Chemoproteomics Reveals the Antiproliferative Potential of Parkinson's Disease Kinase Inhibitor LRRK2-IN-1 by Targeting PCNA Protein

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00325

LRRK2-IN-1, one of the first selective inhibitors of leucine-rich repeat kinase 2 (LRRK2), was serendipitously found to exhibit potent antiproliferative activity in several types of human cancer cells. In this study, we employed a chemoproteomic strategy utilizing a photoaffinity probe to identify the cellular target(s) of LRRK2-IN-1 underlying its anticancer activity. LRRK2-IN-1 was found to induce cell cycle arrest as well as cancer cell death by specifically binding to human proliferating cell nuclear antigen (PCNA) in cancer cells. Our current findings suggest the potential of LRRK2-IN-1 as a novel pharmacological molecule for scrutinizing cell physiology and furnish a logical foundation for the future development of therapeutic reagents for cancer.

Full text of DOI:10.1021/acs.molpharmaceut.8b00325

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Article
Chemoproteomics Reveals the Anti-proliferative Potential of Parkinson’s
Disease Kinase Inhibitor LRRK2-IN-1 by Targeting PCNA Protein
Weichao Li, Yiqing Zhou, Guanghui Tang, Nai-Kei Wong, Mengquan Yang, Dan Tan, and Youli Xiao
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00325 • Publication Date (Web): 11 Jul 2018
Downloaded from http://pubs.acs.org on July 13, 2018
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Chemoproteomics Reveals the Anti-proliferative Potential of Parkinson’s
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Disease Kinase Inhibitor LRRK2-IN-1 by Targeting PCNA Protein
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Weichao Li,1 Yiqing Zhou,1 Guanghui Tang,2 NaiꢀKei Wong,3 Mengquan Yang,1,4 Dan Tan,5 Youli Xiao1,4,*
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1CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences,
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200032, China. Eꢀmail: ylxiao@sibs.ac.cn.
2School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen
518055, China
3State Key Discipline of Infection Diseases, Shenzhen Third People’s Hospital, The Second Affiliated
Hospital, Shenzhen University, Shenzhen 518112, China.
4University of Chinese Academy of Sciences, Beijing 100039, China
5Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
* Corresponding author
Prof. Youli Xiao
CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences,
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200032, China
Phone: 86ꢀ21ꢀ54924226
ylxiao@sibs.ac.cn
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ABSTRACT
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LRRK2ꢀINꢀ1, one of the first selective inhibitors of leucineꢀrich repeat kinase 2 (LRRK2), was
serendipitously found to exhibit potent antiꢀproliferative activity in several types of human cancer cells.
In this study, we employed a chemoproteomic strategy utilizing a photoaffinity probe to identify the
cellular target(s) of LRRK2ꢀINꢀ1 underlying its antiꢀcancer activity. LRRK2ꢀINꢀ1 was found to induce
cell cycle arrest as well as cancer cell death through specifically binding to human PCNA (proliferating
cell nuclear antigen) in cancer cells. Our current findings suggest the potential of LRRK2ꢀINꢀ1 as a
novel pharmacological molecule for scrutinizing cell physiology and furnish a logical foundation for the
future development of therapeutic reagents for cancer.
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KEYWORDS
LRRK2ꢀINꢀ1; click chemistry; chemical proteomics; PCNA; antiꢀproliferative
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INTRODUCTION
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Parkinson’s disease (PD) is a devastating neuronal degeneration disorder afflicting ~1% of
people over 60 years of age worldwide.1 Mutations and aberrant activity of leucineꢀrich repeat kinase 2
(LRRK2), a ~280 kDa multiꢀdomain protein bearing serine/threonine kinase activity, have been
suggested to play significant roles in PD,2 galvanizing much interest in the discovery of LRRK2
inhibitors for the treatment of PD.3ꢀ5 In 2011, significant progress was achieved in this field when
LRRK2ꢀINꢀ1 and CZCꢀ25146 were established as potent and selective LRRK2 inhibitors (Fig. 1A).6,7
However, while these inhibitors are “selective” within a finite kinome based on highꢀthroughput
screening with purified kinases in vitro, the specificity of these compounds across an extended scope of
proteome (e.g., ADP/ATPꢀdependent protein interactome, or even the whole proteome) may vary
considerably. For instance, glyoxalaseꢀ1, has been shown to be an offꢀtarget of CZCꢀ25146 in HeLa
cells.8 Also, other pharmacological effects of LRRK2ꢀINꢀ1 have been reported, including antiꢀcancer
activity, ocular toxicity, and proꢀinflammatory or immunomodulatory activity in neuroꢀinflammation.9ꢀ11
While the primary mechanism of LRRK2ꢀINꢀ1 have been extensively studied, the molecular target(s)
responsible for its offꢀtarget effects, however, have remained elusive, which imposes a realistic obstacle
to translational applications in medicine.
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To tackle the above issues, we examined the antiꢀproliferative activity of LRRK2ꢀINꢀ1 towards a
series of human cancer cell lines, including Jurkat (lymphocyte), HeLa (cervical carcinoma), HCT116
(colon cancer), MCFꢀ7 (breast cancer), and HepG2 (hepatocarcinoma). As shown in Figure 1B,
LRRK2ꢀINꢀ1 doseꢀdependently blunted the growth of these human cancer cells, with efficacy in low
micromolar range in Jurkat cells. Cell cycle analysis revealed that LRRK2ꢀINꢀ1 markedly increased the
proportion of Jurkat cells in G2/M phases (Figure 1C and Figure S4). Because no expression of LRRK2
was detected in Jurkat cells,6 there might be additional functional target(s) of LRRK2ꢀINꢀ1 in this cell
line. Previously, LRRK2ꢀINꢀ1 has been reported to elicit cell cycle arrest by suppressing DCLK1 kinase
activity and inhibiting its mRNA and protein expression, which in turn inhibits downstream cꢀMyc
signaling in colorectal and pancreatic cancer cells.12 However, in human T lymphocyte Jurkat cells, we
found LRRK2ꢀINꢀ1 inhibits cell proliferation without affecting the expression of DCLK1 and cꢀMyc
(Figure S5), suggesting the existence of additional target(s).
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Recently, a number of research groups including us have reported the employment of chemical
proteomics in target identification of bioactive small molecules.13ꢀ16 In the present study, we combined
AfBPP (affinityꢀbased protein profiling) strategy with multiple molecule/cellꢀbased experiments to
identify the molecular targets as well as mode of actions of LRRK2ꢀINꢀ1 in Jurkat cells. Proliferating
cell nuclear antigen (PCNA), a processivity factor in cell replication, recruit proteins involved in
chromatin remodeling, DNA repair, DNA replication, and epigenetics,17 was identified as the potential
offꢀtarget of LRRK2ꢀINꢀ1. Interacting of PCNA by LRRK2ꢀINꢀ1 offers a unifying mechanism to
explain the antiꢀproliferative activities of LRRK2ꢀINꢀ1, which could provide molecular basis for the
development of related antiꢀcancer reagents.
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EXPERIMENTAL METHODS
General Methods. All reagents used for the synthesis were purchased from TCI (Tokyo Kasei
Kogyo, Inc), SigmaꢀAldrich (St. Louis, USA), or MedChemExpress (Shanghai, China) and used without
further purification. All recombinant proteins, enzymes, antibodies, and bioꢀreagents were purchased
from Promega (Madison, USA), Life Technologies (Carlsbad, USA), Abcam (Cambridge, USA),
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Thermo Fisher Scientific (Waltham, USA), or Sangon (Shanghai, China). HꢀNMR and 13CꢀNMR
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spectra were recorded on a Bruker Advance III 500 (1H: 500 MHz, C: 125 MHz) with chemical shifts
in ppm relative to residual Dꢀchloroform (δH 7.26 and δC 77.16) as standard. HRꢀMS analyses were
performed on a Waters Acquity UPLC tandem with a QꢀExactive™ mass spectrometer (Thermo
Scientific). Confocal studies were performed using a laser scan microscopy Leica TCS SP8 STED 3X.
Proteomic analyses were performed on a Thermo Orbitrap Fusion™ Lumos mass spectrometer (Thermo
Scientific).
Kinase enzyme activity assay. The biochemical IC50 values of LRRK2ꢀINꢀ1 and LK2ꢀP1 against
LRRK2 were measured by Invitrogen Adapta® assays. Data analyses were performed with GraphPad
Prism 7.0.
Labeling of recombinant protein. Recombinant LRRK2 kinase (Merck, Inc.), PCNA (Abcam,
Inc.), or BSA (Sangon Biotech) was diluted to 0.1 mg mLꢀ1 in PBS. For inhibitorꢀcompetitive reactions
pretreated with LRRK2ꢀINꢀ1 (100 µM, 10×), MLiꢀ2 (100 µM, 10×) or staurosporine (10 µM) for 30
min, then LK2ꢀP1 (10 µM) was added as indicated for 30 min at on ice. Samples were transferred to 96ꢀ
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well plate in a same line and irradiated with UV (365 nm, 8 Watt) on ice for 20 min. Each of 20 µL
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protein samples were added with 1% SDS and fresh prepared of Tetramethylrhodamine azide
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(TAMRAꢀN3,
100 µM,
Lumiprobe),
CuSO4
(1
mM,
Sigma),
Tris(3ꢀ
hydroxypropyltriazolylmethyl)amine (THPTA, 100 µM, Sigma), and sodium ascorbate (1 mM). The
samples were reacted at 37 oC for 1 hour, then separated by SDSꢀPAGE gel and detected by a FLA 9000
plus DAGE scanner (Fujifilm). Finally, the gel was visualized by Coomassie blue staining or silver
staining.
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Gel-based AfBPP in Jurkat cells. Jurkat cells were incubated respectively with probes or DMSO
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at 37 C (for the enrichment experiment: the cells were treated with DeadꢀDayne (10 µM) or LK2ꢀP1
(10 µM), respectively for 3 hours; for the competitive experiment: the cells were treated with LRRK2ꢀ
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INꢀ1 (50 µM) used for competition or DMSO together with LK2ꢀP1 (10 µM) for 3 hours at 37 C) in
2.0 mL of fresh RPMIꢀ1640 growth medium. After remove the growth medium, the cells then washed
three times with iceꢀcold PBS, followed by irradiated by a UV lamp (365 nm, 8×3 watt) for 20 min. The
cells were harvested by scraping the cells in iceꢀcold lysis buffer (50 mM HEPES, pH 8.0, 150 mM
NaCl, 0.1% Triton Xꢀ100, 0.1 mM EDTA and cOmplete protease inhibitors). The lysed cells were
centrifuged at 14,000 g for 5 min and the soluble fractions were adjusted to 2 mg mLꢀ1 with lysis buffer
containing 1% sodium dodecyl sulfate (SDS, w/v). Click reaction was performed in each sample as
described above.
Mass spectrometry-based AfBPP. The probe incubation and proteome preparation procedures
were same as that of labeling studies mentioned above. Each of 300 µL of proteome sample was mixed
with biotinꢀN3 (500 µM, Biomatrick Inc.), CuSO4 (1 mM), THTPA (100 µM) and sodium ascorbate (1
mM) for 2 hours at 37 oC. The proteomes were then precipitated with adding CH3OH/CHCl3/H2O: 4/1/2
(v/v) and vortexed for 5 min. After centrifuge at 14,000 g for 3 min, the protein disk was washed with
CH3OH (500 µL), airꢀdried and reꢀdissolved in 200 µL of 1% SDS in PBS. 50 µL of Streptavidin
Sepharose High Performance affinity resin (GE Healthcare) were added to each sample and rotated
gently at room temperature for 1 hour. Then the beads were washed sequentially with 1.0 % SDS in
PBS three times, 0.5 M NaCl in PBS three times, 50 mM triethylammonium bicarbonate (TEAB)
containing 4 M Urea two times, and 50 mM TEAB five times. All enriched proteins were subjected to
on beads reduction with 5 mM of dithiothreitol (DTT, 250 µL) at 56 °C for 30 min and alkylation with
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50 mM iodoacetamide (IAA, 250 µL) at 37 °C in dark for additional 30 min. Finally, the beads were
washed three times with 100 mM of TEAB. Bound proteins then were digested by 0.5 µg of trypsin
(Thermo Fisher) dissolved in 30 µL of 50 mM TEAB overnight at 37 °C. All digested peptides were
reacted with TMTꢀsixplexTM Isobaric Label Reagent (Thermo Scientific), based on the manufacturer’s
procedures, respectively (TMT6ꢀ126 for DMSO treated sample; TMT6ꢀ127 for DeadꢀDayne treated
sample; TMT6ꢀ128 for LK2ꢀP1 treated sample; TMT6ꢀ128 for LK2ꢀP1 and LRRK2ꢀINꢀ1 treated
sample). After desalted by Pierce C18 spin columns, the labeled peptides were diluted in 30 µL of
acetonitrile/H2O/formic acid (v:v:v = 5%/94.5%/0.5%). All pulldown experiments were performed in
biological triplicates. A volume of 2 µL of peptides was loaded on a Acclaim PepMap100 column (5
µm, 0.3 mm × 5 mm) and eluted on an Acclaim PepMap RSLC column (30 µm × 10 cm).
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Protein identification and quantification. ProteinGroups files were used for analysis using
Perseus 1.5.1.6. For all files, proteins falling into the following categories were removed: identified only
by site, reverse, contaminant. Data were matched to annotation data downloaded from Uniprot
(http://www.uniprot.org/) in October, 2017 (proteome ID: UP000005640, total sequence 20230). The
mass spectra raw data were analyzed by Proteome Discoverer 2.1 (Thermo Scientific). TMTꢀsixplex
(Lys and Nꢀterminal) and carbamidomethyl (Cys, +57.0215 Da) were used as static modifications,
oxidation (Met, +15.9949 Da) was used as a variable modification. Protein ratios were calculated as the
median of all peptide hits belonging to a certain protein. Statistical analysis was performed with
Perseus. TMT ratios obtained from Proteome Discoverer 2.1 were transformed by log2(x), normalized
using Zꢀscore, ꢀlog10(pꢀvalue) were obtained by a two sided one sample tꢀtest over three biological
replicates. Proteins were ranked according to the sum of the ranking values from TMT ratios and ꢀ
log10(pꢀvalue) across both experiments (enrichment experiment: LK2ꢀP1/DeadꢀDayne, competition
experiment: LK2ꢀP1/LK2ꢀP1+LRRK2ꢀINꢀ1). Only proteins identified have ratios higher than 4.0 and pꢀ
values less than 0.05 were considered as statistical significant targets.
Binding site identification. 1 µg of recombinant PCNA protein (Abcam, Inc.) in PBS (20 µL)
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were incubated with 10 µM of LK2ꢀP1 at 0 C for 1 hour, followed by irradiated by a UV lamp (365
nm, 8×3 watt) for 20 min. After SDSꢀPAGE gel separation and reductive alkylation, 0.5 µg of trypsin
(Thermo Scientific) was added and the sample was incubated at 37 °C overnight. The digests were
desalted by Ziptip desalting column (Pierce), dried on a SpeedVac. The dried peptides were diluted in
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20 µL of acetonitrile/H2O/formic acid (v:v:v = 5%/94.5%/0.5%) with sonication and analyzed by
Thermo Orbitrap FusionTM Lumos proteomic mass spectrometer as mentioned above. The mass spectra
data was analyzed by Proteome Discoverer 2.1 (Thermo Scientific). The database used was the protein
sequence of human PCNA (UniprotKB ID: P12004, sequence download date: June, 2017). The search
parameters used were as follows: Trypsin Digestion; carbamidomethyl (Cys, +57.0215 Da), oxidation
(Met, +15.9949 Da), LK2ꢀP1 probe modified residue mass difference 567.29 amu was specified as
variable modification at serine, valine, glycine, arginine, isoleucine, proline, tryptophan, leucine,
phenylalanine, methionine, cysteine, glutamic acid, tyrosine, lysine, histidine, threonine, alanine and
aspartic acid residues. Fragment Mass Tolerance was 0.02 Da, Peptide Mass Tolerance was 10 ppm and
target false discovery rate (FDR) was 1%. Manual verification was performed to ensure confident
peptide identification
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RESULT AND DISCUSSION
First, a LRRK2ꢀINꢀ1ꢀderived photoaffinity probe, LK2ꢀP1 (Figure 2A) was synthesized by
tagging a minimalꢀsized diazirineꢀcontaining clickable handle to the scaffold (Scheme 1).18 Deadꢀ
Dayne, a negative control probe, was employed to exclude potential nonꢀspecific binding proteins.19 In
vitro kinase assays showed LK2ꢀP1 exhibited comparable activity towards LRRK2 as its parent
compound (Figure 2B). Next, we examined the labeling efficiency of LK2ꢀP1 on recombinant LRRK2
proteins. Competition experiment showed that the labeling was indeed activityꢀbased (Figure 2C). Then,
we carried out cellꢀbased assays to examine the antiꢀproliferative activity of the probe. LK2ꢀP1 inhibited
the growth of Jurkat cells with efficacy similar to that of LRRK2ꢀINꢀ1 (Figure S6), and also caused cell
cycle arrest in the G2/M phase (Figure S4). Collectively, these results demonstrate that LK2ꢀP1 is
indeed a cellꢀpermeable probe that sufficiently retains LRRK2ꢀINꢀ1 activity for exploring potential
binding proteins in living cells.
In order to detect the binding protein(s) of LRRK2ꢀINꢀ1, we performed gelꢀbased labelling
experiments in living Jurkat cells. Cells were incubated with LK2ꢀP1 (10 µM) in the presence or
absence of excess LRRK2ꢀINꢀ1 (50 µM, 5ꢀfold) as a competitor. To discriminate background proteins
of diazirineꢀalkyne moiety, an identically designed experiment was added with the control probe Deadꢀ
Dayne (10 µM). After probe incubation and photocrosslinking, Jurkat cells were lysed. The samples
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were coupling to TAMRAꢀN3 via click chemistry and analyzed by SDSꢀPAGE gelꢀbased fluorescence
(Figure 3A and Figure S7). Proteins of the size around 35 kDa were labelled predominately (lane 2),
with most of these labelled proteins being able to be abolished with excess LRRK2ꢀINꢀ1 (lane 3). Probe
labelling of virtually all detected proteins was UVꢀdependent and few proteins showed up as
photocrosslinkerꢀbinders (lanes 1 and 2). Furthermore, intracellular click reaction was applied to
visualize the subcellular localization of LK2ꢀP1. LK2ꢀP1ꢀ or DeadꢀDayneꢀ (both 10 µM) treated cells.
After UVꢀcrosslinked, fixed and permeabilized, Jurkat cells were conjugated to TAMRAꢀN3. In LK2ꢀ
P1ꢀtreated cells, fluorescent signals were observed in both the nucleus and cytoplasm, while few signals
were observed in DeadꢀDayneꢀtreated cells, suggesting the fluorescence represented proteins labeled by
LK2ꢀP1 (Figure 3B).
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To identify the cellular targets of LK2ꢀP1, we next performed largeꢀscale pullꢀdown/LCꢀMS
experiments by quantitative proteomics. Probeꢀlabelled proteomes prepared same as those in gelꢀbased
experiments were conjugated with biotinꢀN3 and enriched by pullꢀdown with streptavidin sepharose
affinity resin. Enriched proteins were processed for on beads tryptic digestion and the digestion products
were labelled with tandem mass tag (TMT) reagents, respectively, followed by mass spectra analysis.
The proteomics experiment was carried out in biological triplicates. To reduce falseꢀpositives, identified
proteins were ranked in the corresponding volcano plots as a (log2) of enrichment ratio (LK2ꢀP1 versus
DeadꢀDayne) as well as competition ratio (LK2ꢀP1 versus LK2ꢀP1 + LRRK2ꢀINꢀ1) against statistical
significance (log10 pꢀvalue). Proteins enriched or competed by a factor > 4 with pꢀvalue < 0.05 were
considered hits (Figure 4A, 4B and Tables S1, S2). As shown in Figure 4A and 4B, proliferating cell
nuclear antigen (PCNA), a nuclear protein plays significant roles in DNA synthesis and cell
proliferation, especially in cancerous cells, was identified as the most significant hit in both enrichment
and competition experiments.17 Similar proteomics experiments were repeated in HeLa cells, as shown
in Figure S8, PCNA also significantly targeted, confirms PNCA as the offꢀtarget of LRRK2ꢀINꢀ1
(Tables S3, S4)。 Inꢀgel competitive photoꢀlabelling experiments provided evidence that PCNA was
specially labelled in both UVꢀ and LRRK2ꢀINꢀ1ꢀdependent manners (Figure 4C). Enrichment of PCNA
was also confirmed by competition pulldown and immunoblotting experiments as previously described
(Figure 4D). Furthermore, to assess whether PCNA is engaged by LRRK2ꢀINꢀ1 in living cells, a cellular
thermalꢀshift assay (CETSA) was also performed to compared the thermal stability of PCNA between
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drugꢀ and DMSOꢀtreated Jurkat cells.20 PCNA was significantly stabilized upon drug treatment in situ
with ꢁTm of 6.7 °C, as indicated by a significant temperature increase required for protein loss by
precipitation compared to DMSO controls (Figure 4E). Collectively, these results confirmed PCNA as a
specific LRRK2ꢀINꢀ1 binding protein.
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Structurally, three monomers of PCNA configured as a hexagonal structure, form a molecular
sliding clamp around the DNA double helix.17 The interior of the clamp interacts with DNA, while the
outer surface of the clamp contains an interdomain connector loop (IDCL), which was a binding area of
many PCNAꢀinteracting proteins.21 To investigate the binding site of LK2ꢀP1, recombinant PCNA
proteins photoꢀlabelled with LK2ꢀP1 was digested and analyzed by mass spectra.22 The peptide
218ATPLSSTVTLSMSADVPLVVEYK240 was found to be covalently modified by the probe whose
molecular weight increased by an expected increment (567.29 Da, LK2ꢀP1 with loss of N2) relative to
the unmodified peptide fragment. Further analysis of the MS2 spectrum of this peptide localized the
labelling on the side chain carboxyl group of Glu238 (Figure 5A and Figure S9). Based on the identified
binding site, we then performed molecular docking studies to elucidate the interaction at atomic level.
As shown in Figure 5B and Figure S10, the aminopyrimidine scaffold of LRRK2ꢀINꢀ1 is in close
proximity of the IDCL domain (residues Glu124ꢀIle128), a key region responsible for interacting with
binding proteins.23 As a key modulator of DNA synthesis, PCNA specifically binds with two DNA
polymerases, namely DNA polymerases δ (Pol δ) and ε (Pol ε), to regulate DNA replication and repair
in eukaryotes.17, 23ꢀ24 Therefore, we speculated that LRRK2ꢀINꢀ1ꢀcomplexed PCNA becomes relatively
insensitive to the DNA polymerases binding, resulting in the reduction of DNA replication and
synthesis. To verify the hypothesis, proteins immunoprecipitated by antibody of PCNA from both
LRRK2ꢀINꢀ1ꢀ and DeadꢀDayneꢀtreated Jurkat cells were subjected to immunoblot analysis by using
respective antibodies. Both DNA polymerases in LRRK2ꢀINꢀ1ꢀtreated cells showed a weakened binding
affinity to PCNA (Figure 5C), suggesting that LRRK2ꢀINꢀ1 did disrupt the binding between PCNA and
DNA polymerases δ and ε. Because disruption of PCNAꢀDNA polymerases interaction has been
reported to cause the inhibition of DNA synthesis and hence cell apoptosis,25ꢀ27 we thus examined the
level of DNA synthesis upon LRRK2ꢀINꢀ1 treatment by 5ꢀethynylꢀ2'ꢀdeoxyuridine (EdU) incorporation
assay.28 T2AA, a known PCNA inhibitor, was tested in parallel as positive control.26 As shown in
Figure 5D, incorporation of EdU was doseꢀdependently inhibited by LRRK2ꢀINꢀ1 and T2AA with IC50
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values of 0.26 µM and 1.63 µM, respectively, validating our discovery. Furthermore, We utilized short
hairpin RNA (shRNA)ꢀbased knockdown of PCNA to validate its roles in LRRK2ꢀINꢀ1ꢀmediated
inhibition of cell proliferation as previously reported.29 Knockdown of PCNA was confirmed by
Western blot (Figure 5E), and resulted in an obvious cell cycle arrest in G2/M phase (Figure 5F) which
was similar to that of known small molecules PCNA inhibitors, such as p21 and PCNAꢀI1.27,30 Taken
together, these results further confirmed LRRK2ꢀINꢀ1 exhibits its antiꢀproliferative activity through
binding PCNA and acting as a proteinꢀprotein interaction (PPI) inhibitor of PCNA in Jurkat cells,
linking PCNA binding with phenotypic response. MLiꢀ2, a wellꢀknown potent and highly selective
LRRK2 inhibitor, was used in parallel as validated negative control.34 Both in Gelꢀbased competitive
labelling experiment and cell proliferation assay, MLiꢀ2 did not shown the similar activity in
comparisons with LRRK2ꢀINꢀ1 (Figure S11). Together these data strongly link PCNA to the cell
proliferation inhibition response to LRRK2ꢀINꢀ1.
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CONCLUSION
So far, diaminopyrimidine scaffold agents have been extensively applied in medicine
chemistry.31,32 However, the high frequency of offꢀtarget activity of small molecules could lead to
unanticipated outcomes in drug development programs which seriously hindered the development of
this field.33 In the present study, we reported the first proteomeꢀwide profiling of LRRK2ꢀINꢀ1 in living
cancer cells by using a cellꢀpermeable photoaffinity probe, LK2ꢀP1. With LK2ꢀP1, PCNA was
identified as a potential offꢀtarget of LRRK2ꢀINꢀ1 in Jurkat cells. Accordingly, we carried out multiple
molecule/cellꢀbased experiments to validate PCNA as a specific target of LRRK2ꢀINꢀ1. We
demonstrated that LRRK2ꢀINꢀ1 specifically binds PCNA and prevents it from interacting with DNA
polymerases, which reveals a novel mechanism for the antiꢀproliferative activity of LRRK2ꢀINꢀ1.
Regardless additional offꢀtargets that could escape the examination with the AfBPP strategy, this work
emphasize the unique inhibitory effect of PCNA on the cell proliferation inhibition of LRRK2ꢀINꢀ1.
These results should furnish a logical foundation for the future design and development of related kinase
inhibitors as well as antiꢀcancer agents.
ASSOCIATED CONTENT
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Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI:
10.1021/acsmolpharmaceut.xxx.
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Additional details of experiments and material characterization including the synthetic method of LK-
P1, biological experimental details, and quantitative proteomic analyze detail lists (PDF).
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AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: ylxiao@sibs.ac.cn. Tel: +86ꢀ21ꢀ54924226.
ORCID
Weichao Li: 0000ꢀ0001ꢀ6875ꢀ7315
Yiqing Zhou: 0000ꢀ0002ꢀ6391ꢀ3259
Mengquan Yang: 0000ꢀ0001ꢀ6473ꢀ1409
Youli Xiao: 0000ꢀ0002ꢀ4803ꢀ3333
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
This work was supported by grants including XDPB0402 of CAS, NSFC 21572243 and 21502206. We
thank X. Deng from Xiamen University for valuable discussion, and staff members (Y. Liu, Y. Shan, Y.
He, and S. Bu) in the Core Facility Centre of the Institute of Plant Physiology and Ecology for technical
assistance in mass spectrometry, confocal imaging and NMR. We also thank Y. Tao from IHS, CAS for
kind support in cell experiments.
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Figure 1. (A) Structure of LRRK2ꢀINꢀ1. (B) Cell proliferation assays of LRRK2ꢀINꢀ1 against human cancer cell lines.
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(C) Cell cycle analysis in Jurkat cells after LRRK2ꢀINꢀ1 (10 µM) treatment, demonstrating robust G2/M arrest.
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Figure 2. LRRK2ꢀINꢀ1 based photoꢀaffinity probe. (A) Structure of LK2ꢀP1 and DeadꢀDayne. (B) Doseꢀdependent
inhibition of LRRK2 by LRRK2ꢀINꢀ1, LK2ꢀP1 and DeadꢀDayne. Values represent the means ± S.D. of duplicates in
two independent experiments. (C) Profiles for competition of LK2ꢀP1 labelling with LRRK2ꢀINꢀ1 (LKI) and
staurosporine (STA, a panꢀkinase inhibitor). Probeꢀlabelled proteins were visualized by click conjugation to the
TAMRAꢀazide tag, SDS gel separation, and fluorescence scanning. The concentration used for LK2ꢀP1 in labeling
experiments was 10 µM.
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Figure 3. Fluorescence labeling of LK2ꢀP1 in Jurkat cells. (A) Gelꢀbased profiling of LK2ꢀP1 labelled in intact Jurkat
cells. (B) Confocal fluorescence imaging of Jurkat cells. The cells were treated with LK2ꢀP1, irradiated by UV light as
indicated, conjugated with TAMRAꢀN3 (red) and stained with the nuclear DNA dye DAPI (4,6ꢀdiamidinoꢀ2ꢀ
phenylindole, blue) (BF, bright field).
Figure 4. Chemoproteomics profiling of LRRK2ꢀINꢀ1 in Jurkat cells. (A, B) Quantitative mass spectrometryꢀbased
profiling of LRRK2ꢀINꢀ1 binding proteins in intact Jurkat cells. Green and red dots depict selected targets that are
outcompeted by DeadꢀDayne (A) or LRRK2ꢀINꢀ1 (B) (criteria: log2ꢀfold enrichment ≥ 2 and −log10 (pꢀvalue) ≥ 1.33).
(C) Competition of LK2ꢀP1 (10 ꢂM) labeling recombinant PCNA (lanes 1ꢀ3) and BSA (lanes 4ꢀ6), LRRK2ꢀINꢀ1 at a
concentration 5 folds that of LK2ꢀP1. (D) Preliminary validation of PCNA by PD/WB from LK2ꢀP1 (10 ꢂM)ꢀlabelled
Jurkat lysates. The corresponding control experiments were done with DMSO, the negative probe (DeadꢀDayne) and
LRRK2ꢀINꢀ1 at a concentration 5 folds that of LK2ꢀP1 (10×LRRK2ꢀINꢀ1). (E) Cellular thermalꢀshift assay (CETSA).
Stabilization of PCNA after incubating cells with LRRK2ꢀINꢀ1/DMSO, respectively. The band intensity of soluble
o
PCNA at 47 C is set at 100%, the band intensity of the remaining soluble protein at different temperatures were
measured, the data were fitted to obtain apparent ꢁTm values using the Boltzmann Sigmoid equation within GraphPad
Prism 7.0.
Figure 5. Validation of PCNA as an offꢀtarget of LRRK2ꢀINꢀ1. (A) MS2 spectrum of LK2ꢀP1ꢀmodified peptide with
PCNA protein, (LK2, LK2ꢀP1; O, Oxidation). (B) Docking of LRRK2ꢀINꢀ1 into the PIPꢀbox of PCNA (PDB ID code:
3WGW). (C) DNA polymerase δ and ε could not be coꢀimmunoprecipitated with PCNA after LRRK2ꢀINꢀ1 treatment
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in Jurkat cells. (D) Effects of LRRK2ꢀINꢀ1 and T2AA on DNA replication in Jurkat cells. The EdU incorporation and
dose dependence of drug treatment were quantified by fluorescent microplate reader. (E) Validation of PCNA
knockdown by Western blot analysis. Cells treated with scrambled shRNA served as a negative control (Scr). (F) Jurkat cells
were transfected with ShꢀScr or ShꢀPCNA, and analysed via flow cytometry after 24 h.
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Molecular Pharmaceutics
Figure 4
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Molecular Pharmaceutics
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Figure 5
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Molecular Pharmaceutics
SCHEME
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Scheme 1. Synthesis of LK2ꢀP1. Reagents and conditions: (a) 1ꢀEthylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
hydrochloride (EDCI), 1ꢀHydroxybenzotriazole (HOBt), N,NꢀDiisopropylethylamine (DIPEA), DMF, 0°C to rt, 12 hr,
85%; (b) dicyclohexylphosphioꢀ2’,4’,6’ꢀtriꢀisopropylbipheny (XꢀPhos), tris(dibenzylideneꢀacetone)dipalladium
(Pd3(bda)3), K2CO3, tꢀBuOH, 100°C, 4 hr, 40%; (c) 1. TFA, DCM, rt, 1 h, 100%; 2. 3ꢀ(butꢀ3ꢀynꢀ1ꢀyl)ꢀ3ꢀ(2ꢀiodoethyl)ꢀ
3Hꢀdiazirine, K2CO3, DMF, 50°C, 12 hr, 50%.
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