Back Pocket Flexibility Provides Group II p21-Activated Kinase (PAK

Jan 16, 2014 - ABSTRACT: Structure-based methods were used to design a potent and highly selective group II p21-activated kinase. (PAK) inhibitor with...
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Back Pocket Flexibility Provides Group II p21-Activated Kinase (PAK) Selectivity for Type I 1/2 Kinase Inhibitors Steven 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 Drug Metabolism 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 a potent and highly selective group II p21-activated kinase (PAK) inhibitor with a novel binding mode, compound 17. Hydrophobic interactions within a lipophilic pocket past the methionine gatekeeper of group II PAKs approached by these type I 1/2 binders were found to be important for improving potency. A structure-based hypothesis and strategy for achieving selectivity over group I PAKs, and the broad kinome, based on unique flexibility of this lipophilic pocket, is presented. A concentration-dependent decrease in tumor cell migration and invasion in two triple-negative breast cancer cell lines was observed with compound 17.

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INTRODUCTION

Within the group II PAK family, PAK4 is overexpressed and/ or genetically amplified in lung, colon, prostate, pancreas, and breast cancer cell lines and tumor tissues.3,4 Functional studies have implicated PAK4 in cellular transformation4a and Kirsten rat sarcoma viral oncogene homologue (KRAS)-driven xenograft tumor formation in vivo.5 Beyond regulation of the actin cytoskeleton, PAK4 has been implicated in cell proliferation and survival signaling,6 and validated PAK4 effectors include Lim domain kinase 1 (LIMK1), guanine nucleotide exchange factor H1 (GEF-H1), v-Raf-1 murine leukemia viral oncogene homologue 1 (RAF1), and BCL2associated agonist of cell death (BAD).7 Interestingly, group II PAKs are also highly expressed in the brain, and although mice lacking either PAK5 or PAK6 develop normally and are fertile, genetic disruption of PAK48 or PAK5/PAK69results in defective neuronal development. These loss-of-function developmental phenotypes include defects in neural progenitor cell proliferation, dendritic spine morphology, learning, and cognitive function and are thought to arise via group II PAK

Approximately 30% of human tumors harbor somatic gain-offunction mutations in the RAS family of small GTPases that serve to transduce mitogenic signals from cell surface receptor tyrosine kinases to intracellular serine/threonine kinases.1 p21activated kinases (PAKs) are members of the STE20 family of serine/threonine kinases that lie downstream of RAS and regulate many cellular processes that are commonly perturbed in cancer, including migration, polarization, and proliferation.2 The PAK family is comprised of six members and is subdivided into two groups (groups I and II) on the basis of sequence and structural homology. The group I and II PAKs share a number of conserved structural characteristics, such as a p21-binding domain, multiple proline-rich regions, and a carboxyl-terminal kinase domain. However, the kinase domains of group I and II PAKs share only about 50% identity, suggesting that the two groups may recognize distinct substrates and govern unique cellular processes.3 Currently, group I PAKs (PAK1−PAK3) are relatively well characterized, whereas considerably less is known regarding the function and regulation of group II PAKs (PAK4−PAK6). © 2014 American Chemical Society

Received: November 15, 2013 Published: January 16, 2014 1033

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CHEMISTRY General synthetic routes for inhibitors in Tables 1 and 2 are shown in Scheme 1. Triamine i-1 was reacted with ethyl Scheme 1. Synthetic Route for Analogues in Tables 1 and 2 and Figure 3a

Figure 1. Pan-PAK inhibitor 1 and group II PAK selective compound 2.

regulation of the cytoskeleton in the central nervous system.10 PAK5 and PAK6 overexpression has been observed in several tumor types, although robust evidence for a role of these family members in carcinogenesis has not yet been published and would benefit from the availability of better tool reagents to inhibit PAK5 and PAK6 in various model systems.11 Given the roles in tumorgenesis, oncogenic signaling, and embryonic development, there is significant interest in developing tools to further interrogate the biology of group II PAKs as well as potentially targeting PAK4 therapeutically.12 Despite the importance of PAK4 and its upstream regulators in cancer development, there currently are no reported smallmolecule inhibitors with high potency and selectivity for group II PAKs. In a 2010 paper, details regarding clinical PAK4 inhibitor (S)-N-(2-(dimethylamino)-1-phenylethyl)-6,6-dimethyl-3-((2-methylthieno[3,2-d]pyrimidin-4-yl)amino)-4,6dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide (PF3758309, 1) were disclosed by Pfizer (Figure 1).13a However, this compound inhibits both group I and group II PAKs and also potently inhibits a number of additional kinases. Subsequent to this discovery, structure-based methods were used to substantially improve the broad kinase selectivity of this inhibitor class. However, the resultant molecules still displayed pan-PAK activity.13b Therefore, new compounds that selectively inhibit group II PAKs are still needed to further analyze this important signaling pathway in both homeostasis and disease contexts. Herein, we describe the identification of potent group II PAK inhibitors with high selectivity over group I PAKs and the broad kinome. To our knowledge, our study is the first report of selective and potent group II PAK inhibitors, and our work thus provides an important resource to further interrogate the perturbation of group II PAK signaling. Compound 2 was identified from our internal compound collection as having moderate inhibition in a PAK4 biochemical assay. This compound possesses a 2-methylbenzimidazole core with 1-(4-aminotriazine) and 6-propargyl alcohol substituents. Although only modestly potent, it displayed high PAK4 over PAK1 specificity of interest (PAK1 Ki = 15.3 μM, PAK4 Ki = 0.50 μM14). Our goal for optimization was improving group II inhibition activity of 2 while maintaining high selectivity over group I PAKs (PAK4 and PAK1 as surrogates for group II and group I PAKs, respectively). Since our objective was to develop a tool compound for evaluation of group II PAK biology, we were not concerned by the possibility of reactive metabolite generation by the propargyl alcohol functionality contained in 2.15

a

Reagents and conditions: (a) ethyl isothiocyanate, DMF, rt, then CDI, 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 6chloropyrimidin-4-amine, 60 °C, 33% yield; (f) NaH, DMF, then 4chloropyrimidin-2-amine, rt to 50 °C, 23% yield; (g) Sonogashira coupling conditions which varied depending on the substrate and alkyne coupling partner [example conditions are 2-methyl-3-butyn-1ol, PdCl2(PPh3)2, CuI, Et3N, MeCN, 80 °C (see the Experimental Section for details)].

isothiocyanate or acetic anhydride to prepare 2-(ethylamino)or 2-methylbenzimidazole intermediates i-2 and i-3, respectively. 2-(Methoxyethyl)amino substitution was accomplished through intermediacy of the 2-(trichloromethyl)benzimidazole i-5 followed by SNAr substitution with methoxyethylamine. Reaction of 6-iodoindazole (i-7) with sodium hydride followed by 4-amino-6-chloropyrimidine or 2-amino-4-chloropyrimidine gave i-8 and i-9, respectively, in modest yield. Final analogues 1034

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Table 2. Key Data for Compound 17a

were prepared via Sonogashira coupling of tertiary propargyl or homopropargyl alcohols to these aryl bromides or iodides under standard conditions.

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RESULTS AND DISCUSSION The biochemical potency of this class of inhibitors was highly dependent on propargyl alcohol substitution. Table 1 Table 1. Structure−Activity Relationship Associated with Modification of Acetylenic Substitutiona

a Key: *, measured at Genentech (see the Experimental Section for details); **, measured at Invitrogen.

of 1), while 2-thiazolyl incorporation in compound 11 gave affinity similar to that of compound 2 (0.60 μM). In contrast to five-membered heteroaryl substitution, six-membered heteroaryl substitution (i.e., pyrimidine 12, PAK4 Ki > 2.9 μM) was not potent. Demonstrating a privileged feature of this class of inhibitors, none of these analogues had significant affinity for PAK1 (PAK1 Ki > 4.5 μM in all cases). From this substituent scan, 1-alkynylcyclohexanol was chosen for incorporation into additional analogues given the PAK4 activity and overall kinase selectivity (discussed later) of compound 8. a See the Experimental Section for PAK4 and PAK1 Ki assay conditions. An asterisk indicates the assay was run with a higher top concentration.

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

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. 1035

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

The X-ray structure of compound 8 in complex with PAK416 revealed a novel binding mode among PAK inhibitors known in the literature: two-point hinge binding interaction of the aminotriazine with Leu398 and the alkyne directing the propargyl substituent past the gatekeeper residue (Met395, Figure 2). The propargyl alcohol donates a hydrogen bond to Glu366 on the α-C-helix and accepts a hydrogen bond from the backbone NH of Phe459 of the DFG motif. The cyclohexyl substituent fills a lipophilic pocket past gatekeeper Met395. The lipophilic nature of the back pocket, bordered by three methionine residues, is highly consistent with large potency losses occurring when polarity is added to inhibitors directed at this region (i.e., compare compounds 7/8 and 4/5). Given the PAK4 DFG motif and α-C-helix are “in” and yet the ligand extends past the gatekeeper, this binding mode fulfills the colloquial type I 1/2 description proposed recently with the

exception that a hydrogen bond is accepted from the Phe rather than Asp residue backbone NH.17 This binding mode is unprecedented in the PAK literature;18 however, propargyl alcohol-driven type I 1/2 binding has been demonstrated for AKT19 and NIK20 (both also possessing methionine gatekeepers). Compound 8 preferentially donates a hydrogen bond to hinge residue C-terminal Leu398 instead of the proximal Nterminal Glu396.21 This is in stark contrast to the abovementioned AKT and NIK inhibitors19,20 (aminooxadiazole and aminopyrimidine hinge-binding moieties, respectively) which have similar overall binding modes, yet donate a hydrogen bond to the carbonyl of the residue immediately adjacent to the gatekeeper methionine (corresponding to Glu396PAK4). Interestingly, the biaryl torsion angle of 8 is twisted from planarity to 25° presumably to achieve optimal H-bonding to Leu398. The 1036

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

a

Percent inhibition values are an average of two measurements at the given concentration of inhibitor. Boxes colored by percent inhibition range: green, 70%.

low-energy conformation of the unbound ligand (biaryl torsion angle predicted to be 0° or 180°, Figure 3) thus does not match the bound conformation (25°, 0.5 kcal/mol energy difference). We designed a set of analogues to preferentially target either Leu398 or Glu396 by conformational enforcement of this biaryl torsion angle, and the results are presented in Figure 3. We tested indazole-based cores bearing 4- and 2-aminopyrimidine substitution (14 and 15, respectively) as well as a 2aminobenzimidazole core (13 and 16) (Figure 3). On the basis of the calculated lowest energy small-molecule conformation (affected by electron pair repulsion or intramolecular hydrogen-bonding), compounds 14 and 16 (0° and 30° optimal biaryl torsion angles, respectively) are biased to direct a hydrogen bond donor closer to Leu398 than Glu396. With compound 15, the predicted conformational preference biases a hydrogen bond donor toward Glu396. Compounds 2 and 13 are predicted to have two low-energy conformers at 0° and 180° biaryl torsion angle and thus are not energetically restricted toward either residue’s carbonyl (compound 2 is structurally analogous to compound 8).22 Several observations were made from the data in Figure 3. Interestingly, strong inhibitory activity for PAK4 was possible whether the small molecule was biased toward hydrogen-bonding with the internal Glu396 or the external Leu398 (compare 15 and 16, approximately equipotent biochemical activity). This result was surprising because in the X-ray structure of compound 8, the small molecule adopted an apparent nonoptimal conformation to achieve a hydrogen bond with Leu398. Also there appeared to be a preference for matching the low-energy small-molecule conformation with the likely binding conformation to avoid an

energetic penalty associated with hydrogen-bonding to the external Leu398. For example, compound 14 inhibits PAK4 with a Ki of 1.3 μM having an optimal torsion of 0°, while 16 inhibits PAK4 with a Ki of 0.14 μM having an optimal torsion of 30° consistent with the binding mode of 8.23 Although PAK4 inhibitory activity and selectivity over PAK1 were achievable targeting either Glu396 or Leu398, we chose to focus on the aminobenzimidazole scaffold of 16 as the calculated smallmolecule conformation was most consistent with the binding mode of 8. Combination of the 2-aminobenzimidazole motif of 16 with the 1-alkynylcyclohexanol moiety of 8 gave a potent inhibitor with PAK4 Ki = 3.3 nM (compound 17). A selection of in vitro data for compound 17 is presented in Table 2. Most importantly, this compound demonstrates high group II over group I specificity with modest selectivity for PAK4 over the other group II members (PAK5, PAK6). The good enzymatic potency, moderate solubility, and high passive permeability (MDCK Papp = 22.2 × 10−6 cm/s) of 17 make it an attractive in vitro 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 data in Table 3. Compounds 2, 8, 16, and 17 were tested for inhibition against six methionine gatekeeper kinases at Invitrogen. Compound 2 showed weak percent inhibition of PAK4 at 1 μM and actually stronger inhibition of KHS1 and NIK as well as comparable inhibition of MINK1 and MAP4K4. Notably, an increase in propargyl substituent size from gemdimethyl to cyclohexyl (from 2 to 8) improved PAK4 potency and decreased activity against all counter targets tested. 1037

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calculated (Figure 3) and measured values from a smallmolecule crystal structure (30° and 24°, respectively).26 The propargyl alcohol extends into the hydrophobic pocket past gatekeeper Met395. We were motivated to generate a structure-based rationale for not only the high kinase selectivity of compound 17 but especially the selectivity over PAK1 for this entire structural class. For this analysis, we focused on the region of the binding site that was uniquely occupied by these inhibitors relative to less-selective PAK inhibitors: the hydrophobic back pocket past the gatekeeper Met395. Further analysis of PAK4 structures available in the Protein Data Bank (PDB) revealed that substantial differences exist in the shape of this back pocket that are primarily related to positioning of Met370, a residue located on the α-C-helix. A survey of all PDB PAK1 structures indicated that the PAK1 equivalent of Met370PAK4 (Met319PAK1) consistently adopts rotamer conformations that drive its side chain into the back pocket, whereas Met370PAK4 can adopt rotamer conformations that allow the side chain to be further removed from the same region. Exemplifying this difference, the back pockets of the complexes of 1 (PAK4 Ki = 15 nM, PAK1 Ki = 36 nM) in PAK1 and PAK4 are quite distinct (Figure 4b,c). Although compound 1 does not protrude into the region past the methionine 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 back pocket when compound 1 is in complex with PAK1 (Figure 4c, in-house structure, PDB ID 4O0R). In the PAK1 structure, this pocket is filled by the side chain of α-C-helix residue Met319PAK1. In the PAK4 structure of 1, the side chain of the corresponding Met370PAK4 undergoes a C-α/β rotation to create an open back pocket (similar orientation to that observed when compound 17 is bound to PAK4; compare parts a and b to part c of Figure 4). A more readily accessible orientation for Met319PAK1 vs Met370PAK4 could explain why even small propargyl alcohol substitution is not tolerated in PAK1 for our class of inhibitors (i.e., compound 2). We attribute this phenomenon to the elevated main chain conformational flexibility associated with Met370PAK4 and the α-C-helix of PAK4. More specifically, the C-terminal turn of the α-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 following Met319PAK1 is Arg320PAK1, Glu321PAK1, and Asn322PAK1. Asn322PAK1 donates a hydrogen bond to the main chain carbonyl 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 capping motif where a glycine at the C′ position of a helix adopts a lefthanded conformation to donate a hydrogen bond with the backbone carbonyl of the C3 residue.27 The resulting hydrogen bond stabilizes the C-terminal helical turn and increases the energy cost for unwinding. In contrast, PAK4 contains a Tyr residue (Tyr373PAK4) in place of Asn322PAK1, consequently increasing the flexibility in the last helical turn, which includes Met370PAK4.28 We tested this helix-flexibility hypothesis against the previously determined crystal structures of PAK4 and PAK1 deposited in the PDB. All PAK1 structures (nine of nine) possess the final turn of the α-C-helix, whereas 4/12 PAK4 structures have an open pocket as a result of α-C-helix unwinding and Met370PAK4 rotation.29 Our conclusion from this analysis is that an open back pocket, as a result of helix

Installation of a 2-amino substituent in analogue 16 did improve PAK4 activity. However, selectivity over the counter targets in Table 3, although improved, was not sufficient. Combining both structural features in compound 17 improved PAK4 potency and selectivity. Compound 17 displays selectivity not only over the selected methionine gatekeeper kinases in Table 3, but across a broad kinase panel. In a 222kinase panel at Invitrogen at 0.100 μM concentration, it inhibited only PAK4, PAK5, and PAK6 at >60% (see the Supporting Information for details).25 The X-ray structure of inhibitor 17 in PAK4 is displayed in Figure 4a. Conformational enforcement of the desired aminopyrimidine geometry was confirmed as this ligand makes a two-point hinge-binding interaction with Leu398. The intramolecular hydrogen bond between the 2-aminobenzimidazole and the 4-pyrimidyl nitrogen is present (2.9 Å). The biaryl torsion angle is at 25°, consistent with both

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 1 in PAK1 (PDB ID 4O0R). Portions of the proteins are omitted for clarity of visualization. The back pocket of PAK1 is collapsed by the presence of Met319. PAK4 can display an open back pocket, even in the absence of ligand occupancy. Clipped van der Waals surfaces show the shape of the binding pockets. 1038

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Figure 6. X-ray structure of 13 in PAK4 (PDB ID 4O0Y). Met381PAK4 partially occupies the back pocket and makes hydrophobic contacts with the ligand.

Compound 13 donates a hydrogen bond to the proximal Glu396, and the back pocket is partially collapsed by residence of the side chain of Met381PAK4 (from the pre-β4 loop). We believe the smaller propargyl alcohol substitution is responsible for these changes: greater flexibility of the gatekeeper Met395PAK4 allows binding of the ligand to the internal Glu396, and remaining hydrophobic space unoccupied by the ligand is filled by Met381PAK4. Interestingly, this Met381PAK4 orientation is also represented in PAK4 structures in the PDB in cases where this back pocket is completely unoccupied by a ligand (for example, PDB ID 4FII). In PAK1, this residue is Tyr330PAK1. We hypothesize the ability of Met381PAK4 to partially fill the back pocket and make hydrophobic contacts with small propargyl alcohol substitution may provide additional potency for PAK4 when ligands possess small substitution in this region. Our back pocket flexibility hypotheses for selectivity over PAK1 are based on equivalent binding modes for these inhibitors in both PAK1 and PAK4. We thus pursued complexes of compounds 2 and 17 with PAK1 in separate trials. We were successful in obtaining a 2.57 Å structure with compound 17 bound (PDB ID 4O0T), and the binding mode was identical when compared to that of 17 bound to PAK4 (Figure 7). This structure supports our hypothesis that the PAK1 α-C-helix is stable and did not unwind and the hydrogen bond 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. Compound 8 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-helix C-terminal Val318PAK1 is capped by a hydrogen bond with Asn322PAK1 (blue line) that stabilizes the helix and projects Met319PAK1 into a back pocket, preventing compound 8 from binding (compound 8, PDB ID 4O0X, overlaid with PAK1, PDB ID 3FXZ). Tyr373PAK4 cannot form a similar hydrogen bond with the helix backbone to stabilize it in PAK4. (b) When the α-C-helix of PAK4 remains intact, Met370PAK4 projects into the back pocket (compound 8, PDB ID 4O0X, overlaid with PAK4, PDB ID 2QON). (c) Lower helical stability in PAK4 allows its α-C-helix to unwind, leading Met370PAK4 to undergo C-α/β rotation and creating room for compound 8 to bind (PDB ID 4O0X).

unwinding, is likely more energetically accessible in PAK4 than it is in PAK1.30 The crystal structure of 13 in complex with PAK4 revealed further unique flexibility associated with the back pocket region of the protein (Figure 6). Compound 13 retains the 2aminobenzimidazole substitution, similarly to 17 with the aminotriazine hinge-binding element of compound 8. As in the other structures, helical unwinding results in C-α/β rotation of the Met370PAK4 side chain. However, there are two notable changes relative to the PAK4 structures of 17 and 8.

Figure 7. Crystal structure of compound 17 bound to PAK1 (gray) (PDB ID 4O0T). The same ligand bound in PAK4 is shown for comparison (magenta). Met319PAK1 folds against the α-C-helix, creating room for the compound to bind. The hydrogen bond between Asn322 and Val3l8 (blue dotted line) caps the α-C-helix, preventing it from unwinding. Met370PAK4 is shown in magenta for comparison. 1039

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

rotation to fold back against the α-C-helix, creating room for the cyclohexyl group of 17. Met319PAK1 adopted a gauche−, gauche−, gauche+ rotamer that has a 3% chance of being observed in the PDB. In contrast, the rotamer adopted by Met370PAK4 is the most populated at 19%.31 Compared to Met370PAK4, there is likely a higher energy penalty for Met319PAK1 to fold back against the α-C-helix and accommodate compound 17 binding. This energy penalty is reflected in compound 17’s 2.9 μM biochemical binding affinity for PAK1. 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 in primary structure in this position relative to the PAKs. JAK3, Map4K4, and MINK1 all possess a leucine residue in place of Met370PAK4, while KHS1 has a valine, and NIK possesses a cysteine residue.32 Notably, all kinases in Table 3 (with the exception of PAK4) are insulted with increased size of propargyl substitution. This suggests at least partial occupancy of this key α-C-helix residue’s side chain in the back pocket of these kinases. Overall, we believe the identity, conformational flexibility, and lipophilicity of this unique Met395/Met381/Met370 trio of residues in PAK4 (Met370 due to α-C-helix instability) are key to allowing variable propargyl substitution size as well as alternate hinge-binding orientation. In turn, both features are exploited in compound 17 to achieve high kinome and PAK group selectivity. Given that compound 17 represents a novel small molecule that potently and selectively inhibits group II PAKs, we sought to utilize this compound to demonstrate the role of this kinase

subfamily in disease contexts. Previous studies have shown that PAK4 is required for efficient migration and/or invasion of prostate, ovarian, pancreatic, and glioma cancer cell lines.2,3 Cell migration and invasion are multistep processes which are dependent on signaling pathways that regulate rapid reorganization of the cytoskeleton. Migration and invasion contribute to numerous cellular processes, including tissue reorganization, angiogenesis, immune cell trafficking, inflammation, tumorigenesis, and metastasis. We were curious to analyze the effect of compound 17 on migration and invasion of two triple-negative breast cancer cell lines, MDA-MB-436 and MCF10A carrying a PIK3CA(H1047R) knock-in mutation. Expression of PAK4 and PAK6 is elevated in triple-negative breast cancer, and their role in cell motility has not been previously described for this tumor type, which provides further rationale for their use in our inhibitor studies. We also utilized compound 17 to reassess PAK4-dependent phenotypes that were previously reported for 8988T pancreatic adenocarcinoma cells. 8988T has genomic amplification and robust expression of PAK4, and RNA interference-mediated knockdown of PAK4 resulted in significantly diminished migration, invasion, and anchorageindependent growth relative to those of the controls.33 However, the role of PAK4 catalytic activity in regulating 8988T tumor cell phenotypes was not previously examined. We used a wound migration assay and an Essen Bioscience Incucyte platform to collect and analyze relative wound densities from phase-contrast time-lapse images of cells. This method is based on creating a scratch on a confluent cell monolayer and motile cells at the leading edge closing the gap until new cell−cell contacts are re-established.34 The migration and invasion data in Figures 8 and 9, respectively, show time1040

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Figure 9. Compound 17 inhibits MDA-MB-436 and MCF10A PIK3CA Matrigel invasion. (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 = 48 h for 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 50 μM 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 luminescence in 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 DMSO and 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 for compound 17 is plotted against pak4 mRNA reads per kilobase per million (RPKM) derived from RNaseq.

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

dependent relative wound density. MDA-MB-436 wound density was reduced by ∼50% with >10 μM 17 and was robustly inhibited with 50 μM 17 at 20 h (Figure 8a). Representative phase-contrast images of the wound at 0 and 24 h are shown in Figure 8b. Compared to MDA-MB-436 cells, MCF10A PIK3CA cells show a more elongated morphology and migrate into the wounded area more rapidly. Similar to the effect on MDA-MB-436 cells, greater than 10 μM 17 was required to see a ∼50% reduction in wound density at 20 h in the MCF10A PIK3CA cells (Figure 8c,d). No decrease in the rate of cell motility was observed using a group I PAK selective inhibitor (data not shown). Inhibition of 8898T migration following treatment with compound 17 was also comparable to previously reported data using a genetic approach for PAK4 inhibition.33 In invasion assays, MDA-MB-436 and MCF10A PIK3CA cells invaded into a three-dimensional layer of laminin-rich extracellular matrix, Matrigel, that was plated on top of the wounded cell monolayer. MDA-MB-436 cells invaded the Matrigel layer at a slow rate and did not achieve full wound closure 80 h after the experiment was started (Figure 9a). Nonetheless, a concentration-dependent reduction in MDAMB-436 wound density was observed with inhibitor 17. Compound 17 exhibited a more robust effect in invasion assays than in migration assays, and ∼50% inhibition of wound density was observed with 1 μM. Time-lapse images show that MDA-MB-436 cells form multicellular strands when invading into the Matrigel (Figure 9b), which is different from the wound edge morphology observed in migration assays (Figure 8b). MCF10A PIK3CA cells invaded more rapidly than MDAMB-436 cells (Figure 9d), and full wound closure was observed by 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 compound 17 affects proliferation by performing cell growth curves and quantifying the number of metabolically active cells 24 and 48 h after addition of compound 17 (or DMSO control). Both MDA-MB-436 and MCF10A PIK3CA cell viabilities were reduced by ∼50% in the presence of 10 μM compound 17 (parts a and b, respectively, of Figure 10). In the presence of 50 μM 17, MDA-MB-436 and MCF10A PIK3CA viabilities were robustly inhibited. A panel of breast cancer cell lines also showed micromolar sensitivity to compound 17 in proliferation assays (Figure 10c). Importantly, sensitivity correlated with pak4 mRNA expression from RNaseq analysis. Given the correlation in Figure 10c and the identity of the observed phenotypes, we currently believe the above results are driven by group II PAK inhibition since compound 17 does not inhibit PAK1 autophosphorylation in cells and a group I PAK inhibitor does not affect 8988T motility (data not shown). However, we are curious but unable to definitively explain the large shift in concentration between biochemical IC50 and phenotypic inhibition for compound 17. The data for 17 stand in contrast to the strong cellular antiproliferative activity of 1 and other pyrrolopyrazole PAK inhibitors. Compound 1 (PAK4 Ki = 36 nM, PAK1 Ki = 15 nM) is reported to inhibit proliferation of >40 of 92 cell lines at 95% chemical and optical purity, as assayed by HPLC (Waters Acquity UPLC column, 21 × 50 mm, 1.7 μm) with a gradient of 0−90% acetonitrile (containing 0.038% TFA) in 0.1% aqueous TFA or 0.1% ammonium hydroxide, with UV detection at λ = 254 and 210 nm. 4-[1-(4-Amino-1,3,5-triazin-2-yl)-2-methyl-1H-1,3-benzodiazol-6-yl]-2-methylbut-3-yn-2-ol (2). A suspension of 4-(6-bromo2-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), and Pd(PPh3)2Cl2 (140 mg, 0.20 mmol, 0.2 equiv) in triethylamine (1 mL) 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 and extracted with 3 × 100 mL of ethyl acetate. The combined organic layers were washed with 3 × 50 mL of water, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was 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 (500 MHz, 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, 1.5 Hz, 1H), 5.49 (s, 1H), 2.90 (s, 3H), 1.49 (s, 6H); 13C NMR (126 MHz, 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 (150 mg, 0.49 mmol) in N,N-dimethylformamide (3 mL) were added 1,3bis(diphenylphosphino)propane (40 mg, 0.1 mmol), Pd(OAc)2 (10 mg, 0.05 mmol), K2CO3 (207 mg, 1.5 mmol), and 1-ethynylcyclohexanol (124 mg, 1 mmol). Then the solution was sparged with nitrogen for 5 min and heated in a microwave apparatus for 1 h at 120 °C under nitrogen. The reaction mixture was filtered, and the filtrate was purified by flash column chromatography (DCM:MeOH = 10:1) to afford 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-benzodiazol-6-yl]-2-methylbut-3-yn-2-ol (13). A suspension of 1-(4amino-1,3,5-triazin-2-yl)-6-bromo-N-ethyl-1H-1,3-benzodiazol-2amine (i-2; 170 mg, 0.51 mmol), 2-methylbut-3-yn-2-ol (129 mg, 1.53 mmol), and Pd(PPh3)4 (118 mg, 0.10 mmol) in piperidine (2 mL) was 1042

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heated in a microwave reactor under nitrogen for 1.5 h at 90 °C. The resulting solution was diluted with 200 mL of dichloromethane, washed with 2 × 100 mL of water and 2 × 100 mL of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified on a silica gel column eluted with dichloromethane/methanol (10:1). The product was further purified by PrepHPLC 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 1 min, down to 18.0% in 2 min); detector, UV 220 and 254 nm. This resulted in 43.5 mg (25%) of 13 as an off-white solid: 1H NMR (500 MHz, 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). 13 C 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 mixture of 1-(2-aminopyrimidin-4-yl)-6-bromo-N-(2-methoxyethyl)-1H-1,3benzodiazol-2-amine (i-6; 120 mg, 0.33 mmol), 2-methylbut-3-yn-2ol (277.2 mg, 3.30 mmol), Pd(PPh3)2Cl2 (231.67 mg), and triethylamine (2.4 mL) in dimethyl sulfoxide (1 mL) was stirred under nitrogen for 1 h at 70 °C. The reaction mixture was cooled to room 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 over anhydrous 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 colorless solid: 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 suspension of i-6 (250 mg, 0.65 mmol, 95% purity), 1-ethynylcyclohexan1-ol (300 mg, 2.42 mmol), and Pd(PPh3)2Cl2 (250 mg, 0.36 mmol) in DMSO (3 mL) and triethylamine (2 mL) was heated in a microwave for 20 min at 70 °C under a nitrogen atmosphere. The reaction mixture was concentrated under vacuum, and the residue was purified by 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) δ 163.5, 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) protein was assessed in vitro by assay of the phosphorylation of a FRET peptide substrate. The activity/inhibition of PAK enzymes was estimated by measuring the phosphorylation of a FRET peptide substrate (Ser/Thr19) labeled with coumarin and fluorescein using the Z′-LYTE assay platform (Invitrogen). The peptide substrate is a consensus sequence (KKRNRRLSVA) based on various PAK substrates reported in the scientific literature. The 10 μL assay mixtures contained 50 mM HEPES (pH 7.5), 0.01% Brij-35, 10 mM MgCl2, 1 mM EGTA, 2 μM FRET peptide substrate, and 20 pM PAK1-KD or 80 pM PAK4-KD. Incubations were carried out at 22 °C in black polypropylene 384-well plates (Corning Costar). Prior to the assay, PAK1-KD or PAK4-KD, FRET peptide substrate, and serially diluted test compounds were preincubated together in assay buffer (7.5 μL) for 10 min, and the assay was initiated by the addition of 2.5 μL of assay buffer containing 160 μM ATP (4×) for the PAK1 assay or 16 μM ATP (4×) for the PAK4 assay. Following the 60 min

incubation, the assay mixtures were quenched by the addition of 5 μL of Z′-LYTE development reagent, and 1 h later the emissions of coumarin (445 nm) and fluorescein (520 nm) were determined after excitation at 400 nm using an Envision plate reader (Perkin-Elmer). An emission ratio (445 nm/520 nm) was determined to quantify the degree of substrate phosphorylation. Cell Lines. MDA-MB-436 cells (ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 2 mM L-glutamine. MCF10A PIK3CA cells 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 Lglutamine, and 10 mM HEPES. Migration Assays. For migration experiments 40 000 MDA-MB436 or MCF10A PIK3CA cells were added per well of an Essen ImageLock 96-well plate (Essen BioScience, Ann Arbor, MI). After overnight incubation, a uniform scratch was introduced into the cell monolayer using an Essen WoundMaker device (Essen BioScience). Detached or loosely attached cells were removed by washing the wounded monolayer two times with warm medium. After the last wash, 150 μL of medium containing DMSO or compound 17 was added. Images were collected and quantified every 2 h in an IncuCyte system (Essen BioScience). Invasion Assays. ImageLock 96-well plates were coated with 100 μg/mL growth factor reduced Matrigel (BD Biosciences, San Jose, CA) for 3 h at 37 °C. After removal of the diluted Matrigel, 40 000 cells were plated per well and allowed to adhere for 5 min before the plate was moved to a 37 °C incubator. After overnight incubation, a uniform scratch was introduced into the cell monolayer with an Essen WoundMaker device. The wounded monolayers were washed two times, and 50 μL of 2.4 mg/mL growth factor reduced Matrigel was added to each well. The Matrigel was allowed to solidify at 37 °C for 30 min, and then 150 μL of complete medium containing DMSO or compound 17 was carefully added on top of the Matrigel layer. Images were collected and quantified every 3 h in an IncuCyte system. Viability Assays. MDA-MB-436 and MCF10A PIK3CA cells were sparsely plated in white-walled 96-well plates. After the cells adhered, DMSO and compound 17 were added, and the cells were incubated for 24 and 48 h. Celltiter Glo (Promega, Madison, WI) assays were carried out according to the manufacturer’s instructions, and luminescence was measured in a plate reader.

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ASSOCIATED CONTENT

S Supporting Information *

Full kinase selectivity data for compound 17, small-molecule Xray data for compound 17, tabulated EC50 data from the cell viability assays, and protein production/purification and X-ray crystallography conditions. This material is available free of charge via the Internet at http://pubs.acs.org Accession Codes

PDB IDs: 4O0R, 4O0T, 4O0V, 4O0Y, 4O0X.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: (650) 467-3103. *E-mail: hoefl[email protected]. Phone: (650) 225- 6697. Author Contributions ¶

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

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Genentech internship program for support of D.A.R. 1043

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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. Cell Logist. 2012, 2 (2), 95−104. (12) Crawford, J. J.; Hoeflich, K. P.; Rudolph, J. p21-Activated kinase inhibitors: 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-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic 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 the biochemical assays. (15) Kalgutkar, A. S.; Dalvie, D. Obach, R. S.; Smith, D. A. Reactive Drug Metabolites; Wiley-VCH: Weinheim, Germany, 2012. (16) See the Supporting Information for a description of the protein production, purification, and crystallization conditions (PAK1 and PAK4) used for the compounds discussed in this paper. (17) (a) Zuccotto, F.; Ardini, E.; Casale, E.; Angiolini, M. Through the “gatekeeper door”: exploiting the active kinase conformation. J. Med. Chem. 2010, 53, 2681−2694. (b) Angiolini, M. Targeting the DFG-in kinase conformation: a new trend emerging from a patent analysis. Future Med. Chem. 2011, 3, 309−337. (18) A series of Afraxis group I PAK inhibitors extend past the methionine 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)-associated schwannomas. 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)-1ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1H-imidazo[4,5-c]pyridin-4yl)-2-methyl-3-butyn-2-ol (GSK690693), a novel inhibitor of AKT kinase. J. Med. Chem. 2008, 51, 5663−5679. Within this paper, GSK690693 is noted to hit class II PAKs in a selectivity panel, but class 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-kB inducing 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-κBinducing 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 of Glu396 and the 6-H of the 2-aminotriazine is possible (3.6 Å).

ABBREVIATIONS USED: PAK, p-21-activated kinase; RAS, rat sarcoma; NIK, NFκBinducing 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, acetic anhydride; AcOH, acetic acid; MeCN, acetonitrile

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REFERENCES

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dependent on the ATP concentration (for example, a theoretical 250fold shift at standard 1 mM ATP). The efficacious concentrations could be increased by protein binding and typical pathway to phenotype shifts. The lower than expected biochemical to cell shift for other PAK inhibitors in the literature could be caused by group I PAK or other kinase inhibition. Alternatively, it is possible that the inhibition of cellular PAK4 may be weaker for compounds similar to 17. (37) These phenotypic results are similar to those of a recent analysis of PAK4 function in pancreatic ductal adenocarcinoma cell lines with PAK4 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 pathway causes an invasive phenotype which can be reversed or promoted by blocking the pathway at divergent nodes. PLoS One 2012, 7, 36402.

(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 of compound 16 can explain this result as it does not appear to make any significant 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 100 nM. 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 the Supporting Information. (27) Aurora, R.; Srinivasan, R.; Rose, G. D. Rules for α-helix termination 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′ capping position of an α-helix is consistent with this hypothesis, indicating some selection against Tyr (1.39 vs 0.93). (29) Another major difference in primary structure is the DFG − 1 residue (Thr in PAK1 and Ser in PAK4). However, we do not believe that access to the back pocket of group I PAKs is hindered by the larger branched residues. KHS1, Map4K4, and MINK1 all possess a branched valine residue in this position and seem to tolerate this vector of substitution on the basis of our selectivity data (Table 3). Additionally, AKT, which is known to tolerate type I 1/2 binding of inhibitors possessing propargyl alcohols, also contains a threonine residue 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. Four PAK4 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 by homology. (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 of GTPases 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: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2007, 2, 329−333. (35) (a) Tabusa, H.; Brooks, T.; Massey, A. J. Knockdown of PAK4 and PAK1 inhibits the proliferation of mutant KRAS colon cancer cells independently 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 and tumorigenesis in athymic mice. Mol. Cancer Res. 2008, 6, 1215−1224. (c) Tian, Y.; Lei, L.; Minden, A. A key role for Pak4 in proliferation and 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 p21activated kinase 4 inhibitor, suppresses proliferation and invasion in human 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-independent growth. Mol. Cell. Biol. 2001, 21, 3523−3533. (36) One possible rationalization for a high enzyme to cell shift of compound 17 is the low Km,apparent of ATP for the kinase domain of PAK4 (4 μM) such that a high enzyme to cell shift is expected 1045

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