Bioactivity profiles of cytoprotective short-chain quinones

Article abstract of DOI:10.3390/molecules26051382

Short-chain quinones (SCQs) have been investigated as potential therapeutic candidates against mitochondrial dysfunction, which was largely thought to be associated with the reversible redox characteristics of their active quinone core. We recently reported a library of SCQs, some of which showed potent cytoprotective activity against the mitochondrial complex I inhibitor rotenone in the human hepatocarcinoma cell line HepG2. To better characterize the cytoprotection of SCQs at a molecular level, a bioactivity profile for 103 SCQs with different compound chemistries was generated that included metabolism related markers, redox activity, expression of cytoprotective proteins and oxidative damage. Of all the tested endpoints, a positive correlation with cytoprotection by SCQs in the presence of rotenone was only observed for the NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent reduction of SCQs, which also correlated with an acute rescue of ATP levels. The results of this study suggest an unexpected mode of action for SCQs that appears to involve a modification of NQO1-dependent signaling rather than a protective effect by the reduced quinone itself. This finding presents a new selection strategy to identify and develop the most promising compounds towards their clinical use.

Full text of DOI:10.3390/molecules26051382

molecules
Article
Bioactivity Profiles of Cytoprotective Short-Chain Quinones
Zikai Feng 1,2,† , Monila Nadikudi 1,†, Krystel L. Woolley 2, Ayman L. Hemasa 1 , Sueanne Chear 1,
Jason A. Smith 2 and Nuri Gueven 1,
*
1
School of Pharmacy and Pharmacology, University of Tasmania, Hobart, TAS 7005, Australia;
zikai.feng@utas.edu.au (Z.F.); monila.nadikudi@utas.edu.au (M.N.);
ayman.hemasa@utas.edu.au (A.L.H.); sueanne.chear@utas.edu.au (S.C.)
School of Natural Sciences, University of Tasmania, Hobart, TAS 7005, Australia;
krystel.woolley@utas.edu.au (K.L.W.); jason.smith@utas.edu.au (J.A.S.)
Correspondence: nuri.guven@utas.edu.au
2
*
†
These authors contributed equally to this work.
Abstract: Short-chain quinones (SCQs) have been investigated as potential therapeutic candidates
against mitochondrial dysfunction, which was largely thought to be associated with the reversible
redox characteristics of their active quinone core. We recently reported a library of SCQs, some of
which showed potent cytoprotective activity against the mitochondrial complex I inhibitor rotenone
in the human hepatocarcinoma cell line HepG2. To better characterize the cytoprotection of SCQs
at a molecular level, a bioactivity profile for 103 SCQs with different compound chemistries was
generated that included metabolism related markers, redox activity, expression of cytoprotective
proteins and oxidative damage. Of all the tested endpoints, a positive correlation with cytoprotection
by SCQs in the presence of rotenone was only observed for the NAD(P)H:quinone oxidoreductase
1 (NQO1)-dependent reduction of SCQs, which also correlated with an acute rescue of ATP levels.
The results of this study suggest an unexpected mode of action for SCQs that appears to involve a
modification of NQO1-dependent signaling rather than a protective effect by the reduced quinone
itself. This finding presents a new selection strategy to identify and develop the most promising
compounds towards their clinical use.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Feng, Z.; Nadikudi, M.;
Woolley, K.L.; Hemasa, A.L.; Chear, S.;
Smith, J.A.; Gueven, N. Bioactivity
Profiles of Cytoprotective
Short-Chain Quinones. Molecules
2021, 26, 1382. https://doi.org/
10.3390/molecules26051382
Keywords: mitochondrial dysfunction; short-chain quinone; bioactivity; cytoprotection
Academic Editor: Francesca
Giampieri
1. Introduction
Mitochondria are essential organelles involved in many cellular processes such as
Received: 22 January 2021
Accepted: 3 March 2021
Published: 4 March 2021
the control of cell death, Ca2+-signaling as well as redox- and energy-homeostasis [
1–3].
Mitochondria provide about 95% of the cellular chemical energy in the form of adenosine
triphosphate (ATP) via oxidative phosphorylation (OXPHOS). Any insult or genetic predis-
position that impairs mitochondrial function can lead to a range of mitochondrial diseases
such as Leber’s hereditary optic neuropathy (LHON), Leigh syndrome (LS) and domi-
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
nant optic atrophy (DOA) [
vast number of common inflammatory (i.e., ulcerative colitis) [
Alzheimer’s disease, Parkinson’s disease, glaucoma, age-related macular degeneration) [
neuromuscular (i.e., Duchenne muscular dystrophy, multiple sclerosis) [ ], and metabolic
disorders (i.e., diabetes, obesity) [ ], which illustrates that mitochondrial pathology is
4
]. In addition, mitochondrial dysfunction is also present in a
], neurodegenerative (i.e.,
],
5
6
7
8
widespread. However, despite the large number of affected patients, there is an obvious
lack of approved drugs that aim to target mitochondrial function directly. This represents
a significant unmet medical need and thus, new drug candidates are needed that can be
developed into effective and safe medications.
The chemical class of quinones is known for their wide biological activities that are
largely due to the reversible redox characteristics of the quinone core [9–11]. Naturally
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
occurring quinones such as coenzyme Q10 (CoQ10) and vitamin K are described as signal-
ing molecules, enzymatic cofactors and antioxidants that are generally well tolerated and
Molecules 2021, 26, 1382. https://doi.org/10.3390/molecules26051382
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1382
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are known to be required for normal mitochondrial function [12]. Although this could
make these compounds ideal drug candidates to protect normal mitochondrial function,
their generally high hydrophobicity is thought to interfere with drug absorption and dis-
tribution [13]. Therefore, synthetic quinones such as idebenone, EPI-743 [14], MitoQ [15],
SkQ1 [16], KH176 [17] are marketed or are under development. At present, the only syn-
thetic quinone clinically approved for a single mitochondrial disease is the benzoquinone
idebenone [18], which showed some activity to protect against vision loss and restored
visual acuity and color vision in LHON patients [19
well tolerated in vitro (IC50 = 151.7 M, 24 h) [22] and in vivo (2250 mg/d, 14 d) [23], it is
not an ideal drug as it shows low solubility (logP = 1.24, logD = 3.57) and more importantly
is characterized by very low metabolic stability in vitro (t1/2 = 2 h, 40 M) [24] and in vivo
–21]. Although idebenone is safe and
µ
µ
(t1/2 = 3 h, 150 mg) [25], which significantly restrict its therapeutic potential.
To overcome these significant limitations, we previously described a class of short-
chain quinones (SCQs), which centers around a 2,3-disubstituted naphthoquinone core
with an amide linkage on the alkyl side chain [26]. Some of these SCQs showed significantly
higher cytoprotective activity [26], higher metabolic stability [24] and/or lower cytotoxic-
ity [22] in the human hepatocarcinoma cell line HepG2. This cell line, widely employed
in in vitro studies, represents a robust testing platform to offer reproducible outcomes
due to its phenotypic stability and unlimited availability [27]. Two SCQs from our library
also showed significantly better restoration of visual acuity compared to idebenone in
a rat model of diabetic retinopathy [28]. However, in this in vivo model, the two SCQs
differed in their molecular activities; while one significantly reduced vascular leakage, the
other effectively suppressed oxidative damage [28]. Despite these associations, it is unclear,
which bioactivities are causally responsible for the SCQ-dependent cytoprotection observed
in vitro and in vivo, which also applies to all other quinones in development. Therefore,
the current study approached this problem by characterizing multiple in vitro bioactivities
that were previously proposed to be responsible for cytoprotection of quinones. A large
number of closely related SCQ compounds with different cytoprotective activity was tested
in the same cell line to answer the question whether SCQ-induced cytoprotection can be
associated with a single unified mechanism or not. For this purpose, different cellular
responses to 103 SCQs 1–103 (Table 1), including 24 novel SCQs (1–21, 33 and 101–102;
see Table S1 for details) were assessed with regard to metabolism-related markers, redox
activity, expression of cytoprotective proteins, and oxidative damage. These endpoints
were correlated to the specific compound chemistries and to their cytoprotective potentials.
In contrast to studies that employ only a single molecule, using a large range of closely
related compounds allows to correlate varying cytoprotective activities with biological
endpoints, which can link specific activities to a chemical family of compounds. This
approach can provide essential information towards the underlying mode(s) of action to
support the development of selected candidates of this class of compounds towards their
clinical use.

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Table 1. Short-chain quinone (SCQ) test compounds.
Structure
#
1
n
R
References
#
2
n
R
References
#
3
n
R
References
-
-
-
-
-
-
-
4
-
-
5
-
-
6
-
7
-
-
-
-
8
-
-
-
-
9
-
-
-
-
10
11
12
13
16
-
-
-
-
14
17
-
-
-
-
15
18
-
-
-
-
19
-
-
20
-
-
21
-
-
22
25
-
-
[26]
[26]
23
26
-
-
[26]
[26]
24
27
-
-
[26]
[26]
28
-
[26]
29
-
[26]
30
-
[26]
31
-
[26]
32
-
[26]
33
-
-
34
-
[26]
35
-
[22,24,26]
36
-
[26]
37
40
-
-
[26]
[26]
38
41
-
-
[26]
39
42
-
-
[26]
[24,26]
[22,24,26]
43
-
[26]
44
-
[26]
45
-
[26]

Molecules 2021, 26, 1382
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Table 1. Cont.
Structure
#
n
R
References
#
n
R
References
#
n
R
References
46
-
[26]
47
-
[26]
48
-
[26]
[26]
[26]
49
52
-
-
[26]
[26]
50
53
-
-
[26]
[26]
51
54
-
-
55
58
-
-
[26]
[26]
56
-
[26]
57
-
[26]
59
62
65
2
2
2
[24,26]
[22,24,26]
[24,26]
60
63
66
2
2
3
[26]
[22,24,26]
[26]
61
64
67
2
2
4
[26]
[22,24,26]
[26]
68
71
2
2
[22,24,26]
69
72
3
3
[22,24,26]
[26]
70
73
4
4
[24,26]
[26]
[22,24,26,28]
74
77
2
2
[26]
[26]
75
78
2
2
[26]
[26]
76
79
2
2
[26]
[26]
80
83
2
1
[24,26]
[26]
81
84
2
2
[26]
[26]
82
85
2
3
[26]
[26]

Molecules 2021, 26, 1382
5 of 24
Table 1. Cont.
Structure
#
n
R
References
#
n
R
References
#
n
R
References
86
4
[26]
87
2
[26]
88
2
[26]
89
2
[26]
90
93
2
2
[26]
[26]
91
94
2
2
[26]
[26]
92
95
2
2
[22,24,26]
[26]
96
99
1
3
[26]
97
2
4
[22,24,26,28]
[26]
98
2
[26]
[22,24,26]
100
101
102
103
-
-
-
-
-
-
-
-
[29]
SCQs are characterized into seven chemical classes: aliphatic, amino alcohol, acid, amino acid, aliphatic ester, amino ester, and slight polarity (see Tables S1 and S2 for details).

Molecules 2021, 26, 1382
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2. Results
2.1. Cytoprotection against Mitochondrial Dysfunction
In the present study, a transient challenge with the mitochondrial complex I inhibitor
rotenone reduced HepG2 cellular viability to 26.9 7.9% (Figure 1, dotted line) compared
±
to cells not exposed to rotenone (100%). Under these conditions of rotenone-induced
mitochondrial dysfunction, 62 out of the 103 SCQs significantly improved viability (54 com-
pounds, p < 0.001; 4 compounds, p < 0.01; 4 compounds, p < 0.05; Table S2). Of the 62
cytoprotective compounds, 9 compounds (35, 42, 64, 68, 69, 71, 92, 97 and 99, p < 0.001)
significantly improved cell viability in the presence of rotenone to >90% compared to cells
not treated with rotenone. While more than half of the test compounds protected viability
to levels above rotenone-treated cells, 7 compounds (25, 30 and 44, p < 0.001; 48 and 51,
p < 0.01 31 and 32, p < 0.05) were cytotoxic and reduced cell viability more than rotenone
;
alone (Table S2). Of the 7 chemical classes, compounds with an amino alcohol, amino acid,
amino ester (p < 0.001) or acid (p < 0.05) side chains significantly increased viability when
compared to rotenone-treated cells. The highest cytoprotection by compounds with an
amino ester side chain was significantly higher than those with aliphatic (p < 0.001), acid
(p < 0.05), aliphatic ester (p < 0.001) or slight polar (p < 0.01) side chains (Figure 1; Table S3).
Figure 1. Cytoprotection of short-chain quinones (SCQs) belonging to different chemical classes
against mitochondrial dysfunction in HepG2 cells. Each point represents the average responses from
several independent experiments for one SCQ. Black solid lines represent the mean for each chemical
class. Error bars were omitted for clarity (for detailed information see Tables S2 and S3). Gray dotted
line represents the effect of rotenone.
2.2. Metabolism-Related Markers
2.2.1. Acute Rescue of ATP
One of the dominant parameters affected by mitochondrial dysfunction is cellular
ATP synthesis. A mode of action was previously proposed for benzoquinones that relies on
the bypass of complex I to restore mitochondrial electron flow in the presence of rotenone,
which was termed ATP rescue [10
,11
,30]. Compared to non-treated HepG2 cells (100%),
rotenone rapidly reduced cellular ATP levels to 33.6
line). Under these conditions, 54 out of 103 SCQs (52 compounds, p < 0.001; 2 compounds,
p < 0.05; Table S2) significantly rescued ATP levels to levels above those of rotenone-treated
±
11.1% within 1 h (Figure 2a, dotted
cells (Figure 2a). Of the 54 compounds, 12 compounds (39, 40, 61, 64, 67, 68, 70, 71, 83
and 85–87, p < 0.001) significantly rescued ATP levels in the presence of rotenone to >90%
compared to cells not treated with rotenone. Of the 7 chemical classes, compounds with
amino alcohol, amino ester (p < 0.001) or amino acid (p < 0.01) side chains significantly
increased ATP levels when compared to rotenone-treated cells. The highest rescue of ATP
level by compounds with an amino ester side chain was significantly higher than those with
aliphatic, acid, amino acid, aliphatic ester or slight polar (p < 0.001) side chains (Figure 2a
;

Molecules 2021, 26, 1382
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Table S3). A correlation was observed between test compound-induced cytoprotection and
extent of cellular ATP level rescue (R2 = 0.44, Figure 2e).
Figure 2. Effect of SCQs on metabolism-related markers in HepG2 cells. (a) Cellular ATP levels (gray dotted line represents
the effect of rotenone), ( ) extracellular lactate levels of SCQs, ( ) extracellular
b
c
β
-hydroxybutyrate (BHB), and ( ) their
d–f
correlations with SCQ-protected viability. Each point represents the average responses from independent experiments
for one SCQ. Error bars were omitted for clarity (for detailed information see Tables S2 and S3). Linear regression was
generated using GraphPad Prism (version 8.2.1, San Diego, CA, USA).
2.2.2. Extracellular Lactate
Under conditions of mitochondrial dysfunction, NAD+ is required to maintain the
process of glycolysis, which is generated from NADH during the oxidation of pyruvate to
lactate [31]. Quinones are reduced to the hydroquinone form in the presence of cellular
reductases like NAD(P)H:quinone dehydrogenase 1 (NQO1), a process that also oxidizes
NADH to NAD+. This quinone-generated NAD+ could theoretically be used to maintain
glycolysis without the need to generate potentially toxic lactate levels. Alternatively,
SCQs could increase glycolysis further to compensate for reduced mitochondrial ATP
synthesis, which could increase lactate levels. Therefore, the current study tested if SCQs
could affect lactate production of HepG2 cells. In non-treated cells, lactate in cell culture
supernatant was measured as 67.3
compounds altered extracellular lactate levels between 65.1 and 236.6%. Some compounds
with aliphatic (27 29 and 33, p < 0.001; 24, p < 0.01; 13, p < 0.05), amino alcohol (62 66 and
76, p < 0.001; 60, p < 0.01; 72, p < 0.05) or slight polar side chains (51 and 89, p < 0.001; 58
p < 0.05; Table S2) significantly increased lactate levels compared to the non-treated cells.
In contrast, some compounds with aliphatic (26, p < 0.01), acid (39, p < 0.01), amino acid (96
±
8.05 µmol/mg protein (100%; Figure 2b). The test
,
,
,
,
p < 0.01) or slight polar (103, p < 0.05; Table S2) side chains significantly decreased lactate
levels compared to the untreated cells. However, based on the means of the 7 chemical
classes, none significantly altered lactate levels (Figure 2b). No correlation was observed
between cytoprotection and extracellular lactate levels (R2 < 0.1, Figure 2e).
2.2.3. Extracellular β-Hydroxybutyrate
In the absence of carbohydrates, cells also utilize fatty acids to generate ATP via the
β-oxidation of lipids that can lead to the accumulation of ketone bodies [32]. In addition,

Molecules 2021, 26, 1382
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ketone bodies such as
β-hydroxybutyrate (BHB) can initiate bioenergetic and mito-hormetic
signaling pathways by inhibiting histone deacetylases and by reducing mitochondrial
oxidative radical formation [33]. Therefore, the influence SCQs on the accumulation of
BHB in the supernatant of HepG2 cell cultures was measured. In non-treated cells, BHB
levels in cell culture supernatant were measured as 1.75
Figure 2c). In comparison, all test compounds except 33 (1132.1%, Figure 2c, outside y axis)
±
0.41 µmol/mg protein (100%;
with an aliphatic side chain altered cellular BHB levels between 60.2 and 311.8%. Some
compounds with aliphatic (23
64 66 69 71 73 75 and 76, p < 0.001; 35 and 67, p < 0.05), acid (18 and 38, p < 0.001; 39
p < 0.01), amino acid (11 43 and 100, p < 0.001; 93, p < 0.01), amino ester (81 84 and 85
p < 0.001 12 and 80, p < 0.05) or slight polar (51 55
chains significantly increased BHB levels when compared to non-treated cells. In contrast,
some compounds with aliphatic (34, p < 0.01), amino alcohol ( , p < 0.001; , p < 0.01) or
,
24,
32 and 33, p < 0.001), amino alcohol (
2,
14
,
16
,
59,
60
,
,
,
,
,
,
– ,
,
,
;
,
, 56 and 103, p < 0.001; Table S2) side
1
8
aliphatic ester (47, p < 0.05, Table S2) side chains significantly decreased BHB levels when
compared to non-treated cells. Overall, except for compounds with aliphatic or aliphatic
ester side chains, all other 5 classes significantly increased BHB levels in the cell culture
media (Table S3). No correlation was observed between cytoprotection and extracellular
BHB levels (R2 < 0.1, Figure 2f).
2.3. Redox Activity
It was previously reported that benzoquinones are largely (93.9%) reduced from the
quinone to their hydroquinone form by the cytoplasmic enzyme NQO1 [11], while for
naphthoquinones, this information was so far not available. It was generally assumed that
the hydroquinone form is responsible for cytoprotection, antioxidant function and other
beneficial activities. To determine to what extent the reduction of SCQs is NQO1-dependent,
its activity in HepG2 cells was inhibited by dicoumarol as previously described [11]. When
the reduction of SCQs (Figure 3a, Table S2) was compared in the absence and presence of
dicoumarol, significant differences were observed, with NQO1 dependence between 0.2
and 82.1% (Figure 3b, Table S2). Of the 7 chemical classes, the highest NQO1 dependence
for reduction was seen for compounds with an amino alcohol side chain, which was
significantly higher than for those with aliphatic, acid, amino acid or aliphatic ester side
chains (p < 0.001, Figure 3b, Table S3). The highest dependence on other reductases was
detected for compounds with an aliphatic ester side chain and was significantly higher than
those with amino alcohol (p < 0.001), amino ester (p < 0.01) or slight polar (p < 0.05) side
chains (Figure 3c, Tables S2 and S3). No general correlation between cytoprotection and
chemical reduction of test compounds per se was observed (R2 < 0.1, Figure 3d). However, a
mild positive correlation was observed for cytoprotection and NQO1-dependent reduction
of quinones. Conversely, cytoprotection negatively correlated with reduction by non-NQO1
reductases (R2 = 0.23, Figure 3e,f). In addition, a mild positive correlation was also observed
for NQO1-dependent reduction and the acute rescue of cellular ATP levels in the presence
of rotenone. Similar to cytoprotection, reduction by non-NQO1 reductases negatively
correlated with the acute rescue of cellular ATP levels (R2 = 0.39, Figure 3g,h).

Molecules 2021, 26, 1382
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Figure 3. Redox activity of SCQs in HepG2 cells. (
a) Total reduction of quinones (R-Total), (
b) reduction by NQO1 (R-NQO1),
(
c) reduction by other reductases (R-Other) and their correlations with (
d
–f
) SCQ-protected viability and (g,h
) acute rescue
of ATP levels. Each dot represents the average responses from independent experiments for one SCQ. Error bars were
omitted for reasons of clarity (for detailed information see Tables S2 and S3). Linear regressions were generated using
GraphPad Prism (version 8.2.1, San Diego, CA, USA).
2.4. Expression of Cytoprotective Proteins
2.4.1. Cellular Lin28A Expression
Recently, retinal Lin28A protein expression induced by a benzoquinone was reported
to be responsible for the cytoprotective activity observed [34]. This finding was extremely
surprising, as Lin28A is generally not expressed in adult tissues (except for reproductive
tissues), but is known to be involved in differentiation of embryonic tissues. Lin28A
overexpression regulates metabolism by enhancing mitochondrial enzyme production,
glycolysis and mitochondrial OXPHOS to alleviate mitochondrial dysfunction and to
promote tissue repair [35–37]. Since this activity could account for the cytoprotective
effects observed with our test compounds, their effects on Lin28A levels in HepG2 cells
were determined. In our in vitro system, the test compounds only mildly altered Lin28A
levels between 94.7 and 107.0% (Figure 4a). Only 74 (7.0
alcohol side chain and 81 (6.0 3.2%, p < 0.05) with an amino ester side chain significantly
increased Lin28A levels (Table S2). None of the 7 chemical classes significantly increased
±
3.9%, p < 0.01) with an amino
±
Lin28A levels and no statistical significance was observed between the classes (Figure 4a
Table S3). Since only two significant effects were detected, no general correlation was
observed between cytoprotection and Lin28A expression (R2 < 0.1, Figure 4d).
,

Molecules 2021, 26, 1382
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Figure 4. SCQ-induced expression of cytoprotective proteins in HepG2 cells. SCQ-induced (a) Lin28A levels; (b) Hsp70
levels; ( ) acetylated tubulin (acetyl-tubulin) levels, and their ( ) correlations with SCQ-protected viability. Each point
c
d–f
represents the average responses from several independent experiments for one SCQ. Black solid lines represent the mean
for each chemical class. Error bars were omitted for clarity (for detailed information see Tables S2 and S3).
2.4.2. Cellular Hsp70 Expression
Heat shock protein 70 (Hsp70) is essential for mitochondrial function. It chaperones
mitochondrial protein biogenesis, translocates and folds proteins, and prevents their ag-
gregation to maintain mitochondrial proteostasis [38
in mitochondrial DNA (mtDNA) maintenance and replication [42] and protects against
diabetes, which is associated with mitochondrial dysfunction [43 44]. Since some quinones
have been reported to increase Hsp70 levels [45 47], the effects of our test compounds
–41]. Hsp70 also plays critical roles
,
–
on Hsp70 expression in HepG2 cells were assessed. The test compounds altered Hsp70
levels between 92.0 and 167.7% (Figure 4b, Table S2). Some compounds with aliphatic
(
13
and 41, p < 0.001; 39, p < 0.05), aliphatic ester (44, p < 0.05), amino ester (20
84, p < 0.001; 21, p < 0.01) or slight polar (52 54 58 and 103, p < 0.001; 51, p < 0.05) side
,
27 and 30, p < 0.001), amino alcohol (74, p < 0.01; 66 and 76, p < 0.05), acid (17
, 37
,
79, 83 and
,
,
chains showed a significant upregulation of Hsp70, while none of the compounds led to a
significant downregulation of Hsp70 (Table S2). Of the 7 chemical classes, only compounds
with a slight polar side chain (130.0
±
23.7%, p < 0.001) significantly upregulated Hsp70
expression (Figure 4b, Table S3). Comparisons between the chemical classes revealed
that compounds with a slightly polar side chain also showed significantly higher Hsp70
induction levels compared to those with amino alcohol (p < 0.01) or amino acid (p < 0.05)
side chains (Figure 4b, Table S3). However, no general correlation between cytoprotection
and Hsp70 expression was detected (R2 < 0.1, Figure 4e).
2.4.3. Tubulin Acetylation
There is significant evidence that inhibition of histone deacetylase 6 (HDAC6) protects
against mitochondrial dysfunction by increasing mitochondrial biogenesis [48], stabilizing
the cytoskeleton [49], regulating mitochondrial homeostasis [50], increasing oxidative
metabolism [51], restoring mitochondrial transport [52], and increasing mitochondrial
motility and fusion [53] to sustain cell viability in vitro and in vivo. Previous reports
indicated that some short-chain naphthoquinones can inhibit HDAC6 [54]. As HDAC6
inhibition increases tubulin acetylation [55], acetylated tubulin was used as a surrogate

Molecules 2021, 26, 1382
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marker to detect HDAC6 inhibition in HepG2 cells by our SCQs. The test compounds
altered tubulin acetylation between 84.8 and 134.5% (Figure 4c, Table S2). Some compounds
with aliphatic (23
p < 0.05), acid (37 and 40, p < 0.001), amino acid (19, p < 0.001), aliphatic ester (47, p < 0.001;
44 45 and 48, p < 0.01; 46, p < 0.05), amino ester (20 and 83, p < 0.001; 79, p < 0.01; 84 and
85, p < 0.05) or slight polar (51 53 54 and 57, p < 0.001) side chains significantly increased
tubulin acetylation, while only 74 (86.2 7.8%, p < 0.05) with an amino alcohol side
, 24, 27 and 34, p < 0.001; 25 and 33, p < 0.01), amino alcohol (70, p < 0.01; 61,
,
,
,
±
chain significantly reduced tubulin acetylation (Table S2). None of the 7 chemical classes
significantly increased tubulin acetylation and no statistical significance was observed
between the classes (Figure 4c, Table S3). No general correlation was observed between
cytoprotection and tubulin acetylation levels (R2 < 0.1, Figure 4f).
2.5. Effects on Oxidative Damage
2.5.1. Basal Lipid Peroxidation
The mild correlation between cytoprotection and chemical reduction could suggest
that cytoprotection by SCQs against mitochondrial dysfunction involves the redox char-
acteristics of the quinones [10,11]. One mechanistic explanation could be a hormetic
form of protection where SCQs induce a sublethal level of damage that induces cyto-
protective and mitoprotective pathways. This effect, termed mito-hormesis, can involve
the production of oxidative radicals and has been demonstrated in a variety of systems
in vitro and in vivo
characterized as antioxidants and consequently have been reported to prevent lipid peroxi-
dation [58 60], while some reports revealed that SCQs can act as pro-oxidants at higher
concentrations [61 62]. Unlike reported for a range of benzoquinones [10], none of the naph-
thoquinone test compounds in the current study showed any significant changes of basal
[56,57]. Most SCQs that are developed as potential therapeutics are
–
,
lipid peroxidation (BLP) in HepG2 cells when compared to non-treated cells (Figure 5a
Tables S2 and S3). Consequently, no correlation was observed between cytoprotection and
BLP levels (R2 < 0.1, Figure 5d).
,
Figure 5. Effect of SCQs on oxidative damage in HepG2 cells. (a) basal lipid peroxidation, (b) nitrotyrosine levels, (c)
γ
-H2AX-positive cells, and their ( ) correlations with SCQ-protected viability. Each point represents the average responses
d–f
from several independent experiments for one SCQ. Black solid lines represent the mean for each chemical class. Error bars
were omitted for clarity (for detailed information see Tables S2 and S3). Gray dotted lines represent effect on non-treated cells.

Molecules 2021, 26, 1382
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2.5.2. Oxidative Protein Damage
Given that the redox activity of some quinones can include pro-oxidative behaviour [61
,62],
we tested our SCQs for their effects on cellular nitrotyrosine levels as a surrogate marker
for oxidative protein damage in HepG2 cells. In our system, the test compounds altered
oxidized protein levels (100%) between 69.3 and 121.1% (Table S2). Some compounds with
aliphatic (26 and 101, p < 0.001), amino alcohol (36 and 63, p < 0.001; 59 and 64, p < 0.01;
1
and 35, p < 0.05), acid (18 and 39, p < 0.001; 37, p < 0.01), amino acid (11
,
90
,
96 and 100
,
p < 0.001; 92 93 95 and 97, p < 0.01; 94 and 98, p < 0.05), aliphatic ester (47, p < 0.001; 44
,
,
,
p < 0.01), amino ester (50, p < 0.01) or slight polar (57, p < 0.01; 52, p < 0.05) side chains
significantly reduced oxidative damage, while only 58 and 103 (p < 0.05) with slight polar
side chains significantly increased nitrotyrosine levels (Table S2). Of the 7 chemical classes,
only compounds with an amino acid side chain significantly lowered nitrotyrosine levels
(79.6
±
6.2%, p < 0.05), while no statistically significant differences were observed between
the 7 chemical classes (Figure 5b, Table S3). No general correlation between cytoprotection
and oxidative protein damage was detected (R2 < 0.1, Figure 5e).
2.5.3. Oxidative DNA Damage
Since the redox activity of some quinones can include pro-oxidative behaviour [61
we tested our SCQs for their effects on cellular -H2AX levels as a surrogate marker
for DNA damage in HepG2 cells. In our system, test compounds mostly altered the
number of -H2AX-positive cells (1.1 0.8% for non-treated cells, Figure 5c, gray dotted
line) between 0.2 and 7.3% (Table S2), except for 103 with a slightly polar side chain
46.0 ± 10.8% p < 0.001). Of the 103 test compounds, only 43 (6.0 1.9%, p < 0.05) with an
amino acid side chain as well as the epoxide alkylating agents 54 (7.3 3.4%, p < 0.01) and
103 with a slightly polar side chain, significantly increased the number of
cells (Table S2). Overall, compounds with a slight polar side chain (9.2
,62],
γ
γ
±
(
,
±
±
γ
-H2AX-positive
±
15.0%, p < 0.05,
Figure 5c, average line and 103 outside y axis) significantly elevated the percentage of
positive cells on average, while all other chemical classes appeared non-genotoxic (Table
S3). No general correlation between cytoprotection and oxidative DNA damage was
detected (R2 < 0.1, Figure 5f).
2.6. Heatmap of Results
All in vitro bioactivities (Figures 1–5, Table S2) and physical properties (Figure S1,
Table S2) of the test compounds 1–103 are summarized as a heatmap (Figure 6).

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Figure 6. Heatmap of results shown in Figures 1–5 and Figure S1. Each column represents one
parameter and each line represents one short-chain quinone (SCQ). For some compounds, not all
parameters could be assessed (gray boxes). Data expressed as the mean of multiple independent
experiments.
treated, 26.9%);
logP, partition coefficient; logD, distribution coefficient; BHB,
∆
Cytoprotection (%) = SCQ-protected viability (%)—basal viability level (rotenone-
ATP (%) = SCQ-protected ATP level (%)—basal ATP level (rotenone-treated, 33.6%);
-hydroxybutyrate; BLP, basal lipid
∆
β
peroxidation; R-Total, total reduction of quinone; R-NQO1, reduction of quinone by NQO1; R-Other,
reduction by other reductases; γ-H2AX, γ-H2AX-positive cells. Raw data available in Table S2.

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3. Discussion
This study aimed to characterize the in vitro bioactivities of a library of 103 recently
described short-chain naphthoquinones (SCQs) [22 24 26] to investigate the underlying
,
,
mechanism(s) of SCQ-dependent cytoprotection. For this purpose, the current study
employed not only the previously reported endpoints of cytoprotection and normalization
of cellular ATP levels [26], but also assessed additional bioactivities of quinones that are
indicative of mitochondrial function, compound bioactivation, expression or activities of
cytoprotective proteins (Lin28A, Hsp70, HDAC6), oxidative damage and DNA damage.
The results of these endpoint measurements were used to reveal potential correlations
with SCQ-induced cytoprotection. While correlations obtained in vitro do not necessarily
allow a direct translation to the in vivo situation, the current study aimed to provide a first
unbiased insight into the molecular activities of SCQs by utilizing a larger number of test
compounds.
A prior study, based on a small numbers of SCQs, suggested that a combination
of a naphthoquinone core with selected functional groups attached to a side chain, was
responsible for the cytoprotective activity of SCQs [26]. The previous study also concluded
that the lipophilic tail moiety is the major determinant of cytoprotection [26], which was
confirmed by the current study that observed a large range of cytoprotective activities
associated with different side chain chemistries. While most compounds showed some
level of cytoprotection against mitochondrial dysfunction, of particular interest were the
compounds with an amino ester side chain, all of which significantly protected cellular
viability against rotenone with the highest average cytoprotection of all side-chain classes
tested. Based on the results of the current study, the structure-activity relationship (SAR)
with the most active compounds supports the function of the side chain playing a key
role in the activity of the short chain naphthoquinones of this study, which confirms the
results of a prior study [26]. The incorporation of an amide into the side chain significantly
increased the cytoprotective activity compared to the presence of a polar carboxylic acid or
less polar ester linkages. However, at present the exact role of the amide function is not
clear. Future studies have to establish if the amide function just changes the polarity of the
molecule or if it is directly involved in target binding.
Overall, it is surprising that most endpoints assessed in this study did not correlate
with the cytoprotective activity of the test compounds. This suggests that in our test
system most endpoints previously attributed to mitoprotection (i.e., extracellular lactate,
β-hydroxybutyrate, expression of Lin28A or Hsp70, HDAC6 inhibition, basal lipid peroxi-
dation, oxidative protein or DNA damage) are not responsible for the cytoprotection against
rotenone and that other underlying mechanisms are responsible for the cytoprotective
effects. While we acknowledge that our results cannot exclude tissue-specific bioactivities,
such as neuroprotection, the enzymatic reduction of SCQs and the acute redox-dependent
rescue of ATP levels mildly correlated with cytoprotection in the present study. SCQs
are believed to be bioactivated by two-electron reductases such as NQO1 to the hydro-
quinone form upon entering the cell [26]. Despite their reduction in the cytosol, some
hydroquinones can donate electrons to the mitochondrial electron transport chain (ETC) to
restore proton flux, membrane potential and ATP production under conditions of complex
I-deficiency [10,11]. This is achieved by SCQs bypassing the dysfunctional complex I
and feeding electrons to complex III of the ETC (Figure 7). Therefore, both cytosolic and
mitochondrial activities are required for the acute rescue of ATP levels in vitro. Based on
the common quinone core of our test compounds, it was expected that all test compounds
are reduced by NQO1 to a similar extent. However, our test compounds exhibited variable
levels of reduction by NQO1. This indicates that our SCQs are bioactivated not only by
NQO1, but also other reductases that could include vitamin K epoxide reductase (VKOR),
as predicted by the shared naphthoquinone moiety with vitamin K [63,64]. Surprisingly,
SCQ reduction by NQO1 was positively correlated with cytoprotection, while reduction
by other reductases was negatively correlated with SCQ-induced cytoprotection. This
was entirely unexpected, as it should not matter how SCQs are bioactivated or where the

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electrons for their reduction originate from, since only the hydroquinone form was so far
thought to be responsible for the protective effects. Our data significantly question this
view and suggest a different interpretation that it is not the reduced SCQs themselves that
are responsible for the cytoprotective activity but instead their effect on NQO1 and its
substrates.
Figure 7. Schematic representation of ligand-induced structural change of NQO1 and hypothetical
mechanism for SCQ-induced cytoprotection. Q, short-chain quinone; NQO1, NAD(P)H:quinone
oxidoreductase 1; QH2, short-chain hydroquinone.
One possible explanation could be that SCQs, by activating NQO1, alter the levels
of NQO substrates such as cytoplasmic NADPH, which is subsequently responsible for a
cytoprotective activity. For another quinone, dunnione, this mode of action was proposed
to be responsible to ameliorate acute pancreatitis on the basis that lower levels of NADPH
would result in reduced NADPH oxidase (NOX)-dependent ROS production and reduced
tissue damage [65]. Although we did not test cytoplasmic NADPH/NADP+ ratios in our
cells, it has to be noted that cytoplasmic NADPH is essential for a variety of essential cellular
function such as nucleotide synthesis and reactivation of glutathione [66,67]. Therefore,
reduced NADPH levels should under physiological conditions lower the concentrations
of the most important cellular antioxidant, and consequently increase oxidative stress.
In contrast, we did not find any evidence that oxidative damage is associated with the
cytoprotective activity of the test compounds. In addition, it is not easily conceivable how
reduced cytoplasmic NADPH levels could protect against mitochondrial rotenone toxicity.
Therefore, this possibility might apply to certain pathological conditions, while it does not
seem to be valid for the test system of this study.
Alternatively, SCQs could affect cellular NADH levels by their interaction with NQO1.
Their NQO1-dependent reduction could increase cellular NAD+ levels. A previous study
observed the activation of the NAD+-dependent deacetylase sirtuin 2 (Sirt2) in response
to the NQO1 substrate β-lapachone [68]. Although Sirt2 suppresses inflammation [69],

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stimulate the pentose phosphate pathway [70], it is also associated with neurodegenerative
and metabolic diseases and cancer [71]. In this report, Sirt2-dependent deacetylation of
microtubules was dependent on NQO1-generated NAD+ [68]. Based on our observation
that SCQ-induced tubulin acetylation did not correlate with their cytoprotective effects
and that overall only minor effects on tubulin acetylation were observed, it is unlikely that
the SCQs in the present study affected Sirt2 activity and therefore the NADH/NAD+ ratio.
It is also unclear how increased NAD+ levels, if present at all, could protect cells against
rotenone toxicity, without at the same time upregulating lactate levels.
Another explanation why selective reduction of SCQs by NQO1 correlated with
their cytoprotective activity could involve a direct modification of NQO1 activities by
SCQs. While the current study did not assess the effect of SCQs on NQO1 enzymatic
activity, it is important to note that inhibition of NQO1 activity has never been reported
as cytoprotective. In contrast, NQO1 inhibition increased the sensitivity of cells to a host
of stressors [72] and hence it is unclear how a possible inhibition of NQO1 activity per
se, could be responsible to protect against rotenone exposure [73,74]. Therefore, our data
suggests that SCQs might either activate NQO1 or provide it with additional functionality
(Figure 7). NQO1 is a cytoprotective enzyme that not only detoxifies xenobiotics and
displays endogenous superoxide dismutase activity but is also involved in a multitude of
cellular signaling events. By acting as proteasomal gatekeeper [75], NQO1 controls protein
levels of key signaling molecules such as the tumor suppressor p53 [76], and HIF1 [77]. It
is possible that binding of selected SCQs to NQO1 could alter the 3D structure of NQO1,
which was described for another NQO1 substrate β-lapachone [68]. The authors of this
study proposed that NQO1 acts as a sensor of the cellular redox environment by binding to
specific substrates based on an altered protein structure. In this study, NQO1 co-localized
with acetylated tubulin during mitosis and it was suggested that this activity would
mediate cytoprotection by modifying acetylated tubulin dynamics [68]. Although the
present study did not observe major changes to tubulin acetylation, we did not investigate
local changes such as on the mitotic spindle. Altered NQO1 structure and activity based
upon ligand binding provides a completely novel view on how SCQs could modify cellular
signaling by activating NQO1 and controlling its binding to selective proteins to mediate
cytoprotection. Similar to the binding of selected proteins, NQO1 also selectively binds to
certain mRNAs, a function that for example increases the translation of α1-antitrypsin [78].
Therefore, future studies will investigate if exposure to the cytoprotective SCQs described
in this study can mediate the pleiotropic effects of NQO1 by affecting NQO1 binding to
proteins or RNAs. We previously reported that the redox activity of the quinone moiety
is required but not sufficient for cytoprotection by SCQs [26]. Instead, the cytoprotective
activity was localized to the structure of the side chain [26]. Together with the results of the
present study, this could suggest that it is the SCQ side chain that generates selectivity for
NQO1 to alter its interaction with RNA or protein molecules. This hypothesis will have to
be verified in future studies in molecular detail using additional test compounds.
Our results also confirm previous reports that quinone-dependent rescue of ATP levels
is dependent on NQO1 [10,11]. Most SCQs that rescued ATP levels were cytoprotective,
and consistently, most that left ATP levels unaffected or even decreased them further were
not. It is important to note that this does not necessarily imply that rescuing ATP levels
is cytoprotective per se. Instead, it is more likely that the rescue of ATP levels is merely a
reflection of the ability of quinones to interact with NQO1 and to get reduced. The reduced
hydroquinones subsequently bypass complex I by shuttling electrons to complex III of the
mitochondrial ETC, which enables ATP production in the presence of rotenone [11].
Collectively, this study attempted a pharmacological approach to characterize chemi-
cally closely related SCQs and identified several promising SCQs with interesting in vitro
bioactivities. This study aimed to identify a possible mode of action (or lack thereof)
by correlating different bioactivities with the corresponding cytoprotective effects for
each compound and compound chemical class. The results of the present study question
whether SCQ-induced cytoprotection is caused by previously suggested bioactivities such

Molecules 2021, 26, 1382
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as their antioxidant function. Instead, the results suggest that the mode of action of SCQs
could involve a so far unidentified direct modification of NQO1-dependent signaling. The
detailed mode of action of SCQs will require confirmation in the presence of rotenone
in combination with selected SCQs in future experiments. The current results not only
serve as a starting point to elucidate this NQO1-dependent form of cytoprotection, but
if confirmed, also enables the future optimization of mitoprotective SCQs. Based on the
current status, understanding this mode of action, in combination with detailed in vivo
pharmacokinetic and efficacy studies will identify the most mitoprotective, stable and
safe candidates that could be developed for a large range of mitochondrial diseases and
disorders associated with mitochondrial dysfunction.
4. Materials and Methods
4.1. Chemicals and Reagents
All SCQ test compounds were synthesized in-house (Chemistry, School of Natural
Sciences, University of Tasmania, Hobart, TAS, Australia) with purities > 95% determined by
NMR analysis. Dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle Medium (DMEM),
sodium bicarbonate, glutamic pyruvic transaminase, monopotassium phosphate (KH2PO4),
phenazine methosulphate (PMS), dichlorophenolindophenol (DCPIP), nicotinamide adenine
dinucleotide (NAD+), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), mena-
dione, celastrol, shikonin, tubastatin, paraformaldehyde (PFA), Tween 20, rat tail collagen,
and bovine serum albumin (BSA), rabbit polyclonal anti-Lin28A antibody (SAB2702125),
and mouse monoclonal anti-acetyl-tubulin antibody (T7451) were purchased from Sigma-
Aldrich (Ryde, NSW, Australia). Fetal bovine serum (FBS), penicillin-streptomycin, ethylene-
diaminetetraacetic acid (EDTA), trypsin, phosphate buffered saline (PBS), Hanks Balanced
Salt Solution (HBSS), BODIPY C11581/591, Triton X-100, 40,6-diamidino-2-phenylindole
(DAPI), goat anti-mouse Alexa Fluor 594 secondary antibody (A-11072), and goat anti-
mouse Alexa Fluor 488 secondary antibody (A-11029) were obtained from ThermoFisher
Scientific (Scoresby, VIC, Australia). D-luciferin and luciferase were obtained from Promega
(Alexandria, NSW, Australia). DC Protein Assay Kit was purchased from BioRad Labo-
ratories (Gladesville, NSW, Australia). Water-soluble tetrazolium salt (WST-1) was from
Cayman Chemical (Redfern, NSW, Australia). Lactate dehydrogenase was from Cell Sci-
ences (Newburyport, MA, USA). Rabbit monoclonal anti-Hsp70 antibody (EP1007Y) and
mouse monoclonal anti-3-nitrotyrosine antibody (ab61392) were from Abcam (Melbourne,
VIC, Australia). Mouse monoclonal anti-phospho-Histone H2AX antibody (05-636-I) was
from Merck (Kilsyth, VIC, Australia). Cell culture plastics were obtained from Corning
(Mulgrave, VIC, Australia), if not stated otherwise.
For all assays, stock solutions of test compounds (SCQs) and reference compounds
◦
(10 mM in DMSO) were prepared as single use aliquots and stored at
−
20 C until used. All
test compounds were used at a final concentration of 10 µM, as previously published [10,79].
4.2. Cell Culture
The human hepatocellular carcinoma cell line HepG2 (HB-8065) was obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultivated
in DMEM (10% FBS, 1 g/L glucose, 3.7 g/mL sodium bicarbonate, 100 U/mL penicillin-
streptomycin, and 0.584 g/L glutamine) under standard conditions (37 ◦C, 5% CO2, 95%
humidity). The cells were routinely passaged twice weekly in T25 cell culture flasks.
4.3. Cytoprotection against Mitochondrial Dysfunction
Cytoprotection against mitochondrial dysfunction was measured as previously de-
scribed [80]. Briefly, 5
with test compounds (10
chondrial complex I inhibitor rotenone (1
×
103 cells/well were preincubated in DMEM, in 96-well plates
µM in DMEM) for 2 days prior to being challenged by the mito-
µM in HBSS, 7 h). After post-incubation with
only test compounds (10 µM in HBSS) for an additional 24 h, cell viability was quantified
by analyzing ATP content per well using a luciferase-based reaction as previously de-

Molecules 2021, 26, 1382
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scribed [80]. Data was standardized on the non-treated control (no rotenone) and expressed
as mean ± standard deviation (SD) of 6 replicates from 3 independent experiments.
4.4. Acute Rescue of ATP Levels
Acute rescue of ATP levels in the presence of rotenone was measured as previously
described [10]. Briefly, 1.5
24 h, cells were simultaneously incubated with test compounds (10
(10 M) or rotenone alone for 1 h in glucose-free DMEM before ATP levels were measured
using a luciferase-based reaction as previously described [10]. Data was standardized on
×
104 cells/well were seeded in 96-well plates in DMEM. After
µM) and rotenone
µ
the non-treated control (no rotenone) and expressed as mean
6 replicates from 3 independent experiments.
±
standard deviation (SD) of
4.5. Extracellular Lactate
To quantify the effects of the test compounds on extracellular lactate levels, cells were
seeded at a density of 1
×
105 cells/well in 6-well plates in DMEM. After 24 h, media
was replaced with challenge media (DMEM supplemented with 25 mM glucose, 8 mM
L-glutamine, 100 U/mL penicillin-streptomycin and 1 mM pyruvate). After 48 h, cell
culture media was collected for lactate measurement and the remaining cells were lysed at
room temperature (RT) in lysis buffer (0.5% Triton X-100/PBS) for protein measurement
using the DC Protein Assay as recommended by the manufacturer. Lactate was measured
as previously described [11]. Briefly, 10
90
µL of collected cell culture media was added to
µ
L of reaction buffer (10 mM KH2PO4, pH 7.8, 1 mg/mL BSA, 0.5 mM PMS, 2 mM
EDTA, 0.3 mM DCPIP, 0.08 mM NAD+, 0.5 U/mL glutamic pyruvic transaminase, 1.5 mM
glutamate, 1.25 U/mL lactate dehydrogenase) in transparent 96-well plates including
lactate standards from 3 to 23 mM. Absorbance at 600 nm was measured using a plate
reader (Multiskan Go, ThermoFisher Scientific, Scoresby, VIC, Australia) every 2 min over
◦
a period of 100 min at 30 C. Data was standardized on protein levels and was expressed as
percentage Lactate/Protein compared to non-treated cells. Data represents the mean ± SD
of 6 replicates from 3 independent experiments.
4.6. Extracellular β-Hydroxybutyrate
Effect of test compounds on
was assessed by seeding 1
104 cells/well in 96-well plates in DMEM. After 24 h, media
was replaced with DMEM containing test compounds. After 72 h, supernatant was col-
lected. To 50
L of reaction mixture (containing 0.5 mM PMS, 2.5 mM NAD+, 0.00625 U
BHB-dehydrogenase, 10 M DCPIP in 100 mM Tris-HCl buffer), 50 L of cell supernatant
β-hydroxybutyrate (BHB) in the cell culture supernatant
×
µ
µ
µ
was added to initiate the reaction. Absorbance at 600 nm was measured every 30 s for
10 min at RT using a plate reader (Multiskan Go., ThermoFisher Scientific, Scoresby, VIC,
Australia). A BHB standard curve with BHB concentrations from 0.15 to 5 mM was used to
calculate BHB levels in the supernatant of test compounds-treated cells. Data represents
the mean ± SD of 6 replicates from 3 independent experiments.
4.7. Reduction of Test Compounds
To measure cellular reduction of test compounds in vitro, 1
seeded in 96-well plates in DMEM. After 6 h, media was replaced with DMEM containing
0.3 g/L glucose and 2% FBS and incubated for 18 h. Subsequently, media was replaced with
×
104 cells/well were
DMEM with/without test compounds with/without dicoumarol (10
µM) and incubated
for 1 h. Finally, media was replaced with HBSS with/without test compounds containing a
water-soluble, cell-impermeable redox tetrazolium dye (WST-1, 450
at 450 nm was measured every 2 min for 2 h at 37 C using a plate reader (Multiskan
µ
M) and absorbance
◦
Go, ThermoFisher Scientific, Scoresby, VIC, Australia) as previously described [81,82].
Total reduction of test compounds = maximum absorbance in the absence of dicoumarol.
Reduction by NQO1 (%) = maximum absorbance in the presence of dicoumarol divided by
that in the absence of dicoumarol,
×
100%. Reduction by other reductases (%) = 100%
−

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Reduction by NQO1 (%). Data represents the mean
experiments.
±
SD of 6 replicates from 3 independent
4.8. Cellular Lin28A Expression
To measure cellular Lin28A protein levels, 5
×
103 cells/well were seeded in 100
Clear, Greiner, Ryde, NSW, Australia)
pre-coated with rat tail collagen (1:20 in HBSS, pH 7.4, 50 L, 45 min) and left to adhere
overnight. Subsequently, cells were treated with test compounds (in HBSS, 100 L) for
24 h. After fixation (4% PFA/PBS, 50 L, 10 min), permeabilization (0.5% Triton X-100/PBS,
50 L, 10 min) and blocking (5% FBS + 5% BSA in PBS, 50 L, 1 h), cells were exposed to
rabbit polyclonal anti-Lin28A antibody (1:500 in blocking buffer, overnight). After washing
with PBST (0.1% Tween-20/PBS, 50 L, 5 min, 3 ), cells were exposed to goat anti-rabbit
Alexa Fluor 594 secondary antibody (1:10,000 in PBST, 15 L, 1 h) in the dark. After another
washing with PBST, nuclei were counter-stained with DAPI (1:10,000 in PBST, 15 L,
2 min; Figure S2). After 3 washing with PBST, cells were stored in 50 L PBS for high con-
tent imaging using an INCell 2200 analyzer (10 magnification, GE Healthcare, Rydalmere,
µL
serum-free DMEM in 384-well plates (781091,
µ
µ
µ
µ
µ
µ
µ
×
µ
3
×
µ
×
µ
×
NSW, Australia). Average cellular Lin28A intensity was automatically quantified on each
acquired image using IN Carta image analysis software (GE Healthcare, Rydalmere, NSW,
Australia). Data was standardized on the non-treated control (100%) and expressed as
mean
±
SD of at least quadruplicates from one assay. At least 1
×
103 cells were analyzed
separately per treatment.
4.9. Cellular Hsp70 Expression
To measure cellular Hsp70 protein levels, cells were seeded, treated, fixed, and per-
meabilized as described under 4.6. Celastrol [83] and shikonin [84] were used as positive
control compounds. After blocking, cells were exposed to rabbit monoclonal anti-Hsp70
antibody (1:1000 in blocking buffer, 15
Alexa Fluor 594 secondary antibody (1:10,000, 15
and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10
magnification) and analyzed using IN Carta image analysis software as described above
(GE Healthcare, Rydalmere, NSW, Australia). Average cellular Hsp70 intensity was auto-
matically quantified for each acquired image. Data were standardized on the non-treated
µ
L, overnight). After exposure to goat anti-rabbit
µL, 1 h), cells were stained using DAPI
×
control (100%) and expressed as mean
±
SD of at least quadruplicates from one assay. At
least 1 × 103 cells were analyzed for each treatment.
4.10. Quantification of Acetylated Tubulin
To measure cellular acetyl-tubulin levels, cells were seeded, treated, fixed, and perme-
abilized as described above. Tubastatin was used as a positive control [85]. After blocking,
cells were exposed to mouse monoclonal anti-acetyl-tubulin antibody (1:1000 in blocking
buffer, 15
antibody (1:10,000, 15
images were aquired using an INCell 2200 analyzer (10
using IN Carta image analysis software as described above (GE Healthcare, Rydalmere,
NSW, Australia). Average cellular acetyl-tubulin intensity was automatically quantified for
each acquired images. Data were standardized over the non-treated control (100%) and
µ
L, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary
L, 1 h), cells were stained using DAPI and stored in PBS (Figure S2),
magnification) and analyzed
µ
×
expressed as mean
were analyzed for each treatment.
±
SD of at least quadruplicates from one assay. At least 1
×
103 cells
4.11. Basal Lipid Peroxidation
To assess the effects of the test compounds on basal levels of lipid peroxidation,
2 × 104 cells/well were seeded in black 96-well plates in DMEM. After 24 h, the media was
removed, and cells were loaded with 10 µM dye solution (1% BODIPY C11581/591 in 100 µL
HBSS per well) for 30 min. Subsequently, the dye solution was replaced with 100
µL HBSS
with/without test compounds and incubated for 1 h. After the cells were washed 3
×
with

Molecules 2021, 26, 1382
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PBS, fluorescence was measured (Ex/Em 490/520 and Ex/Em 490/600 in 50
µL PBS) using
a plate reader (Fluoroskan Ascent, ThermoFisher Scientific, Scoresby, VIC, Australia) [10].
Fluorescence ratios were calculated and presented as percentage of non-treated control
cells. Data represents the mean ± SD of 6 replicates from 3 independent experiments.
4.12. Quantification of Oxidative Protein Damage
To assess if the test compounds induce oxidative damage, cells were seeded, treated,
fixed, and permeabilized as described above. Shikonin was used as a positive control [86].
After blocking, cells were exposed to mouse monoclonal anti-3-nitrotyrosine antibody
(1:500 in blocking buffer, 15
488 secondary antibody (1:10,000, 15
PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10
and analyzed using IN Carta image analysis software as described above (GE Healthcare,
Rydalmere, NSW, Australia). Average cellular nitrotyrosine intensity was automatically
quantified for each acquired images. Data were standardized over the non-treated control
µ
L, overnight). After exposure to goat anti-mouse Alexa Fluor
L, 1 h), cells were stained using DAPI and stored in
magnification)
µ
×
(100%) and expressed as mean
cells were analyzed for each treatment.
±
SD of at least 8 replicates from one assay. At least 2
×
103
4.13. Quantification of Oxidative DNA Damage
To assess if the test compounds induce some level of genotoxicity, cells were seeded
as previously described and treated with 10 M test compounds. Since -H2AX signal can
µ
γ
decrease over time due to DNA repair, this assay captured SCQ-induced DNA damage
after a short treatment period of 4 h. Celastrol was used as a positive control [87]. After
fixation, permeabilization and blocking, cells were exposed to mouse monoclonal anti-
phospho-Histone H2AX antibody (1:1000 in blocking buffer, 15
exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15
cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using
an INCell 2200 analyzer (10 magnification) and analyzed using IN Carta image analysis
software as described above (GE Healthcare, Rydalmere, NSW, Australia). The number of
µL, overnight). After
µL, 1 h),
×
γ
-H2AX-positive cells was automatically quantified for all acquired images. Percentage
-H2AX-positive cells was expressed as mean SD of at least quadruplicates from one
γ
±
assay. At least 500 cells were analyzed for each treatment.
4.14. Statistical Analysis
One- or two-way ANOVA followed by Dunnett’s multiple comparison post-test was
performed using GraphPad Prism (version 8.2.1, San Diego, CA, USA) to compare test
compounds and control(s) or between chemical classes: *** p < 0.001, ** p < 0.01, * p < 0.05,
otherwise non-significant (Tables S2 and S3).
Supplementary Materials: The following are available online. Figure S1: Physical properties of
short-chain quinone (SCQ) test compounds, Figure S2: Exemplary images of stained HepG2 cells,
Table S1: Synthesis of novel SCQs, Table S2: Bioactivity profiles and physical properties of SCQs,
Table S3: Bioactivity profiles and physical properties of SCQs belonging to different chemical classes.
Author Contributions: Conceptualization, investigation, project administration and funding acquisi-
tion, J.A.S. and N.G.; writing, Z.F., J.A.S. and N.G.; methodology and validation, Z.F., M.N. and N.G.;
data curation, Z.F., M.N., A.L.H. and S.C.; software, formal analysis and visualization, Z.F.; resources,
K.L.W., J.A.S. and N.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Data is available in Tables S1–S3.
Acknowledgments: Z.F. and M.N. are thankful to the University of Tasmania for receiving Tasmanian
Graduate Research Scholarships.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2021, 26, 1382
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