37 and 45 ꢀC were tested.
Our data indicated that the enzyme is temperature sensitive and
most active between 37–45 ꢀC as the optimal temperature for the
enzyme activity. However, 37 ꢀC was maintained for further experi-
ments as it represents physiological conditions. The investigation
of the reaction’s efficiency at six different buffer pH revealed the
stability of the enzyme’s activity in lower pH ranges and increased
instability of the assay above pH 8. Under basic conditions, the
substrate is subject to autohydrolysis, making the reaction inde-
pendent of the enzyme’s activity and which is characteristic for
ester bonds. Therefore, the selected reaction conditions were
approximated to physiological conditions at pH 7, since they
show the robustness and optimal reaction requirements.
Subsequently, several parameters were adjusted separately for
both substrates. While the concentration of the substrate was
3.3. Analysis of CRL extract
The used enzyme extract CRL, obtained from Deerland was further
characterised. First, we investigated, which of the isoforms were
present in the extract and second, which other components could
be present in the mixture. A gas-chromatography analysis (GCMS)
revealed the presence of only CRL isoforms 1, 2, 3, and 4,
although 4 had solely one fragment detected indicating a low
abundance (see Supplementary Table S1). Furthermore, the stabil-
ity of the enzyme in water was determined over six days at room
temperature to identify potential impurities degrading the enzyme
in solution (Figure 3).
Our data showed that both methods, SDS page and GCMS,
revealed a pure enzyme extract, which was used for the investiga-
tion of CRL’s enzyme activity (Supplementary Figure S2 and
Supplementary Table S1). However, the activity assay showed a
slightly decreasing activity over the six-day period indicating auto-
degradation or presence of proteases that were below the detec-
tion limit of the method used for evaluation. Thus, the identifica-
tion of mainly the 4 isoforms of CRL in the extract and only a
slight degradation over 6 days, deemed the enzyme extract to be
pure and stable in water.
Figure 1. 4-MU standard curve. (A) Plot of several 4-MU concentrations
(0.125–125 mM) to determine the shape of the standard curve. The sigmoidal
shape was analysed with a 4-parameter logistic fit. (B) The linear range for the
standard curve was determined between the Lower Limit of Quantification
(LLOQ) and Upper Limit of Quantification (ULOQ), which is in the linear range at
concentrations ranging from 0.5 to 625.5 mM.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1477
Table 1. Validation of the enzyme activity assay for both substrates 4-MUB (A) and 4-MUP (B). Samples were measured in trip-
licates each day allowing to determine Precision (Intra-Day and Inter-Day CV%) and Accuracy (%) for the validation.
A
Precision (CV) (%)
Analytes
(Substrate: 4-MUB)
QC1a
QC2b
QC3c
Concentration (mM)
(n ¼ 3)
Accuracy (%)
(n ¼ 3)
Intra-day (n ¼ 3)
Inter-day (n ¼ 3)
12.8 0.6
16.7 1.2
74.2 3.2
0.5–6.5
1.3–3.3
0.9–2.6
4.6
6.9
4.4
0.7%
1.7%
B
Analytes
(Substrate: 4-MUP)
QC1a
QC2b
QC3c
6.5 0.4
9.8 0.6
56.8 1.9
0.9–2.2
0.6–4.2
0.8–2.6
5.6
6.0
3.4
2.0%
0.6%
aQC1: CRL and substrate.
bQC2: CRL, substrate and a low spike of 4-MU (3.13 mM).
cQC3: CRL, substrate and a high spike of 4-MU (50 mM).
Figure 2. Determination of assay robustness under application of altered conditions. (A) Temperature variation (4–45ꢀC) with buffer pH 7. (B) Buffer pH variation (pH
4–9) with reaction temperature 37 ꢀC.
3.4. Identification of an effective inhibitor
Subsequently, we used the in-vitro assay to verify the allosteric
modulator potential of Tropolone, a compound, which was deter-
mined in an in-silico screening approach (Glide, Schroedinger, XP
GScore: -5.603)24. Tropolone was used to demonstrate the under-
lying workflow (Supplementary method S1). Tropolone was inves-
tigated in both developed in-vitro assays in a concentration range
of 2 mM to 10 mM with both substrates showing a concentration-
dependent inhibition (Figure 4(A)). We calculated the correspond-
ing IC50 values (4-MUP: 3.75 ꢃ 10ꢄ4 M; 4-MUB: 3.95 ꢃ 10ꢄ4 M) by
plotting the logarithmic Tropolone concentration against the
activity of CRL (%), which indicates CRL’s inhibition being slightly
more efficient in 4-MUP samples with
(Figure 4(B)).
a lower IC50 value
In addition, we investigated reported CRL inhibitors and their
IC50 values. The reduction of the concentration of CRL by 10-fold
in the assay allowed the measurement of IC50 values of weak
inhibitors. The measured IC50 values in the in-house activity assay
were close to the reported IC50s of Trigonelline, b-Aescin and
Figure 3. Investigation of CRL’s stability throughout six days in water at room
temperature. The in-house developed activity assay was applied to determine for
each sampled day the impact on the enzyme’s activity, which slightly decreased
over time.
Berberine (Supplementary Figure S4)25,26
.
and applied in 8 different concentrations over a 50-min time span
by measuring the increase of relative fluorescent units over time.
The subsequent plot of initial velocity against the substrate con-
centration (Michaelis–Menten plot) allowed the determination of
Vmax (0.54 0.03 mM/min) and Km (0.46 0.06 mM) for the butyrate
substrate (Figure 5(A,B)).
3.5. Michaelis–Menten kinetics
Finally, we determined the kinetic parameters of the CRL extract.
The in-house developed assay with the substrate 4-MUB was used

1478
A. MENDEN ET AL.
The additional analysis of Km and Vmax after addition of with its relatively low IC50 (compared to b-Aescin) indicating a
Tropolone in two different concentrations (100 and 400 mM),
respectively, determined Tropolone’s mode of inhibition. While
Km did not show a clear trend of decline or increase due to
inhibitor addition, Vmax decreased with increasing inhibitor con-
centration (Figure 5(A,B)). This experimental trend and the analysis
of the Lineweaver-Burk plot (data not shown), confirmed the in-
silico analysis, which identified Tropolone as an allosteric modulator
of CRL and identified the inhibition as non-competitive.
Furthermore, we examined Trigonelline and b-Aescin together with
Tropolone using biolayer-interferometry (Supplementary method
S2) to investigate the binding of CRL with these compounds.
more specific interaction. However, the relatively modest IC50 is
consistent with Tropolone’s deemed mechanism of action, namely
allosteric modulation. Finally, Trigonelline showed relatively high
KD and IC50, indicating low or unspecific interaction with CRL
(Supplementary Figure S5).
4. Discussion
In this study, we developed and applied an enzyme activity assay
for two different substrate specificities (4-MUB (short-chain fatty
acids) and 4-MUP (long-chain fatty acids)) to verify the mode of
action (allosteric modulation) of Tropolone. The in-vitro assay con-
ditions were validated and showed reproducibility, robustness,
and stability. The assay is a reliable and cost-effective enzymatic
assay tool, which is easily implemented. In particular, the consider-
ation of a reagent to stop the reaction, which differentiates this
from other published assays, is pivotal for reproducibility within
and between different reads27,28. Although the current assay con-
ditions were optimised for Deerland’s CRL extract, the assay allows
adjustments for any enzyme’s specificity as 4-MU cannot only be
synthesised to other fatty acids, but also to substrates of glucosi-
dases or phosphatases. The use of 4-MUB and 4-MUP demon-
strated the assay’s adaptability to different substrates. In future
experiments, different fatty acids (substrates) and combinations
could be investigated. However, we acknowledge the limitations
of the assay such as the intrinsic decrease in solubility of the
hydrophobic fatty acid tail with length and the autocatalysis of
the substrate in the basic pH range.
b-Aescin’s KD was determined to be relatively low but was
inconsistent with high IC50 suggesting non-specific binding to
CRL. By contrast, Tropolone’s determined KD value was consistent
In addition, the assay allows measurement of IC50s and kinetic
parameters and characterisation of the enzyme’s performance.
Similarly, shifts in kinetic parameters can be investigated after the
addition of enhancers or inhibitors, which further characterises
their activity (competitive, non-competitive and uncompetitive) as
Figure 4. Analysis of Tropolone’s inhibitory effect on CRL activity in the devel-
oped enzyme activity assay with 4-MUP and 4-MUB. (A) Plot of replicated IC50
curves for 4-MUP and 4-MUB with Tropolone concentrations between 2 lM and
10 mM. (B) Analysis and determination of the IC50 values for both substrates.
IC50 values were determined by plotting the logarithmic Tropolone concentration
against the percentage of CRL activity, which allows the determination of IC50
values: 4-MUP: 3.75ꢃ 10ꢄ4 M; 4-MUB: 3.95 ꢃ 10ꢄ4 M and standard deviations of
1.65 ꢃ 10ꢄ5 and 2.19 ꢃ 10ꢄ5, respectively.
exemplified by Tropolone as
a
non-competitive inhibitor.
Although we determined that the used extract was enriched in
CRL1 and CRL3 through MS analysis, the determined kinetic
parameters are specific for this particular extract composition. In
conclusion, each isoform needs to be investigated individually to
acquire specific kinetic parameters.
Figure 5. Michaelis–Menten kinetics to investigate the performance of CRL with the in-house developed enzyme activity assay and various 4-MUB concentrations over
time. We used 0, 100, or 400mM Tropolone in the experiment to measure the impact on Vmax and Km by the inhibitor. While Km was maintained after inhibitor add-
ition, Vmax was decreasing with increasing inhibitor concentration. Therefore, our results suggest that Tropolone is a non-competitve inhibitor.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1479
Moreover, the assay was used to verify the impact of the new
in-silico determined allosteric modulator Tropolone on CRL activity.
Tropolone is a seven-carbon aromatic ring, which is a common
motif in naturally occurring compounds. It was shown to inhibit
the grape polyphenol oxidase as well as the mushroom tyrosin-
ase29,30. So far, it has not been reported that Tropolone can
inhibit lipases, although other natural compound classes such as
flavonoids, alkaloids, and saponins are known to inhibit CRL’s
activity with varying efficacy depending on the used
Funding
This work was supported by the Roskamp Institute Inc. and by a
Sponsored Research Agreement between The Roskamp Institute,
Inc and Enzymedica, Inc.
ORCID
concentration25,26
.
Ariane Menden
Davane Hall
Venkatarian Mathura
Michael Mullan
Ghania Ait-Ghezala
http://orcid.org/0000-0002-0108-6243
http://orcid.org/0000-0002-5063-1666
Both assays, 4-MUP and 4-MUB, were tested to show that CRL’s
activity is inhibited by Tropolone in a concentration-dependent
manner. The substrate independent inhibition and the similarity of
the determined IC50s indicate that Tropolone could interfere with
CRL’s enzymatic activity, which has been further investigated by
biolayer-interferometry and Michaelis–Menten kinetics. The results
suggested that Tropolone is a non-competitive inhibitor. Given
the slight differences in the assay conditions between 4-MUB and
4-MUP, we were not able to directly compare the efficiency of
Tropolone in inhibiting CRL in those assays. However, each assay
individually is inter-comparable between runs and samples and
can be applied for high-throughput screening.
Finally, to validate our enzyme activity assay performance, in
comparison to IC50s of the published CRL inhibitors Berberine,
b-Aescin and Trigonelline (which had been determined by high-
performance liquid chromatography (HPLC)), we measured the
IC50s of the published compounds in our assay25,26. The selected
inhibitors showed similar IC50s in the enzyme activity assay as
compared to the HPLC method, which confirmed the assays
competitiveness.
In addition, using biolayer-interferometry to determine the KD
of Tropolone, Trigonelline and b-Aescin, Tropolone showed the
most specific interaction with the strongest KD, while Trigonelline
and b-Aescin indicated weaker KDs and/or unspecific interaction
with the enzyme. Although the results are partly different from
the enzyme activity assay’s results, the two assays investigate dif-
ferent parameters: activity and interaction. Our results highlight
Tropolone’s mode of action as non-competitive CRL inhibitor and
that our newly developed assay is comparable to other methods
such as HPLC.
http://orcid.org/0000-0003-3337-7122
http://orcid.org/0000-0002-1473-7527
http://orcid.org/0000-0002-9836-8617
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Disclosure statement
The authors report no conflict of interest.

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