Back Pocket Flexibility Provides Group II p21-Activated Kinase (PAK) Selectivity for Type I 1/2 Kinase Inhibitors

Back Pocket Flexibility Provides Group II p21-Activated Kinase (PAK)Selectivity for Type I 1/2 Kinase InhibitorsSteven T. Staben,*,†,¶ Jianwen A. Feng,†,¶ Karen Lyle,△ Marcia Belvin,‡ Jason Boggs,§ Jason D. Burch,†

Ching-ching Chua,# Haifeng Cui,∇ Antonio G. DiPasquale,○ Lori S. Friedman,‡ Christopher Heise,∥

Hartmut Koeppen,△ Adrian Kotey,# Robert Mintzer,∥ Angela Oh,⊥ David Allen Roberts,† Lionel Rouge,⊥

Joachim Rudolph,† Christine Tam,⊥ Weiru Wang,⊥ Yisong Xiao,◆ Amy Young,‡ Yamin Zhang,∇

and Klaus P. Hoeflich*,‡

†Department of Discovery Chemistry, ‡Department of Translational Oncology, △Department of Pathology, §Department of DrugMetabolism and Pharmacokinetics, ∥Department of Biochemical and Cellular Pharmacology, and ⊥Department of Structural Biology,Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States#Medicinal Chemistry, Evotec, Abingdon, Oxfordshire OX144SA, United Kingdom∇Pharmaron-Beijing, 6 Taihe Road, Beijing 100176, People’s Republic of China○X-ray Crystallography Facility, University of California, Berkeley, California 94720, United States◆Wuxi AppTec, 288 Fute Zhong Road, Shanghai 200131, People’s Republic of China

*S Supporting Information

ABSTRACT: Structure-based methods were used to design apotent and highly selective group II p21-activated kinase(PAK) inhibitor with a novel binding mode, compound 17.Hydrophobic interactions within a lipophilic pocket past themethionine gatekeeper of group II PAKs approached by thesetype I 1/2 binders were found to be important for improvingpotency. A structure-based hypothesis and strategy forachieving selectivity over group I PAKs, and the broadkinome, based on unique flexibility of this lipophilic pocket, ispresented. A concentration-dependent decrease in tumor cellmigration and invasion in two triple-negative breast cancer cell lines was observed with compound 17.

■ INTRODUCTION

Approximately 30% of human tumors harbor somatic gain-of-function mutations in the RAS family of small GTPases thatserve to transduce mitogenic signals from cell surface receptortyrosine kinases to intracellular serine/threonine kinases.1 p21-activated kinases (PAKs) are members of the STE20 family ofserine/threonine kinases that lie downstream of RAS andregulate many cellular processes that are commonly perturbedin cancer, including migration, polarization, and proliferation.2

The PAK family is comprised of six members and is subdividedinto two groups (groups I and II) on the basis of sequence andstructural homology. The group I and II PAKs share a numberof conserved structural characteristics, such as a p21-bindingdomain, multiple proline-rich regions, and a carboxyl-terminalkinase domain. However, the kinase domains of group I and IIPAKs share only about 50% identity, suggesting that the twogroups may recognize distinct substrates and govern uniquecellular processes.3 Currently, group I PAKs (PAK1−PAK3)are relatively well characterized, whereas considerably less isknown regarding the function and regulation of group II PAKs(PAK4−PAK6).

Within the group II PAK family, PAK4 is overexpressed and/or genetically amplified in lung, colon, prostate, pancreas, andbreast cancer cell lines and tumor tissues.3,4 Functional studieshave implicated PAK4 in cellular transformation4a and Kirstenrat sarcoma viral oncogene homologue (KRAS)-drivenxenograft tumor formation in vivo.5 Beyond regulation of theactin cytoskeleton, PAK4 has been implicated in cellproliferation and survival signaling,6 and validated PAK4effectors include Lim domain kinase 1 (LIMK1), guaninenucleotide exchange factor H1 (GEF-H1), v-Raf-1 murineleukemia viral oncogene homologue 1 (RAF1), and BCL2-associated agonist of cell death (BAD).7 Interestingly, group IIPAKs are also highly expressed in the brain, and although micelacking either PAK5 or PAK6 develop normally and are fertile,genetic disruption of PAK48 or PAK5/PAK69results indefective neuronal development. These loss-of-function devel-opmental phenotypes include defects in neural progenitor cellproliferation, dendritic spine morphology, learning, andcognitive function and are thought to arise via group II PAK

Received: November 15, 2013Published: January 16, 2014

Article

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© 2014 American Chemical Society 1033 dx.doi.org/10.1021/jm401768t | J. Med. Chem. 2014, 57, 1033−1045

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regulation of the cytoskeleton in the central nervous system.10

PAK5 and PAK6 overexpression has been observed in severaltumor types, although robust evidence for a role of these familymembers in carcinogenesis has not yet been published andwould benefit from the availability of better tool reagents toinhibit PAK5 and PAK6 in various model systems.11 Given theroles in tumorgenesis, oncogenic signaling, and embryonicdevelopment, there is significant interest in developing tools tofurther interrogate the biology of group II PAKs as well aspotentially targeting PAK4 therapeutically.12

Despite the importance of PAK4 and its upstream regulatorsin cancer development, there currently are no reported small-molecule inhibitors with high potency and selectivity for groupII PAKs. In a 2010 paper, details regarding clinical PAK4inhibitor (S)-N-(2-(dimethylamino)-1-phenylethyl)-6,6-di-methyl-3-((2-methylthieno[3,2-d]pyrimidin-4-yl)amino)-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide (PF-3758309, 1) were disclosed by Pfizer (Figure 1).13a However,this compound inhibits both group I and group II PAKs andalso potently inhibits a number of additional kinases.Subsequent to this discovery, structure-based methods wereused to substantially improve the broad kinase selectivity of thisinhibitor class. However, the resultant molecules still displayedpan-PAK activity.13b Therefore, new compounds that selec-tively inhibit group II PAKs are still needed to further analyzethis important signaling pathway in both homeostasis anddisease contexts.Herein, we describe the identification of potent group II PAK

inhibitors with high selectivity over group I PAKs and the broadkinome. To our knowledge, our study is the first report ofselective and potent group II PAK inhibitors, and our work thusprovides an important resource to further interrogate theperturbation of group II PAK signaling.Compound 2 was identified from our internal compound

collection as having moderate inhibition in a PAK4 biochemicalassay. This compound possesses a 2-methylbenzimidazole corewith 1-(4-aminotriazine) and 6-propargyl alcohol substituents.Although only modestly potent, it displayed high PAK4 overPAK1 specificity of interest (PAK1 Ki = 15.3 μM, PAK4 Ki =0.50 μM14). Our goal for optimization was improving group IIinhibition activity of 2 while maintaining high selectivity overgroup I PAKs (PAK4 and PAK1 as surrogates for group II andgroup I PAKs, respectively). Since our objective was to developa tool compound for evaluation of group II PAK biology, wewere not concerned by the possibility of reactive metabolitegeneration by the propargyl alcohol functionality contained in2.15

■ CHEMISTRYGeneral synthetic routes for inhibitors in Tables 1 and 2 areshown in Scheme 1. Triamine i-1 was reacted with ethyl

isothiocyanate or acetic anhydride to prepare 2-(ethylamino)-or 2-methylbenzimidazole intermediates i-2 and i-3, respec-tively. 2-(Methoxyethyl)amino substitution was accomplishedthrough intermediacy of the 2-(trichloromethyl)benzimidazolei-5 followed by SNAr substitution with methoxyethylamine.Reaction of 6-iodoindazole (i-7) with sodium hydride followedby 4-amino-6-chloropyrimidine or 2-amino-4-chloropyrimidinegave i-8 and i-9, respectively, in modest yield. Final analogues

Figure 1. Pan-PAK inhibitor 1 and group II PAK selective compound2.

Scheme 1. Synthetic Route for Analogues in Tables 1 and 2and Figure 3a

aReagents and conditions: (a) ethyl isothiocyanate, DMF, rt, thenCDI, 80 °C, 60% yield; (b) Ac2O, AcOH, 100 °C, 66% yield; (c)methyl trichloroacetimidate, AcOH, 45 °C, used crude in step d; (d)methoxyethylamine, Cs2CO3, rt, 42% yield; (e) NaH, DMF, then 6-chloropyrimidin-4-amine, 60 °C, 33% yield; (f) NaH, DMF, then 4-chloropyrimidin-2-amine, rt to 50 °C, 23% yield; (g) Sonogashiracoupling conditions which varied depending on the substrate andalkyne coupling partner [example conditions are 2-methyl-3-butyn-1-ol, PdCl2(PPh3)2, CuI, Et3N, MeCN, 80 °C (see the ExperimentalSection for details)].

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were prepared via Sonogashira coupling of tertiary propargyl orhomopropargyl alcohols to these aryl bromides or iodidesunder standard conditions.

■ RESULTS AND DISCUSSIONThe biochemical potency of this class of inhibitors was highlydependent on propargyl alcohol substitution. Table 1

represents the structure−activity relationship (SAR) for aseries of selected analogues. Both PAK4 and PAK1 Ki data arepresented to estimate group II specificity relative to that ofcompound 1. Homologation of the propargyl alcohol was nottolerated (3), nor was hydroxyl substitution on one of themethyl groups (4). Aliphatic and cycloaliphatic substitution wasalso examined (compounds 5−9). Cyclopropylmethyl sub-stitution (5) did not affect PAK4 affinity or selectivitysubstantially (PAK4 Ki = 0.67 μM, PAK1 Ki > 4.5 μM). 3-Oxetyl (6) and 3-pyranyl (7) substitution was not tolerated;however, cyclohexyl (8) and bicyclo[2.2.1]heptyl (9) analoguesshowed 7.4- and 2.6-fold greater PAK4 affinity, respectively,relative to that of compound 2. Propargyl alcohols derived frommethyl heteroaryl ketones were also incorporated. (R)-3-(5-Methylisoxazolyl) analogue 10 gained significant PAK4 potency(PAK4 Ki = 0.032 μM, 15.6-fold improvement relative to that

of 1), while 2-thiazolyl incorporation in compound 11 gaveaffinity similar to that of compound 2 (0.60 μM). In contrast tofive-membered heteroaryl substitution, six-membered hetero-aryl substitution (i.e., pyrimidine 12, PAK4 Ki > 2.9 μM) wasnot potent. Demonstrating a privileged feature of this class ofinhibitors, none of these analogues had significant affinity forPAK1 (PAK1 Ki > 4.5 μM in all cases). From this substituentscan, 1-alkynylcyclohexanol was chosen for incorporation intoadditional analogues given the PAK4 activity and overall kinaseselectivity (discussed later) of compound 8.

Table 1. Structure−Activity Relationship Associated withModification of Acetylenic Substitutiona

aSee the Experimental Section for PAK4 and PAK1 Ki assayconditions. An asterisk indicates the assay was run with a higher topconcentration.

Table 2. Key Data for Compound 17a

aKey: *, measured at Genentech (see the Experimental Section fordetails); **, measured at Invitrogen.

Figure 2. X-ray structure of 8 in complex with PAK4 (PDB ID 4O0X).Hydrogen bond interactions are indicated by blue dashed lines.Portions of the protein were omitted for clarity of visualization.Clipped van der Waals surfaces show the shape of the binding pocket.



The X-ray structure of compound 8 in complex with PAK416

revealed a novel binding mode among PAK inhibitors known inthe literature: two-point hinge binding interaction of theaminotriazine with Leu398 and the alkyne directing thepropargyl substituent past the gatekeeper residue (Met395,Figure 2). The propargyl alcohol donates a hydrogen bond toGlu366 on the α-C-helix and accepts a hydrogen bond from thebackbone NH of Phe459 of the DFG motif. The cyclohexylsubstituent fills a lipophilic pocket past gatekeeper Met395. Thelipophilic nature of the back pocket, bordered by threemethionine residues, is highly consistent with large potencylosses occurring when polarity is added to inhibitors directed atthis region (i.e., compare compounds 7/8 and 4/5). Given thePAK4 DFG motif and α-C-helix are “in” and yet the ligandextends past the gatekeeper, this binding mode fulfills thecolloquial type I 1/2 description proposed recently with the

exception that a hydrogen bond is accepted from the Phe ratherthan Asp residue backbone NH.17 This binding mode isunprecedented in the PAK literature;18 however, propargylalcohol-driven type I 1/2 binding has been demonstrated forAKT19 and NIK20 (both also possessing methionine gate-keepers).Compound 8 preferentially donates a hydrogen bond to

hinge residue C-terminal Leu398 instead of the proximal N-terminal Glu396.21 This is in stark contrast to the above-mentioned AKT and NIK inhibitors19,20 (aminooxadiazole andaminopyrimidine hinge-binding moieties, respectively) whichhave similar overall binding modes, yet donate a hydrogenbond to the carbonyl of the residue immediately adjacent to thegatekeeper methionine (corresponding to Glu396PAK4). Inter-estingly, the biaryl torsion angle of 8 is twisted from planarity to25° presumably to achieve optimal H-bonding to Leu398. The

Figure 3. Small-molecule conformational effects on PAK4 and PAK1 biochemical potency. Notes: (a) dihedral minima were calculated on the smallmolecule alone by Jaguar’s relaxed coordinate scan program using density functional theory (B3LYP/6-31G**);24 (b) conformations predicted to beequal in energy; (c) 0° within 1 kcal/mol relative to 180°; (*) run with a higher top concentration.



low-energy conformation of the unbound ligand (biaryl torsionangle predicted to be 0° or 180°, Figure 3) thus does not matchthe bound conformation (25°, 0.5 kcal/mol energy difference).We designed a set of analogues to preferentially target eitherLeu398 or Glu396 by conformational enforcement of this biaryltorsion angle, and the results are presented in Figure 3. Wetested indazole-based cores bearing 4- and 2-aminopyrimidinesubstitution (14 and 15, respectively) as well as a 2-aminobenzimidazole core (13 and 16) (Figure 3). On thebasis of the calculated lowest energy small-molecule con-formation (affected by electron pair repulsion or intramolecularhydrogen-bonding), compounds 14 and 16 (0° and 30°optimal biaryl torsion angles, respectively) are biased to directa hydrogen bond donor closer to Leu398 than Glu396. Withcompound 15, the predicted conformational preference biases ahydrogen bond donor toward Glu396. Compounds 2 and 13are predicted to have two low-energy conformers at 0° and180° biaryl torsion angle and thus are not energeticallyrestricted toward either residue’s carbonyl (compound 2 isstructurally analogous to compound 8).22 Several observationswere made from the data in Figure 3. Interestingly, stronginhibitory activity for PAK4 was possible whether the smallmolecule was biased toward hydrogen-bonding with theinternal Glu396 or the external Leu398 (compare 15 and 16,approximately equipotent biochemical activity). This result wassurprising because in the X-ray structure of compound 8, thesmall molecule adopted an apparent nonoptimal conformationto achieve a hydrogen bond with Leu398. Also there appearedto be a preference for matching the low-energy small-moleculeconformation with the likely binding conformation to avoid an

energetic penalty associated with hydrogen-bonding to theexternal Leu398. For example, compound 14 inhibits PAK4with a Ki of 1.3 μM having an optimal torsion of 0°, while 16inhibits PAK4 with a Ki of 0.14 μM having an optimal torsionof 30° consistent with the binding mode of 8.23 Although PAK4inhibitory activity and selectivity over PAK1 were achievabletargeting either Glu396 or Leu398, we chose to focus on theaminobenzimidazole scaffold of 16 as the calculated small-molecule conformation was most consistent with the bindingmode of 8.Combination of the 2-aminobenzimidazole motif of 16 with

the 1-alkynylcyclohexanol moiety of 8 gave a potent inhibitorwith PAK4 Ki = 3.3 nM (compound 17). A selection of in vitrodata for compound 17 is presented in Table 2. Mostimportantly, this compound demonstrates high group II overgroup I specificity with modest selectivity for PAK4 over theother group II members (PAK5, PAK6). The good enzymaticpotency, moderate solubility, and high passive permeability(MDCK Papp = 22.2 × 10−6 cm/s) of 17 make it an attractive invitro tool for evaluation of group II PAK pathway biology.Progressive improvement in PAK4 potency and kinome

selectivity leading to compound 17 is demonstrated by the datain Table 3. Compounds 2, 8, 16, and 17 were tested forinhibition against six methionine gatekeeper kinases atInvitrogen. Compound 2 showed weak percent inhibition ofPAK4 at 1 μM and actually stronger inhibition of KHS1 andNIK as well as comparable inhibition of MINK1 and MAP4K4.Notably, an increase in propargyl substituent size from gem-dimethyl to cyclohexyl (from 2 to 8) improved PAK4 potencyand decreased activity against all counter targets tested.

Table 3. Percent Inhibition Values for a Selection of Met Gatekeeper Kinasesa

aPercent inhibition values are an average of two measurements at the given concentration of inhibitor. Boxes colored by percent inhibition range:green, <30%; orange, 30−70%; red, >70%.



Installation of a 2-amino substituent in analogue 16 didimprove PAK4 activity. However, selectivity over the countertargets in Table 3, although improved, was not sufficient.Combining both structural features in compound 17 improvedPAK4 potency and selectivity. Compound 17 displaysselectivity not only over the selected methionine gatekeeperkinases in Table 3, but across a broad kinase panel. In a 222-kinase panel at Invitrogen at 0.100 μM concentration, itinhibited only PAK4, PAK5, and PAK6 at >60% (see theSupporting Information for details).25

The X-ray structure of inhibitor 17 in PAK4 is displayed inFigure 4a. Conformational enforcement of the desiredaminopyrimidine geometry was confirmed as this ligandmakes a two-point hinge-binding interaction with Leu398.The intramolecular hydrogen bond between the 2-amino-benzimidazole and the 4-pyrimidyl nitrogen is present (2.9 Å).The biaryl torsion angle is at 25°, consistent with both

calculated (Figure 3) and measured values from a small-molecule crystal structure (30° and 24°, respectively).26 Thepropargyl alcohol extends into the hydrophobic pocket pastgatekeeper Met395.We were motivated to generate a structure-based rationale

for not only the high kinase selectivity of compound 17 butespecially the selectivity over PAK1 for this entire structuralclass. For this analysis, we focused on the region of the bindingsite that was uniquely occupied by these inhibitors relative toless-selective PAK inhibitors: the hydrophobic back pocket pastthe gatekeeper Met395. Further analysis of PAK4 structuresavailable in the Protein Data Bank (PDB) revealed thatsubstantial differences exist in the shape of this back pocket thatare primarily related to positioning of Met370, a residue locatedon the α-C-helix.A survey of all PDB PAK1 structures indicated that the PAK1

equivalent of Met370PAK4 (Met319PAK1) consistently adoptsrotamer conformations that drive its side chain into the backpocket, whereas Met370PAK4 can adopt rotamer conformationsthat allow the side chain to be further removed from the sameregion. Exemplifying this difference, the back pockets of thecomplexes of 1 (PAK4 Ki = 15 nM, PAK1 Ki = 36 nM) inPAK1 and PAK4 are quite distinct (Figure 4b,c). Althoughcompound 1 does not protrude into the region past themethionine gatekeeper (Met395PAK4), the complex in PAK4(PDB ID 2X4Z, Figure 4b) possesses an open back pocket.This is in contrast to the observed orientation of the backpocket when compound 1 is in complex with PAK1 (Figure 4c,in-house structure, PDB ID 4O0R). In the PAK1 structure, thispocket is filled by the side chain of α-C-helix residueMet319PAK1. In the PAK4 structure of 1, the side chain ofthe corresponding Met370PAK4 undergoes a C-α/β rotation tocreate an open back pocket (similar orientation to thatobserved when compound 17 is bound to PAK4; compareparts a and b to part c of Figure 4). A more readily accessibleorientation for Met319PAK1 vs Met370PAK4 could explain whyeven small propargyl alcohol substitution is not tolerated inPAK1 for our class of inhibitors (i.e., compound 2). Weattribute this phenomenon to the elevated main chainconformational flexibility associated with Met370PAK4 and theα-C-helix of PAK4. More specifically, the C-terminal turn of theα-C-helix in PAK4 appears to be flexible, allowing Met370PAK4

to be displaced, as opposed to a stable helical structure in PAK1(Figure 5). The amino acid sequence of PAK1 followingMet319PAK1 is Arg320PAK1, Glu321PAK1, and Asn322PAK1.Asn322PAK1 donates a hydrogen bond to the main chaincarbonyl of Val318PAK1 and serves as the helix-capping residue,neutralizing the dipole moment developed within the α-C-helix(Figure 5a). This is analogous to the common α-helix cappingmotif where a glycine at the C′ position of a helix adopts a left-handed conformation to donate a hydrogen bond with thebackbone carbonyl of the C3 residue.27 The resulting hydrogenbond stabilizes the C-terminal helical turn and increases theenergy cost for unwinding. In contrast, PAK4 contains a Tyrresidue (Tyr373PAK4) in place of Asn322PAK1, consequentlyincreasing the flexibility in the last helical turn, which includesMet370PAK4.28 We tested this helix-flexibility hypothesis againstthe previously determined crystal structures of PAK4 and PAK1deposited in the PDB. All PAK1 structures (nine of nine)possess the final turn of the α-C-helix, whereas 4/12 PAK4structures have an open pocket as a result of α-C-helixunwinding and Met370PAK4 rotation.29 Our conclusion fromthis analysis is that an open back pocket, as a result of helix

Figure 4. (a) Crystal structure of 17 in PAK4 (PDB ID 4O0V). (b)Crystal structure of 1 in PAK4 (PDB 2X4Z). (c) Crystal structure of 1in PAK1 (PDB ID 4O0R). Portions of the proteins are omitted forclarity of visualization. The back pocket of PAK1 is collapsed by thepresence of Met319. PAK4 can display an open back pocket, even inthe absence of ligand occupancy. Clipped van der Waals surfaces showthe shape of the binding pockets.



unwinding, is likely more energetically accessible in PAK4 thanit is in PAK1.30

The crystal structure of 13 in complex with PAK4 revealedfurther unique flexibility associated with the back pocket regionof the protein (Figure 6). Compound 13 retains the 2-aminobenzimidazole substitution, similarly to 17 with theaminotriazine hinge-binding element of compound 8. As in theother structures, helical unwinding results in C-α/β rotation ofthe Met370PAK4 side chain. However, there are two notablechanges relative to the PAK4 structures of 17 and 8.

Compound 13 donates a hydrogen bond to the proximalGlu396, and the back pocket is partially collapsed by residenceof the side chain of Met381PAK4 (from the pre-β4 loop). Webelieve the smaller propargyl alcohol substitution is responsiblefor these changes: greater flexibility of the gatekeeperMet395PAK4 allows binding of the ligand to the internalGlu396, and remaining hydrophobic space unoccupied by theligand is filled by Met381PAK4. Interestingly, this Met381PAK4

orientation is also represented in PAK4 structures in the PDBin cases where this back pocket is completely unoccupied by aligand (for example, PDB ID 4FII). In PAK1, this residue isTyr330PAK1. We hypothesize the ability of Met381PAK4 topartially fill the back pocket and make hydrophobic contactswith small propargyl alcohol substitution may provide addi-tional potency for PAK4 when ligands possess smallsubstitution in this region.Our back pocket flexibility hypotheses for selectivity over

PAK1 are based on equivalent binding modes for theseinhibitors in both PAK1 and PAK4. We thus pursuedcomplexes of compounds 2 and 17 with PAK1 in separatetrials. We were successful in obtaining a 2.57 Å structure withcompound 17 bound (PDB ID 4O0T), and the binding modewas identical when compared to that of 17 bound to PAK4(Figure 7). This structure supports our hypothesis that thePAK1 α-C-helix is stable and did not unwind and the hydrogenbond between Asn322PAK1 and Val318PAK1 remained intact(Figure 7, black dotted line). Met319PAK1 still undergoes C-α/β

Figure 5. PAK4 and PAK1 α-C-helix length and stability. Compound8 is modeled in (a) and (b) to illustrate steric clash with Met319PAK1

and Met370PAK4 when the α-C-helix stays intact. (a) PAK1’s α-C-helixC-terminal Val318PAK1 is capped by a hydrogen bond with Asn322PAK1

(blue line) that stabilizes the helix and projects Met319PAK1 into a backpocket, preventing compound 8 from binding (compound 8, PDB ID4O0X, overlaid with PAK1, PDB ID 3FXZ). Tyr373PAK4 cannot form asimilar hydrogen bond with the helix backbone to stabilize it in PAK4.(b) When the α-C-helix of PAK4 remains intact, Met370PAK4 projectsinto the back pocket (compound 8, PDB ID 4O0X, overlaid withPAK4, PDB ID 2QON). (c) Lower helical stability in PAK4 allows itsα-C-helix to unwind, leading Met370PAK4 to undergo C-α/β rotationand creating room for compound 8 to bind (PDB ID 4O0X).

Figure 6. X-ray structure of 13 in PAK4 (PDB ID 4O0Y). Met381PAK4

partially occupies the back pocket and makes hydrophobic contactswith the ligand.

Figure 7. Crystal structure of compound 17 bound to PAK1 (gray)(PDB ID 4O0T). The same ligand bound in PAK4 is shown forcomparison (magenta). Met319PAK1 folds against the α-C-helix,creating room for the compound to bind. The hydrogen bondbetween Asn322 and Val3l8 (blue dotted line) caps the α-C-helix,preventing it from unwinding. Met370PAK4 is shown in magenta forcomparison.



rotation to fold back against the α-C-helix, creating room forthe cyclohexyl group of 17. Met319PAK1 adopted a gauche−,gauche−, gauche+ rotamer that has a 3% chance of beingobserved in the PDB. In contrast, the rotamer adopted byMet370PAK4 is the most populated at 19%.31 Compared toMet370PAK4, there is likely a higher energy penalty forMet319PAK1 to fold back against the α-C-helix and accom-modate compound 17 binding. This energy penalty is reflectedin compound 17’s 2.9 μM biochemical binding affinity forPAK1.We also believe the residue on the α-C-helix (equivalent to

Met370PAK4) is important for controlling kinome selectivity.Notably, all non-PAK kinases listed in Table 3 have a change inprimary structure in this position relative to the PAKs. JAK3,Map4K4, and MINK1 all possess a leucine residue in place ofMet370PAK4, while KHS1 has a valine, and NIK possesses acysteine residue.32 Notably, all kinases in Table 3 (with theexception of PAK4) are insulted with increased size ofpropargyl substitution. This suggests at least partial occupancyof this key α-C-helix residue’s side chain in the back pocket ofthese kinases.Overall, we believe the identity, conformational flexibility,

and lipophilicity of this unique Met395/Met381/Met370 trioof residues in PAK4 (Met370 due to α-C-helix instability) arekey to allowing variable propargyl substitution size as well asalternate hinge-binding orientation. In turn, both features areexploited in compound 17 to achieve high kinome and PAKgroup selectivity.Given that compound 17 represents a novel small molecule

that potently and selectively inhibits group II PAKs, we soughtto utilize this compound to demonstrate the role of this kinase

subfamily in disease contexts. Previous studies have shown thatPAK4 is required for efficient migration and/or invasion ofprostate, ovarian, pancreatic, and glioma cancer cell lines.2,3

Cell migration and invasion are multistep processes which aredependent on signaling pathways that regulate rapid reorgan-ization of the cytoskeleton. Migration and invasion contributeto numerous cellular processes, including tissue reorganization,angiogenesis, immune cell trafficking, inflammation, tumori-genesis, and metastasis. We were curious to analyze the effect ofcompound 17 on migration and invasion of two triple-negativebreast cancer cell lines, MDA-MB-436 and MCF10A carrying aPIK3CA(H1047R) knock-in mutation. Expression of PAK4and PAK6 is elevated in triple-negative breast cancer, and theirrole in cell motility has not been previously described for thistumor type, which provides further rationale for their use in ourinhibitor studies. We also utilized compound 17 to reassessPAK4-dependent phenotypes that were previously reported for8988T pancreatic adenocarcinoma cells. 8988T has genomicamplification and robust expression of PAK4, and RNAinterference-mediated knockdown of PAK4 resulted insignificantly diminished migration, invasion, and anchorage-independent growth relative to those of the controls.33

However, the role of PAK4 catalytic activity in regulating8988T tumor cell phenotypes was not previously examined.We used a wound migration assay and an Essen Bioscience

Incucyte platform to collect and analyze relative wounddensities from phase-contrast time-lapse images of cells. Thismethod is based on creating a scratch on a confluent cellmonolayer and motile cells at the leading edge closing the gapuntil new cell−cell contacts are re-established.34 The migrationand invasion data in Figures 8 and 9, respectively, show time-

Figure 8. Compound 17 inhibits MDA-MB-436 and MCF10A PIK3CA cell migration. (a) MDA-MB-436 time-dependent relative wound density(%) in the presence of DMSO and 0.1, 1, 10, and 50 μM 17. (b) Phase-contrast images showing wounded monolayers at t = 0 h and t = 24 h forMDA-MB-436 cell migration. (c) MCF10A PIK3 CA time-dependent relative wound density (%) in the presence of DMSO and 0.1, 1, 10, and 50μM 17. (d) Phase-contrast images showing wounded monolayers at t = 0 h and t = 24 h for MCF10A PIK3CA cell migration.



Figure 9. Compound 17 inhibits MDA-MB-436 and MCF10A PIK3CA Matrigel invasion. (a) MDA-MB-436 time-dependent relative wounddensity (%) in the presence of DMSO and 0.1, 1, 10, and 50 μM 17. (b) Phase-contrast images showing wounded monolayers at t = 0 h and t = 48 hfor MDA-MB-436 cell invasion. (c) MCF10A PIK3 CA time-dependent relative wound density (%) in the presence of DMSO and 0.1, 1, 10, and 50μM 17. (d) Phase-contrast images showing wounded monolayers at t = 0 h and t = 48 h for MCF10A PIK3CA cell invasion.

Figure 10. Compound 17 inhibits MDA-MB-436 and MCF10A PIK3CA viabilities. (a) MDA-MB-436 time-dependent Celltiter Glo luminescencein the presence of DMSO and 0.1, 1, 10, and 50 μM 17. (b) MCF10A PIK3 time-dependent Celltiter Glo luminescence in the presence of DMSOand 0.1, 1, 10, and 50 μM 17. (c) Correlation of pak4 mRNA expression and compound 17 sensitivity in cell viability assays. The absolute EC50 forcompound 17 is plotted against pak4 mRNA reads per kilobase per million (RPKM) derived from RNaseq.



dependent relative wound density. MDA-MB-436 wounddensity was reduced by ∼50% with >10 μM 17 and wasrobustly inhibited with 50 μM 17 at 20 h (Figure 8a).Representative phase-contrast images of the wound at 0 and 24h are shown in Figure 8b. Compared to MDA-MB-436 cells,MCF10A PIK3CA cells show a more elongated morphologyand migrate into the wounded area more rapidly. Similar to theeffect on MDA-MB-436 cells, greater than 10 μM 17 wasrequired to see a ∼50% reduction in wound density at 20 h inthe MCF10A PIK3CA cells (Figure 8c,d). No decrease in therate of cell motility was observed using a group I PAK selectiveinhibitor (data not shown). Inhibition of 8898T migrationfollowing treatment with compound 17 was also comparable topreviously reported data using a genetic approach for PAK4inhibition.33

In invasion assays, MDA-MB-436 and MCF10A PIK3CAcells invaded into a three-dimensional layer of laminin-richextracellular matrix, Matrigel, that was plated on top of thewounded cell monolayer. MDA-MB-436 cells invaded theMatrigel layer at a slow rate and did not achieve full woundclosure 80 h after the experiment was started (Figure 9a).Nonetheless, a concentration-dependent reduction in MDA-MB-436 wound density was observed with inhibitor 17.Compound 17 exhibited a more robust effect in invasionassays than in migration assays, and ∼50% inhibition of wounddensity was observed with 1 μM. Time-lapse images show thatMDA-MB-436 cells form multicellular strands when invadinginto the Matrigel (Figure 9b), which is different from thewound edge morphology observed in migration assays (Figure8b). MCF10A PIK3CA cells invaded more rapidly than MDA-MB-436 cells (Figure 9d), and full wound closure was observedby 40 h (Figure 9c). In our MCF10A PIK3CA invasion assays,>10 μM was required for a ∼50% reduction in wound density.PAK4 has been shown to regulate cell proliferation and

survival in several cell types.35 We assessed whether compound17 affects proliferation by performing cell growth curves andquantifying the number of metabolically active cells 24 and 48 hafter addition of compound 17 (or DMSO control). BothMDA-MB-436 and MCF10A PIK3CA cell viabilities werereduced by ∼50% in the presence of 10 μM compound 17(parts a and b, respectively, of Figure 10). In the presence of 50μM 17, MDA-MB-436 and MCF10A PIK3CA viabilities wererobustly inhibited. A panel of breast cancer cell lines alsoshowed micromolar sensitivity to compound 17 in proliferationassays (Figure 10c). Importantly, sensitivity correlated withpak4 mRNA expression from RNaseq analysis.Given the correlation in Figure 10c and the identity of the

observed phenotypes, we currently believe the above results aredriven by group II PAK inhibition since compound 17 does notinhibit PAK1 autophosphorylation in cells and a group I PAKinhibitor does not affect 8988T motility (data not shown).However, we are curious but unable to definitively explain thelarge shift in concentration between biochemical IC50 andphenotypic inhibition for compound 17. The data for 17 standin contrast to the strong cellular antiproliferative activity of 1and other pyrrolopyrazole PAK inhibitors. Compound 1(PAK4 Ki = 36 nM, PAK1 Ki = 15 nM) is reported to inhibitproliferation of >40 of 92 cell lines at <10 nM EC50 (i.e., MDA-MB-436 proliferation IC50 = 0.79 nM).13a,36

In summary, a biochemically potent and highly selectivegroup II PAK inhibitor has been identified in compound 17. Anovel structure-based hypothesis and strategy for achievingselectivity over the group I PAK family and kinome were

presented on the basis of the flexibility of the back pocket ofgroup II PAKs leveraged by these novel type I 1/2 binders. Incombination with good biochemical potency and selectivity,compound 17 possesses good solubility and passive perme-ability. As such, 17 has proved to be a useful in vitro tool forfurther elucidation of the function of PAK family kinases as wellas their strength as targets for oncological or other applications.Indeed, concentration-dependent catalytic inactivation of groupII PAKs in two triple-negative breast cancer cell lines, MDA-MB-436 and MCF10A PIK3CA, resulted in a decrease in tumorcell migration and invasion.37

■ EXPERIMENTAL SECTIONAll chemicals were purchased form commercial suppliers and used asreceived. Moisture- or oxygen-sensitive reactions were conductedunder an atmosphere of argon or nitrogen gas. Flash chromatographywas carried out with prepacked SiO2 cartridges on automatedchromatography systems (for example, ISOC Companion chromatog-raphy system). NMR spectra were recorded on a Bruker AV III 400 or500 NMR spectrometer and referenced to tetramethylsilane. Thefollowing abbreviations are used: br = broad signal, s = singlet, d =doublet, dd = doublet of doublets, t = triplet, q = quartet, m =multiplet. Preparative HPLC was performed eluting with mixtures ofwater/acetonitrile. High-resolution mass spectra were recorded on aWaters LCT Premiere XC in ES+ mode. All final compounds werepurified to >95% chemical and optical purity, as assayed by HPLC(Waters Acquity UPLC column, 21 × 50 mm, 1.7 μm) with a gradientof 0−90% acetonitrile (containing 0.038% TFA) in 0.1% aqueous TFAor 0.1% ammonium hydroxide, with UV detection at λ = 254 and 210nm.

4-[1-(4-Amino-1,3,5-triazin-2-yl)-2-methyl-1H-1,3-benzodia-zol-6-yl]-2-methylbut-3-yn-2-ol (2). A suspension of 4-(6-bromo-2-methyl-1H-1,3-benzodiazol-1-yl)-1,3,5-triazin-2-amine (i-3; 305 mg,1.00 mmol), 2-methylbut-3-yn-2-ol (252 mg, 3.00 mmol), andPd(PPh3)2Cl2 (140 mg, 0.20 mmol, 0.2 equiv) in triethylamine (1mL) and DMSO (1 mL) was heated in a microwave for 30 min at 90°C. The resulting solution was diluted with 10 mL of water andextracted with 3 × 100 mL of ethyl acetate. The combined organiclayers were washed with 3 × 50 mL of water, dried over anhydroussodium sulfate, filtered, and concentrated under vacuum. The residuewas purified on a silica gel column with ethyl acetate/petroleum ether(5:1) to give 73 mg (24%) of 2 as a light yellow solid: 1H NMR (500MHz, DMSO-d6) δ 8.66 (s, 1H), 8.38 (d, J = 1.5 Hz, 1H), 8.07 (br s,1H), 8.01 (br s, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 8.2, 1.5Hz, 1H), 5.49 (s, 1H), 2.90 (s, 3H), 1.49 (s, 6H); 13C NMR (126MHz, DMSO) δ 167.9, 167.2, 162.9, 154.6, 142.6, 133.6, 127.5, 119.2,118.9, 117.9, 95.6, 81.7, 64.1, 32.2, 19.5; LC−MS (ES, m/z) 309 [M +H]+.

1-((1-(4-Amino-1,3,5-triazin-2-yl)-2-methyl-1H-benzo[d]-imidazol-6-yl)ethynyl)cyclohexanol (8). To a solution of i-3 (150mg, 0.49 mmol) in N,N-dimethylformamide (3 mL) were added 1,3-bis(diphenylphosphino)propane (40 mg, 0.1 mmol), Pd(OAc)2 (10mg, 0.05 mmol), K2CO3 (207 mg, 1.5 mmol), and 1-ethynylcyclohex-anol (124 mg, 1 mmol). Then the solution was sparged with nitrogenfor 5 min and heated in a microwave apparatus for 1 h at 120 °C undernitrogen. The reaction mixture was filtered, and the filtrate waspurified by flash column chromatography (DCM:MeOH = 10:1) toafford the desired compound 8 (100 mg, 58%): 1H NMR (500 MHz,DMSO-d6) δ 8.66 (s, 1H), 8.38 (d, J = 1.4 Hz, 1H), 8.06 (br s, 1H),8.01 (br s, 1H) 7.58 (d, J = 8.2 Hz, 1H), 7.32 (dd, J = 8.2, 1.7 Hz, 1H),5.43 (s, 1H), 2.89 (s,3H), 1.94 − 1.79 (m, 2H), 1.71 − 1.12(overlapping m, 10H); 13C NMR (126 MHz, DMSO) δ 167.9, 167.2,162.9, 154.5, 142.6, 133.6, 127.6, 119.2, 118.7, 118.0, 94.5, 83.8, 67.4,55.4, 25.4, 23.2, 19.5; LC−MS (ESI, m/z) 349 [M + H]+.

4-[1-(4-Amino-1,3,5-triazin-2-yl)-2-(ethylamino)-1H-1,3-ben-zodiazol-6-yl]-2-methylbut-3-yn-2-ol (13). A suspension of 1-(4-amino-1,3,5-triazin-2-yl)-6-bromo-N-ethyl-1H-1,3-benzodiazol-2-amine (i-2; 170 mg, 0.51 mmol), 2-methylbut-3-yn-2-ol (129 mg, 1.53mmol), and Pd(PPh3)4 (118 mg, 0.10 mmol) in piperidine (2 mL) was



heated in a microwave reactor under nitrogen for 1.5 h at 90 °C. Theresulting solution was diluted with 200 mL of dichloromethane,washed with 2 × 100 mL of water and 2 × 100 mL of brine, dried overanhydrous sodium sulfate, and concentrated under vacuum. Theresidue was purified on a silica gel column eluted with dichloro-methane/methanol (10:1). The product was further purified by Prep-HPLC with the following conditions (1#-Pre-HPLC-005 (Waters)):column, XBridge Shield RP18 OBD column, 5 μm, 19 × 150 mm;mobile phase, water with 10 mmol of NH4HCO3 and CH3CN (18.0%CH3CN to 43.0% in 10 min, to 95.0% in 1 min, hold at 95.0% for 1min, down to 18.0% in 2 min); detector, UV 220 and 254 nm. Thisresulted in 43.5 mg (25%) of 13 as an off-white solid: 1H NMR (500MHz, DMSO-d6) δ 8.93 (t, J = 5.8 Hz, 1H), 8.62 (s, 1H), 8.39 (d, J =1.5 Hz, 1H), 8.12 (br s, 1H), 8.04 (br s, 1H), 7.18 (m, 1H), 7.15 (m,1H), 5.41 (s, 1H), 3.53 (m, 2H), 1.48 (s, 6H), 1.27 (t, J = 7.2 Hz, 3H).13C NMR (126 MHz, DMSO) δ 167.5, 166.1, 162.7, 155.5, 144.1,131.7, 127.9, 119.0, 115.6, 113.5, 94.2, 82.3, 64.1, 37.8, 32.3, 15.3;LC−MS (ES, m/z) 338 [M + H]+.4-[1-(2-Aminopyrimidin-4-yl)-2-[(2-methoxyethyl)amino]-

1H-1,3-benzodiazol-6-yl]-2-methylbut-3-yn-2-ol (16). A mixtureof 1-(2-aminopyrimidin-4-yl)-6-bromo-N-(2-methoxyethyl)-1H-1,3-benzodiazol-2-amine (i-6; 120 mg, 0.33 mmol), 2-methylbut-3-yn-2-ol (277.2 mg, 3.30 mmol), Pd(PPh3)2Cl2 (231.67 mg), andtriethylamine (2.4 mL) in dimethyl sulfoxide (1 mL) was stirredunder nitrogen for 1 h at 70 °C. The reaction mixture was cooled toroom temperature, and the solid material was removed by filtration.The filtrate was diluted with 5 mL of water and then extracted with 3× 30 mL of ethyl acetate. The combined organic layers were dried overanhydrous sodium sulfate, filtered, and concentrated under vacuum.The residue was purified by Prep-HPLC with the following conditions(HPLC): column, Xbridge; mobile phase, acetonitrile/water; detector,UV 220 and 254 nm. This resulted in 5.0 mg (4%) of 16 as a colorlesssolid: 1H NMR (300 MHz, DMSO-d6) δ 8.43 (d, J = 5.4 Hz, 1H), 8.21(d, J = 5.4 Hz, 1H), 7.47 (s, 1H), 7.25 (s, 1H), 7.16−7.12 (m, 3H),6.91 (d, 1H), 5.40 (s, 1H), 3.63−3.57 (m, 4H), 3.35 (s, 3H), 1.47 (s,6H); LC−MS (m/z) 367 [M + H]+.1-[2-[1-(2-Aminopyrimidin-4-yl)-2-[(2-methoxyethyl)amino]-

1H-1,3-benzodiazol-6-yl]ethynyl]cyclohexan-1-ol (17). A sus-pension of i-6 (250 mg, 0.65 mmol, 95% purity), 1-ethynylcyclohexan-1-ol (300 mg, 2.42 mmol), and Pd(PPh3)2Cl2 (250 mg, 0.36 mmol) inDMSO (3 mL) and triethylamine (2 mL) was heated in a microwavefor 20 min at 70 °C under a nitrogen atmosphere. The reactionmixture was concentrated under vacuum, and the residue was purifiedby HPLC on a C18 column eluted with CH3CN/H2O (5:95 to 80:20)to give 100 mg (37%) of 17 as a yellow solid: 1H NMR (500 MHz,DMSO-d6) δ 8.43 (d, J = 5.5 Hz, 1H), 8.22 (t, J = 5.5 Hz, 1H), 7.47(d, J = 1.6 Hz, 1H), 7.25 (m, 1H), 7.16 (m, 1H), 7.14 (s, 2H), 6.91 (d,J = 5.5 Hz, 1H), 5.36 (s, 1H), 3.60 (overlapping m, 4H), 3.28 (s, 3H),1.88 1.19 (overlapping m, 10H); 13C NMR (126 MHz, DMSO) δ163.5, 161.9, 157.0, 155.3, 144.0, 131.8, 127.3, 116.3, 113.9, 113.8,100.3, 93.3, 84.2, 70.7, 67.4, 58.5, 42.5, 25.4, 23.3; LC−MS (ES, m/z)407 [M + H]+.PAK1 and PAK4 Ki Biochemical Assay Protocol. Activity of

human recombinant PAK1 and PAK4 (KD, kinase domain) proteinwas assessed in vitro by assay of the phosphorylation of a FRETpeptide substrate. The activity/inhibition of PAK enzymes wasestimated by measuring the phosphorylation of a FRET peptidesubstrate (Ser/Thr19) labeled with coumarin and fluorescein using theZ′-LYTE assay platform (Invitrogen). The peptide substrate is aconsensus sequence (KKRNRRLSVA) based on various PAKsubstrates reported in the scientific literature. The 10 μL assaymixtures contained 50 mM HEPES (pH 7.5), 0.01% Brij-35, 10 mMMgCl2, 1 mM EGTA, 2 μM FRET peptide substrate, and 20 pMPAK1-KD or 80 pM PAK4-KD. Incubations were carried out at 22 °Cin black polypropylene 384-well plates (Corning Costar). Prior to theassay, PAK1-KD or PAK4-KD, FRET peptide substrate, and seriallydiluted test compounds were preincubated together in assay buffer(7.5 μL) for 10 min, and the assay was initiated by the addition of 2.5μL of assay buffer containing 160 μM ATP (4×) for the PAK1 assay or16 μM ATP (4×) for the PAK4 assay. Following the 60 min

incubation, the assay mixtures were quenched by the addition of 5 μLof Z′-LYTE development reagent, and 1 h later the emissions ofcoumarin (445 nm) and fluorescein (520 nm) were determined afterexcitation at 400 nm using an Envision plate reader (Perkin-Elmer).An emission ratio (445 nm/520 nm) was determined to quantify thedegree of substrate phosphorylation.

Cell Lines. MDA-MB-436 cells (ATCC, Manassas, VA) werecultured in Dulbecco’s modified Eagle’s medium (DMEM) containing10% fetal bovine serum and 2 mM L-glutamine. MCF10A PIK3CAcells carry one endogenous allele of the PIK3CA(H1047R) gene.38

These cells were cultured in DMEM/F12 medium containing 5%horse serum, 5 μg/mL insulin, 1 μg/mL hydrocortisone, 2 mM L-glutamine, and 10 mM HEPES.

Migration Assays. For migration experiments 40 000 MDA-MB-436 or MCF10A PIK3CA cells were added per well of an EssenImageLock 96-well plate (Essen BioScience, Ann Arbor, MI). Afterovernight incubation, a uniform scratch was introduced into the cellmonolayer using an Essen WoundMaker device (Essen BioScience).Detached or loosely attached cells were removed by washing thewounded monolayer two times with warm medium. After the lastwash, 150 μL of medium containing DMSO or compound 17 wasadded. Images were collected and quantified every 2 h in an IncuCytesystem (Essen BioScience).

Invasion Assays. ImageLock 96-well plates were coated with 100μg/mL growth factor reduced Matrigel (BD Biosciences, San Jose,CA) for 3 h at 37 °C. After removal of the diluted Matrigel, 40 000cells were plated per well and allowed to adhere for 5 min before theplate was moved to a 37 °C incubator. After overnight incubation, auniform scratch was introduced into the cell monolayer with an EssenWoundMaker device. The wounded monolayers were washed twotimes, and 50 μL of 2.4 mg/mL growth factor reduced Matrigel wasadded to each well. The Matrigel was allowed to solidify at 37 °C for30 min, and then 150 μL of complete medium containing DMSO orcompound 17 was carefully added on top of the Matrigel layer. Imageswere collected and quantified every 3 h in an IncuCyte system.

Viability Assays. MDA-MB-436 and MCF10A PIK3CA cells weresparsely plated in white-walled 96-well plates. After the cells adhered,DMSO and compound 17 were added, and the cells were incubatedfor 24 and 48 h. Celltiter Glo (Promega, Madison, WI) assays werecarried out according to the manufacturer’s instructions, andluminescence was measured in a plate reader.

■ ASSOCIATED CONTENT

*S Supporting InformationFull kinase selectivity data for compound 17, small-molecule X-ray data for compound 17, tabulated EC50 data from the cellviability assays, and protein production/purification and X-raycrystallography conditions. This material is available free ofcharge via the Internet at http://pubs.acs.org

Accession CodesPDB IDs: 4O0R, 4O0T, 4O0V, 4O0Y, 4O0X.

■ AUTHOR INFORMATION

Corresponding Authors*E-mail: [email protected]. Phone: (650) 467-3103.*E-mail: [email protected]. Phone: (650) 225- 6697.

Author Contributions¶S.T.S. and J.A.F. contributed equally to this work.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We thank the Genentech internship program for support ofD.A.R.



http://pubs.acs.org

mailto:[email protected]

mailto:[email protected]

■ ABBREVIATIONS USED:PAK, p-21-activated kinase; RAS, rat sarcoma; NIK, NFκB-inducing kinase; AKT, protein kinase B; KHS1 or MAP4K4,mitogen-activated protein kinase kinase kinase kinase 5;MINK1, misshapen-like kinase 1; JAK3, Janus kinase 3; CDI,carbonyldiimidazole; DMF, dimethylformamide; Ac2O, aceticanhydride; AcOH, acetic acid; MeCN, acetonitrile

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(10) Kreis, P.; Barnier, J.-V. PAK signalling in neuronal physiology.Cell Signalling 2009, 21, 384−393.(11) Minden, A. PAK4−6 in cancer and neuronal development. CellLogist. 2012, 2 (2), 95−104.(12) Crawford, J. J.; Hoeflich, K. P.; Rudolph, J. p21-Activated kinaseinhibitors: a patent review. Expert Opin. Ther. Pat. 2012, 22, 293−310.(13) (a) Murray, B. W.; Guo, C.; Piraino, J.; Westwick, J. K.; Zhang,C.; Lamerdin, J.; Dagostino, E.; Knighton, D.; Loi, C. M.; Zager, M.;Kraynov, E.; Popoff, I.; Christensen, J. G.; Martinez, R.; Kephart, S. E.;Marakovits, J.; Karlicek, S.; Bergqvist, S.; Smeal, T. Small-moleculep21-activated kinase inhibitor PF-3758309 is a potent inhibitor ofoncogenic signaling and tumor growth. Proc. Natl. Acad. Sci. U.S.A.2010, 107, 9446−9451. (b) Guo, C.; McAlpine, I.; Zhang, J.;Knighton, D. D.; Kephart, S.; Johnson, M. C.; Li, H.; Bouzida, D.;Yang, A.; Dong, L.; Marakovits, J.; Tikhe, J.; Richardson, P.; Guo, L.C.; Kania, R.; Edwards, M. P.; Kraynov, E.; Christensen, J.; Piraino, J.;Lee, J.; Dagostino, D.; Del-Carmen, C.; Deng, Y.; Smeal, T.; Murray,B. W. Discovery of pyrroloaminopyrazoles as novel PAK inhibitors. J.Med. Chem. 2012, 55, 4728−4739.(14) See the Experimental Section for a description of thebiochemical assays.(15) Kalgutkar, A. S.; Dalvie, D. Obach, R. S.; Smith, D. A. ReactiveDrug Metabolites; Wiley-VCH: Weinheim, Germany, 2012.(16) See the Supporting Information for a description of the proteinproduction, purification, and crystallization conditions (PAK1 andPAK4) used for the compounds discussed in this paper.(17) (a) Zuccotto, F.; Ardini, E.; Casale, E.; Angiolini, M. Throughthe “gatekeeper door”: exploiting the active kinase conformation. J.Med. Chem. 2010, 53, 2681−2694. (b) Angiolini, M. Targeting theDFG-in kinase conformation: a new trend emerging from a patentanalysis. Future Med. Chem. 2011, 3, 309−337.(18) A series of Afraxis group I PAK inhibitors extend past themethionine gatekeeper but reach a different area of the kinase; see:Licciulli, S.; Maksimoska, J.; Zhou, C.; Troutman, S.; Kota, S.; Liu, Q.;Duron, S.; Campbell, D.; Chernoff, J.; Field, J.; Marmorstein, R.; Kissil,J. L. FRAX597, a small molecule inhibitor of the p21-activated kinases,inhibits tumorigenesis of neurofibromastosis type 2 (NF2)-associatedschwannomas. J. Biol. Chem. 2013, 288, 29105−29114.(19) (a) Heerding, D. A.; Rhodes, N.; Leber, J. D.; Clark, T. J.;Keenan, R. M.; Lafrance, L. V.; Li, M.; Safonov, I. G.; Takata, D. T.;Venslavsky, J. W.; Yamashita, D. S.; Choudhry, A. E.; Copeland, R. A.;Lai, Z.; Schaber, M. D.; Tummino, P. J.; Strum, S. L.; Wood, E. R.;Duckett, D. R.; Eberwein, D.; Knick, V. B.; Lansing, T. J.; McConnel,R. T.; Zhang, S.; Minthorn, E. A.; Concha, N. O.; Warren, G. L.;Kumar, R. Identification of 4-(2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1H-imidazo[4,5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol (GSK690693), a novel inhibitor of AKTkinase. J. Med. Chem. 2008, 51, 5663−5679. Within this paper,GSK690693 is noted to hit class II PAKs in a selectivity panel, butclass I PAK data are not provided. (b) Rouse, M. B.; Seefeld, M. A.;Leber, J. D.; McNulty, K. C.; Sun, L.; Miller, W. H.; Zhang, S.;Minthorn, E. A.; Concha, N. O.; Choudhry, A. E.; Schaber, M. D.;Heerding, D. A. Aminofurazans as potent inhibitors of AKT kinase.Bioorg. Med. Chem. Lett. 2009, 19, 1508−1511.(20) (a) de Leon, G.; Bowman, K. K.; Feng, J. A.; Crawford, T.;Everett, C.; Franke, Y.; Oh, A.; Stanley, M.; Staben, S. T.; Starovasnik,M. A.; Wellweber, H. J. A.; Wu, J.; Wu, L. C.; Johnson, A.; Hymowitz,S. G. The crystal structure of the catalytic domain of the NF-kBinducing kinase reveals a narrow but flexible active site. Structure 2012,20, 1704−1714. (b) Li, K.; McGee, L. R.; Fisher, B.; Sudom, A.; Liu, J.;Rubenstein, S. M.; Anwer, M. K.; Cushing, T. D.; Shin, Y.; Ayres, M.;Lee, F.; Eksterowicz, J.; Faulder, P.; Waszkowycz, B.; Plotnikova, O.;Farrelly, E.; Xiao, S.-H.; Chen, G.; Wang, Z. Inhibiting NF-κB-inducing kinase (NIK): discovery, structure-based design, synthesis,structure-activity relationship, and co-crystal structures. Bioorg. Med.Chem. Lett. 2013, 23, 1238−1244.(21) A potential nonclassical H-bond between the carbonyl ofGlu396 and the 6-H of the 2-aminotriazine is possible (3.6 Å).



(22) For a similar strategy in conformational restriction see: Bryan,M. C.; Fasey, J. R.; Frohn, M.; Riechelt, A.; Yao, G.; Bartberger, M. D.;Bailis, J. M.; Zalameda, L.; San Miguel, T.; Doherty, E. M.; Allen, J. G.N-substituted azaindoles as potent inhibitors of Cdc7 kinase. Bioorg.Med. Chem. Lett. 2013, 23, 2056−2060.(23) We do not believe that the methoxyethyl substitutent ofcompound 16 can explain this result as it does not appear to make anysignificant interactions in the X-ray complex of 17 in PAK4.(24) Suite 2012: Jaguar, version 7.9; Schrodinger LLC: New York,2012.(25) In the kinase panel for 17, EphB1 was inhibited 100% at 100nM. However, no measurable IC50 was seen in a follow-up assay (IC50

>10 μM). We thus believe the initial data to be incorrect.(26) The small-molecule X-ray data for 17 are presented in theSupporting Information.(27) Aurora, R.; Srinivasan, R.; Rose, G. D. Rules for α-helixtermination by glycine. Science 1994, 264, 1126.(28) Aurora, R.; Rose, G. D. Helix capping. Protein Sci. 1998, 7, 21.The normalized frequency of Asn vs Tyr residues at the C′ cappingposition of an α-helix is consistent with this hypothesis, indicatingsome selection against Tyr (1.39 vs 0.93).(29) Another major difference in primary structure is the DFG − 1residue (Thr in PAK1 and Ser in PAK4). However, we do not believethat access to the back pocket of group I PAKs is hindered by thelarger branched residues. KHS1, Map4K4, and MINK1 all possess abranched valine residue in this position and seem to tolerate thisvector of substitution on the basis of our selectivity data (Table 3).Additionally, AKT, which is known to tolerate type I 1/2 binding ofinhibitors possessing propargyl alcohols, also contains a threonineresidue in this position (identical to group I PAKs).(30) Nine PAK1 structures in the PDB that have an intact α-C-helix:1F3M, 1YHV, 1YHW, 3FXZ, 3FY0, 3Q4Z, 3Q52, 3Q53, 4DAW. FourPAK4 structures that have a partially unwound α-C-helix: 2BVA, 2JOI,2X4Z, 4APP. Eight PAK4 structures that have an intact α-C-helix:2CDZ, 2Q0N, 4FIE, 4FIF, 4FIG, 4FIH, 4FII, 4FIJ.(31) Lovell, S. C.; Word, J. M.; Richardson, J. S.; Richardson, D. C.The penultimate rotamer library. Proteins 2000, 40, 389−408.(32) Residue assignments for KHS1 and MINK1 were done byhomology.(33) Kimmelman, A. C.; Hezel, A. F.; Aguirre, A. J.; Zheng, H.; Paik,J. H.; Ying, H.; Chu, G. C.; Zhang, J. X.; Sahin, E.; Yeo, G.; Ponugoti,A.; Nabioullin, R.; Deroo, S.; Yang, S.; Wang, X.; McGrath, J. P.;Protopova, M.; Ivanova, E.; Zhang, J.; Feng, B.; Tsao, M. S.; Redston,M.; Protopopov, A.; Xiao, Y.; Futreal, P. A.; Han, W. C.; Klimstra, D.S.; Chin, L.; DePinho, R. A. Genomic alterations link Rho family ofGTPases to the highly invasive phenotype of pancreas cancer. Proc.Natl. Acad. Sci. U.S.A. 2008, 105, 19372−19377.(34) Liang, C.-C.; Park, A. Y.; Guan, J.-L. In vitro scratch assay: aconvenient and inexpensive method for analysis of cell migration invitro. Nat. Protoc. 2007, 2, 329−333.(35) (a) Tabusa, H.; Brooks, T.; Massey, A. J. Knockdown of PAK4and PAK1 inhibits the proliferation of mutant KRAS colon cancer cellsindependently of RAF/MEK/ERK and PI3K/AKT signaling. Mol.Cancer Res. 2013, 11, 109−121. (b) Liu, Y.; Xiao, H.; Tian, Y.;Nekrasova, T.; Hao, X.; Lee, H. J.; Suh, N.; Yang, C. S.; Minden, A.The pak4 protein kinase plays a key role in cell survival andtumorigenesis in athymic mice. Mol. Cancer Res. 2008, 6, 1215−1224.(c) Tian, Y.; Lei, L.; Minden, A. A key role for Pak4 in proliferationand differentiation of neural progenitor cells. Dev. Biol. 2011, 353,206−216. (d) Zhang, J.; Wang, J.; Guo, Q.; Wang, Y.; Zhou, Y.; Peng,H.; Cheng, M.; Zhao, D.; Li, F. LCH-7749944, a novel potent p21-activated kinase 4 inhibitor, suppresses proliferation and invasion inhuman gastric cancer cells. Cancer Lett. 2012, 317, 24−32. (e) Qu, J.;Cammarano, M. S.; Shi, Q.; Ha, K. C.; de Lanerolle, P.; Minden, P.Activated PAK4 regulates cell adhesion and anchorage-independentgrowth. Mol. Cell. Biol. 2001, 21, 3523−3533.(36) One possible rationalization for a high enzyme to cell shift ofcompound 17 is the low Km,apparent of ATP for the kinase domain ofPAK4 (4 μM) such that a high enzyme to cell shift is expected

dependent on the ATP concentration (for example, a theoretical 250-fold shift at standard 1 mM ATP). The efficacious concentrationscould be increased by protein binding and typical pathway tophenotype shifts. The lower than expected biochemical to cell shift forother PAK inhibitors in the literature could be caused by group I PAKor other kinase inhibition. Alternatively, it is possible that theinhibition of cellular PAK4 may be weaker for compounds similar to17.(37) These phenotypic results are similar to those of a recent analysisof PAK4 function in pancreatic ductal adenocarcinoma cell lines withPAK4 genomic amplification: see ref 33.(38) Wallin, J. J.; Guan, J.; Edgar, K. A.; Zhou, W.; Francis, R.;Toress, A. C.; Haverty, P. M.; Eastham-Anderson, J.; Arena, S.;Bardelli, A.; Griffen, S.; Goodall, J. E.; Grimshaw, K. M.; Hoeflich, K.P.; Torrance, C.; Belvin, M.; Friedman, L. S. Active PI3K pathwaycauses an invasive phenotype which can be reversed or promoted byblocking the pathway at divergent nodes. PLoS One 2012, 7, 36402.



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Back Pocket Flexibility Provides Group II p21-Activated Kinase (PAK) Selectivity for Type I 1/2 Kinase Inhibitors