This document describes a screen of 50,000 compounds to identify inhibitors of hepatocyte growth factor (HGF)-induced epithelial scattering. Several structurally distinct compounds were identified that had not previously been reported to have biological activity. Further analysis revealed that many of the compounds broadly inhibit cancer cell proliferation by arresting cell division in G2/M phase with minimal induction of apoptosis. This effect is consistent with microtubule-targeting agents. Biochemical assays showed that several compounds directly inhibit microtubule polymerization. Some pharmacokinetic properties of the compounds were also assessed.

1. Journal of Biomolecular Screening
1­–9
© 2016 Society for Laboratory
Automation and Screening
DOI: 10.1177/1087057116651850
jbx.sagepub.com
Original Research
Introduction
Hepatocyte growth factor (HGF) binds the c-met receptor
tyrosine kinase,1
driving increased proliferation, migration,
and epithelial scattering,2
as well as enhanced cell survival.3
Detachment of individual cells from epithelial tissues to
yield invasive and solitary mesenchymal cells that can colo-
nize distant tissues occurs in tightly regulated instances
throughout development, where it is termed epithelial-
mesenchymal transition (EMT). It is thought that HGF sig-
naling drives cancer metastasis in part by triggering EMT,
resulting in detachment of cells from the primary tumor,
their increased migration and invasion, and colonization of
distant sites.
Given the connection of HGF signaling and cancer pro-
gression, there has been intense interest in developing ther-
apeutics that inhibit cellular events triggered by HGF
signaling.4
Ligand receptor binding and the enzymatic
activity of the c-met receptor tyrosine kinase have been the
principal targets of rational therapeutic design, while fewer
efforts have targeted downstream components of the HGF
signaling network for potential therapeutic intervention. We
previously reported efforts to identify components and sys-
tems required for epithelial scattering using phenotypic
screening of a small-molecule library.5
We used an assay
system that measures MDCK cells’ scattering response to
HGF, which is a well-established model system for HGF-
induced epithelial scattering. In fact, epithelial scattering
was first described using this model system.6
In our prior publication, we reported a number of screen
compounds with known biological activity, including
651850JBXXXX10.1177/1087057116651850Journal of Biomolecular ScreeningHoj et al.
research-article2016
1
Brigham Young University, Provo, UT, USA
2
Baylor College of Medicine, Houston, TX, USA
3
Samford University, Birmingham, AL, USA
Received Mar 9, 2016, and in revised form Apr 19, 2016. Accepted for
publication Apr 29, 2016.
Supplementary material for this article is available on the Journal of
Biomolecular Screening Web site at http://jbx.sagepub.com/supplemental.
Corresponding Author:
Marc D. H. Hansen, Department of Physiology and Developmental
Biology, Brigham Young University, 4005 LSB, Provo, UT 84602, USA.
Email: marchansen@byu.edu
Small Molecules Revealed in a Screen
Targeting Epithelial Scattering Are
Inhibitors of Microtubule Polymerization
Taylor H. Hoj1
, Ryan J. Robinson1
, Jason C. Burton2
,
Rachel A. Densley-Ure1
, Tyler V. Olson1
, Logan K. Williams1
,
Lori Coward3
, Greg Gorman3
, and Marc D. H. Hansen1
Abstract
Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in the detachment of cell-
cell junctions and initiation of cell migration. Instead of coordinating collective cell behavior within a tissue, cells become
solitary and have few cell-cell interactions. Since epithelial scattering is recapitulated in cancer progression and since HGF
signaling drives cancer metastasis in many cases, inhibitors of HGF signaling have been proposed to act as anticancer
agents. We previously sought to better understand critical components required for HGF-induced epithelial scattering
by performing a forward chemical genetics screen, which resulted in the identification of compounds with no previously
reported biological activity that we report here. In efforts to determine the mechanism of these compounds, we find that
many compounds have broad antiproliferative effects on cancer cell lines by arrest of cell division in G2/M with minimal
induction of apoptosis. This effect is reminiscent of microtubule-targeting agents, and we find that several of these scaffolds
directly inhibit microtubule polymerization. Compounds are assessed for their toxicity and pharmacokinetics in vivo.
The identification of novel small-molecule inhibitors of microtubule polymerization highlights the role of the microtubule
cytoskeleton in HGF-induced epithelial scattering.
Keywords
cell-based assays, phenotypic drug discovery, oncology, microtubule
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2. 2 Journal of Biomolecular Screening 
multiple scaffolds that target the microtubule cytoskeleton.5
The screening assay uses quiescent cells to limit any com-
pound effects on cell growth. We showed that microtubules
are required for mobilization of calcium channels to the
plasma membrane shortly after HGF stimulation, which
explains why microtubule-targeting agents were found in
our chemical screen.
Here we report the structures of a number of structurally
distinct compounds from our previous phenotypic, cell-
based screen for inhibitors of HGF-induced epithelial scat-
tering but with no previously ascribed biological activity.
We assess the effects of these compounds in cellular assays,
revealing how they affect cell behavior that might be con-
sistent with microtubule targeting. Biochemical assays
reveal that a number of compounds directly inhibit microtu-
bule polymerization. Some drug-like properties of these
scaffolds are also reported.
Materials and Methods
Chemical Screening
Cell-based assays measuring HGF-induced EMT were used
as described.5
Briefly, MDCK cells, grown in Dulbecco’s
modified Eagle’s medium (DMEM) + 10% fetal bovine
serum, were seeded on the upper chamber of a 96-well
Transwell filter plate and cultured for 24 h to form a conflu-
ent monolayer with minimal cell division. Compounds were
then added from 100× DMSO stocks to a final screening
assay concentration of 5 µM, ensuring that DMSO concen-
trations did not exceed 1% in the assay. Cells were then
stimulated with HGF and cultured for an additional 24 h. At
the conclusion of the assay, Transwell filters were fixed with
4% paraformaldehyde on ice for 15 min and stained with
crystal violet. After initial visual inspection for plates with
biologically active compounds, quantitation of the assay was
performed by swabbing the upper surface of the Transwell
filter of cells and then measuring light transmission through
the filter. Negative control assays without drug treatment
and positive control experiments lacking HGF stimulation
and drug treatment were used to establish the assay range for
eachplate.Thisscreeningassaywasappliedtoa50,000-com-
pound chemical library representing drug-like small mole-
cules (ChemBridge, San Diego, CA).To minimize variability
between assays, we used an Eppendorf EpMotion 5070
(Eppendorf, Hamburg, Germany) liquid handling robotics
system to initiate, treat, and process all screening assays.
Cell Proliferation Assays
Cancer cell proliferation assays were performed by seeding
cancer cell lines in the wells of a 96-well plate. After 24 h,
a set of control wells was fixed and stained as described
below. Other wells were treated with compounds or vehicle,
then cultured for 48 h. Cells were fixed by addition of
trichloroacetic acid to a final concentration of 20% (v/v),
washed with 1% (v/v) acetic acid, air dried, and stained
with 0.4% (w/v) sulforhodamine B in 1% acetic acid.
After washing with 1% acetic acid, stain was released
with a 10-mM Trizma (Sigma, St. Louis, MO) base and
assay values were quantified by measuring absorbance at
a wavelength of 515 nm. In all cell-based assays, seeding,
treatment, and stimulation was performed using an
Eppendorf EpMotion 5070 robotics station to maximize
reproducibility.
Flow Cytometry
Analysis of cells’ distribution through the cell cycle was per-
formed on MDA-MB-231 cells after 24 h of treatment with the
indicated compound at a final concentration of 10 µM DMSO.
Cells were trypsinized and fixed in suspension with ice-cold
absolute ethanol and subsequent staining with propidium
iodide in the presence of Triton X-100 and RNase-free DNase.
Analysis of apoptosis in cell populations was performed on
MDA-MB-231 cells after 24 h of treatment with the indicated
compound at a final concentration of 10 µM or with DMSO.
Trypsinized cells were processed for flow cytometry after
staining with propidium iodide and FITC–Annexin V using a
kit from BD Biosciences (San Jose, CA). Cells were analyzed
by flow cytometry using the Attune Acoustic Focusing
Cytometer (Applied Biosystems, Foster City, CA, USA).
Microtubule Polymerization Assays
Analysis of microtubule polymerization was performed
usingtheprotocolandreagentsintheTubulinPolymerization
Assay Kit from Cytoskeleton (Denver, CO, USA). In sum-
mary, purified tubulin was maintained in a buffer under
nonpolymerizing conditions. Compounds were added to
each assay from DMSO stocks and buffer, and then polym-
erization was initiated by addition of a polymerization buf-
fer. Fluorescence during polymerization was measured in
real time using a BioTek (Winooski, VT) Synergy HT plate
reader.
Bioanalytical Method Development
To measure compound levels in biological samples, analy-
sis was conducted using a Shimadzu LC20-AD HPLC
interfaced to an AB Sciex 4000 QTRAP (Kyoto, Japan)
equipped with an electrospray ionization source (ESI) oper-
ated in the positive ion mode (Framingham, MA, USA).
Separation of the analytes and the internal standard from the
plasma matrix was performed with a Thermo Aquasil C18,
100 × 2-mm 5-µm particle column (Thermo Fisher,
Waltham, MA) operated at ambient temperature using a
gradient elution profile. The mobile phase consisted of
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3. Hoj et al. 3
5 mM ammonium acetate with 0.1% formic acid (A) and
acetonitrile with 0.1% formic acid (B). The starting compo-
sition was 60/40 A:B, which was ramped linearly to 5/95
A:B over 4 min, held for 1 min and then returned to 60/40
A:B, and equilibrated for 2.5 min. A 10-µL injection vol-
ume was used with a flow rate of 400 µL. To prepare sam-
ples, a 50-µL aliquot of each biological sample was placed
into individually labeled 1.5-mL microcentrifuge tubes, and
500 µL of 5 ng/mL terfenadine in acetonitrile was added.
Each sample was vortexed well and then centrifuged for 5
min at 13,000 × g. The resulting supernatant was transferred
to an autosampler vial for analysis by liquid chromatogra-
phy–tandem mass spectrometry (LC-MS/MS).
Pharmacological Assays
Maximum tolerable acute toxicity was determined by formu-
lating compound in 5% DMSO, 20% Tween 80, and 75%
saline. Doses of 400, 200, and 100 mg/kg were each injected
into mice via the intraperitoneal route. Mice were monitored
for 2 weeks, including measuring of body weights, to assess
signs of toxicity. Pharmacokinetic properties of each com-
pound tested were determined by injecting mice or rabbits
via the intraperitoneal route. Blood was withdrawn from
three animals at each designated time point following injec-
tion. Blood was collected in EDTA-coated polypropylene
tubes and plasma was generated by centrifugation at 2000 ×
g for 15 min at 4 °C. Plasma was frozen on dry ice until pro-
cessing. The amount of compound in each sample was mea-
sured using bioanalytical methods described above.
Plasma and Microsomal Stability
The plasma stability of compound 3 was determined in mul-
tiple species of plasma (rat, rabbit, hamster, guinea pig, ger-
bil, mouse, and human) collected with K2EDTA. Each
species of plasma was spiked at 2 µg/mL and incubated at
37 °C for up to 1 h. Samples were taken at 5, 15, 30, and 60
min and analyzed for remaining amount of the parent com-
pound using the above-described method. Metabolic stabil-
ity of various compounds was determined in human liver
microsomes (HLMs) at a 10-µM concentration of the sub-
strate in a total reaction volume of 0.5 mL at 37 °C in a
shaking water bath. The reaction mixtures consisted of
RapidStart (XenoTech, Kansas City, KS, USA) NADPH
regenerating solution consisting of 1.0 mM nicotinamide
adenine dinucleotide phosphate (NADP+), 5 mM glucose-
6-phosphate, 1 U/mL glucose-6-phosphate dehydrogenase;
UGT reaction mix solution A consisting of 2 mM UDPGA;
and UGT reaction mix solution B containing 50 mM Tris-
HCl (pH 7.5), 8 mM magnesium chloride, and 25 µg/mL
alamethicin. The total organic solvent concentration in the
reaction mixture was kept at less than 1%. Aliquots of the
reaction mixture were quenched with an equal volume of
acetonitrile at the start of the reaction (0 min) and after incu-
bation for 5, 15, 30, and 60 min and analyzed as described
above.
Results
Compound Structures and Synthesis
As described in a prior publication,5
we developed a pheno-
typic screen for inhibitors of HGF-induced epithelial scat-
tering in attempt to find small molecules whose biological
activity would elucidate molecular components required for
this process. The screen assay accurately measures the
potency of a known c-met inhibitor and distinguishes
between positive and negative controls with a z′ of 0.48.5
The screen assay uses MDCK cells, which form an epithe-
lial monolayer on a Transwell filter at a high confluency
with minimal cell division. Maximum throughput of this
assay is a 96-well plate format, a result of limitations in the
Transwell plate format. MDCK monolayers are treated with
individual compounds, then stimulated with HGF in the
lower culture chamber. HGF treatment normally stimulates
epithelial scattering, resulting in individual cells detaching
from the monolayer on the upper surface of the filter and
actively migrating through the filter. After 24 h, cells on
both sides of the filter are stained with crystal violet.
Staining on the underside indicates productive HGF stimu-
lation, and staining intensity is a quantitative readout of epi-
thelial scattering. In contrast, reduced staining on both the
lower and upper sides of the filter indicates toxicity, an out-
come that is easily distinguished from inhibition of produc-
tive HGF signaling and whose assay results can be ignored.
We previously deployed this screen against a 50,000-
compound library of drug-like molecules and reported seven
active scaffolds with known mechanism of action.5
Additional
active compounds, specifically those without a previously
reported biological mechanism, are shown in Figure 1.
Multiple analogues with high structural similarity are grouped
to represent a subset of scaffolds identified in this screening
step, while other scaffolds are represented by single deriva-
tives. Compounds 1 and 2 are quinazolinones, examples of
which were synthesized and previously assessed for anti-
inflammatory activity7
but also shown to have antitumor activ-
ity.8,9
Biphenylcarboxamide compound 5 was synthesized and
assessed for antimicrobial activity.10
Compound 6 belongs to
a class of thienpyrimidones, members of which have been
previously reported to have antiviral activity11
and for
17β-hydroxysteroid dehydrogenase inhibition as antitumor
agents.12
Dihydroquinolines similar to compounds 8 to 10
have been attributed with activity against α-synuclein in a
yeast model for Parkinson disease amelioration13
and for use as
anticonvulsant agents.14
Compounds 11 and 12 are tetrahydro-
indolone esters that are structurally similar to several mole-
cules that have been previously reported to have antitumor
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4. 4 Journal of Biomolecular Screening 
activity through an undefined mechanism,15,16
although micro-
tubule inhibition is observed with some structurally related
compounds.17
Given the provenance of the molecules from a chemical
library, we made the compounds using the synthetic routes
indicated (Suppl. Fig. 1). Compounds were retested for
biological activity in the original screening assay, and the
newly synthesized molecules were used for subsequent
analysis.
Cellular and Biochemical Activity
To better understand the cellular function of the compounds
depicted in Figure 1, we assessed their activity in arresting
proliferation of cancer cell lines from the NCI-60 panel.18
We reasoned that any compounds that target microtubules
would prevent cell proliferation in a broad panel of cell
lines, which is precisely what we observed for the entire
panel of molecules tested (Fig. 2). There was only a single
instance of a cell line being totally refractory to one of the
compounds (HOP92 cells to compound 1). Most cell lines
responded to all the compounds with similar levels of sensi-
tivity, with the EC50
values of each compound falling within
an order of magnitude for all compounds tested. The gen-
eral trend was that cell lines were similarly sensitive to all
of the compounds tested; besides cell lines where a single
compound had a markedly altered potency than the others,
few cell lines showed a high degree of variability in sensi-
tivity to the panel of compounds (HOP92 and UACC257).
Interestingly, compound 12 was typically more potent than
compound 11, despite compound 11 generating a stronger
effect in our screening assay.
We also reasoned that molecules targeting the microtu-
bule cytoskeleton would arrest cell proliferation in ana-
phase.Totestthis,westainedthenucleiofcompound-treated
MDA-MD-231 cells and determined the distribution of
cells across G1, S, and G2/M phases of the cell cycle using
flow cytometry (Table 1). We found that an accumulation
Figure 1. Compounds
identified in screening and
their screening assay potency.
The structures of compounds
identified in the original
phenotypic screen are depicted.
Values in parentheses indicate
the inhibition of epithelial
scattering by each compound in
the screening assay.
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5. Hoj et al. 5
of cells in the G2/M phase was clearly observed for cultures
treated with compounds 6, 8, and 11. Treatment with com-
pounds 1, 3, and 7 yielded an increase in the proportion of
cells in G2/M relative to G1, but there were also increases
in the number of cells in the S phase or what appeared to be
cells or cellular debris with minimal DNA staining.
Nevertheless, both experimental outcomes are consistent
with activity that targets microtubule function. Cells treated
with compound 5, in contrast, were predominantly in G1
with a small number of cells in the S phase and no accumu-
lation of cells in G2/M, although there was an increase in
cells or cell fragments that stained with less than a full copy
of DNA.
Since accumulation of cells in the G2/M phase might
represent arrest resulting from a DNA damage response or
arrest in mitosis, and since we did observe an accumulation
of cell or cellular debris that had low DNA staining, we also
assessed compounds for activity in inducing apoptosis.
Typically, compounds that induce a G2 arrest as a result of
a DNA damage response immediately enter apoptosis,
whereas cells arrested in mitosis only undergo apoptosis at
low levels during early treatment. We treated cultures of
cells with analogues of each scaffold for 24 h and then mea-
sured apoptosis by flow cytometry of propidium iodide and
Annexin V–stained MDA-MB-231 cells (Table 1). Of
untreated cells, 5.7% stained for Annexin V but not prop-
idium iodide, indicating that this portion of the population
was apoptotic. For all of the compounds tested, we saw only
minimal increases in the numbers of apoptotic cells.
Compound 3 showed the largest increase in the proportion
of apoptotic cells, reaching 11.5% of the population, while
the proportion of apoptotic cells in cultures treated with
other compounds ranged from 5.4% for compound 8 to
9.0% for compound 6. This suggests that compounds gener-
ating G2/M arrest act by mitotic arrest and not by induction
of a G2 phase cell cycle arrest that leads to apoptosis.
We finally sought to directly assess compounds for activ-
ity in microtubule polymerization assays using purified
Figure 2. Compounds’
antiproliferative potencies in NCI-60
cancer cell lines. Plots show the EC50
of growth inhibition for each cell
line and compound tested. No bar
indicates the cell line was not tested
for that compound. Cell lines are
grouped by cancer type.
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6. 6 Journal of Biomolecular Screening 
tubulin, which would assess the specific activity of each
compound in preventing polymerization or, less likely, sta-
bilizing microtubules. In control assays, polymerization
began after a lag period, reaching a maximal rate and slow-
ing to a plateau. Compounds 6, 7, and 8 had no effect on
microtubule polymerization, even at the highest concentra-
tion tested. Compounds 1, 3, 5, and 7 reduced microtubule
polymerization in a concentration-dependent manner; the
maximal rate of polymerization at different concentrations
allowed calculation of IC50
values for each compound, and
compounds 3, 5, and 11 bore potencies similar to that of
vincristine (Fig. 3), a known microtubule targeting agent
that is also an approved drug. Given that these polymeriza-
tion assays used purified tubulin dimers only, these com-
pounds must directly inhibit microtubule polymerization.
Pharmacology
We next sought to determine the pharmacology of com-
pounds identified in screening. Acute toxicity was deter-
mined by administering intraperitoneal injections of this
compound to mice at a series of doses. Mice were observed
for 2 weeks, including the monitoring of body weight. No
toxicity, including significant weight loss, was observed for
any of the three compounds tested, even at the highest tested
dose of 400 mg/kg.
Table 1. Cellular Activity: Cell Cycle Effects and Induction of Apoptosis.
Cell Cycle Distribution, %
G1 S G2/M Apoptotic
Control 56.5 16.7 25.9 5.70
Compound 1 47.8 18.7 30.1 6.41
Compound 3 23.6 20.7 51.5 11.45
Compound 5 49.3 21.4 25.9 5.69
Compound 6 16.4 16.3 61.7 8.98
Compound 7 31.0 19.6 44.5 8.32
Compound 8 17.6 14.4 64.1 5.40
Compound 11 19.1 14.1 56.6 7.36
Distribution of MDA-MB-231 cells in G1, S, or G2/M phases of the cell cycle, as measured by propidium iodide fluorescence and flow cytometry. The
percentage of apoptotic cells, determined by Annexin V staining and lack of propidium iodide staining in a separate flow cytometry experiment, is also
included. Cells were treated with the indicated compound or with DMSO (control) for 24 h.
Figure 3. Biochemical activity: inhibition of microtubule polymerization. The maximal rate of microtubule polymerization in
biochemical assays for different concentrations of each indicated compound is plotted. IC50
values for each compound are calculated
from the fitted data curve (red).
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7. Hoj et al. 7
Our next effort was to determine their plasma stability and
pharmacokinetic profile. We first developed an extraction
method based on protein precipitation with acetonitrile to
recover each compound from biological samples. We then
developed a bioanalytical method using LC-MS/MS to deter-
mine compound concentrations using internal standard quanti-
tation, the parameters of which are listed in Table 2 for each
compound. With bioanalytical methods in place, we deter-
mined the pharmacokinetic profiles of these compounds. Mice
were injected with compound and plasma levels determined at
several time points following injection. Where possible, we
used intravenous injections (compounds 1, 7, and 8), but poor
solubility forced us to rely on intraperitoneal injections for
most compounds. Not surprisingly, the pharmacokinetics of
the compounds differed significantly, both in bioavailability
following injection and in the persistence of compound in cir-
culation. A number of compounds showed very limited bio-
availability. The exceptions were compounds 6, 7, and 11,
which achieved biologically relevant plasma levels that per-
sisted at detectable levels until 8 or 24 h after injection, respec-
tively, although the rate of clearance of compound 7 was very
fast (Fig. 4A).
Given the rapid clearance of a number of compounds, we
examined their stability when incubated with liver micro-
somes.All compounds experienced metabolism, with ~20%
or less remaining after 1 h of incubation in human liver
microsomes in all cases (Fig. 4B). Compound 1 was the
most stable, although significant amounts of irreversible
Figure 4. Pharmacokinetics and
metabolism. (A) Plasma levels of
compounds following intravenous
(compound 7) or intraperitoneal
(compounds 6 and 11) dosing.
Only compounds that achieved
maximal plasma levels in excess
of 100 ng/mL at any time after
dosing are shown. (B) Plots indicate
the amount of each compound
remaining after incubation with
human liver microsomes. (C) Plots
show the amount of compound 3
remaining after incubation in plasma
at room temperature as a function
of time.
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8. 8 Journal of Biomolecular Screening 
binding to protein in liver microsomes were observed for
compound 5 and might account for some of its depletion in
this assay.
We also sought to better understand the instability of com-
pound 3 in plasma. Metabolism resulted from cleavage of the
nitrogen-nitrogen bond of the linker region of compound 3,
and we found that protease inhibitors prevented plasma insta-
bility (data not shown) and sought to examine the stability of
this compound in plasma from different species. In contrast
to the rapid metabolism observed in mouse plasma, greater
stability was observed in other species tested, with the high-
est stability observed in humans (Fig. 4C). Given the high
plasma stability of this molecule in rabbits, we therefore
sought to examine the pharmacokinetics of compound 3 in
rabbits. Analysis of plasma levels after intraperitoneal injec-
tion shows that bioavailability was very low for this com-
pound, never exceeding plasma concentrations of 100 ng/mL
at any time point following administration.
Discussion
The HGF pathway is clearly associated with cancer progres-
sion, and developing agents that target HGF, its receptor, or
downstream signaling has been the subject of therapeutic inter-
est for some time.3
Here we report the identity of a series of
small molecules that inhibit HGF-induced epithelial scattering
discovered in phenotypic screening. The compounds represent
seven clearly distinct scaffolds.
Given that microtubule targeting agents are active in this
assay,5
it is not surprising that these compounds are antiprolif-
erative in a broad array of cancer cell lines and that, with the
exception of compound 5, they generally induce cell cycle
arrestinG2/Mwithminimalimmediateapoptosis.Biochemical
assays using purified tubulin reveal that compounds, excepting
6 and 8, directly inhibit microtubule polymerization, suggest-
ing that they bind and sequester tubulin dimers, although any
effect of compound 1 appears to be quite weak. That com-
pound 5 directly targets microtubule polymerization without
inducing cell cycle arrest in G2/M suggests polypharmacology
or significant off-target effects, resulting in pleiotropic effects
in cell culture assays. Similarly, additional effects besides
G2/M arrest seen by flow cytometry of DNA-stained cells sug-
gest that compounds 1, 3, and 7 may have additional effects on
cells besides those that may be attributed from inhibition of
microtubule polymerization alone. Compounds 6, 8, and 11
have the clearest effects on the distribution of treated cells
through the cell cycle, which suggests the highest degree of
selectivity, but only compound 11 targets the microtubule cyto-
skeleton. Whether compounds 6 and 8 affect microtubules
indirectly or whether these compounds generate G2/M arrest
by a completely different mechanism and an unrelated target
remains to be seen.
A key point here is that molecules that bear activity in
preventing microtubule polymerization also prevent HGF-
induced epithelial scattering, the basis of the screening
assay used to identify these molecules. Importantly, this cel-
lular assay was performed at high confluence that mini-
mizes cell proliferation, suggesting that disruption of the
microtubule cytoskeleton is blocking scattering another
way. One way is by limiting cell migration in cells without
an intact microtubule cytoskeleton. Our prior work suggests
that disruption of the microtubule cytoskeleton might also
limit critical vesicle trafficking steps necessary to enact cell
behavior changes initiated by HGF stimulation, including
influxes of calcium into the cell.4
Regardless, the identifica-
tion of microtubule targeting agents with high frequency in
our phenotypic screen demonstrates a critical role for
microtubules in ways that are independent of their role in
mitosis. Given the large number of microtubule targeting
agents revealed in this screen, we suggest that compounds 6
and 8 also generate G2/M arrest by targeting microtubules,
although through a mechanism that is not direct.
While not a major focus of this work, we also present
information showing some drug-like properties and phar-
macology of the compounds identified in screening. Most
compounds have poor pharmacokinetics and are subjected
Table 2. Bioanalytical Method Parameters.
Compound No.
Detection
Range, ng/mL
Reproducibility:
Correlation
Coefficient (from
Composite Curve)
Method
Accuracy, %
Method Precision
(Coefficient of
Variability), %
Plasma
Recovery, %
Plasma Stability at Room
Temperature
1 5–5000 0.9995 85–115 15 95 107% remains after 30 h
3 2–5000 0.9995 85–115 15 86.9 Unstable without protease
inhibitors
5 2–5000 0.9997 85–115 15 90.4 89.4% remains after 39 h
6 2–5000 0.9962 85–115 15 101 97.5% remains after 28 h
7 5–5000 0.9976 85–115 15 97.2 100% remains after 31 h
8 5–5000 0.9996 85–115 15 91.2 96.7% remains after 31 h
11 5–5000 0.9994 85–115 15 91.5 96.7% remains after 31 h
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9. Hoj et al. 9
to significant metabolism. Efforts to optimize these mole-
cules in any way must include those directed toward
improving these properties and not only increasing potency.
In summary, we report novel biological activities, as
inhibitors of microtubule polymerization, for a series of
compounds identified in a screen for inhibitors of HGF-
induced epithelial scattering. This mechanism of action
highlights a growing role for microtubules in epithelial
morphogenesis.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: J.B.
was supported by a BYU Cancer Center summer undergraduate
research internship.
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