Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain

ABSTRACT p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various

human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit

PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of

full-length PAKs and are selective for PAK1 and PAK3 _in vitro_ and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and

may also be used as lead molecules in the development of more selective and potent PAK inhibitors. SIMILAR CONTENT BEING VIEWED BY OTHERS DISCOVERY OF SMALL MOLECULE INHIBITORS THAT

EFFECTIVELY DISRUPT IQGAP1-CDC42 INTERACTION IN BREAST CANCER CELLS Article Open access 17 October 2022 ALTERATIONS TO THE BROAD-SPECTRUM FORMIN INHIBITOR SMIFH2 MODULATE POTENCY BUT NOT

SPECIFICITY Article Open access 08 August 2022 EXPLORING PURINE ANALOGUES AS INHIBITORS AGAINST KATANIN, A MICROTUBULE SEVERING ENZYME USING MOLECULAR MODELING APPROACH Article Open access

30 December 2024 INTRODUCTION p21-activated kinases (PAKs) are Ser/Thr kinases that are classified into two groups on the basis of their structural and functional features: group I (PAK1–3)

and group II (PAK4–6).1 Group I PAKs have an auto-inhibitory domain (also called an inhibitory switch domain) and a kinase domain (catalytic domain, CD) and are activated by the binding of

the active (that is, GTP-bound) forms of Rho GTPases, such as Cdc42 and Rac1. Group II PAKs have no auto-inhibitory domains and are not activated by active Rho GTPases. PAK1, the

best-characterized member of group I PAKs, forms auto-inhibited homodimers, in which the active site of the kinase domain of one monomer is blocked by the inhibitory switch domain (residues

87–136) of the other; the inhibitory switch domain partially overlaps with the p21-binding domain (PBD, residues 67–150; Figure 1). When Cdc42•GTP or Rac1•GTP interacts with the PBD of PAK1,

PAK1 is converted to a monomeric form, leading to a conformational change of its CD that restores its kinase activity.2, 3 This event induces the autophosphorylation of Thr423 followed by

the autophosphorylation of multiple residues in PAK1. Because the deregulation of PAKs is closely associated with various human diseases,4, 5 small-molecule inhibitors of these kinases have

great potential as therapeutic agents.6 In addition, these compounds can also be used as powerful tools in studies aimed at understanding the PAK signaling pathway. To date, several PAK

inhibitors have been developed for use as biological probes and therapeutic agents. Most of these inhibitors target the ATP-binding sites of PAKs and are referred to as ATP-competitive

inhibitors. Although ATP-competitive inhibitors may have relatively poor selectivity because of the similarity between the ATP-binding pockets of kinases of the same family, PAK-selective

ATP-competitive inhibitors have been recently identified.7, 8, 9, 10 An allosteric inhibitor, IPA-3 (2,2′-dihydroxy-1,10-dinaphthyldisulfide), has also been discovered; it binds to the

regulatory domains of PAKs rather than to their ATP-binding sites. Allosteric kinase inhibitors have the potential to be more selective than ATP-competitive inhibitors.11, 12 IPA-3 shows

enhanced specificity to group I PAKs; however, it has a disulfide bond that can be reduced under reducing conditions. We therefore sought to develop novel allosteric inhibitors of PAKs.

Herein, we describe naphtho(hydro)quinone (N(H)Q)-based small molecules that allosterically inhibit PAK activity by binding to the regulatory domains (PBDs) rather than to the ATP-binding

sites. The compounds that we developed selectively inhibit the activities of the group I PAKs, PAK1 and PAK3. MATERIALS AND METHODS MATERIALS [γ-32P] ATP was purchased from Perkin-Elmer

(Waltham, MA, USA), SuperSignal West Pico Chemiluminescent substrate kit from Thermo Fisher Scientific Inc. (Waltham, MA, USA) and polyvinylidene difluoride membranes (HybondTM) from GE

Healthcare Life Sciences (Pittsburgh, PA, USA). Horseradish peroxidase (HRP)-conjugated anti-glutathione _S_-transferase (GST) antibodies were purchased from Abcam (Cambridge, MA, USA),

anti-phospho-PAK antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), anti-total myelin basic protein (MBP) antibodies from Millipore (Billerica, MA, USA) and anti-myc

antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Plasmids to express the YFP-PBD and CFP-V12Cdc42 fusion proteins for fluorescence resonance energy transfer (FRET)

analysis were a gift from Dr Michiyuki Matsuda.13 THE CHEMICAL LIBRARY The Korea Chemical Bank of the Korea Research Institute of Chemical Technology (Daejeon, Korea) provided 1280 small

molecules whose molecular weights are less than 300 Da. CHEMICALS 1,4-naphthohydroquinone (1,4-NHQ; compound 1), 1,4-naphthoquinone (NQ; compound 7), 2-hydroxy-1,4-NQ (compound 9),

2-methyl-1,4-NQ (compound 10) and 2-methoxy-1,4-NQ (compound 11) were purchased from Sigma-Aldrich (St Louis, MO, USA). Other compounds used in this study (Table 1) were synthesized

(Supporting Information). CLONING, EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS The cDNAs for Cdc42 (wild type or constitutively active V12Cdc42) and active PAK1 (T423E and H83L/H86L

T423E) were subcloned into pGEX-4T to produce GST fusion proteins. The cDNA for wild-type PAK1 (Wt-PAK1) was subcloned into pCMV-myc to produce a myc fusion protein. The cDNA for the Wt-PBD

of PAK1 and the mutated PBD (H83L/H86L) were cloned into pGEX-4T to produce GST fusion proteins and into pET-24a to produce His-tagged proteins. These cDNAs were amplified by PCR and were

ligated into each plasmid vector. The plasmids were expressed in _Escherichia coli_, and GST- and His-tagged proteins were purified by glutathione-Sepharose and Ni2+-agarose affinity

chromatography, respectively. Wt-PAK1 was obtained from Abcam. ELISA-BASED SCREENING OF SMALL-MOLECULE INHIBITORS A 96-well microplate was coated with His-tagged PBD (PBD-His; final

concentration of 1 μg ml−1) in a carbonate–bicarbonate buffer (pH 9.6) at 4 °C overnight. The plate was washed three times with Tris-buffered saline that contained Tween-20 (TBS-T, 25 mM

Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20). To prepare Cdc42•GTP, GST-Cdc42 was treated with GTPγS in a Tris buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 1 mM

dithiothreitol, 5% glycerol) for 0.5 h at room temperature (RT). GST-Cdc42(GTPγS) (100 ng ml−1) was incubated with small molecules (40 μM) from the library for 0.5 h at RT and then for 1 h

at 37 °C. After wells were washed with TBS-T three times, an HRP-conjugated anti-GST antibody (final concentration: 200 ng ml−1) was added to the wells and incubated for 1 h at 37 °C. Wells

were then washed with TBS-T three times and the substrate solution, which contained 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in 0.1 M citrate–phosphate buffer (pH 5.6), was

added. After 15–30 min, the reaction was terminated by the addition of TBS-T containing 1% SDS. Absorbance at 405 nm was then measured. Colorimetry was performed using a SpectraMax M2

instrument (Molecular Devices, Sunnyvale, CA, USA). Compounds that reduced the Cdc42(GTPγS)–PBD interaction by at least 60% were selected as ‘hits’ for subsequent studies. SURFACE PLASMON

RESONANCE (SPR) ANALYSIS SPR analysis of the binding between small molecules and the PBD was performed with a BIAcore 2000 instrument (Uppsala, Sweden). GST (control) and GST-PBD (wild-type

or mutated LL (H83/L and H86/L)) were covalently immobilized on CM5 chips (GE Healthcare Life Sciences) through amine-coupling methods with

_N_-ethyl-_N_-(dimethylaminopropyl)carbodiimide/_N_-hydroxysuccinimide according to the manufacturer’s instructions. The final immobilization levels were ~10 000 resonance units. Analysis

was performed at 25 °C using a running buffer (10 mM HEPES, 150 mM NaCl, 0.005% P20, 0.4% dimethylsulphoxide (DMSO), pH 7.4). Various concentrations (12.5, 25, 50 or 100 μM) of each chemical

were injected into the cells at a flow rate of 20 μl min−1. The equilibrium dissociation constant (_K_d=_k_d/_k_a) was derived from the calculated dissociation rate constant (_k_d) and the

association rate constant (_k_a) using BIAevaluation software, version 3.0.2 (GE Healthcare Life Sciences). All curves were fitted using a 1:1 Langmuir association/dissociation model. CELL

CULTURES HeLa and 293T cells were cultured in minimum essential medium and Dulbecco’s modified Eagle’s medium, respectively, which were supplemented with 10% fetal bovine serum (Invitrogen,

Carlsbad, CA, USA). The culture media contained 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. CELL VIABILITY HeLa cells were cultured in a 96-well plate at a density of 2 × 105 cells

per ml. Test compounds were added to cell cultures at a final concentration of 20 μM and incubated for 2, 4, 6, 8 or 12 h at 37 °C. After the indicated time periods, each well was loaded

with WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolum, monosodium salt] at a final concentration of 10% (v/v) and incubated for 2 h at 37 °C.

Absorbance was read at a 450-nm wavelength, followed by colorimetry using a SpectraMax M2. PAK ASSAYS For the _in vitro_ PAK1 assay, purified GST-tagged active PAK1 (T423E mutant, 400 ng)

and dephosphorylated MBP(2.5 μg) were mixed in a kinase buffer (20 mM 3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.2, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM

dithiothreitol). This mixture was pre-incubated with various concentrations of each inhibitor for 0.5 h at 30 °C and was further incubated with 5 μCi [γ-32P] ATP for 0.5 h at 30 °C. The

reaction was terminated by the addition of SDS-polyacrylamide gel electrophoresis sample buffer. Reaction mixtures were separated on 15% SDS-polyacrylamide gels, transferred onto

polyvinylidene difluoride membranes and autoradiographed by exposure to X-ray film. To monitor PAK1 and PAK3 activation in cultured cells, HeLa cells were transfected with 2 μg of cDNA

encoding pCMV-PAK1 or pCMV-PAK3 using Lipofectamine 2000 (Invitrogen) for 24 h. Cells were pre-incubated with inhibitors for 2 h and then stimulated with 0.4 M sorbitol for 0.5 h to

stimulate PAK kinase activity. Cells were then extracted with a lysis buffer (50 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 200 mM Na3VO4 and 100 mM NaF).

Activated PAK1 and PAK3 were detected by immunoblotting with an antibody that recognized phospho-PAK1. Activation of a downstream effector, MEK1, was monitored by immunoblotting for MEK1

phosphorylated at S298 (pMEK1S298). IMMUNOBLOTTING Proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane in TGM

buffer (25 mM Tris-base, 200 mM glycine, 20% methanol). Membranes were blocked with 3% bovine serum albumin in TBS-T for 0.5 h, incubated with each primary antibody for 1 h at RT and washed

three times with TBS-T. Then, membranes were blotted with a secondary HRP-conjugated antibody for 1 h at RT and washed five times with TBS-T. Signals were visualized using the SuperSignal

West Pico Chemiluminescent substrate kit. FRET ANALYSIS HeLa cells were plated onto poly-L-lysine-coated 18-mm glass coverslips and transfected with a plasmid for subsequent FRET analysis;

12 h after transfection, cells were treated with DMSO or 20 μM inhibitor, and FRET efficiency was determined over a period of 1.5 h. Cells were imaged with an Olympus IX81-ZDC inverted

microscope equipped with a cooled CCD camera (Cascade 512B, Photometrics, Tucson, AZ, USA) and controlled by MetaMorph software, version 7.1.7 (Molecular Devices). At an excitation

wavelength of 433 nm, increases and decreases in FRET were observed at 530 and 475 nm, respectively, using a 440AF21 excitation filter, a 455DRLP dichroic mirror and two emission filters

(480AF30 for CFP and 535AF26 for FRET) (Omega Optical, Brattleboro, VT, USA). Cells were illuminated with a 75-W xenon lamp through a 12% ND filter and a × 40 UPlanSApo objective. The

exposure time for 2 × 2 binning was 200 ms to obtain the images of CFP and FRET. After background subtraction from the captured images, FRET/CFP ratio images were generated by dividing each

FRET image value by the corresponding CFP image value using the MetaMorph software, and the results were used to represent FRET efficiency. To calculate the relative ratios, the values of

FRET at time zero were arbitrarily set to 1. DATA CONVERSION For graphic conversion, the data from the ELISA and FRET analyses and cell viability tests were transformed using SigmaPlot 10.0

(Systat Software Inc, San Jose, CA, USA). RESULTS SCREENING FOR COMPOUNDS THAT DISRUPT THE CDC42–PBD INTERACTION TO IDENTIFY POTENTIAL PAK INHIBITORS To search for small-molecule inhibitors

that inhibit PAK1, we developed a screening protocol involving an ELISA based on a strong Cdc42–PBD interaction. For this assay, Cdc42 was expressed as a GST fusion protein (GST-Cdc42) and

the PBD as a His-tagged protein (PBD-His). GST-Cdc42•GTP was added to a 96-well plate coated with PBD-His in the presence of small molecules (1280 compounds) with molecular weights less than

300 Da. The disruption of the Cdc42–PBD interaction caused by the small molecules was assessed using an HRP-conjugated anti-GST antibody. Initial screening identified two compounds,

1,4-naphthohydroquinone (compound 1, 1,4-NHQ) and 2-methoxycarbonyl-1,4-naphthohydroquinone (compound 2, 2-Mc-1,4-NHQ), which effectively inhibited the Cdc42–PBD interaction _in vitro_.

Compounds 1 and 2 blocked the association of Cdc42 with the PBD and had IC50 values of 7.25 and 6.59 μM, respectively (Table 1). We refer to these compounds as Cdc42–PBD dissociators

hereafter. STRUCTURE-ACTIVITY RELATIONSHIP OF CDC42–PBD DISSOCIATORS To examine the effects of substituents on the ability of the inhibitors to block the Cdc42–PBD interaction, we

synthesized a variety of 1,4-NHQ derivatives and their oxidized forms and evaluated their inhibitory potencies. Interestingly, 1,4-naphthoquinone (compound 7, 1,4-NQ) and

2-methoxycarbonyl-1,4-naphthoquinone (compound 8, 2-Mc-1,4-NQ) had inhibitory activities that were similar to those of the reduced forms of compounds 1 and 2, respectively (Table 1 and

Supplementary Figure 1). Notably, 1,4-NHQ and the 1,4-NQ derivatives with electron-withdrawing groups (compounds 2, 6, 8, 13 and 14) showed high inhibitory activities. However, derivatives

with electron-donating groups (compounds 3–5 and 9–12) had no substantial inhibitory activities. These results suggest that two substituents, regardless of the 1,4-NQ and 1,4-NHQ scaffolds,

are important for inhibitory activity. Because Rac1 can also bind to the PBD,14 we hypothesized that the same inhibitors might also interfere with the Rac1–PBD interaction. Indeed, similar

experiments performed with Rac1•GTP instead of Cdc42•GTP showed that these compounds also inhibited Rac1’s interaction with the PBD but were less effective than for the Cdc42–PBD interaction

(Table 1 and Supplementary Figure 2). ANALYSIS OF BINDING BETWEEN CDC42–PBD DISSOCIATORS AND THE PBD To determine whether the Cdc42–PBD dissociators directly bound to the PBD, their binding

affinities were measured using SPR spectroscopy. For these studies, purified GST (control) and GST-PBD were immobilized on a modified gold surface, and various concentrations of each

Cdc42-PBD dissociator were subsequently applied. The dissociation constants (_K_d values) for the interactions of compounds 1, 2, 7 and 8 with the PBD were determined to be less than 7 μM

(Figures 2a, b and Supplementary Figure 3). Although reliable _K_d values for the interactions of halogen-containing inhibitors (compounds 13 and 14) with the PBD were not obtained from the

SPR analysis, we did observe that these compounds effectively interfered with the Cdc42–PBD interaction (Supplementary Figure 4), which was consistent with the ELISA results. In contrast, no

substantial binding was observed between the PBD and the other derivatives that did not affect the Cdc42–PBD or Rac1–PBD interaction. Because these Cdc42–PBD dissociators directly bind to

the PBD, we refer to them as PBD binders (PBD-binding compounds) hereafter. The PBD binders reversibly suppress the Cdc42–PBD interaction because after binding to the PBD they could be

readily washed out during the SPR experiments. To further examine their reversible binding to the PBD, we performed a washout experiment in which a PBD-coated microplate was washed after its

incubation with the PBD binders, and then Cdc42 was applied. Washing away the tested PBD binders almost completely restored Cdc42 binding to the PBD compared with the binding observed in

the DMSO-treated control (Supplementary Figure 5), indicating that binding was reversible. We also performed a MALDI-TOF analysis of the incubation mixtures containing the PBD and PBD

binders (compounds 1 and 2) to investigate whether binding was covalent or non-covalent and did not find any adducts (Supplementary Figure 6). Collectively, these results support a

reversible association between the PBD binders examined and the PBD. The Cdc42/Rac1-interactive binding (CRIB) motif (residues 75–90; PAK1 numbering) of the PBD is important for its

interaction with Cdc42 and Rac1.14, 15 To test whether this motif was important for the PBD interaction with the PBD binders, we constructed a mutated PBD in which His83 and His86 were

replaced with Leu (thus named PBD-LL) and whose Rho GTPase-binding ability was abolished.16 If the CRIB motif is involved in the PBD interaction with the PBD binders, the binding affinity to

the mutant PBD-LL should be attenuated. We therefore used SPR analysis to compare the binding abilities of the PBD binders (compounds 1, 2, 7, and 8) to the wild-type PBD (Wt-PBD) and

PBD-LL. As shown in Figure 2c and Supplementary Figure 7, the binding affinities of the PBD binders to PBD-LL were significantly reduced (to approximately half of those for Wt-PBD binding).

EFFECTS OF PBD BINDERS ON THE ACTIVITY OF FULL-LENGTH PAK _IN VITRO_ AND IN LIVING CELLS We next examined if the PBD-binding ability of the PBD binders affected the kinase activity of

full-length PAK1, which would be important for their biological and therapeutic applications. Initially, we investigated their ability to suppress the Cdc42-dependent activation of

full-length PAK1. The kinase activity of PAK1 can be readily monitored by measuring the phosphorylation of its substrates, such as MBP. Most PBD binders (except compound 8) suppressed MBP

phosphorylation in a dose-dependent manner (Figure 3a), which demonstrates their ability to block Cdc42-dependent PAK1 activation. We investigated the potential for a direct suppression of

PAK1 kinase activity by PBD binders using PAK1T423E, which is constitutively active even without Cdc42 binding.17 MBP phosphorylation was strikingly suppressed by all the PBD binders except

compound 8 (Figure 3b). These results strongly support the hypothesis that PBD binders act as allosteric inhibitors of full-length PAK1 _in vitro_. To assess the functional implication of

the reduced affinity of the PBD binders to PBD-LL (Figure 2c), we examined their inhibitory effects on the kinase activity of the mutant form of full-length PAK1 (PAK1-LL; H83L/H86L). If the

PBD binders require the CRIB motif for their interaction with PAK1, they should have little inhibitory effect on the kinase activity of PAK1-LL because of their reduced binding. We prepared

constitutively active PAK1T423E with the LL mutation (PAK1T423E/LL) because PAK1-LL does not interact with Rho GTPases and thus is not activated by Cdc42. In DMSO-treated controls, MBP was

phosphorylated by both PAK1T423E and PAK1T423E/LL (Figure 3c). PBD binders (compounds 1, 2, 6 and 7) markedly suppressed PAK1T423E activity but had little inhibitory effect on PAK1T423E/LL.

Even a single substitution (H83L or H86L) was sufficient to abrogate the inhibitory effects of the PBD binders (Figure 3d). This observation, together with the results of SPR studies using

PBD-LL, suggests that PBD binders inhibit PAK1 activity, at least in part, by interacting with the CRIB motif; this interaction may stabilize the inactive conformation of PAK1. We next

evaluated the inhibitory potency of the PBD binders in living cells. First, to determine whether the compounds suppressed the Cdc42–PBD interaction, we performed FRET analysis with cells

expressing the YFP-PBD and CFP-V12Cdc42 fusion proteins (Figures 4a–c).13 V12Cdc42 is constitutively active because of its defective GTPase activity. Thus, V12Cdc42 should constitutively

interact with the PBD in cells. In the absence of PBD binders, high FRET should be observed due to the proximity of YFP and CFP caused by the V12Cdc42–PBD interaction (Figure 4a). However,

if PBD binders prevent the V12Cdc42–PBD interaction by binding to the PBD, FRET efficiency should be reduced as a result of the longer distance between YFP and CFP. Changes in FRET

efficiency were determined in the absence and presence of the compounds. When cells were exposed to 20 μM of compounds 1, 2 or 7, no significant cytotoxicity was observed (data not shown),

but the FRET efficiency decreased (Figure 4b; quantified in Figure 4c). In contrast, cells treated with the inactive compound 3 did not exhibit any detectable change in FRET efficiency

(Figures 4b and c). Unfortunately, we could not perform FRET analysis with compounds 6, 13 and 14 because of their light-induced cytotoxicity. However, these compounds did inhibit PAK1

kinase activity in cells (described below). These results indicate that PBD binders can block the Cdc42–PBD interaction in cultured cells in a manner similar to that observed _in vitro_.

Finally, the effects of PBD binders on the kinase activity of full-length group I PAKs (1–3) in HeLa cells were investigated. Because PAK2 is the major isoform in this cell line (Figure 4d),

PAK1 and PAK3 (myc-tagged forms) were ectopically expressed. Cells that expressed the three PAKs were separately exposed to each compound (compounds 1, 2, 6, 7, 13 or 14) for 2 h and then

treated with 0.4 M sorbitol.18 Their inhibitory effects were assessed using an anti-phospho-group I PAK antibody, which detects autophosphorylation of PAK1 at T423, PAK2 at T402 and PAK3 at

T421. The autophosphorylation of this residue in each PAK reflects its active state. In the absence of the compounds, the autophosphorylation of each of the three PAKs increased upon

sorbitol treatment (Figure 4d, lanes 1 and 2). Importantly, PAK1 autophosphorylation was greatly suppressed by compounds 1, 2, 6, 7 and 14 but was only weakly suppressed by compound 13,

whereas PAK3 autophosphorylation was greatly suppressed by compounds 1, 2 and 7 but not by compounds 6, 13 and 14 (Figure 4d, top and bottom panels). In contrast, the tested compounds had no

significant inhibitory effects on endogenous PAK2 activity (Figure 4d, middle panel). These findings are in agreement with the results of _in vitro_ inhibition studies using full-length

PAKs (Supplementary Figure 8). Because MEK has been reported to function downstream of PAK1,19 we examined whether compounds 1, 2 and 7 would affect MEK activation. The pMEK1S298 levels were

markedly decreased after inhibition of pPAK1T423 (Figure 4e). These results suggest that the tested compounds may be useful tools for elucidating PAK signaling pathways. ANALYSIS OF THE

ISOFORM SELECTIVITY OF ALLOSTERIC PAK INHIBITORS To understand the molecular basis of inhibitor selectivity, we performed a kinase domain-swapping experiment in which the PAK1 or PAK3 CDs

were swapped (Figure 5a), and the inhibitory effects of the PBD binders on these kinases were assessed. Sorbitol activated both PAK1 and R1K3, in which the PAK1 CD was replaced by the PAK3

CD, and both kinases were almost completely inhibited by compounds 7 and 14 (Figure 5b, left). PAK3 was specifically inhibited by only compound 7 (Figure 5b, right). R3K1, in which the PAK3

CD was replaced by the PAK1 CD, showed a similar inhibitory response (Figure 5b, right) to that of PAK3. These results suggest that the inhibitory potential of both compounds depends on the

PAK regulatory domain. To test the selectivity of the inhibitors toward other members of the PAK family, full-length PAKs (1–6) were incubated with Cdc42•GTP, [γ-32P]-ATP and MBP in the

presence of each inhibitor (40 μM), and MBP phosphorylation was analyzed. Interestingly, compounds 1, 2 and 7 suppressed the activities of both PAK1 and PAK3 to a similar degree, whereas

halogen-containing compounds 6, 13 and 14 showed selective inhibition of PAK1 (Supplementary Figure 8). However, none of the tested compounds inhibited PAK2 or PAK4–6. We also used compound

2 to test 30 other kinases representing most kinase families. Compound 2 did not show significant inhibitory activity against any of these kinases with the exception of myosin light chain

kinase (~50% inhibition; Supplementary Figure 9). DISCUSSION Despite enormous efforts to develop PAK-specific inhibitors as therapeutic drugs, no compound is currently being studied in a

clinical trial, because PF-3758309 did not proceed beyond phase I studies.8 The large ATP-binding pocket with high N-lobe mobility in PAKs has impeded the identification of high-affinity

ATP-competitive inhibitors. Structural similarities in the ATP-binding pockets between PAK isoforms has made it even more difficult to find isoform-selective inhibitors. In parallel with

studies targeting ATP-binding sites, studies on targeting allosteric sites have been conducted and have resulted in the identification of IPA-3.11, 12 In the present study, we developed a

new ELISA-based high-throughput screening system to search for potential allosteric inhibitors of group I PAKs. Our screening is based on the ability of the compounds to disrupt the

interactions between Cdc42 or Rac1 and the PBD of PAK1. In addition, we showed that the identified compounds directly bound to the PBD and inhibited the activities of group I PAKs, except

PAK2, but not those of group II PAKs. Thus, we demonstrated the validity of our high-throughput screening for the identification of allosteric inhibitors of PAKs. Modification of this

high-throughput screening for the screening of compounds that directly bind to the PBD by using a chip-based chemical library would also be feasible. However, allosteric inhibitors,

including IPA-3, have a lower potency than ATP-competitive inhibitors, which is an issue that must be resolved in the future. The Cdc42/Rac1–PBD interaction involves the CRIB motif, which

contains the critical residues His83 and His86.14, 15 In accordance with the importance of these histidines, a single substitution of any of these residues with Leu appeared to be sufficient

to disrupt the interaction. Consistent with this result, the allosteric inhibitors bound the double-mutant form of PBD, PBD-LL, less efficiently than they bound the Wt-PBD, and did not

inhibit the activity of full-length PAK1-LL. Because the CRIB motif is almost identical in all group I PAKs, one might expect that the allosteric inhibitors would be effective against all

three PAKs of this group. However, they had no inhibitory effect on the activity of PAK2 (Figure 4d and Supplementary Figure 8), suggesting the involvement of additional residues surrounding

the CRIB motif. The N-terminus of PAK2 is shorter than those of PAK1 and PAK3; thus, PAK2 may lack critical residues that stabilize the PBD–inhibitor interaction. This further suggests a

potential for the identification of isoform-selective compounds that are able to discriminate between PAK1 and PAK3. Indeed, compounds 6, 13 and 14 specifically inhibited PAK1 but not PAK3

_in vitro_ and in cultured cells. Several steps are required for the full activation of group I PAKs3, 20: the initial disruption of the inactive dimeric state, phosphorylation at T423 (PAK1

numbering) and subsequent autophosphorylation at multiple sites. PAK1T423E is thus considered to be an intermediate active form. The allosteric inhibitors identified in the present study

suppressed the activity of PAK1T423E (Figure 3b) and also the Cdc42-dependent (Figure 3a) and sorbitol-induced (Figure 4d) activation of Wt-PAK1. IPA-3 has been reported to effectively

inhibit the Cdc42-dependent activation of wild-type group I PAKs in an inactive conformation; thus, PAK1T423E is not susceptible to inhibition by IPA-3. These differences indicate the

presence of distinct inhibitory mechanisms for IPA-3 and compounds identified in the present study. Analysis of the X-ray crystal structures of the N-termini of group I PAKs in a complex

with allosteric inhibitors would reveal the detailed mechanisms of these interactions. 1,4-NQ derivatives are found in a large number of natural products, and some of them have been used as

herbal medicines and show cytotoxicity against cancer cells and microbes21, 22. Thus, 1,4-NQ-based small molecules are attractive compounds for clinical use because of their potential

application as anticancer and antimicrobial agents, although the scientific rationale for their use at the molecular level has not yet been clearly defined. Naphthoquinone compounds have

been suggested to exert their cellular effects by modulating cellular redox cycling, alkylating DNA and breaking DNA strands.23 Recent studies have expanded the list of their intracellular

targets, which now also includes DNA topoisomerase,24, 25 epidermal growth factor receptor26 and Cdc25 phosphatase.27 However, the target proteins for most 1,4-naphthoquinone derivatives

still remain to be identified; such information would be necessary for their therapeutic use. The present study provides evidence that group I PAKs are novel targets of naphthoquinone

compounds. In recent years, group I PAKs, especially PAK1, have emerged as therapeutic targets in diverse types of cancer.28, 29 Given that NQs and their derivatives may target group I PAKs,

they may be useful as lead compounds in drug discovery. The cytotoxicity of NQ-based compounds is mostly due to their thiol reactivity; thus, chemical modifications to reduce this

reactivity may generate non-toxic inhibitors30 and may also reduce unwanted side effects arising from interactions with non-target proteins. In summary, we identified small molecules that

allosterically inhibit PAK activity by interacting with their regulatory domains (PBDs) rather than with their ATP-binding sites, both _in vitro_ and in living cells. The identified

inhibitors selectively suppressed the activity of group I PAKs, particularly PAK1 and PAK3. The comparison of the inhibitors identified in the present work with those reported previously

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289: 7702–7717. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by a National Research Foundation of Korea (NRF) grant

funded by the Korean government (MSIP; No. 2007-0054930). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Research Institute and Quality Management Team, NanoPharm Corp.,, Jincheon-gun, Korea

Duk-Joong Kim * Division of Planning and Research, Korea National Institute of Health, KCDC, Osong Health Technology Administration Complex, Cheongju, Korea Chang-Ki Choi * Livestock

Products Standard Division, Food Standard Planning Office, Food Safety Policy Bureau, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju, Korea

Chan-Soo Lee * Division of Medical Science Knowledge Management, Center of Biomedical Sciences, Korea National Institutes of Health, Osong Health Technology Administration Complex, Cheongju,

Korea Mee-Hee Park * Department of Chemistry, Yanbian University, Yanji, China Xizhe Tian * New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Korea Nam Doo

Kim * Green Chemistry Division, Korea Research Institute of Chemical Technology, Daejeon, Korea Kee-In Lee, Joong-Kwon Choi & Jin Hee Ahn * Department of Biochemistry, College of

Medicine and Medical Research Center, Chungbuk National University, Cheongju, Korea Eun-Young Shin & Eung-Gook Kim * Department of Chemistry, Center for Biofunctional Molecules, Yonsei

University, Seoul, Korea Injae Shin Authors * Duk-Joong Kim View author publications You can also search for this author inPubMed Google Scholar * Chang-Ki Choi View author publications You

can also search for this author inPubMed Google Scholar * Chan-Soo Lee View author publications You can also search for this author inPubMed Google Scholar * Mee-Hee Park View author

publications You can also search for this author inPubMed Google Scholar * Xizhe Tian View author publications You can also search for this author inPubMed Google Scholar * Nam Doo Kim View

author publications You can also search for this author inPubMed Google Scholar * Kee-In Lee View author publications You can also search for this author inPubMed Google Scholar * Joong-Kwon

Choi View author publications You can also search for this author inPubMed Google Scholar * Jin Hee Ahn View author publications You can also search for this author inPubMed Google Scholar

* Eun-Young Shin View author publications You can also search for this author inPubMed Google Scholar * Injae Shin View author publications You can also search for this author inPubMed 

Google Scholar * Eung-Gook Kim View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to Injae Shin or Eung-Gook Kim.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Supplementary Information accompanies the paper on Experimental & Molecular

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http://creativecommons.org/licenses/by-nc-nd/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Kim, DJ., Choi, CK., Lee, CS. _et al._ Small molecules that allosterically

inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain. _Exp Mol Med_ 48, e229 (2016). https://doi.org/10.1038/emm.2016.13 Download citation * Received: 20

October 2015 * Revised: 29 December 2015 * Accepted: 30 December 2015 * Published: 29 April 2016 * Issue Date: April 2016 * DOI: https://doi.org/10.1038/emm.2016.13 SHARE THIS ARTICLE Anyone

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