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Selective Inhibition of Human AKR1B10 by n-Humulone, Adhumulone and

Cohumulone Isolated from Humulus lupulus Extract

Article in Molecules · November 2018

DOI: 10.3390/molecules23113041

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 molecules
Article
Selective Inhibition of Human AKR1B10 by
n-Humulone, Adhumulone and Cohumulone Isolated
from Humulus lupulus Extract
Jan Moritz Seliger 1, * , Serhat Sezai Cicek 2 , Lydia T. Witt 3 , Hans-Jörg Martin 1 ,
Edmund Maser 1 and Jan Hintzpeter 1
1 Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein,
Campus Kiel, Brunswikerstr. 10, D-24105 Kiel, Germany; martin@toxi.uni-kiel.de (H.-J.M.);
maser@toxi.uni-kiel.de (E.M.); hintzpeter@toxi.uni-kiel.de (J.H.)
2 Department of Pharmaceutical Biology, Faculty of Mathematics and Natural Sciences,
Christian-Albrechts-Universität zu Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany;
scicek@pharmazie.uni-kiel.de
3 Department of Pharmaceutical Chemistry, Faculty of Mathematics and Natural Sciences,
Christian-Albrechts-Universität zu Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany;
lwitt@pharmazie.uni-kiel.de
* Correspondence: seliger@toxi.uni-kiel.de; Tel.: +49-4131-500-30901; Fax: +49-431-500-30904
Academic Editor: Marcello Locatelli



Received: 17 October 2018; Accepted: 19 November 2018; Published: 21 November 2018


Abstract: Hop-derived compounds have been subjected to numerous biomedical studies investigating
their impact on a wide range of pathologies. Isomerised bitter acids (isoadhumulone, isocohumulone
and isohumulone) from hops, used in the brewing process of beer, are known to inhibit members
of the aldo-keto-reductase superfamily. Aldo-keto-reductase 1B10 (AKR1B10) is upregulated in
various types of cancer and has been reported to promote carcinogenesis. Inhibition of AKR1B10
appears to be an attractive means to specifically treat RAS-dependent malignancies. However,
the closely related reductases AKR1A1 and AKR1B1, which fulfil important roles in the detoxification
of endogenous and xenobiotic carbonyl compounds oftentimes crossreact with inhibitors designed to
target AKR1B10. Accordingly, there is an ongoing search for selective AKR1B10 inhibitors that do
not interact with endogeneous AKR1A1 and AKR1B1-driven detoxification systems. In this study,
unisomerised α-acids (adhumulone, cohumulone and n-humulone) were separated and tested for
their inhibitory potential on AKR1A1, AKR1B1 and AKR1B10. Also AKR1B10-mediated farnesal
reduction was effectively inhibited by α-acid congeners with Ki -values ranging from 16.79 ± 1.33 µM
(adhumulone) to 3.94 ± 0.33 µM (n-humulone). Overall, α-acids showed a strong inhibition with
selectivity (115–137 fold) for AKR1B10. The results presented herein characterise hop-derived α-acids
as a promising basis for the development of novel and selective AKR1B10-inhibitors.

Keywords: aldo-keto reductases; cancer; tight-binding inhibition; selective inhibition; humulone;

farnesal reduction; hops; humulus lupulus; alpha-acids

1. Introduction
In (phyto-)pharmacology and nutritional medicine, beer and its constituents have been subject
to numerous epidemiological and molecular studies, not least in order to evaluate the manifold
effects of its main flavouring ingredient, the female inflorescences of the hop plant (Humulus
lupulus). In particular, hop-derived chalcones (xanthohumol), prenylflavonoids (isoxanthohumol,
8-prenylnaringenin) and (iso-) α- and β-bitter acids ((iso-)humulone and (iso-)lupulone) are among

Molecules 2018, 23, 3041; doi:10.3390/molecules23113041 www.mdpi.com/journal/molecules

Molecules 2018, 23, 3041 2 of 12

the bioactive compounds accounting for various modes of action in the prevention or potential
treatment of many (lifestyle) diseases. These include metabolic and inflammatory diseases as well as
carcinogenesis [1–5].
Iso-α acids (isoadhumulone, isocohumulone, isohumulone) derived from thermic isomerization of
their precursors (adhumulone (compound 1), cohumulone (compound 2), n-humulone (compound 3))
during wort boiling are counted among the most abundant classes of phenolics in beer with
concentrations ranging from 0.6 to 100 mg/L (Figure 1) [4,6]. Unisomerized α-acids are comparatively
less abundant (1.7 mg/L) [6], their concentration might, however, increase due to processes called late
or dry-hopping, where hop cones or pellets are added either near the end of the boiling process or
even later, at low temperatures, before the product is packaged [6].
The chemopreventive effect of α- and iso-α-acids on biological systems has been investigated in
earlier studies and extensively reviewed by Gerhäuser [4]. Apart from their antibiotic capacities,
and antiangiogenic and antidiabetic properties, α-acids have been reported to interfere with
carcinogenesis. For example, in human hepatocarcinoma cells, α-acids significantly reduced
phosphorylation of NfκB as well as AP-1 and ERK1/2 activity, thus reducing migration and
proliferation [1].
Prenylation of GTP-binding proteins, a process occurring further upstream of the involved
RAS-RAF-MEK-ERK (MAPK) pathway, may cause aberrant activation of RAS or RAS-like proteins,
which contributes to the development of different malignancies including glioblastoma, hepatocellular
carcinoma and pancreatic cancer [7–9]. As the mechanism of protein prenylation requires intermediates
from the cholesterol metabolism, it is, among other reactions, strongly dependent on the reduction
of the isoprenoids geranylgeranyl and farnesal in order to provide proper covalent bonding to the
C-terminal cysteines of the target proteins [10,11]. These isoprenoids fall within the specific substrate
spectrum of aldo-keto reductase member 1B10 (AKR1B10), an NADPH-dependent oxidoreductase that
has been shown to play a pivotal role in the prenylation-dependent activation of KRAS and RAS-like
proteins by mediating the reduction of geranylgeranyl and farnesal to their respective alcohols [9,12].
Hence, AKR1B10 has not only become a biomarker (as it is upregulated in various types of cancer),
but has also turned into an attractive pharmacological target in cancer prevention and treatment.
Accordingly, numerous studies have been conducted in the search for new selective inhibitors for
AKR1B10 [13–16].
Apart from the aforementioned lipids and unlike other members of the aldo-keto reductase family
that accept sugar and lipid aldehydes, steroid hormones, prostaglandins and xenobiotics as their
substrates, AKR1B10 only reduces selected carbonyls and retinal (retinaldehyde) to their corresponding
alcohols [13,17]. However, due to its capacities of detoxifying reactive carbonyl compounds
(which would otherwise induce apoptosis) and carbonyl group containing chemotherapeutics,
it contributes to different resistance mechanisms in cancer cells when upregulated [18–21]. By reducing
retinal to retinol, AKR1B10 prevents retinal from entering the retinoic acid pathway, thereby diminishing
the cellular potential to regulate differentiation and proliferation [22–24].
Two other members of the same superfamily AKR1B1 and AKR1A1 are closely related to AKR1B10,
sharing around 71% and 48% of sequence identity on the protein level, respectively [25]. Furthermore,
they are both involved in other detoxifying mechanisms and are necessary to maintain homeostasis
of the glucose metabolism [26]. Hitherto, cross-inhibition is a major pitfall in the development of
inhibitors specific to either one of these enzymes as it oftentimes accounts for severe side effects in
their clinical application [14,27].
Herein, we report the in vitro inhibitory effects of the three most prevalent, hop-derived α-acids
(compound 1–3) on the catalytic activity of human AKR1B10, AKR1B1 and AKR1A1. The selective
binding behaviour for AKR1B10 renders these natural-based compounds promising structural
analogues of new AKR1B10 inhibitors.
Molecules 2018, 23, 3041 3 of 12
Molecules 2018, 23, x FOR PEER REVIEW 3 of 12

Figure Structures of
Figure 1.1. Structures α-acids (n-humulone
of α-acids (n-humulone == compound
compound 3, 3, cohumulone
cohumulone == compound
compound 22 and
and
adhumulone = compound 1) and iso-α-acids (isohumulone, isocohumulone, and isoadhumulone)
adhumulone = compound 1) and iso-α-acids (isohumulone, isocohumulone, and isoadhumulone) after
thermal isomerisation
after thermal through
isomerisation wort boiling.
through wort boiling.
2. Results and Discussion
2. Results and Discussion
In recent research hop-derived prenylflavonoids, including the most prominent hop-compounds
In recent research hop-derived prenylflavonoids, including the most prominent
xanthohumol and 8-prenylnaringenin, have been subject to a variety of studies in order to elucidate
hop-compounds xanthohumol and 8-prenylnaringenin, have been subject to a variety of studies in
the beneficial effects of these substances in certain disease models. However, experimental data on
order to elucidate the beneficial effects of these substances in certain disease models. However,
the biological interaction potential of hop bitter acids, such as (iso-)-α-acids, are relatively scarce [4].
experimental data on the biological interaction potential of hop bitter acids, such as (iso-)-α-acids,
This is especially true for non-isomerized α-acids, which are up to 10-fold (≈10 µM) enriched in many
are relatively scarce [4]. This is especially true for non-isomerized α-acids, which are up to 10-fold
late- or dry-hopped types of beer [28]. Lately, these techniques have become more prominent in the
(≈10 µM) enriched in many late- or dry-hopped types of beer [28]. Lately, these techniques have
craft beer industry, which, in the future, might lead to increased plasma levels of α-acids following the
become more prominent in the craft beer industry, which, in the future, might lead to increased
consumption of certain types of beer.
plasma levels of α-acids following the consumption of certain types of beer.
In this study, mixtures of iso-α-acids and α-acids and the three purified α-acids, compound 1, 2
In this study, mixtures of iso-α-acids and α-acids and the three purified α-acids, compound 1,
and 3, were evaluated as inhibitors of the three related human aldo-keto reductases AKR1A1, AKR1B1
2 and 3, were evaluated as inhibitors of the three related human aldo-keto reductases AKR1A1,
and AKR1B10. For comparability reasons and in order to investigate selectivity, DL-glyceraldehyde
AKR1B1 and AKR1B10. For comparability reasons and in order to investigate selectivity,
served as a common test substrate (Tables 1–3). Compared to the iso-α-acid mixture, the mixture
DL-glyceraldehyde served as a common test substrate (Table 1–3). Compared to the iso-α-acid
of α-acids showed superior inhibitory effects with respect to all enzymes tested (Table 1). A slight
mixture, the mixture of α-acids showed superior inhibitory effects with respect to all enzymes
selectivity for AKR1B1 was observed with the iso-α-acid mixture (Table 1).
tested (Table 1). A slight selectivity for AKR1B1 was observed with the iso-α-acid mixture (Table 1).
Table 1. IC50 values of an iso-α-acid solutions and an α-acid mixture for the respective reductases.
Table 1. IC50 values of an iso-α-acid solutions and an α-acid mixture for the respective reductases.
IC50 values are presented as mean ± SD of at least three experiments.
IC50 values are presented as mean ± SD of at least three experiments.
Enzyme Iso-α-Acid Solution IC50 [µg/mL] α-Acid Mixture IC50 [µg/mL]
Enzyme Iso-α-acid solution IC50 [µg/mL] α-acid mixture IC50 [µg/mL]
AKR1B10 127.90 ± 9.79 0.42 ± 0.02
AKR1B1
AKR1B10 100.30
127.90 ± 6.03
± 9.79 57.47 ± 1.76± 0.02
0.42
AKR1A1 163.00 ± 8.96 48.23 ± 1.81
AKR1B1 100.30 ± 6.03 57.47 ± 1.76
Table 2. IC50 and Ki values of the
AKR1A1 isolated
163.00 hop-compounds for the respective48.23
± 8.96 reductases.
± 1.81 IC50 and Ki
values are presented as mean ± SD of at least three experiments. (n. d. = not determined).
Table 2. IC50 and Ki values
Enzyme of the isolated hop-compounds
AKR1A1 AKR1B1 for the respective reductases.
AKR1B10 IC50 and Ki
values are
Substrate presented as mean ± SD of
Glyceraldehyde [3.6 mM] at least three experiments.
Glyceraldehyde [50 µM](n. d. = not determined).
Glyceraldehyde [4.0 mM]
Parameter IC50 Ki (Morrison) IC50 Ki (Morrison) IC50 Ki (Morrison)
Enzyme AKR1A1 AKR1B1 AKR1B10
Compound 1 ≥100 µM n. d. >125 µM n. d. 5.41 ± 0.42 µM 3.27 ± 0.52 µM
Substrate
Compound 2 Glyceraldehyde
>100 µM [3.6
n. d. mM] Glyceraldehyde
>125 µM n. [50
d. µM] 1.35 ±
Glyceraldehyde
0.07 µM 0.70 ±[4.0
0.09mM]
µM
Compound 3 >100 µM n. d. >125 µM n. d. 1.94 ± 0.10 µM 0.98 ± 0.12 µM
Parameter IC50 Ki (Morrison) IC50 Ki (Morrison) IC50 Ki (Morrison)

Compound 1 ≥ 100 µM n. d. > 125 µM n. d. 5.41 ± 0.42 µM 3.27 ± 0.52 µM

However, AKR1B10 inhibition was up to 115 (ratio AKR1A1/AKR1B10)–137 times (ratio AKR1B1/
Compound
AKR1B10) 2 > 100
stronger thanµM n. of
inhibition d. AKR1A1
> 125 µMAKR1B1,
and n. d. 1.35 ±when
respectively, 0.07 µM 0.70 ± 0.09
a mixture µM
of α-acids
wasCompound
applied (Table 3).
3 > 100 µM n. d. > 125 µM n. d. 1.94 ± 0.10 µM 0.98 ± 0.12 µM
Molecules 2018, 23, 3041 4 of 12

Table 3. AKR1B10 selectivity of the α-acid-mixture and its isolated compounds expressed as IC50 -ratios
of AKR1A1/AKR1B10 and AKR1B1/AKR1B10.

Compounds Ratio AKR1A1/AKR1B10 Ratio AKR1B1/AKR1B10

α-acid
115 137
mixture
Compound 1 ≥19 >23
Compound 2 >74 >93
Compound 3 >52 >64

Shindo et al. [29] report on the inhibitory effect of iso-α-acids on AKR1B1 at lower concentrations
(48% inhibition at 33 µg/mL). IC50 values of iso-α-acids for AKR1B1 in the present study were
somewhat higher (100.30 ± 6.03 µg/mL) than reported by Shindo et al. The observed discrepancy
to the present study might have been due to the source and quality of the inhibitor as well as due
to different purification conditions of the recombinant enzyme. In the present study a 30% (w/w)
prediluted standardised solution of iso-α-acids produced from CO2 hop extract has been used, whereas
Shindo et al. used international calibration standard iso-α-acids. Moreover, the concentrations of
the single iso-α-acid congeners in the mixture were not further evaluated in both studies and might
have influenced the respective IC50 values as well. Unlike in the present study, Shindo et al. used
recombinant AKR1B1 from a eukaryotic expression system, which might have affected the binding
behaviour of the inhibitor through posttranslational modifications that are not present in an enzyme
derived from the expression system used in this study.
Additionally, potential synergistic effects with other substances found in the hop extract
could have also affected AKR1B1 activity. In fact, hop-specific compounds such as xanthohumol,
isoxanthohumol and 8-prenylnaringenin have been reported to be strong inhibitors of AKR1B1 and its
related reductase AKR1B10 [30].
Due to the promising results of the α-acid extract, AKR-inhibition of the single compounds
was of particular interest. UHPLC analysis of the extract showed three dominating peaks (Figure 2)
corresponding to the major α-acids (n-humulone (3), cohumulone (2) and adhumulone (1)) found
in hops [28]. Thereupon, α-acids were separated by preparative column chromatography, yielding
compound 2 as well as a mixture of compounds 1 and 3 in a first step. Subsequent semi-preparative
HPLC led to the isolation of the remaining two main compounds, 1 and 3. All three substances were
further analysed for their inhibitory potential.
Unlike the iso-α-mixture, IC50 determination of the single hop compounds isolated from the
α-acid-mixture showed AKR1B10 selectivity (Table 3). Among the three compounds tested, AKR1B10
inhibition by compound 2 was the strongest (IC50 = 1.35 ± 0.07 µM). In case of AKR1A1 and AKR1B1,
inhibition was less than 50 % at 100 and 125 µM, respectively (Table 2). Similarly, humulone from beer
hop extract has been shown to also selectively inhibit the inflammatory modulator cyclooxygenase-2
(IC50 = 1.60 µM), whereas homologous cyclooxygenase-1 was not inhibited at concentrations below 10
µM [31]. Interestingly, arachidonic acid, the primary substrate of cyclooxygenase-1 and 2, effectively
inhibits AKR1B10 at nanomolar concentrations (Ki = 0.26 µM) [32]. Hence, similarities in the binding
behaviour of humulone and unsaturated fatty acids to these enzymes might as well pinpoint towards
common inhibitory mechanisms underlying the effects observed with AKR1B10.
Molecules 2018, 23, 3041 5 of 12
Molecules 2018, 23, x FOR PEER REVIEW 5 of 12

Figure 2.2.Chromatogram
Figure Chromatogramof α-acid separation.
of α-acid Compound
separation. 1 = adhumulone;
Compound compound compound
1 = adhumulone; 2 = cohumulone;
2 =
cohumulone; n-humulone.
compound 3 =compound See text for further
3 = n-humulone. details.
See text for further details.

We further determined inhibition parameters of compounds 1, 2 and 3 during farnesal reduction

We further determined inhibition parameters of compounds 1, 2 and 3 during farnesal
by AKR1B10, a process that has been postulated to be crucial for the prenylation and thus
reduction by AKR1B10, a process that has been postulated to be crucial for the prenylation and thus
activation of several RAS- or RAS-like proteins during carcinogenesis [9]. Thus, inhibiting one of
activation of several RAS- or RAS-like proteins during carcinogenesis [9]. Thus, inhibiting one of
the key enzymes in protein prenylation constitutes an opportunity to interfere with RAS-driven
the key enzymes in protein prenylation constitutes an opportunity to interfere with RAS-driven
carcinogenesis. Remarkably, an isovaleryl group at the C4 position of compound 3 seems to
carcinogenesis. Remarkably, an isovaleryl group at the C4 position of compound 3 seems to
improve its inhibitory performance on AKR1B10: IC50 values were approximately 4-fold lower
improve its inhibitory performance on AKR1B10: IC50 values were approximately 4-fold lower with
with
compoundcompound3 (7.783± (7.78
0.43 µM)± 0.43 µM) compared
compared to its relatedto its related compounds
compounds 1 (29.27 ± 1.53 1 (29.27
µM) and ± 21.53(29.78µM) ±
and 2 (29.78 ± 1.72 µM), which show slightly different acylic moieties at the C4 position
1.72 µM), which show slightly different acylic moieties at the C4 position (Figure 1). Also,
(Figure 1). Also, prenylation of the inhibitor seems to positively influence binding behaviour:
prenylation of the inhibitor seems to positively influence binding behaviour: a ring contraction
a ring contraction occurring during isomerisation of α-acids leads to the introduction of
occurring during isomerisation of α-acids leads to the introduction of a carbonyl group at the
a carbonyl group at the C6-prenyl residue, which might explain the comparatively lower inhibition
C6-prenyl residue, which might explain the comparatively lower inhibition capacities of
iso-α-acids. Aiso-α-acids.
capacities of A similar
similar effect effect observed
has been has been observed with γ-mangostin,
with γ-mangostin, another another potent
potent natural
natural AKR1B10-inhibitor from mangosteen (Garcinia mangostana)
AKR1B10-inhibitor from mangosteen (Garcinia mangostana) (IC50 = 0.018 µM), and two related (IC 50 = 0.018 µM), and two
xanthones 1,5-dihydroxy-2-isoprenyl-3-methoxyxanthone [(1,5-DIMX) (IC50 > 50 10 µM) and
related xanthones 1,5-dihydroxy-2-isoprenyl-3-methoxyxanthone [(1,5-DIMX) (IC > 10 µM) and
1,7-dihydroxy-2-isoprenyl-3-methoxyxanthone (1,7-DIMX) (IC 50 = 0.85 µM)].
1,7-dihydroxy-2-isoprenyl-3-methoxyxanthone (1,7-DIMX) (IC50 = 0.85 µM)]. In this case, the loss of In this case, the loss
of an
an isoprenyl
isoprenyl group
group inin1,5-DIMX
1,5-DIMXand and1,7-DIMX
1,7-DIMXmight mighthavehavealso also contributed
contributed to to deteriorating
deteriorating the the
inhibitory effect when compared to the parent substance
inhibitory effect when compared to the parent substance [16]. [16].
With respect
With respect toto the
the mechanism
mechanism of of inhibition,
inhibition, Zhang
Zhang et et al.
al. suggested
suggested aa moremore accessible
accessible anionic
anionic
site at TRP112, allowing the entrance of more bulky and rigid inhibitors
site at TRP112, allowing the entrance of more bulky and rigid inhibitors at the broader active at the broader active sitesite
of
of AKR1B10 [33]. Molecular docking experiments have been performed
AKR1B10 [33]. Molecular docking experiments have been performed for the three reductases in for the three reductases
in order
order to to clarify
clarify thethe binding
binding behaviour
behaviour and and specificity
specificity of of
thethe respective
respective inhibitors.
inhibitors. EvenEven though
though in
in silico analyses indicated a strong inhibition at the active sites of the
silico analyses indicated a strong inhibition at the active sites of the enzymes, the mechanism enzymes, the mechanism
favouring AKR1B10
favouring AKR1B10 inhibition
inhibition over
over inhibition
inhibition of of the
the other
other twotwo enzymes
enzymes could could not
not ultimately
ultimately be be
resolved (data not shown).
resolved (data not shown).
Among the
Among the natural-based
natural-based derivatives
derivatives serving
serving as as selective
selective AKR1B10
AKR1B10 inhibitors,
inhibitors, hop-derived
hop-derived
α-acids have
α-acids have not
notbeen
beeninvestigated
investigatedsosofar.
far.For thethe
For substances
substances tested in this
tested study,
in this non-competitive
study, non-competitive and
competitive modes of inhibition have been observed (Table 4, Figures 3
and competitive modes of inhibition have been observed (Table 4, Figures 3,4). With regard to the and 4). With regard to the
inhibition of
inhibition of AKR1B10
AKR1B10 mediated
mediated farnesal
farnesal reduction
reduction (K (KM = 5 µM), compound 3 was the best inhibitor
M = 5 µM), compound 3 was the best inhibitor
with relatively low K i (3.94 ±
with a relatively low Ki (3.94 ± 0.33 µM) when compared to
a 0.33 µM) when compared to its
its congeners,
congeners,compound
compound22(K 16.53±
(Ki i==16.53 ±
1.74 µM) and 1 (K = 16.79 ±
1.74 µM) and 1 (Kii = 16.79 ± 1.33 µM).1.33 µM).
Molecules 2018, 23, x FOR PEER REVIEW 6 of 12

Molecules 2018, 23, x FOR PEER REVIEW 6 of 12

Molecules 2018, 23, 3041 6 of 12

Figure 3.
Figure 3. IC
IC5050-values
-valuesofofthe
theisolated
isolatedhop
hopcompounds
compoundsfor forAKR1B10-catalysed
AKR1B10-catalysedfarnesal
farnesalreduction
reductionasasa
function
a function
Figure of substrate
of50substrate
3. IC concentration
-values ofconcentration [compound
[compound
the isolated hop 1 (adhumulone)
1 (adhumulone)
compounds (circles), compound
(circles), compound
for AKR1B10-catalysed 2 (cohumulone)
(cohumulone)
farnesal2reduction as a
(squares)
(squares) and
and compound
compound 3
3 (n-humulone)
(n-humulone) (triangles)].
(triangles)].
function of substrate concentration [compound 1 (adhumulone) (circles), compound 2 (cohumulone)
(squares) and compound 3 (n-humulone) (triangles)].

Figure 4. Determination
Determination of inhibition mode. To To determine
determine the mode of inhibition the normalized
velocity4.isisDetermination
velocity
Figure plottedasas
plotted a function
a function of inhibitor
of inhibitor
of inhibition concentration
concentration
mode. To as mode
exemplified
as exemplified
determine the in
ofthe in thediagram
upper
inhibition upper
the diagram
(farnesal
normalized
(farnesal
= 1.25 µM). = 1.25
The µM).
data The
weredata were
fitted to vfitted
velocity is plotted as a function of iinhibitor/v 0 to
= v
1/(1
i /v +
0 = 1/(1 +
([I]/IC
concentration ([I]/IC
)) and50 )) and
lines lines
are are
drawn drawn
from from
[I]
50 as exemplified in the upper diagram = 0 [I] =0
to the
intersection
(farnesal of vi /v
= 1.25 0 = The
µM).
1/2 and so on. The intersection of the dotted lines with the x-axis define the constant
data were fitted to vi/v0 = 1/(1 + ([I]/IC50)) and lines are drawn from [I] = 0
Molecules 2018, 23, 3041 7 of 12

K as indicated [34,35]. This was done for all used farnesal and inhibitor concentrations and K
was plotted as a function of substrate concentration (lower diagram). This secondary plot shows
an increase of K with substrate concentration, which is characteristic for a competitive inhibitor
(adhumulone). In the case of a non-competitive inhibitor K would be independent of substrate
concentration (humulone and cohumulone).

Table 4. IC50 and Ki values of AKR1B10 mediated farnesal reduction with isolated hop-compounds.
IC50 and Ki values are presented as mean ± SD of at least three experiments.

Enzyme AKR1B10
Substrate Farnesal [5 µM]
Mode of inhibition
Parameter IC50 Ki
Compound 1 29.27 ± 1.53 µM 16.79 ± 1.33 µM competitive
Compound 2 29.78 ± 1.72 µM 16.53 ± 1.74 µM non-competitive
Compound 3 7.78 ± 0.43 µM 3.94 ± 0.33 µM non-competitive

Based on the IC50 and Ki values stated herein, inhibition of AKR1B10 through α-acids during
glyceraldehyde or farnesal reduction, might also be compared to the inhibitory efficiency of unsaturated
fatty acids on this enzyme (Ki -values ranging from 0.24 to 1.1 µM). Unsaturated fatty acids show
a competitive inhibition pattern with a specificity towards AKR1B10 [32]. Accordingly, the mechanism
of action suggested by Hara et al. [32], which involves the presence of relatively long chain of
carbon-carbon double bonds interacting with the enzyme’s active site, might in parts also apply
for humulone and its three isoprenoid side chains. However, X-ray diffraction experiments would
help to further clarify the actual binding mechanism.
Under physiological conditions, the inhibitory effect might be further strengthened with increasing
uptake of lipophilic congeners. In this context, a study by Cattoor et al. reported efficient epithelial
absorption of α-acids, which might as well point towards a rather high bioavailability [36,37]. Data on
the bioavailability on α-acids in animal models are currently lacking; however, the metabolically
relevant concentrations (Ki , IC50 ) stated herein, fall within the bioavailable spectrum of iso-α-acids
reported by others: a study on the bioavailability of iso-α-acids in rabbits report of cumulative
iso-α-acid concentrations between 7 and 20 µM [37]. These concentrations seem sufficient for having
an impact on AKR1B10-mediated farnesal reduction. In conjunction with the development of functional
foods, there is increasing evidence that prenylation of a target compound raises its bioavailability [38].
As prenylation occurs in both unisomerised and isomerised α-acids, an overall higher bioavailability
of these compounds might be expected. In vivo, steadily high concentration levels would be especially
important when a competitive mode of inhibition is observed (Table 4, Figures 3 and 4).
In general, research has made great advances in terms of designing new, effective AKR1B10
inhibitors. Unfortunately, though, the clinical safety of their use has in many cases not been evaluated
yet [14]. In contrast, hops and hop-derived bitter acids are considered free for consumption and
generally recognized as safe for oral intake [28,39,40]. Therefore, α-acids may yield the potential to
serve as an alternative basis for the development of AKR1B10-inhibitors.
In conclusion, the results presented in this study identify α-acids as potent inhibitors with
a selectivity for AKR1B10 versus homologous AKR1A1 and AKR1B1. Of the three α-acid congeners
tested, inhibition by compound 3 showed the strongest inhibition. Moreover, there is evidence of
isoprenoid side chains tending to affect the binding behaviour of AKR1B10 inhibitors. With regard
to AKR1B10 selectivity, our results provide a structural basis for the development of future QSAR
models and new drugs/inhibitors targeting cancers characterized by AKR1B10-specific actions or
AKR1B10 upregulation.
Molecules 2018, 23, 3041 8 of 12

3. Materials and Methods

3.1. Chemicals and Reagents

Organic solvents for chromatography, MS grade water and MS grade formic acid were obtained
from VWR (Darmstadt, Germany). Organic solvents used for preparative, semi-preparative and
analytical chromatography were of gradient grade quality and water was bi-distilled water. Solvents
used for LC-MS analyses were of MS grade quality. Formic acid used for chromatography was of
MS grade quality. NADPH was obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany).
DL-glyceraldehyde and farnesal were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
A standardised solution of iso-α-acids produced from CO2 hop extract (30% w/w) was obtained from
Barth Haas UK Limited (Tonbridge, UK). Mixtures of α-acids were kindly provided by Dr. Martin
Biendl (Hopsteiner—HHV GmbH, Mainburg, Germany).

3.2. Isolation and Identification of α-Acids

Preparative LC was accomplished using a Büchi PrepChrom C-700 and Büchi PrepChrom C18
column (15 µm, 250 × 30.0 mm) (Büchi, Germany). Semi-preparative chromatography was carried
out on a Waters Alliance e2695 Separations Module equipped with an Alliance 2998 PDA detector
and a WFC III fraction collector (Waters, Milford MA, USA) using a Phenomenex Aqua column (5 µm.
250 × 10.0 mm). Fractions and pure compounds were analysed by a VWR-Hitachi Chromaster Ultra
RS (VWR, Darmstadt, Germany) using a Nucleodur C18 Pyramid column (5 µm, 250 × 4.6 mm).
LC-MS was conducted with a Shimadzu Nexera X2 UHPLC system and a Shimadzu LC-MS 8030
triple quadrupole mass spectrometer using electrospray ionization (Shimadzu, Kyoto, Japan) and
a Nucleodur C18 Gravity-SB column (1.8 µm, 100 × 2.0 mm). LC-conditions: isocratic elution with
0.4 mL/min at 30 ◦ C using 0.1% formic acid (30%) and acetonitrile (70%). MS-conditions: nebulizer
gas 3 L/min, drying gas 15 L/min, DL temperature 250 ◦ C, heat block temperature 400 ◦ C. Mass range
was 100 to 1000 m/z.
900 mg of α-acids extract were dissolved in 15 mL of 85% methanol and subjected to preparative
chromatography using 0.025% formic acid in water and methanol (15:85) yielding 5 fractions. Fraction
2 contained 125 mg of cohumulone (2) while fraction 4 yielded 67 mg of n-humulone (3) and 28 mg
of adhumulone (1) after separation by semi-preparative chromatography using 0.025% formic acid
in water and acetonitrile (30:70). Compounds were assigned according to their molecular masses
and their retention times in comparison with a reference chromatogram [41], as well as their relative
amounts in the extract used for isolation.

3.3. Preparation of Recombinant Proteins

The carbonyl-reducing enzymes AKR1A1, AKR1B1, AKR1B10 were prepared in an Escherichia coli
expression system according to previously published methods: plasmids of AKR1A1 and AKR1B1 were
friendly gifts from Prof. Dr. Vladimir Wsol [42] and Dr. Nina Kassner; information about production
and purification of AKR1B10 [19] has been published before (sequences of all obtained plasmids
containing the specific inserts were verified by sequencing (MWG Eurofins)). The plasmids were then
transformed in E. coli BL21 (DE3) cells. For overexpression of 6× His-tagged enzymes, a 400 mL culture
(containing the appropriate antibiotic; plasmid dependent) was grown to optical density of 0.6 at
600 nm at 37 ◦ C. Expression was induced by adding isopropyl-1-thio-galactopyranoside to the culture
medium (final concentration of 1 mM). After 3 h, cells were harvested by centrifugation (6000× g,
15 min) and resuspended in 20 mL PBS-I buffer (20 mM Na2 H2 PO4 , 500 mM NaCl, 10 mM imidazole,
10% v/v glycerol, pH 7.4). Cell disruption was performed by ultrasonication with cooling on ice to
avoid heating. The sample was subsequently centrifuged at 100,000× g at 4 ◦ C for 1 h. The obtained
supernatants containing the respective enzymes were purified using Ni-affinity chromatography
(ÄKTA-Purifier; Amersham Pharmacia, Uppsala, Sweden) using PBS-II buffer (20 mM Na2 H2 PO4 ,
500 mM NaCl, 500 mM imidazole, 10% v/v glycerol, pH 7.4). Purification progress was monitored
Molecules 2018, 23, 3041 9 of 12

by SDS-PAGE of the obtained fractions (not shown). Enzyme concentrations were determined using
a Qubit 2.0 fluorometric quantitation system (Life Technologies, Carlsbad, CA, USA) according to the
manufacturer’s instructions.

3.4. Determination of Inhibition Parameters Using Test Substrates

Catalytic properties were determined by measuring the decrease in absorbance at 340 nm
(Cary 100 scan photometer, Varian, CA, USA). A reaction mixture without inhibitor consisted of
different concentrations of DL-glyceraldehyde or farnesal, 200 µM NADPH, 0.1 M NaH2 PO4 buffer
(pH 7.4) and an appropriate amount of enzyme in a total assay volume of 0.8 mL. Final enzyme
concentrations in the assay ranged from 222 nM (AKR1A1) to 899 nM (AKR1B10). KM values were
obtained by fitting the kinetic data (mean ± SD from at least three experiments) to the Michaelis-Menten
model, as implemented in GraphPad Prism6 (GraphPad Software Inc., La Jolla, CA, USA).
For inhibition studies, stock solutions of inhibitors were prepared in H2 O (iso-α-acid mixture)
and DMSO (α-acid mixture and compounds 1–3 purified from the same mixture). The final
concentration of DMSO in the assay was ≤ 1% and did not affect enzyme activity. When collecting
data for dose–response curves initial velocities of DL-glyceraldehyde or farnesal reduction (substrate
concentration at KM ) in the presence of inhibitors were assayed as described above. The percentage of
inhibition was calculated considering the activity in the absence of inhibitor to be 100%.
Initially, the half maximal inhibitory concentrations (IC50 values) were determined for each
inhibitor in presence of each enzyme, using the shared substrate DL-glyceraldehyde (set to their
specific KM ; 3.6 mM, 50 µM and 4 mM for AKR1A1, AKR1B1 and AKR1B10, respectively) to assess
specificity amongst the structurally similar members of the AKR-superfamily.
For IC50 determination, experimental data were normalised and fitted to a sigmoidal curve
as implemented in GraphPad Prism6 (GraphPad Software Inc., La Jolla, CA, USA). Whenever
tight-binding inhibition was observed, the inhibition constant Ki was determined by fitting
inhibition data to the Morrison equation [43]. In order to verify the inhibitory potency, farnesal as
an enzyme-specific physiological substrate for AKR1B10 (farnesal; KM = 5 µM) was used to determine
inhibition parameters. Enzyme inhibition parameters were assayed as described above. The inhibition
mechanism of each compound for AKR1B10 was analysed by plotting IC50 -values at different substrate
concentrations (at least five inhibitor and substrate concentrations) [43,44]. All data obtained were
plotted and analysed using GraphPad Prism6 (GraphPad Software Inc., La Jolla, CA, USA).

Author Contributions: Conceptualization, J.M.S., S.S.C. and J.H.; Investigation, J.M.S., S.S.C. and J.H.;
Methodology, J.M.S., S.S.C., L.T.W., H.-J.M. and J.H.; Project administration, E.M.; Resources, E.M.; Software,
L.T.W.; Supervision, H.-J.M. and J.H.; Visualization, L.T.W.; Writing—original draft, J.M.S.; Writing—review
& editing, S.S.C., H.-J.M., E.M. and J.H.
Funding: This research was funded by Land Schleswig-Holstein within the funding programme Open
Access Publikationsfonds.
Acknowledgments: The authors gratefully acknowledge the provision of α-acid mixtures by Martin Biendl
(Hopsteiner—HHV GmbH, Mainburg, Germany).
Conflicts of Interest: The authors declare no conflicts of interest.

Abbreviations
1,5-DIMX 1,5-dihydroxy-2-isoprenyl-3-methoxyxanthone
1,7-DIMX 1,7-dihydroxy-2-isoprenyl-3-methoxyxanthone
AKR Aldo-keto reductase
AP-1 Activator protein 1
DMSO Dimethyl sulfoxide
ERK-1/2 Extracellular signal-regulated kinase 1/2
GTP Guanosine triphosphate
HPLC High-performance liquid chromatography
KRAS KRAS proto-oncogene
Molecules 2018, 23, 3041 10 of 12

LC Liquid chromatography
LC-MS Liquid chromatography-mass spectrometry
MAPK Mitogen-activated protein kinase
MEK Mitogen-activated protein kinase
NADPH Nicotinamide adenine dinucleotide phosphate
NFκB Nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells
QSAR Quantitative structure-activity relationship
RAF Rapidly accelerated fibrosarcoma
RAS Rat sarcoma
UHPLC Ultra-high-performance liquid chromatography

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Sample Availability: Samples of the compounds are not available from the authors.

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