Discovery of MINC1, a GTPase-activating protein small molecule inhibitor, targeting MgcRacGAP

Article abstract of DOI:10.2174/1386207318666141205112730

The Rho family of Ras superfamily small GTPases regulates a broad range of biological processes such as migration, differentiation, cell growth and cell survival. Therefore, the availability of small molecule modulators as tool compounds could greatly enhance research on these proteins and their biological function. To this end, we designed a biochemical, high throughput screening assay with complementary follow-up assays to identify small molecule compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1% with the majority of those affecting the primary readout, an enzyme-coupled GDP detection assay. After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells.

Full text of DOI:10.2174/1386207318666141205112730

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Combinatorial Chemistry & High Throughput Screening, 2015, 18, 3-17
3
Discovery of MINC1, a GTPase-Activating Protein Small Molecule
Inhibitor, Targeting MgcRacGAP
Arjan J. van Adrichem1, Annika Fagerholm2, Laura Turunen1, Anna Lehto1, Jani Saarela1,
Ari Koskinen2, Gretchen A. Repasky1 and Krister Wennerberg*,1,3
1Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland
2Laboratory of Organic Chemistry, Department of Chemistry, School of Chemical Technology, Aalto University, Espoo,
Finland
3Drug Discovery Division, Southern Research Institute, Birmingham, AL, USA
Abstract: The Rho family of Ras superfamily small GTPases regulates a broad range of biological
processes such as migration, differentiation, cell growth and cell survival. Therefore, the
availability of small molecule modulators as tool compounds could greatly enhance research on
these proteins and their biological function. To this end, we designed a biochemical, high
throughput screening assay with complementary follow-up assays to identify small molecule
compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in
cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house
screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the
NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1%
with the majority of those affecting the primary readout, an enzyme-coupled GDP detection assay.
After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with
other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined
with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in
addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells.
Keywords: Biochemical assays, cytokinesis, HTS, MgcRacGAP, Rac1, small molecule inhibitor.
specifically target a function of interest leaving the other
functions untouched; however, controlled temporal
regulation is not easily achieved and non-physiological
concentrations of the mutant proteins can distort the delicate
balance needed for normal cellular behavior.
INTRODUCTION
Rho GTPases are regulated molecular binary switches
that cycle between active, GTP-loaded and inactive, GDP-
loaded states. This protein family controls a wide array of
cellular functions such as cell proliferation, apoptosis and
cell morphology in health and disease [1, 2], and therefore
are interesting targets for pharmacological inhibition. Small
molecule modulators could have many advantages over
conventional molecular biology tools such as mutant
proteins and RNA interference (RNAi), which, although
having provided global insight into cell biological function,
seemingly led to controversial results in the Rho GTPase
research field [3, 4]. Small molecule inhibitors can easily be
applied in a dose-dependent, temporal or domain-specific
manner, inhibiting one particular function, whereas RNAi
methods completely remove the protein, disrupting possible
protein-protein interactions that are mediated through
domains other than the one of interest. Therefore,
knockdown of proteins that have multiple functions often
leads to different phenotypes observed, based on different
levels of depletion. Introduction of mutant proteins might
For the last couple of decades, protein kinases have been
the most important target class in cancer drug discovery, and
the majority of inhibitors achieve their activity by competing
for binding with the phosphate donor substrate ATP. Like
protein kinases, Rho GTPases are nucleotide-binding
enzymes; therefore, it is conceivable that they could be
inhibited by nucleotide-competitive small molecules.
However, unlike protein kinases, which have moderate
binding affinities toward ATP, Rho GTPases and most other
small GTPases have very high affinity toward guanine
nucleotides making them difficult to target with nucleotide-
competitive small molecule inhibitors [5].
Furthermore, many of the Rho GTPases are involved in
multiple cellular processes and general inhibition of a Rho
GTPase may therefore have nonspecific effects. Instead, by
targeting Rho protein regulators, the biological effect is
expected to be more selective toward specific physiological and
pathological processes. Illustrating this principle, for the 20
mammalian Rho GTPases there are more than 80 guanine
nucleotide exchange factors (GEFs) that facilitate guanine
nucleotide release and more than 60 GTPase activating proteins
(GAPs) that stimulate the intrinsic GTPase activity [6, 7].
*Address correspondence to this author at the Institute for Molecular
Medicine Finland FIMM, Nordic EMBL Partnership for Molecular
Medicine, Biomedicum Helsinki 2U, P.O. Box 20 (Tukholmankatu 8), FI-
00014, University of Helsinki, Finland; Tel: +358 50 415 4900;
E-mail: krister.wennerberg@fimm.fi
1875-5402/15 $58.00+.00
© 2015 Bentham Science Publishers

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Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
van Adrichem et al.
Despite the biological and therapeutic potential of small
development, when the MgcRacGAP/MKLP1/Ect2 complex
proteins are expressed at high levels, their function has been
linked to controlling polarity and driving epithelial-to-
mesenchymal transition [26-30]. Second, it was described
that high MgcRacGAP mRNA expression in high-risk early
breast cancer as well as hepatocellular carcinoma is
associated with poor disease-free survival and has a greater
prognostic significance than traditional mitotic markers [31,
32]. Last, MgcRacGAP, together with Ect2, Rac1 and
Cdc42, regulates centromere maintenance and stable
incorporation of CENP-A after mitosis [33]. Thus, it appears
that MgcRacGAP is involved in a number of physiological
and pathological processes.
GTPase-modulating tool compounds, only a few useful
compounds have been described to date, and none target a
small GTPase GAP. For example, from an NIH Molecular
Libraries Program screening campaign, (PubChem AID
1772) probe compounds that either antagonize or stabilize
nucleotide binding in a selective or nearly selective fashion
have been reported [8-12]. Specifically, one class of these
modulators selectively inhibited the Rho family GTPase
Cdc42 in biochemical assays and interfered with Cdc42-
dependent cellular processes, such as formation of filopodia
in response to bradykinin or growth factor stimulation [8,
10]. From other studies, a compound that was originally
found as an inhibitor of β-amyloid processing, EHT-1864,
was subsequently shown to act as an antagonist of nucleotide
binding on all Rac isoforms but no other Rho GTPases [13,
14]. As a nucleotide binding inhibitor of Rac1, it blocked
GEF-mediated nucleotide exchange, as well as downstream
effector binding and inhibited Rac-dependent processes in
cells [14]. Furthermore, a Rac1-GEF interaction inhibitor,
NSC23766, was discovered by Goa and colleagues using a
structure-based virtual screening approach for compounds
that bind in a pocket of Rac1 that is important for GEF
specific regulation [13, 15]. This compound inhibited Rac-
specific GEF activity by Trio and Tiam1 in a dose-dependent
manner, but not exchange activity stimulated by the
promiscuous GEF Vav1, the interaction between Rac1 and
the GAP BCR or the interaction between other Rho proteins,
such as RhoA and Cdc42, and their GEFs. Finally, brefeldin
A blocks the activity of the Arf GEF Sec7 by stabilizing the
complex between Sec7 and Arf1, thereby blocking
nucleotide exchange [16]. In summary, inhibitors of small
GTPases themselves, GEFs and effectors have been
reported, but prior to our present study no selective
modulators of small GTPase GAP activity have been
described.
With the uncertainties about the role of MgcRacGAP, as
well as the lack of chemical tools to probe its function, we
set out to identify an MgcRacGAP inhibitor that would
provide insight into the biological role of MgcRacGAP. To
this end, we designed a high throughput screening strategy to
identify compounds inhibiting MgcRacGAP and report here
the discovery of MINC1, a selective MgcRacGAP inhibitor
that interferes with MgcRacGAP function biochemically and
appears to interfere with its function in cells. Since no small
molecule inhibitors of MgcRacGAP or any other small
GTPase GAP have been described previously, this
compound and future similar chemical probes can be
powerful tools for the research community to better define
the complete function of MgcRacGAP. Finally, the
discovery of this tool compound suggests that development
of inhibitors of other small GTPase GAPs is possible.
MATERIALS AND METHODS
Protein Expression and Purification
Expression vector for MgcRacGAP GAP domain was
constructed by ligating the cDNA corresponding to codons
for residues 345-618 of human MgcRacGAP (NCBI
accession no. NM_013277) between the EcoR I and Xho I
restriction sites of pGEX-4T-1 (GE Healthcare). Expression
vectors for BCR GAP domain (residues 1010-1271 of human
BCR (NP_004318)), p50RhoGAP GAP domain (residues
205-439 of mouse p50RhoGAP (NP_001139374)), PAK1
PBD domain (residues 72-132 of human PAK1 (AAC50590)
and Rac1 Q61L (NP_008839) were analogous to constructs
that have been described previously [34, 35]. The F28L
variant of human Rac1 was produced by PCR-based Phusion
Site-Directed Mutagenesis (Thermo Scientific) and
subcloned into the bacterial expression vector pGEX-4T-1
[35]. All DNA constructs were confirmed by DNA
sequencing.
Male germ cell Rac GTPase-activating protein
(MgcRacGAP) is essential for proper regulation of
cytokinesis, a highly complex cellular process that requires
tight spatial and temporal control and in which Rho GTPases
and their regulators and effectors are key regulatory
components [17]. At the end of anaphase, MgcRacGAP,
together with Mitotic Kinesin-Like Protein 1 (MKLP1)
forms centralspindlin, an evolutionarily conserved hetero-
tetrameric complex [18-20]. Next, the RhoGEF Ect2
localizes to the central spindle and associates with
centralspindlin to form the MgcRacGAP/MKLP1/Ect2
complex that stimulates and controls RhoA, resulting in the
correct localization of the contractile ring and the subsequent
completion of cytokinesis [19, 21]. While this process is
well-studied, the detailed functional role of MgcRacGAP, its
relevant target GTPase and its GAP activity in cytokinesis
have been debated and are not yet fully resolved [22-24].
The controversies and conflicting results on the role of
MgcRacGAP and its GAP activity reported in the literature
could very well arise from differential functions in different
cell types and species [3, 25], but are also a likely outcome
of studying a highly temporally controlled process by using
genetic and molecular biology tools that are not temporally
controlled and cannot easily be dosed.
E. coli DH5α were transformed with the pGEX
constructs, and recombinant proteins were purified as
described by García-Mata et al. [36]. Protein concentrations
were determined using a NanoDrop photospectrometer
(Thermo Scientific) and Bradford protein assay, while
protein purities were analyzed by SDS-PAGE and
Coomassie Blue staining.
Biochemical GAP Activity Assays
The primary GAP assay was performed with 600 nM
Rac1(F28L) in 15 mM Hepes (pH 7.5), 20 mM NaCl, 1 mM
Furthermore, MgcRacGAP appears to play essential roles
in other cellular processes. First, in both cancer and

Discovery of MINC1, a GTPase-Activating Protein
Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
5
EGTA, 0.02% Tween 20, 0.1 mg/mL bovine serum albumin,
2% DMSO and 150 µM GTP, in the presence or absence of
GAP domain protein at room temperature for 2 h. GAP
domain construct concentrations were titrated for use in a
linear range and concentrations used were normalized to
keep similar GAP activity as judged by signal window:
MgcRacGAP(345-618) at 2 nM, BCR(1010-1271) at
200 nM and p50RhoGAP(205-439) at 50 nM. DMSO up to
4% had no adverse effect on assay performance. The assay
was run in 96-, 384- and 1536-well format at 20, 5 and
2.5 µL volumes, respectively.
Synthesis of MINC1
Reagents were obtained from Sigma–Aldrich, TCI
Europe, and Merck, and solvents used were of pro analysis
quality. Ethylene diamine dihydrochloride was prepared
from ethylene diamine by adding 10% HCl, filtered and
dried in vacuo. Nuclear magnetic resonance spectra data can
be found in supplementary material (see Supplementary
data).
n-BuOH (3.7 mL) and H2O (19 mL) were added to
dimethyl naphthalene-2,6-dicarboxylate (3.66 g, 15 mmol,
100 mol-%). Aq. NaOH (6.25 M, 4.3 mL, 210 mol-%) was
added carefully, and the suspension formed was refluxed at
100 °C for 1h. After a clear solution was obtained the
reaction was cooled for 10 min. The organic and aqueous
phases were separated, and H2SO4 (0.98 mL, 17.4 mmol,
116 mol-%) was added to the aqueous phase. The resulting
precipitate was filtered, washed with H2O, and dried in
vacuo to produce naphthalene-2,6-dicarboxylic acid (2.94 g,
91%) as a white solid.
To measure the amount of GTP hydrolysis, the ADP
Hunter Plus assay kit (DiscoveRx) was used according to the
manufacturer’s instructions at half-volumes. Resorufin
fluorescence was measured at excitation at 530 nm and
emission at 590 nm using a PHERAstar FS (BMG Labtech)
multilabel plate reader.
Inorganic phosphate detection was performed with a
malachite green assay (Biomol Green, Enzo Life Sciences)
under primary assay conditions. To create a substantial light
path for absorbance detection, the assay was run in 384-well
half-area plate format at 15 µL volume and 15 µL of Biomol
Green was added to the sample. After 30 min of incubation,
the absorbance was read at 620 nm.
To a suspension of naphthalene-2,6-dicarboxylic acid
(865 mg, 4.0 mmol, 100 mol-%) in ethylene glycol (3 mL),
ethylene diamine (0.53 mL, 8.0 mmol, 200 mol-%), ethylene
diamine dihydrochloride (1.06 g, 8.0 mmol, 200 mol-%), and
p-toluenesulfonic acid monohydrate (63 mg, 0.33 mmol,
8.3 mol-%) were added sequentially. The reaction was
heated to 190 °C, forming a clear solution, which was stirred
for 15 h, returning to a suspension in the end. The reaction
was cooled to room temperature, and aq. HCl (20.6 mL,
267 mM) was added resulting in a clear solution to which aq.
NaOH (8 mL, 12.5 M) was added. The resulting yellow
precipitate was filtered, washed with H2O and dried in
vacuo. The hydrochloride salt was obtained by slowly
adding 10% HCl solution to a suspension of the product in
MeOH (8.5 mL). The precipitated final product was filtered
and dried in vacuo to yield 2,6-bis(4,5-dihydro-1H-imidazol-
2-yl)naphthalene dihydrochloride, MINC1, (428 mg, 38%)
as a yellow solid.
GTP hydrolysis by wild type Rac1 was measured using
the GTPase/GAP/GEF-Glo Bioluminescent Assay System
(Promega-prototype kit) as recommended by the
manufacturer. Briefly, GTPase and different concentrations
of MINC1 were prepared as a 2x solution containing 2 µM
Rac1 and 2 µM dithiothreitol in GTPase/GAP Reaction
Buffer. Next, MgcRacGAP was diluted serially into the
GTPase solution (0.1-100 nM), and this solution was
incubated at RT. After 2h, 2x GTP solution (10 µM) was
added and the solution incubated for an additional 2h to
allow the GTPase reaction. The reaction was terminated by
the addition GTPase-Glo Reagent and incubation for 30 min
followed by addition of Detection Solution. After 10 min,
luminescence was measured using a PHERAstar FS (BMG
Labtech) multilabel plate reader.
Data Analysis
Small Molecule Compounds
Z’-factor values were calculated for each plate, after
which plates with Z’-factor values lower than 0.5 were
excluded from further analysis [37]. The positive and
negative control values for each plate were normalized as
100% and 0% GAP inhibition, respectively, and used to
calculate a normalized activity of each compound tested.
Compounds showing inhibition greater than three standard
deviations (SDs) for the primary assay and more than 30%
for the orthogonal assay were considered as hits. In
selectivity screening, compounds showing inhibition greater
than 50% for the primary assay and smaller than 15% in
counter assay were considered as hits. High throughput
screening (HTS) data derived from the compounds screened
under NIH Molecular Libraries Program screening campaign
were analyzed using ActivityBase software (IDBS, Inc). All
other HTS data were processed with programs written in
Matlab (R2010a, MathWorks). Four parameter dose-
response curves were fit and constrained by 0% and 100%
inhibition as minima and maxima, respectively using
GraphPad Prism 5 (GraphPad Software). The graphs were
The 20,480 compounds from a chemical diversity
collection (ChemDiv) were transferred by the Echo 550
Liquid Handler (Labcyte) from 1 mM stock solutions into
1536-well assay plates for single dose testing (see
Supplementary Table 2 for complete protocol). Compounds
identified in single dose screening were subjected to dose
response testing at a 0.1-100 µM concentration range against
MgcRacGAP, p50RhoGAP and BCR using the primary
assay conditions and readout. The 342,046 compounds from
the NIH Molecular Libraries Small Molecule Repository
were screened in a similar fashion (See PubChem AID
624330 for complete protocol). MINC1 (PubChem CID
744230) in the ChemDiv small molecule library (4135-0021)
was reordered for confirmatory testing from Asinex (BAS
07310573) and ChemBridge (6035924) as well as
resynthesized in our laboratories (Supplementary Fig. S2, S3
for mass spectrometry spectra and Supplementary Table 3
for MINC1 compound usage). MINC2 was reordered from
NCI/DTP (NSC72365).

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Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
van Adrichem et al.
constrained by 0% and 100% inhibition as minima and
maxima, respectively.
FUCCI Cell Cycle Reporter Assays
Premo FUCCI Cell Cycle Reporter (Life Technologies,
P36237) was used according to manufacturer’s
recommendations to follow cell cycle progression and
division. Briefly, cells were plated in 6-well plates at 50,000
cells/well, and 24 h later Premo gemini-GFP and Premo Cdt-
RFP were added 1:1 to the cell culture medium. After
overnight incubation, cells were trypsinized, subsequently
plated at 1,600 cells/well and imaged every hour with phase
contrast, green and red fluorescence using an IncuCyte
ZOOM microscope (Essen Bioscience). 80 µM MINC1,
DMSO (negative control) or 20 µM benzethonium chloride
(positive for cell killing) were added 14 h later and cells
were continuously followed every h.
MgcRacGAP - Rac1 Q61L Binding Assay
Protein-protein
interaction
dynamics
between
MgcRacGAP and Rac1 (Q61L) were analyzed by bio-layer
interferometry measured with an Octet Red 384 (ForteBio)
using 384-well tilted-bottom plates (ForteBio, 18-5076).
GST-MgcRacGAP(345-618) (100 ng/µL, in 1x PBS with
2% DMSO) was immobilized on Anti-GST Dip and Read
Biosensor (ForteBio, 18-5096) for 5 min. Subsequently, the
sensor was submerged for 5 min in a solution containing
Rac1Q61L (50 ng/µL, in 1x PBS with 2% DMSO) and 10
min in 1x PBS with 2% DMSO to determine dissociation
rates, in the presence of 12.5-50 µM MINC1 or DMSO. All
steps were conducted at room temperature. The data were
obtained with Octet Data Analysis Software (version
7.1.0.35, ForteBio) and curves were plotted using GraphPad
Prism 5.
Cell Cycle Synchronization Using a Double Thymidine
Block
HeLa cells were plated in the presence of 2 mM
thymidine. After 19 h, the cells were washed three times
with 1x PBS before fresh culture medium was added. The
second 2 mM thymidine block was applied 9 h after release.
The cells were washed with 1x PBS 16 h later and released
in the presence or absence of MINC1 (10-80 µM) or
nocodazole (165 nM). The cells were followed by live cell
imaging using an IncuCyte ZOOM microscope.
Cytotoxicity and Cell Proliferation Assays
HeLa (cervical epitheloid carcinoma), MDA-MB-231
(mammary gland epithelial adenocarcinoma), A549 (alveolar
basal epithelial adenocarcinoma) and RWPE-1 (HPV-18
immortalized prostate) cells, obtained from ATCC, were
cultured in Eagle's minimal essential medium (EMEM),
Dulbecco’s modified Eagle’s medium (DMEM) or Roswell
Park Memorial Institute (RPMI) 1640 medium, with 10%
fetal bovine serum and plated in replicate sets at 1,600
cells/well in 384-well plates 6-12 h prior to drugging. Cells
were exposed for up to 72 h to 5-80 µM MINC1, DMSO
(negative control) or 20 µM benzethonium chloride (positive
cell killing control) to identify compounds that are directly
toxic to cells. CellTox Green (Promega) was added to the
Immunofluorescence Analysis
HeLa and A549 cells were plated on fibronectin-coated
coverslips or chamber slides (Nunc, 154534), respectively, at
equal densities and treated for 3-48 h with either DMSO or
25 µM MINC1. Next, HeLa cells were fixed with ice-cold
methanol and incubated with mouse anti-α-tubulin (Sigma
Aldrich, T9026) and goat anti-MgcRacGAP (Abcam,
ab2270), using goat anti-mouse Alexa Fluor 488 (Life
Technologies, A11017) or donkey anti-goat Alexa Fluor 647
(Life Technologies, A-21447) to detect the primary
antibodies, respectively. A549 cells were fixed and stained
as described previously [38], using Alexa-555 Phalloidin
(Life Technologies, A34055), Hoechst 33342 (Life
Technologies, H1399) and the abovementioned antibodies.
Coverslips were mounted with reagent containing DAPI
when appropriate (Life Technologies, P36931 and P36930)
and imaged using a Nikon 9i fluorescence microscope and
NIS elements AR software (Nikon). Color balance was
normalized for all images using ImageJ (NIH). The cells
were scored for four different morphologies, normal
(mononuclear), mitotic (rounded up), multinuclear and dead
cells, using at least eight randomly chosen fields per
condition.
growth
medium
according
to
manufacturer’s
recommendations, and the cells were followed by live cell
imaging using an IncuCyte FLR microscope (Essen
Bioscience) to determine toxicity (CellTox Green
fluorescence, Promega) and proliferation (phase contrast
confluency). These image data were quantified with the
IncuCyte application software (version 2011A rev2) and
extracted cell toxicity and proliferation data were plotted
with GraphPad Prism.
PAK-PBD Rac1-GTP Pull Down
MDA-MB-231 cells were transfected 24 h after plating
with pBABEpuro MgcRacGAP using FuGENE HD. The
next day, cells were treated with either 30 µM MINC1 or
growth medium only and after 6 h incubation, the cells were
lysed in 20 mM Hepes (pH 7.5), 150 mM NaCl, 1% Triton,
1 mM MgCl2 and protease inhibitor cocktail set III
(Millipore, 539134). Subsequently, 30 µg of GST-PAK-PBD
on sepharose beads was added to the concentration-equalized
lysates and incubated for 30 min at 4°C. The bead pellet was
washed three times with the lysis buffer and subsequently
resuspended in SDS-PAGE sample buffer. The proteins were
separated on a 12% SDS-PAGE gel, transferred to a PVDF
membrane and subsequently blotted for using anti-Rac1
antibody (BD Transduction Labs, 610650).
Cellular Movement
A549 cells were plated in 96-well ImageLock plates
(Essen Bioscience, cat. #4379) at 10,000 cells/well and after
6 h treated with either DMSO or 25 µM MINC1. The cells
were imaged every five minutes with an IncuCyte ZOOM
(Essen Bioscience) microscope using a 20x objective for a
total time of 24 h. Afterwards, randomly selected cells were
analyzed by Cell-IQ Analyser software (CM Technologies)
to track individual cell movement.

Discovery of MINC1, a GTPase-Activating Protein
Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
7
Wound Healing
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MgcRacGAP was depleted from MDA-MB-231 cells by
RNAi. Specifically, cells were transfected, at a density of
350,000 cells in a single well of a 6-well plate, with siRNAs
for MgcRacGAP (12.5 ng, pooled Qiagen SI00101178 and
SI04954880) or a scrambled sequence control (Qiagen,
1027280) by Lipofectamine 2000. The following day, the
cells were trypsinized and plated at 40,000 cells/ well in a
Matrigel precoated 96-well ImageLock plate (Essen
Bioscience, cat. #4379). After 16 h, the near confluent cell
monolayers were scratched, washed and subsequently
covered with 50 µL Matrigel (8 mg/mL, BD Bioscience
356231). After 30 min incubation at 37°C, growth medium
containing either DMSO or MINC1 (final concentration 10-
40 µM) was added. Normal growth medium was added to
the siRNA treated cells. The plate was left in an IncuCyte
microscope with the Scratch Wound assay protocol running.
Using the Scratch Wound analysis software, relative wound
density was determined for each image and subsequently
plotted with GraphPad Prism.
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RESULTS
Development of
a Robust Miniaturized Screening
Strategy for MgcRacGAP
As opposed to many ATP-binding proteins that have
been successfully targeted in drug discovery, small GTP-
binding proteins typically have very high affinity for
nucleotide (micromolar versus picomolar, respectively)
making them more challenging targets [5]. Specifically,
since nucleotide binding is very tight, a biochemical assay
with wild type small GTPases effectively becomes a near-
single turnover assay, which is not practical for HTS. In the
case of heterotrimeric G-proteins, Zielinski and coworkers
overcame the challenge of high affinity nucleotide binding
by constructing a variant of the Gαi1 GTPase subunit with a
mutation in the conserved TCAT loop that contacts the
guanine ring. This mutant showed increased GDP
dissociation, which enabled measuring multiple rounds of
turnover of GTP to GDP facilitated by the RGS4 (Regulators
of G-protein Signaling 4) GAP in a competitive fluorescence
polarization immunoassay [39].
ꢀꢁꢂꢃꢄꢅꢆ
ꢀꢁꢂꢃꢄꢇꢈꢉꢊ
Fig. (1). Design and validation of an HTS-compatible assay
measuring the biochemical activity of MgcRacGAP. A) Wild
type Rac1 cycles between GDP- and GTP-bound forms, but owing
to a very high affinity towards the guanine nucleotides, the
exchange step is highly rate limiting (red arrow). B) Rac1(F28L)
exhibits a reduced affinity for nucleotide, and therefore, a higher
rate of nucleotide exchange compared to wild type Rac1. Thus,
with Rac1(F28L) the GTPase step is rate limiting (red arrow)
producing
a sensitive, multiple turnover GAP assay. C) A
comparison of fluorescence-mediated detection of GDP released
from the GAP assay using wild type Rac1 or Rac1(F28L) shows
that the fast-cycling mutant Rac1(F28L) allows for sensitive
detection of GDP (GAP activity). Error bars represent SD (n=7 for
each condition; p<0.0001, ANOVA) (color image is available online).
To study the function of MgcRacGAP in a biochemical
assay we substituted the target GTPase, wild type Rac1, with
a mutant Rac1 (F28L) with a lowered nucleotide affinity,
typically referred to as a fast-cycling mutant [40]. With this
substitution, we overcame the single turnover dynamics,
creating an HTS-compatible GTPase assay dependent on
GAP-mediated GTP hydrolysis (Fig. 1). Specifically in this
setup, GDP dissociated from the GTPase in the absence of a
GEF, creating a continuous GTPase cycle in a GTP-rich
environment, with the GTPase step as the rate-limiting
factor, that enhanced sensitivity of the hydrolysis assay
significantly and appeared well-suited for HTS (Fig. 1C).
The Rac1 (F28L) protein allowed for a 3-4-fold signal
window between intrinsic and GAP-stimulated GTPase
activities while remaining in a linear response range of GAP
concentrations. Notably, MgcRacGAP did not exhibit GAP
activity toward RhoA (data not shown), consistent with
previous studies suggesting that MgcRacGAP is a GTPase-
activating protein for Rac1 and not RhoA [3].
The GAP-stimulated Rac1 GTPase reaction could be
measured effectively using both malachite green to detect
production of inorganic phosphate and the ADP Hunter Plus
assay kit from DiscoveRx to detect production of GDP.
When the two assays were compared in terms of Z’-factor
values [37], the ADP Hunter Plus assay gave higher and
more consistent Z'-factors than the malachite green assay.
Average Z’-factor values were 0.75 and 0.55 for the ADP
Hunter Plus assay and malachite green readouts,
respectively. Therefore, we chose to use the ADP Hunter
Plus assay as the primary detection assay method. The
malachite green assay, on the other hand, was chosen as an
orthogonal validation screening strategy given that it both
detects a different analyte and is based on a different
detection readout (absorbance vs fluorescence).

8
Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
van Adrichem et al.
After confirmation of Rac1 F28L as a fast-cycling mutant
showed robust selective inhibition of MgcRacGAP (>50%,
vertical red line, Fig. 2D) and weak inhibition of BCR GAP
activity (<15%, horizontal red line, Fig. 2D), when used at
10 µM. These nine compounds were then tested against the
GAP domain of MgcRacGAP, BCR and p50RhoGAP in a
dose response fashion, and two compounds showed high
selectivity for MgcRacGAP over BCR and p50RhoGAP.
When obtained from alternate sources and resynthesized, one
of the two selective compounds (CID 744230, Fig. 3A)
showed reproducible, MgcRacGAP-selective dose response
curves with an IC50 of 15 5 µM (Hill slope 0.9) and no
detectable activity towards BCR and p50RhoGAP (Fig. 3C).
We named this compound MINC1 (MgcRacGAP Inhibitor
Compound 1). Thus, from a screening campaign of 20,480
compounds, we identified and confirmed one compound that
showed selective biochemical inhibition of MgcRacGAP.
and the readout methods used, the MgcRacGAP-Rac1 assay
(Fig 1) was optimized for incubation times as well as protein
and GTP concentrations in a 96-well format to make it HTS
compatible. To mimic 100% inhibition of the GAP activity,
MgcRacGAP was omitted from the assay mix. The
biochemical assay was successfully miniaturized to 384- and
1536-well formats using automated liquid handling, showing
consistent Z'-factor values ≥ 0.6 (Table 1) and confirming
that the assay was HTS-compatible.
Table 1. Optimization and validation of the HTS assay.
96-Well
384-Well
1536-Well
-
+
-
+
-
+
CV
6.7%
6.9%
5.0%
4.8%
6.8%
4.2%
To
identify
additional
MgcRacGAP-inhibitory
compounds, the primary assay was screened against 342,046
compounds from the NIH Molecular Libraries Small
Molecule Repository chemical collection (PubChem AID
624330). Using the same active threshold of three standard
deviations over the mean, we identified 4,243 compounds as
active. After the primary assay, compounds determined
previously as active in another Molecular Libraries Program
screen (PubChem AID 1903) utilizing the ADP Hunter Plus
detection system were filtered out as likely false positives.
The remaining 2,362 compounds were screened with the
orthogonal inorganic phosphate detection counter screen to
further filter the positive hits, resulting in 557 compounds
that showed >30% inhibition. Although not all Molecular
Libraries Small Molecule Repository compounds could be
computationally filtered, the filtering brought the orthogonal
assay confirmation ratio from 15% (ChemDiv 37/245) to
almost 25% (Molecular Libraries Small Molecule Repository
577/2362). Subsequently, we focused on the 117 most potent
compounds (> 50% inhibition) and checked for promiscuity
by examining activity in other bioassays reported in
PubChem. As a result, we identified 13 compounds that
tested positive in a maximum of 2.5% of the previously
reported bioassays, and these compounds were chosen for
testing in a dose response assay against MgcRacGAP, BCR
and p50RhoGAP using the orthogonal malachite green
assay. From these 13 compounds, one, MINC2 (CID
251705, Fig. 3B) exhibited selectivity for MgcRacGAP over
BCR and p50RhoGAP, and selectively was confirmed in
screens with reordered compound (Fig. 3D). In conclusion,
after screening more than 360,000 compounds, we
discovered two compounds of which MINC1 was the most
promising hit and, therefore, used for followed-up studies.
1
2
Signal window
2.9
3.7
2.2
Z'-factor
CV
0.58
n/a
0.75
0.61
5.5%
5.4%
5.7%
4.4%
Signal window
Z'-factor
n/a
3.7
3.2
n/a
0.72
0.73
The biochemical GAP activity assay was optimized for
incubation times, protein and GTP concentrations in a 96-
well format and subsequently miniaturized to 384- and 1536-
well formats using automated HTS liquid handling (Z'-factor
values ≥ 0.6). Negative and positive controls are referred to
as - and +, respectively. The coefficient of variation (CV) is
the ratio of the standard deviation to the mean whereas the
signal window is the ratio of both means. The 96-well assay
validation was not repeated, (indicated as n/a in the table).
High Throughput Screening Identifies Selective
MgcRacGAP Inhibitors
After optimization we performed a high throughput
screen using a chemical diversity set from ChemDiv at
20 µM final concentrations. Compound responses from the
assay plates with Z’-factor values between 0.65 - 0.81 were
analyzed (Fig. 2A). As expected, the majority of the 20,480
compounds analyzed showed little or no GAP inhibition,
(Fig. 2B). We set the active threshold at three standard
deviations over the mean, resulting in 245 active compounds
(Fig. 2B).
The hit compounds from the primary screen were
retested in an orthogonal malachite green assay where 37
showed at least 30% inhibition (3.5xSD, Fig. 2C,
supplementary Table 1) and therefore, were considered
MINC1 Acts on the Rac1-MgcRacGAP Complex
We observed that if the proteins were pre-incubated with
MINC1 before GTP was added to start the GTPase reaction,
the dose response curves shifted and the IC50 decreased
almost one order of magnitude to 2 1 µM (Hill slope 0.7)
while no detectable activity was seen towards BCR or
confirmed hits. To exclude the compounds that target Rac1
directly or inhibit GAPs non-selectively, the 37 compounds
were examined in a counter screen using the RhoGAP
domain of BCR in place of MgcRacGAP. Nine compounds

Discovery of MINC1, a GTPase-Activating Protein
Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
9
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ꢀꢁꢂꢃꢄꢃꢅꢃꢆꢁꢄꢌꢍꢎꢈꢏꢎꢐꢑꢒꢄꢉꢊꢋ
Fig. (2). Identification of an MgcRacGAP inhibitor through a novel type of screening assay. A) Z’-factor values from plates used in the
primary screen. One plate with Z’-factor value lower than 0.5 was excluded from further analysis (#14, Z’-factor -13.55). All of the
remaining plates showed Z’-factor values >0.68. B) A screen of 20,480 chemically diverse compounds (ChemDiv) at 20 μM. The dotted
black line represents the three SDs hit cutoff. MINC1 is shown as green dot and red dots show the remaining 244 primary hit compounds. C)
Orthogonal screening results to exclude hits resulting from inhibition of reagents of the primary assay detection system. Malachite green was
used to detect GTPase turnover, and 37 compounds, shown as green (MINC1) and red dots, resulted in ≥ 30% inhibition (3.5xSD,
represented by the dotted black line). D) Counter screen for selectivity. BCR GAP domain was used to exclude compounds that target either
Rac1 or RhoGAPs in general. Of the 37 compounds testing positive through the orthogonal screen (C), 8 compounds, at 10 μM, shown as
red dots and MINC1 as green dot, resulted in high inhibition of MgcRacGAP (>50%, vertical dotted black line) and low inhibition of BCR
GAP (<15%, horizontal dotted black line) (color image is available online).
p50RhoGAP (Fig. 3E). By changing the composition of pre-
incubation mix, we determined that the shift only occurred
when Rac1 was included in the pre-incubation mix (Fig. 3F)
and that the pre-incubation with MgcRacGAP had no
potentiating effect. However, we observed no effects on GTP
hydrolysis when MgcRacGAP was substituted with a
different GAP (Supplementary Fig. S1A) or omitted from
the reaction mix (data not shown), suggesting that although
the binding to Rac1 appears to be slow and the interaction
with MgcRacGAP is fast, MINC1 acts selectively on the
Rac1-MgcRacGAP complex. This phenomenon was further
observed when we used GTP-loaded wild type Rac1 protein.
Since the single turnover assay was quick (10-20 min) and
started by adding GTP-loaded Rac1, there was no effect on
the MgcRacGAP GAP activity by addition of MINC1 to the
reaction (Supplementary Fig. S1B). Moreover, in multiple-
turnover assays using the wild type Rac1 protein, where
nucleotide exchange therefore was the rate-limiting factor,
we did not detect any notable inhibition of MgcRacGAP-
stimulated activity by MINC1 (data not shown).
To further investigate the effect of MINC1 on the Rac1-
MgcRacGAP complex we used a label-free interaction
analysis system to characterize the protein-protein binding
interactions in the presence or absence of MINC1. In these
experiments, we observed that pre-treatment of the complex
with MINC1 slowed down the dissociation process in a dose
dependent matter (Fig. 3G), suggesting that MINC1
stabilized the Rac1-MgcRacGAP complex and thereby
inhibited a GAP-catalyzed GTPase activity.
MINC1 Treated Cells Fail to Complete Cell Division
Since MgcRacGAP is necessary to complete cytokinesis
and has been shown to play an important role in centromere
maintenance, an MgcRacGAP inhibitor is predicted to have
a significant effect on cell growth over time [3, 22, 33].
However, the expectation is that such reduced cell growth is
due to MgcRacGAP inhibition and not cytotoxicity. Thus, to
discriminate between MgcRacGAP inhibition and general
cell toxicity, MINC1 was examined in dose response

10 Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
van Adrichem et al.
Fig. (3). Selective, dose-dependent biochemical inhibition of MgcRacGAP by MINC1. A) Molecular structure of MINC1, CID 744230.
B) Molecular structure of MINC2, CID 251705. C) MINC1, identified from single dose testing, was subjected to dose response analyses
(0.1-100 μM) against MgcRacGAP (red), p50RhoGAP (green) and BCR GAP (blue) in the biochemical assay. MINC1 exhibited a highly
selective inhibition of MgcRacGAP, IC50 15 5 µM. Error bars represent SD (n=8 for each condition). D) MINC2 exhibited a selective
inhibition of MgcRacGAP, IC50 18 7 µM. Error bars represent SD (n=3 for each condition). E) Dose response curves shift if the proteins are
pre-incubated with MINC1 before GTP addition, resulting in a decreased IC50 by almost 1-order of magnitude. IC50, no incubation 15 5 µM; IC50,
2 1 µM. Error bars represent SD (n=8 for each condition). E) MINC2 identified from single dose testing was subjected to dose
incubation
response analyses (0.1-100 μM) against MgcRacGAP (red), p50RhoGAP (green) and BCR GAP (blue) in the biochemical assay. F) Dose
response curves shift due to pre-incubation of Rac1 with MINC1. The presence of MgcRacGAP in the mixture had no additive effect on the
inhibition. Error bars represent SD (n=3 for each condition). G) MINC1 stabilizes Rac1-MgcRacGAP complex. Pre-treatment of the
complex with MINC1 slowed down the dissociation process in dose dependent matter. Error bars not presented (n=2 for each condition)
(color image is available online).

Discovery of MINC1, a GTPase-Activating Protein
Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1 11
analysis with various cell lines using the CellTox Green cell
death assay as a toxicity readout coupled with live-cell
imaging to determine cell proliferation rates. We noted that
treatment with MINC1 (≥20 µM) caused cell proliferation to
cease without detectable cell toxicity in highly proliferative
cells (Fig. 4) compared to negligible effects on cell viability
in slow growing cells (Supplementary Fig. S4A, B).
Therefore, in the subsequent experiments we used MINC1
concentrations between 20 and 30 µM to stay within the
“effective window” and minimize cytotoxic side effects.
Since cell death is associated with prolonged mitotic arrest
[41, 42], we used live-cell imaging data to determine
whether cell death occurred upon addition of MINC1 or at a
later stage of the treatment. Initially, we did not observe any
cell death, but as the MINC1 treatment continued and cell
proliferation ceased, the percentage of cell death started to
rise (Fig. 4B, C, Supplementary Fig. S4C, F). Although
MINC1 failed to inhibit MgcRacGAP in the wild type Rac1
biochemical assays, these results suggest that MINC1 may
have the potential to inhibit MgcRacGAP in cells.
levels using a PAK-PBD pull-down assay in cells [43]
overexpressing MgcRacGAP in the absence or presence of
MINC1. Unfortunately, overexpression of MgcRacGAP
failed to detectably lower overall Rac1 GTP levels,
suggesting that even when overexpressed, MgcRacGAP only
acts on a small sub-pool of active Rac1 and that its activity
therefore is not easily measured with a PAK-PBD pull-down
assay (data not shown).
In normal division of adherent cells, cell rounding occurs
upon entering mitosis, and cells spread again after the
completion of mitosis [44]. When treated with MINC1, cells
rounded up, but failed to complete cell division. Cell death,
however, was observed only under conditions of high
concentrations of MINC1 (80 µM; data not shown) for
prolonged periods of time in HeLa and MDA-MB-231 cells.
Interestingly, A549 cells could tolerate such high
concentrations of MINC1. Therefore, we treated A549 cells
with 80 µM MINC1 and followed cell cycle progression and
division with the Premo FUCCI Cell Cycle Reporter to
detect cells actively cycling or those arrested. Initially, very
few cells displayed a fluorescence signal, but 6 h after
MINC1 exposure, cells continuously fluoresced green
indicating that they were arrested in G2/M-phase (Fig. 5). To
further determine whether cell rounding occurs upon
entering mitosis or at any given time after the administration
of MINC1, we synchronized HeLa cells using double
thymidine block [45] and released them in either the
presence or absence of MINC1. Unlike non-synchronized
HeLa cells, where cells start to round up within
approximately 3 h of MINC1 addition, MINC1-treated
synchronized cells rounded up 8-10 h after release, similar to
the release of nocodazole-treated cells (data not shown),
indicating that the rounding occurs upon entering mitosis.
These results show that the MINC1 treated cells fail to pass
G2/M-phase, which is in agreement with MgcRacGAP
inhibition.
ꢀ
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ꢄꢁ
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ꢀꢁꢂꢃꢄꢄꢅ ꢀꢆ
ꢏ
ꢀ
50
40
30
20
10
0
40 ꢁM MINC1
DMSO
To examine more deeply the effects of cell division
arrest, HeLa and A549 cells were treated with 25 µM
MINC1 for up to 48 h and fixed at different time points for
imaging by immunofluorescence microscopy. Although
plated at similar densities, MINC1-treated cells showed
lower confluency in addition to multinucleation and
abnormalities in cell division compared with control cells
(Figs. 6, 7). To illustrate, a subpopulation of the MINC1-
100
BzCl
50
0
12
24
36
48
12
24
36
48
treated cells progressed into cytokinesis, but failed to
complete the process, resulting in multinucleated cells (Fig.
6F, asterisk), whereas others revealed abnormal mitotic
positioning of the nuclear DNA (Figs. 6D, 7C, D, arrows).
Images of MINC1-treated A549 cells taken at different time
points were used to quantify for average percentage of
normal, mitotic, multinucleated and dead cells (Fig. 6G).
This quantification demonstrated that the number of cells
representing a mitotic phenotype peaked within the first 9 h
of treatment, compared with the control cells, and the
percentage of multinucleated cells increased over time
during the 24h treatment. Furthermore, high-resolution
images of the cells with the mitotic phenotype, revealed
extensive membrane protrusions rather than the rounded
phenotype seen in untreated mitotic cells, suggesting a loss
of cortical actin rigidity (Fig. 7C, D, arrow with round end).
Thus, as predicted for an MgcRacGAP inhibitor, we
observed that MINC1 treated cells showed increased
Time (h)
Time (h)
Fig. (4). Reduced rate of human tumor-derived cell
proliferation without toxicity upon MINC1 treatment. A)
MINC1 at ≥20 µM caused MDA-MB-231 cell proliferation to
cease. MDA-MB-231 cells were subjected to a 48 h dose response
treatment of MINC1 (5-80 µM), using DMSO and benzethonium
chloride (20 µM) as negative and positive control, respectively.
IC50 17 µM; TC50 41 µM; effective window: 2.4. B-C) MDA-MB-
231 cells were treated with 40 µM MINC1 (blue), DMSO (green)
or 20 µM benzethonium chloride (BzCl, red) and followed by live
cell imaging for 48 h for proliferation (B, as determined by
confluency) or cell toxicity (C). Error bars represent SEM (n=6 for
each conditions) (color image available online) (color image is
available online).
To examine whether MgcRacGAP activity in cells is
inhibited by MINC1, we examined the GTP-loaded Rac1

12 Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
van Adrichem et al.
50
MINC1
40
DMSO
30
20
10
0
12
24
36
48
60
72
84
Time (h)
Fig. (5). MINC1-treated cells arrest in G2-/ M-phase. A549 cells transiently expressing FUCCI Cell Cycle Reporter proteins gemini-GFP
(G2-/ M-phase) and Cdt-RFP (G1-/ S-phase) were treated with MINC1 (80 µM). Six hours post treatment initiation (indicated by arrow),
cells started to express a green signal that did not fade, indicating that the cells were arrested in G2/M-phase. Camptothecin treatment (165
nM) was used as a positive reference.
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30
20
10
0
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death
DMSO
3 h
6 h
9 h
24 h
MINC1(25ꢇM)
Fig. (6). Impaired cytokinesis in MINC1 treated cells. A-F) Representative immunofluorescence images of HeLa cells treated for 48 h
with DMSO (A-C) or 25 µM MINC1 (D-F) showing DNA (left panels), α-tubulin (middle panels) and merge (right panels, DNA in blue and
α-tubulin in green). MINC1 treated cells showed more multinucleated cells (asterisks panel F) as well as mitotic cells with collapsed spindles
(arrows panel D) than control cells. Bar, 50 µm. G) Immunofluorescence images of A549 cells treated with 25 µM MINC1 were quantified
for average percentage of normal, mitotic, multinucleated and death cells per time point and results are plotted as a bar graph. Error bars
represent SEM (n>230 cells ’24 h MINC1 treated’ condition and n>320 all other conditions) (color image is available online).
numbers of multinucleated cells, but also abnormally
positioned DNA.
surface, resulting in the loss of the loosely adherent
population. Indeed, MINC1 treatment led to an increased
population of cells that failed to attach well to the tissue
culture treated plastic (data not shown). However, another
As a result of the decreased number of rounded cells, we
speculated that some cells dislodged from the culture

Discovery of MINC1, a GTPase-Activating Protein
Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1 13
ꢅꢉꢆꢈ
MINC1
ꢓ
ꢅꢉꢊ
ꢁꢄꢋꢌꢊꢌꢍꢎꢏ
ꢉꢐꢋꢌꢍꢋꢑꢊꢒ
ꢊ
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ꢅ
Fig. (7). Abnormal spindle morphology in the metaphase in MINC1 treated cells. Representative 60x immunofluorescence images of
mitotic and cytokinetic HeLa cells treated for 48 h with DMSO (A-B) or 25 µM MINC1 (C-D) stained with antibodies to MgcRacGAP (red)
and α-tubulin (green). DNA (blue) was visualized with Hoechst stain. MINC1 treated cells lacked the metaphase spindles originating from
opposite poles resulting in a collapse spindle (C-D, indicated by arrows) and were found less frequent in cytokinetic phase compared to
control cells (A-B). Extensive blebbing was observed in rounded-up MINC1 treatment cells (C-D, indicated by round ended arrows). Bar,
10 µm (color image is available online).
possibility was that the rounded cells re-adhered, which
would directly explain the increased number of
multinucleated cells. To determine the fate of the loosely
adherent cells, we created a series of time-lapse movies in
which we captured the membrane protrusions that occurred
in the MINC1-treated mitotic cells (supplementary Movie
S1-2). We noticed that part of the population of rounded
cells indeed re-adhered to the surface, whereas others stayed
round for a prolonged period of time. After approximately
20-24h, a small part of this rounded cell population
underwent apoptosis. In addition, we observed that MINC1-
treated cells lost almost all motility whereas the majority of
DMSO-treated cells were highly motile (Fig. 8A). To
evaluate whether the MINC1-induced loss of motility on
tissue culture treated plastic also extended to migration
through more complex three-dimensional extracellular
matrices, we performed a scratch-wound Matrigel invasion
assay with MDA-MB-231 cells treated with different
concentrations of MINC1. Cells treated with increasing
concentrations of MINC1 displayed decreased wound-
healing rates (Fig. 8B). Likewise, cells treated with multiple
siRNAs targeting MgcRacGAP showed decreased motility
compared to mock-treated counterparts (Fig. 8C). These data
demonstrate that MINC1 treatment led to abnormal cortical
actin activity, failed cell division and thereby an increased
population of multinucleated cells as well as a loss of cell
motility.
Taken together our results show that we were able to
design an HTS-compatible assay that measures the activity
of MgcRacGAP and utilize this assay to identify
MgcRacGAP selective inhibitors. In cell-based follow-up
assays, we showed that MINC1 is not cytotoxic and that
treatment of cells with MINC1 reduced cell proliferation by
inhibition of cell division, consistent with inhibition of
MgcRacGAP. However differences between the MINC1-
induced and siRNA-mediated MgcRacGAP knockdown
phenotypes suggest either that GAP inhibition of
MgcRacGAP has different implications than loss of the
protein or that MINC1 also affects other cell division factors
than MgcRacGAP in cells.

14 Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
van Adrichem et al.
DISCUSSION
ꢀ
1500
1000
500
0
In this study using a new HTS approach, we report the
discovery of a Rho GTPase GAP inhibitor, a tool compound
with selectivity for MgcRacGAP that can prove important
for exploring the function of this protein in cells.
Specifically, we show that this compound, MINC1, leads to
a dose responsive, selective inhibition of MgcRacGAP
biochemical activity as well as effects on the cellular
pathways downstream of Rho family GTPase function
including reduction in cell division, G2/M phase arrest,
impaired cytokinesis, and reduced motility. Importantly, we
believe that the MINC1 inhibitor demonstrates the success of
the HTS-adapted MgcRacGAP biochemical assay and opens
the door for further discovery and development of selective
Rho family GAP inhibitors.
DMSO
25 ꢇM MINC1
Previously, others have described high throughput format
GAP assays. For example, Sun et al. designed a high
throughput fluorescence polarization assay measuring the
enzymatic activity of GTPase-activating proteins of ADP-
ribosylation factors, and Zielinski et al. developed an assay
for the GAP activity of RGS4 towards the heterotrimeric G-
protein Gαi1 subunit [39, 46]. While both of these GAP
studies reported that small screens had been performed, no
hits for the GAP proteins were described. In our study, we
screened over 360,000 compounds and identified two
compounds that inhibited MgcRacGAP with high selectivity
over BCR or p50RhoGAP in biochemical GAP assays. The
most potent of these hits, a compound we termed MINC1,
inhibited cell proliferation and impaired cytokinesis,
suggesting that the inhibitor is active towards MgcRacGAP
in cells. Based on our results, we conclude that small
GTPase GAPs are highly challenging but putatively
druggable targets.
ꢔ
100
80
60
40
20
0
DMSO
10 ꢇM MINC1
20 ꢇM MINC1
40 ꢇM MINC1
12
24
36
48
60
Time (h)
-20
ꢀ
In studying the mechanism of action of the MINC1
inhibitor, we determined that while it is selective for
MgcRacGAP-mediated GTP hydrolysis by Rac1, it appears
to affect the GTPase-GAP complex with a slow binding to
Rac1. Furthermore, we noted that complete inhibition of
enzymatic activity could not be obtained with MINC1 and
that dose response curves instead reached a maximum at 80-
90% inhibition. Together, these data may suggest that
MINC1 does not necessarily block the GAP activity of
MgcRacGAP, but rather stabilizes the Rac1-MgcRacGAP
complex and, by slowing down the turnover, inhibits
MgcRacGAP activity, similar to what has been described for
the Arf GEF inhibitor brefeldin A [16]. In support of this
idea, we detected a decreased dissociation rate of the Rac1-
MgcRacGAP complex in the presence of MINC1. This
complex mechanism of action might also explain why we
failed to detect inhibition of MgcRacGAP-mediated GTP
hydrolysis in single turnover GTPase assays using wild type
Rac1.
100
80
60
40
20
0
siMock
siMgcRacGAP
12
24
36
48
60
Time (h)
-20
Fig. (8). Loss of cell motility in MINC1 treated cells. A) MINC1
treated A549 cells lose the ability to move freely in 2D culture.
Cells were imaged every 5 min, and images were used to track cell
movement over 24 h. B-C) Cells treated with increasing
concentrations of MINC1 display a decreasing ability to migrate,
and siRNA transfected cells show increased wound-healing times.
MDA-MB-231 cells were treated with an increasing dose of
MINC1 (B) or two pooled siRNAs targeting MgcRacGAP (C).
Error bars represent SEM (n=4) (color image is available online).
MINC1 is represented in PubChem as compound ID
744230, and it has been included in 23 reported bioassays.
The only reported bioactivity is a marginal activity, at
80 µM, against the essential replicative helicase of
Staphylococcus aureus (AID 485395). No close structural
analogs of MINC1 were present in our compound collection

Discovery of MINC1, a GTPase-Activating Protein
Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1 15
or commercially available; therefore, we have not explored
the structure activity relationship of MINC1 with MgcRacGAP
in this study.
polarity and migration. For example, Jacquemet and
colleagues have shown that MgcRacGAP can locally
suppress the activity of Rac1, resulting in upregulation of
RhoA activity, thereby promoting invasive migration and
that removal of MgcRacGAP by RNAi leads to the loss of
invasive migration [30]. In our experiments, we
demonstrated that MINC1-treated cells lost their invasive
migratory properties in a dose dependent matter (Fig. 8).
Furthermore, MINC1 treatment induced extensive blebbing
in mitotic cells (Fig. 7C, D), in agreement with a high
Rac1/low RhoA activity and loss of cortical actin rigidity
resulting from blocking MgcRacGAP activity. These
observations further support the idea that MINC1 affects the
MgcRacGAP-Rac1 complex in cells.
MgcRacGAP, as well as many other small GTPase
GAPs, is a key component of highly dynamic and spatially
and temporally controlled cellular processes. In the past,
studies of the cellular functions of MgcRacGAP have been
limited to methods using RNAi or overexpression of GAP-
activity-defective and other functional mutants of the protein
[20, 47, 48]. Neither of these methods can be easily applied
to a dose or temporal dependency and have a drawback of
resulting in non-physiological concentrations, artificially
high or low, of the protein in cells. Controversies in the
literature regarding
a specific role and function of
MgcRacGAP in cytokinesis as well as other processes [3, 4]
might be explained by the challenging interpretation of
results of knockdown and overexpression studies. Therefore,
we expected that the development of potent and selective
small molecule inhibitors of MgcRacGAP would be critical
for defining the detailed function and role of this protein.
In conclusion, we identified MINC1 as a putative
MgcRacGAP inhibitor from a small molecule screen of over
360,000 compounds in a biochemical assay adapted for HTS.
Future studies aim to use this chemical scaffold to identify
compounds with higher affinity and faster binding kinetics,
as well as exploring inhibitors with selectivity towards other
GAPs and potential targets. Thus, although RhoGAPs are
challenging small molecule targets, our study shows that
they are screenable and appear druggable, and we expect that
optimized analogs of MINC1 will allow us to improve the
understanding of the function of MgcRacGAP in cells and
investigate its role in both physiological processes and
human malignancy.
An interesting example case is that of Polo-like kinase 1
(PLK1). PLK1 is an essential serine/threonine kinase that
has multiple important functions in early mitotic processes,
including the direct regulation of MgcRacGAP [49, 50].
Removal of PLK1 by RNAi leads to a cell-cycle arrest in the
metaphase/anaphase transition with resulting monopolar
spindle cells [51-53], but controversial results, which could
be caused by incomplete PLK1 depletion, were obtained.
The development of potent and selective PLK1 inhibitors
permitted examination of the role of partial versus complete
inhibition [54, 55]. Furthermore, the possibility to temporally
control the inhibition allowed for detailed analysis of PLK1
in different phases of the cell cycle, demonstrating that
inhibition of PLK1 induced a loss of interaction between
Ect2 and MgcRacGAP in anaphase, preventing localization
of Ect2 to the central spindle during late mitosis and thereby
preventing initiation of cytokinesis [56].
AUTHOR CONTRIBUTIONS
AA performed laboratory studies, analyzed the data, and
wrote the manuscript. AF, LT, AL and JS performed
laboratory studies. AK coordinated the chemical synthesis.
GR performed laboratory studies and wrote the manuscript.
KW coordinated the project, analyzed the data, and wrote the
manuscript.
ABBREVIATIONS
Our studies with MINC1 revealed both expected and
unexpected effects on cell division. As expected, we saw an
accumulation of multinucleated cells; however, we also
DMSO
GAP
GDP
GEF
= Dimethyl sulfoxide
= GTPase-activating protein
= Guanosine diphosphate
observed
a
phenotype that resembled
a
failure of
prophase/pro-metaphase (Figs. 6, 7), with the chromosomes
unevenly spaced from the spindle pole, similar to a PLK1-
inhibited phenotype [57]. Moreover, the MgcRacGAP
distribution in these prophase/pro-metaphase cells seemed to
follow the α-tubulin staining, which concurs with findings of
Hirose et al. that MgcRacGAP co-localizes with the
microtubules [48]. It has been shown that inhibition of
MgcRacGAP is important for the correct formation of the
mitotic spindle during the metaphase. Specifically,
overexpression of an MgcRacGAP mutant, whose GAP
activity could not be inhibited, resulted in a lack of normal
bipolar spindles [58]. Taken together with our observation of
MINC1-mediated inhibition of MgcRacGAP, the results may
suggest that the GAP activity of MgcRacGAP plays an
important role in mitosis and that this function may be
regulated by PLK1. Alternatively, our results may result
from additional, yet unknown, MINC1-induced effects on
the cell division machinery through other targets.
= Guanine nucleotide exchange factor
= Glutatione S-transferase
GST
GTP
= Guanosine triphosphate
HTS
= High throughput screening
= Isopropyl β-D-1-thiogalactopyranoside
IPTG
MgcRacGAP = Male germ cell Rac GTPase-activating protein
MINC1
MKLP1
PLK1
PMSF
RNAi
SD
= MgcRacGAP inhibitor compound 1
= Mitotic kinesin-like protein 1
= Polo-like kinase 1
= Phenylmethylsulfonyl fluoride
= RNA interference
= Standard deviation
In addition to the role of MgcRacGAP in cytokinesis,
several studies have shown that it plays a role in cellular

16 Combinatorial Chemistry & High Throughput Screening, 2015, Vol. 18, No. 1
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CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
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ACKNOWLEDGEMENTS
This work was financially supported by the Jane and
Aatos Erkko Foundation and a NIH Molecular Libraries
Program grant (1 R03 DA033983) (KW) and by the Helsinki
Biomedical Graduate Program (AA). We thank the FIMM
Technology Center High Throughput Biomedicine unit and
Marja Hagström from Centre for Drug Research and
Division of pharmaceutical biosciences, both part of the
Biocenter Finland Drug Discovery and Chemical Biology
infrastructure platform for their technical support and the use
of their instrumentation. We also acknowledge the technical
expertise, support and use of instruments of the HTS center
at Southern Research Institute. We thank Subhanjan Mondal
and Said Goueli at Promega for the early access of the
GTPase/GAP/GEF-Glo Bioluminescent Assay System.
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Received: June 27, 2014
Revised: November 12, 2014
Accepted: December 3, 2014