ENOblock does not inhibit the activity of the glycolytic enzyme enolase

Article abstract of DOI:10.1371/journal.pone.0168739

Inhibition of glycolysis is of great potential for the treatment of cancer. However, inhibitors of glycolytic enzymes with favorable pharmacological profiles have not been forthcoming. Due to the nature of their active sites, most high-affinity transition-state analogue inhibitors of glycolysis enzymes are highly polar with poor cell permeability. A recent publication reported a novel, non-active site inhibitor of the glycolytic enzyme Enolase, termed ENOblock (N-[2-[2- 2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]amino] 1,3,5-triazin-2-yl]amino]benzeneacetamide). This would present a major advance, as this is heterocyclic and fully cell permeable molecule. Here, we present evidence that ENOblock does not inhibit Enolase enzymatic activity in vitro as measured by three different assays, including a novel ³¹P NMR based method which avoids complications associated with optical interferences in the UV range. Indeed, we note that due to strong UV absorbance, ENOblock interferes with the direct spectrophotometric detection of the product of Enolase, phosphoenolpyruvate. Unlike established Enolase inhibitors, ENOblock does not show selective toxicity to ENO1-deleted glioma cells in culture. While our data do not dispute the biological effects previously attributed to ENOblock, they indicate that such effects must be caused by mechanisms other than direct inhibition of Enolase enzymatic activity.

Full text of DOI:10.1371/journal.pone.0168739

RESEARCH ARTICLE
ENOblock Does Not Inhibit the Activity of the
Glycolytic Enzyme Enolase
Nikunj Satani1,2, Yu-Hsi Lin2, Naima Hammoudi2, Sudhir Raghavan2, Dimitra K. Georgiou2,
Florian L. Muller2*
1 Department of Neurology, McGovern Medical School, UTHealth, Houston, TX, United States of America,
2 Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX,
United States of America
* fmuller@mdanderson.org
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Abstract
Inhibition of glycolysis is of great potential for the treatment of cancer. However, inhibitors of
glycolytic enzymes with favorable pharmacological profiles have not been forthcoming. Due
to the nature of their active sites, most high-affinity transition-state analogue inhibitors of gly-
colysis enzymes are highly polar with poor cell permeability. A recent publication reported a
novel, non-active site inhibitor of the glycolytic enzyme Enolase, termed ENOblock (N-[2-[2-
2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]amino]
1,3,5-triazin-2-yl]amino]benzeneacetamide). This would present a major advance, as this is
heterocyclic and fully cell permeable molecule. Here, we present evidence that ENOblock
does not inhibit Enolase enzymatic activity in vitro as measured by three different assays,
including a novel 31P NMR based method which avoids complications associated with opti-
cal interferences in the UV range. Indeed, we note that due to strong UV absorbance, ENO-
block interferes with the direct spectrophotometric detection of the product of Enolase,
phosphoenolpyruvate. Unlike established Enolase inhibitors, ENOblock does not show
selective toxicity to ENO1-deleted glioma cells in culture. While our data do not dispute the
biological effects previously attributed to ENOblock, they indicate that such effects must be
caused by mechanisms other than direct inhibition of Enolase enzymatic activity.
OPEN ACCESS
Citation: Satani N, Lin Y-H, Hammoudi N,
Raghavan S, Georgiou DK, Muller FL (2016)
ENOblock Does Not Inhibit the Activity of the
Glycolytic Enzyme Enolase. PLoS ONE 11(12):
e0168739. doi:10.1371/journal.pone.0168739
Editor: Pankaj K. Singh, University of Nebraska
Medical Center, UNITED STATES
Received: July 31, 2016
Accepted: November 20, 2016
Published: December 28, 2016
Copyright: © 2016 Satani et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. The raw data are stored electronically on the
institutional computer of the PI, using a cloud
based backup.
Introduction
The inhibition of glycolytic/gluconeogenic enzymes is of interest in diverse area of medicine,
including treatment of diabetes [1], the treatment of cancer [2] as well as the development of
novel antimicrobials [3]. While high potency, transition state analogue active-site inhibitors of
Enolase, and several other glycolytic enzymes have been described [4, 5], their utility is limited
by poor cell permeability and otherwise poor pharmacological properties [6]. A recent report
by Jung et al. described an altogether different glycolysis inhibitor: a heterocyclic, cell perme-
able inhibitor of Enolase that apparently binds outside the active site [7]. Jung et al. identified
(N-2-2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]
amino]1,3,5-triazin-2-yl]amino]benzeneacetamide hydrochloride) also known as AP-III-a4 as
Funding: This work was supported by: 1) CPRIT
Grant: RP140612 (Ronald DePinho); 2) NIH CDP
SPORE Grant: P50CA12700107 (F.L.M.); 3)
Research Scholar Grant: RSG1514501CDD
(F.L.M.). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
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ENOblock Does Not Inhibit Enolase
Competing Interests: N.S., Y.H.L. and F.L.M. are
inventors on a patent application concerning
SF2312 and its derivatives as inhibitors of Enolase.
a compound selectively toxic to cancer cells under hypoxic conditions [7]. Further experi-
ments led to the conclusion that AP-III-a4 exerts its effect by direct inhibition of the glycolytic
enzyme Enolase with a reported IC50 of ~0.6 μM, hence it was dubbed ‘ENOblock’. This report
piqued our interest as we had recently demonstrated that glycolytic enzymes can be targets of
personalized cancer therapy if they belong to a family of paralogues, where one member is
homozygously deleted in cancer. Specifically, we showed that passenger deletion of the 1p36
locus covering Enolase 1 (ENO1; αEnolase), leads to dramatic selective sensitization to ablation
of Enolase 2 (ENO2; γEnolase) [8]. Enolase is an enzyme that catalyzes the second to last step
in glycolysis, reversibly converting 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate
(PEP). While inhibitors of Enolase have been described, such as Phosphoacetohydroxamate [9,
10] and the natural antibiotic SF2312 [11–13], these compounds have very poor cell perme-
ability and would make poor clinical candidates. As such, a cell permeable Enolase inhibitor
would be of great utility as a potential clinical candidate for treating tumors with ENO1-homo-
zygous deletion, as well as potentially other tumors that are heavily dependent on glycolysis.
Because of the potential utility of a cell permeable Enolase inhibitor for molecular targeted
therapy of ENO1-deleted tumors, we systematically evaluated ENOblock as an inhibitor of
Enolase in vitro and for selective killing of ENO1-deleted cancer cells. We find that ENOblock
does not inhibit Enolase in in-vitro enzymatic assays and that it does not show selective toxicity
towards ENO1-deleted cancer cells. We suggest that this discrepancy with conclusions in the
paper by Jung et al. stems from the fact that the strong UV absorption of ENOblock interferes
with the Enolase activity assay utilized in that publication [7], which relies on the rather weak
UV absorption of PEP, the product of the Enolase reaction.
Materials and Methods
Enolase enzymatic activity
Native lysates of human cell lines were prepared using 20 mM Tris-HCl, 1 mM EDTA, and 1
mM β-mercaptoethanol at pH 7.4 and sonicated ten times for a period of 30s followed by cool-
ing period of 30s, after which the lysates were cleared by centrifugation at 20,000g for 10 min.
Enolase activity was measured using two different methods, either by 1) a fluorometric
NADH-linked assay or 2) a direct spectrophotometric assay via formation of PEP. In the fluo-
rometric assay, enolase activity was measured via NADH oxidation in a pyruvate kinase–lac-
tate dehydrogenase coupled assay as previously described [8]. The assay is conducted in 10
mM KCl, 5 mM MgSO4, 100 mM triethanolamine at pH 7.4, with 400 μM NADH and 2 mM
ADP. 2-Phosphoglycerate (2-PGA), pyruvate kinase (PK) and lactate dehydrogenase (LDH)
are provided in excess, with conversion of 2-PGA to PEP by enolase being rate limiting. PEP
(with ADP) is substrate of PK; pyruvate formed by this reaction is linked to NADH oxidation
by LDH. Enolase activity is determined by measuring oxidation of NADH fluorescently by
excitation at 340 nm and emission at 460 nm. The substrate concentration, if not otherwise
indicated, was 2.5 mM 2-PGA. Fluorescence was measured using Omega Fluorescence Plate
Reader (BMG Labtech). Alternatively, in a direct spectrophotometric assay, enolase activity
was measured via the conversion of by 2-PGA to PEP by measuring absorption at 240 nm. The
assay medium was the same, except that all the auxiliary reagents (PK/LDH, NADH, ADP)
were omitted. Both assays were conducted in a 96-well plate format with the direct assay per-
formed in UV-transmissible plates.
In addition, we repeated the direct spectrophotometric assay by utilizing the 50mM imidaz-
ole-HCl (pH 6.8), 2.0mM MgSO4 and 400 mM KCl buffer as described by Jung et al [7]. The
reaction was initiated by adding 2.5 mM 2-PGA and optical density (OD) was measured at 240
nm using Omegastar Plate reader (BMG Labtech).
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ENOblock Does Not Inhibit Enolase
Cell culture
The cell line D423-MG was kindly provided by D. Bigner [14]. The 1p36 homozygous deletion
in D423 includes the genes from CAMTA1 to SLC25A33, which includes ENO1. Isogenic
ENO1 ectopically rescued lines were described previously (pCMV ENO1 5X, [8]). An ENO1-
intact cell line (LN319) was used as a control for sensitivity to enolase inhibitors. Cells were
routinely cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
bovine serum.
Proliferation assays
Cell Proliferation was determined by crystal violet staining. We used D423 cell line (ENO1-
deleted), D423 ENO1 (overexpressing ENO1) and LN319 (control cell line). Briefly, glioma
cells were seeded in 96-well plates and treated with different concentrations of ENOblock or
SF2312 for 7 days. Cells were then washed with PBS, fixed with 10% formalin and stained with
0.05% crystal violet. Washed and dried plates were dye-extracted using 10% acetic acid, and
absorbance was measured at 595 nm using Omegastar Plate Reader (BMG Labtech). To test
drug’s efficacy on cell proliferation under hypoxia, 1 x 104 cells were plated in 96-well plates,
treated with ENOblock and SF2312 and incubated for 3 days in a hypoxia station (Don Whit-
ley Scientific, Shipley, UK) set at 0.1% O2 and 5% CO2. Crystal violet staining was then per-
formed as described above.
NMR determination of enolase activity
Reactions were conducted in a standard quartz NMR tube in 500 uL of Imidazole enolase
activity buffer with 2 mM of 2-PGA added as a substrate. Phosphorus spectra (1300 transients)
were acquired at the M.D. Anderson NMR Core in the proton decoupled mode on a 300 MHZ
Bruker instrument.
Enolase inhibitors
SF2312 was prepared by the M.D. Anderson Chemistry core using a slightly modified proce-
dure as previously described [15]. A complete description of the SF2312 and its role as an Eno-
lase inhibitor has been described by our group previously [12]. AP-III-a4 (ENOblock) was
purchased from Sellekchem (Catalog No.S7443, CAS# 1177827-73-4). The identity of this
product with the published material was confirmed by high resolution mass spec conducted at
the Baylor College of Medicine Mass Spec Core. calculated [M + H]+ C13H43FN8O3 595.3515,
observed [M + H] + 595.3543; versus 595.3538 observed by Jung et al.
Recombinant enolase proteins
Recombinant ENO1 and ENO2 proteins used in our experiments were generated by the Core
for Biomolecular Structure and Function. Purity of both recombinant ENO1 and ENO2 pro-
teins was verified using Ponceau and Coomasie Blue staining. In addition, western blots were
carried out to verify the purity of recombinant ENO1 and ENO2 isoforms using following
antibodies: anti-ENO1 (1:1000 dilution, Abcam ab155102), anti-ENO2 (1:1000 dilution, Dako
M087301-2) and Pan-Enolase antibody (1:1000 dilution, Abcam ab189891).
Statistical analysis
Generalized linear regression model was used to evaluate the inhibitory effect of ENOblock
and SF2312 in NMR determination of Enolase activity over time. Generalized estimating equa-
tion method was used to measure correlation within samples at different time-points.
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ENOblock Does Not Inhibit Enolase
Statistical analysis for this linear regression model was performed using SAS 9.4. Repeated
measures one-way ANOVA with Bonferroni correction was used to estimate the inhibitory
effects of a dose response with ENOblock and SF2312 on different cell lines. To evaluate differ-
ences in cell proliferation between normoxic and hypoxic conditions, multiple t-tests were
used at specified dose. Data with p-values < 0.05 were considered significantly different. All
ANOVA and t-tests were performed using GraphPad Prism 7.
Results and Discussion
The effect of ENOblock and other inhibitors on Enolase activity in vitro was determined using
an NADH-coupled assay utilizing lysates of cancer cell lines overexpressing ENO1 and ENO2
(D423-ENO1 and D423-ENO2 respectively). In this assay, the formation of PEP (from 2-PGA
supplemented in the assay) is linked to NADH oxidation via Lactate dehydrogenase and Pyru-
vate Kinase [16]. We find that concentrations up to 500 μM ENOblock fail to inhibit the oxida-
tion of NADH, i.e. do not inhibit Enolase activity (Fig 1A and 1B). In contrast, as little as 50
Fig 1. Fluorometric NADH-linked assay for measuring Enolase activity. The effect on ENOblock (a, b) and SF2312 (b, d) on Enolase activity in
lysates from the D423 cell line expressing ENO2 (red diamonds) or ENO1 (blue squares) was determined using the NADH-linked assay. Panel b shows a
representative trace of NADH fluorescence over time of vehicle control (gray symbols) and 100 μM ENOblock (orange symbols), while Panel d shows
traces of vehicle control (gray symbols), with 50 nM SF2312 (red symbols). Panel a and c shows enolase activity normalized to vehicle control and
expressed as a function of inhibitor concentration. Each data point represents mean of N = 6 (a) and N = 4 (c) ± S.D.
doi:10.1371/journal.pone.0168739.g001
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ENOblock Does Not Inhibit Enolase
nM of the active site inhibitor, SF2312, decreased the rate of NADH oxidation (Enolase activ-
ity) by >80% (Fig 1C and 1D). We also measured the effect of ENOblock on enolase activity
using a direct assay, where the appearance of PEP was monitored by UV absorption of its dou-
ble bond (240 nm) [17]. Indeed, in the work of Jung et al., the only data directly supporting
inhibition of Enolase activity by ENOblock was an end-point assay detecting PEP at 240 nm
(before and after 10 minutes following addition of 2-PGA) using recombinant enzyme [7]. We
aimed to reproduce their exact methodology, however, rather than performing an end-point
measurement, we performed this measurement kinetically. During the course of these experi-
ments we found that ENOblock dramatically raises the baseline UV absorption, before sub-
strate is added (Fig 2). This is not surprising as conjugated aromatic systems such as those in
ENOblock are well known to strongly absorb in that region of the UV spectrum. We found
that measurements had very high variability, especially at higher concentration of inhibitor,
and it proved difficult to reach a firm conclusion as to whether the ENOblock yielded genuine
Fig 2. Effect of ENOblock on Enolase activity using spectrophotometric detection of PEP. The effect on ENOblock (a) and SF2312 (c) on
Enolase activity in purified recombinant ENO1 and ENO2 was determined using the direct PEP-detection assay. Panels a and c show enolase activity
normalized to vehicle control and expressed as a function of inhibitor concentration. Each data point represents mean of N = 4 ± S.D. Panel b shows a
representative traces of absorption at 240 nm over time of vehicle control; 62,000 nM ENOblock and 100 nM SF2312. Note the increased baseline with
ENOblock. Panel d shows western blots for recombinant proteins blotted with ENO 1, ENO2 and pan-ENO antibodies.
doi:10.1371/journal.pone.0168739.g002
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ENOblock Does Not Inhibit Enolase
Fig 3. ENOblock does not inhibit the conversion of 2-PGA to PEP as measured by 31P NMR Experimental conditions were the same as for the
experiments presented in Fig 2, except that 10% D2O was added for signal lock and the experiment was performed at room temperature. 31
P
NMR scanning was performed with 1300 transients, totaling 1 hour of measurement with the first time point being taken as half the time of measurement
(0.5hr). (a) The 2-PGA peak (3.5 ppm, e) was stable for >24hrs in the absence of enzyme, but rapidly decreased with a concomitant increase in the PEP
peak (-1ppm, e) in the presence of 10 nM recombinant hENO2. (b) Pre-incubation with 500 μM ENOblock did not slow the conversion of 2-PGA to PEP,
but 20 μM SF2312 eliminated it Panel (c). (d)The ratio of integrals of 2-PGA and PEP as a function of time are shown for vehicle (grey), 500 μM ENOblock
(orange), and 20 μM SF2312 (red). Each data point represents mean of N = 4 ± S.D. Differences between vehicle and ENOblock was not significant
(n.s. = not significant; p = 0.69); while that between vehicle and SF2312 was highly significant (p<0.0001; Generalized linear regression model). Panel (e)
shows the structure of 2-PGA and PEP with the published chemical shifts in the 31P spectrum.
doi:10.1371/journal.pone.0168739.g003
enzymatic activity inhibition (Fig 2A and 2B). At the same time, the active site Enolase inhibi-
tor SF2312 inhibited PEP formation with similar potency as determined in the NADH coupled
assay (Fig 2C). We verified the purity of our recombinant proteins to exclude the possibility of
any impurities causing this variability (Fig 2D, Figure A in S2 Fig). To avoid this complication
of the spectrophotometric assay, we developed a novel enolase activity assay, where the conver-
sion of 2-PGA to PEP was measured by 31P NMR utilizing identical conditions but performing
the experiment in a standard quartz NMR tube. 2-PGA is evident as a peak at 31P 3.5 ppm
[18]; upon addition of 10 nM recombinant Enolase 2, the 2-PGA peak gradually decreased
with concomitant appearance of a peak at -1.0 ppm (Fig 3A), consistent with literature values
for PEP [18]. Addition of 500uM of ENOblock with recombinant enolase failed to inhibit the
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ENOblock Does Not Inhibit Enolase
appearance of 31P NMR PEP peak (p = 0.69) (Fig 3B). On the other hand, 20 μM SF2312 with
recombinant Enolase fully abrogated the appearance of the PEP peak (p<0.0001) (Fig 3C),
providing direct evidence for inhibition of Enolase enzymatic activity.
Taken together these data, from the three different assays, all unequivocally demonstrate
Enolase inhibition by SF2312, providing a “positive control” of how an Enolase inhibitor is
expected to function. ENOblock fails to inhibit enolase activity as measured by the fluoromet-
ric NADH linked assay, while providing highly variable results with the spectroscopic PEP
detection assay. However, 31P NMR clearly shows absence of inhibition by ENOBlock, and
thus the most parsimonious explanation is that ENOblock interferes with the UV absorption
assay. This interference of ENOblock with the spectrophotometric detection of PEP would not
have been obvious using an endpoint method assay as described by Jung et al [7]. We per-
formed these experiments with ENOblock purchased from Sellekchem; the same experiments
were also repeated with ENOblock shared by Jung et al., with the same results (data not
shown). Taking these findings into consideration, we conclude that the spectroscopic assay is
not an appropriate assay to measure inhibition of Enolase activity by ENOBlock.
Previous work from our group demonstrated that glioma cells with deletion of ENO1 show
>90% decrease in total Enolase activity and dramatic selective sensitivity to the pan-Enolase
inhibitor, Phosphonoacetohydroxamate [8]. To determine whether or not ENOBlock might
exhibit selective toxicity towards ENO1-deleted glioma cells we tested ENOBlock on the same
cell lines. Treatment with ENOblock over the course of 7 days, showed near equal toxicity to
D423 ENO1-deleted glioma cells and an isogenic rescued cell line ectopically re-expressing
ENO1, as well as ENO1-intact glioma cell lines (LN319). ENOblock at concentrations >25 μM
eradicated glioma cells regardless of ENO1 status (Fig 4A and 4B). As a positive control, we
determined the effects of the active site Enolase inhibitor SF2312 on D423 ENO1-deleted, an
isogenic rescued cell line ectopically over-expressing ENO1, and ENO1-intact glioma cells
(LN319). SF2312 showed strong selective toxicity towards ENO1-deleted glioma cells with
ENO1-intact glioma cells being minimally affected at up to 100 μM inhibitor (p<0.05) (Fig 4C
and 4D). We also performed short term, 3-day, treatments to determine the effect of hypoxia
on toxicity of ENOblock and SF2312. Consistent with published work [7], hypoxia increased
the toxicity of ENOblock towards all glioma cell lines, regardless of ENO1-deletion status
(Figure A and B in S1 Fig). The selective toxicity of SF2312 towards ENO1-deleted glioma cells
was evident at normoxia, but the effect was dramatically potentiated under hypoxia (Figure C
and D in S1 Fig). At concentrations >12.5 μM SF2312 completely eradicated ENO1-deleted
glioma cells under hypoxia. Under normoxia, similar concentrations inhibited proliferation
resulting in lower cell density, but not complete eradication. This increased sensitivity of
ENO1-deleted cells to the enolase inhibitor SF2312 under hypoxia is fully expected, given that
energy generation by the mitochondrial respiratory chain is expected to be impaired under
these conditions, resulting in increased reliance on glycolysis.
In conclusion, our data quite unequivocally indicate that ENOblock does not inhibit the
enzymatic activity of Enolase. That said, in at least one aspect, sensitization to hypoxia, our
data agree with the biological effects of ENOblock reported by Jung et al. Indeed, our results
do not challenge the validity of the many interesting biological effects of ENOblock that Jung
et al reported. However, identifying the correct mechanism will likely prove challenging.
While our data indicate that ENOBlock does not inhibit the enzymatic activity of Enolase, they
do not dispute that ENOblock may bind to Enolase (Figure 2a in [7]). However, no additional
data, such as X-ray structures, Cellular thermal shift assays or mutational analysis, which
would indicate the specific binding site of ENOBlock on Enolase were presented in Jung et al.
Furthermore, ENOblock appears to bind to several additional proteins (Figure 2a in [7])
besides Enolase. Thus, while we can conclude that ENOBlock acts through a mechanism other
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ENOblock Does Not Inhibit Enolase
Fig 4. Non-selective toxicity of ENOBlock to ENO1-deleted glioma cells. A representative plate of cancer cells treated with ENOblock is shown in
panel a, with quantification shown in panel b A plate treated with SF2312 is shown in panel c, with quantification shown in panel d. Cell were treated for
7 days. (b, d) D423 ENO1-deleted (red diamonds), D423 ENO1-rescued (blue squares) and LN319 ENO1 WT (grey circles) were treated with the
indicated doses of ENOblock in panel b (N = 4 ± S.D) or SF2312 in panel d (N = 4 ± S.D). Cell density was quantified by crystal violet and expressed
relative to vehicle control as a function of inhibitor concentration. At high concentrations, SF2312 selectively killed D423 ENO1-deleted cells as
compared to D423 ENO1-rescued cells (p<0.05, Repeated Measures one-way ANOVA with Bonferroni correction). ENOblock failed to show such
selectivity regardless of dose.
doi:10.1371/journal.pone.0168739.g004
than direct inhibition of the enzymatic activity of Enolase, this mechanism remains unknown
and determining how ENOBlock exerts its reported biological effects is not immediately clear
and will likely require further extensive experimentation.
Supporting Information
S1 Fig. Effect of Hypoxia on sensitivity to ENOblock and SF2312. D423 ENO1-deleted (red
diamonds), D423 ENO1-rescued (blue squares) and LN319 ENO1-WT (grey circles) glioma
cells were treated with indicated ENOblock doses (Panel a and b) or SF2312 (Panel c and d)
and incubated either at 21% O2 indicated as Normoxia or 0.1% O2 indicated as Hypoxia for 3
days. Cell density was quantified by crystal violet and expressed relative to vehicle control as a
function of inhibitor concentration (Panels b and d). Each data point represents mean of
N = 4 S.D. Differences between hypoxic and normoxic conditions for ENO1-deleted glioma
cells significant to at least p<0.01 are indicated (unpaired t-test with Bonferroni correction).
(TIF)
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ENOblock Does Not Inhibit Enolase
S2 Fig. Purity analysis of recombinant ENO1 and ENO2. Panel a shows purity of recombi-
nant ENO1 and ENO2 proteins by Ponceau staining and Coomasie staining. Panel b shows
uncropped western blots from Fig 2 (Red rectangle indicates the blots used in Fig 2 for recom-
binant ENO1 and ENO2 proteins blotted with their respective antibodies (ENO1 antibody,
1:1000, Abcam ab155102; ENO2 antibody, 1:1000, Dako M087301-2 and Pan-Enolase anti-
body, 1:1000, Abcam ab189891).
(TIF)
Acknowledgments
We thank Dr. Kumar Kaluarachchi and Dr. John McMurray for assistance with NMR mea-
surements. We thank Dr. Vivekananda Shetty for mass spec measurements. We thank Rafal
Zielinski for assistance with hypoxia experiments. F.L.M. was supported by a Research Scholar
Grant RSG-15-145-01-CDD from the American Cancer Society, NIH CDP SPORE
P50CA127001-07 and CPRIT RP140612. We thank Paul G. Leonard, Todd M. Link and Gil-
bert Lee (Core for Biomolecular Structure and Function) for sharing recombinant human
ENO1 and ENO2. We thank the Department of Biostatistics at the University of Texas MD
Anderson Cancer Center for providing statistical support and guidance.
Author Contributions
Conceptualization: FLM NS.
Data curation: NS.
Formal analysis: NS YHL SR.
Funding acquisition: FLM.
Investigation: FLM NS.
Methodology: FLM NS YHL NH SR.
Project administration: FLM.
Supervision: FLM.
Validation: FLM NS NH YHL.
Visualization: FLM NS.
Writing – original draft: FLM NS.
Writing – review & editing: NS DKG.
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