Inhibitors of p21-Activated Kinases (PAKs) - Journal of Medicinal

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Inhibitors of p21-Activated Kinases (PAKs) Miniperspective Joachim Rudolph,*,† James J. Crawford,† Klaus P. Hoeflich,§ and Weiru Wang‡ †

Discovery Chemistry, and ‡Structural Biology, Genentech, 1 DNA Way, South San Francisco, California 94080, United States § Department of Biology, Blueprint Medicines, 215 First Street, Cambridge, Massachusetts 02142, United States S Supporting Information *

ABSTRACT: The p21-activated kinase (PAK) family of serine/threonine protein kinases plays important roles in cytoskeletal organization, cellular morphogenesis, and survival, and members of this family have been implicated in many diseases including cancer, infectious diseases, and neurological disorders. Owing to their large and flexible ATP binding cleft, PAKs, particularly group I PAKs (PAK1, -2, and -3), are difficult to drug; hence, few PAK inhibitors with satisfactory kinase selectivity and druglike properties have been reported to date. Examples are a recently discovered group II PAK (PAK4, -5, -6) selective inhibitor series based on a benzimidazole core, a group I PAK selective series based on a pyrido[2,3-d]pyrimidine-7-one core, and an allosteric dibenzodiazepine PAK1 inhibitor series. Only one compound, an aminopyrazole based pan-PAK inhibitor, entered clinical trials but did not progress beyond phase I trials. Clinical proof of concept for pan-group I, pan-group II, or PAK isoform selective inhibition has yet to be demonstrated.

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BACKGROUND

activators of mammalian mitogen activated protein kinase pathways, including ERK1/2, JNK, and p38 MAPK pathways. PAK1 is the most well characterized family member and is highly expressed in brain, muscle, and spleen in normal adult animals.8 PAK2 and PAK4 expression is relatively ubiquitous, whereas PAK3, PAK5, and PAK6 expression is enriched in neuronal tissues.8 PAK genes are conserved through evolution from yeast to human9 and have been studied genetically in metazoan, invertebrate, and vertebrate model systems. In mice, PAK1 gene disruption exhibits relatively subtle defects in the central nervous system and in regulating immunity5 and metabolism.6 For example, bone marrow-derived mast cells derived from PAK1-deficient animals display impaired F-actin depolymerization and reduced cell degranulation in a model of allergen stimulation.5 In addition, despite normal cardiac development of PAK1 knockout mice, PAK1 plays important roles in cardiac physiology, such as the regulation of cardiac ion

The p21-activated kinases (PAKs) are Rac1 and Cdc42 effectors that have generated significant interest as therapeutic targets in cancer.1,2 The PAK family is comprised of six members and is subdivided into two groups (groups I and II) based on sequence and structural homology. Group I and II PAKs have been shown to contain an amino terminal Cdc42/ Rac interaction/binding (CRIB) regulatory domain required for interacting with Rho family GTPases (Figure 1).3 Binding to active Ras-related C3 botulinum toxin substrate 1 (Rac) and cell division control protein 42 (Cdc42) GTPases stimulates kinase activity by relieving an intramolecular interaction between the kinase domain and an autoinhibitory domain. PAKs are currently among the most well characterized effector proteins of Rac and Cdc42. The kinase domains of group I and II PAKs share approximately 50% identity (Figure 2), suggesting that the two groups may recognize some distinct substrates and govern unique cellular processes, although a number of effectors are overlapping.4 The PAK catalytic domains share homology with additional members of the sterile-20 (STE20) subfamily of the kinome that are upstream © XXXX American Chemical Society

Special Issue: New Frontiers in Kinases Received: October 18, 2014

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dx.doi.org/10.1021/jm501613q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. The domain structures of PAK1 and PAK4 are highlighted as representative members of groups I and II. P21-binding domain (PBD) for GTPase association, overlapping autoinhibitory domain (AID), catalytic domain, basic residue cluster for phospholipid binding (Basic), and the PAK-interacting exchange factor (PIX) domain are shown. Black bars represent proline-rich (PXXP) domains.

reperfusion injury.7 PAK2 is highly expressed in endothelial cells and is required for angiogenesis during development.1 PAK2 knockout mice were reported to die early in embryogenesis because of multiple developmental abnormalities, most prominently involving defective vascularization,8,10 but no experimental data have been published to date. Loss-of-function mutations in PAK3 are associated with familial cognitive disorders in humans, and the corresponding mouse model recapitulates behavioral and cellular phenotypes.11 To test redundancy with other PAK family members, PAK1 and PAK3 dual knockout mice were also generated.12 These mice had deficits in learning and memory as well as reduced dendrite formation. Knockout mice have also been developed for all group II PAK family members. Disruption of PAK4 results in lethality prior to embryonic day 11.5 due to defective development of extraembryonic tissues and severe abnormalities in the heart.13 A tissue-specific conditional knockout model has also demonstrated that self-renewal of neuronal progenitor cells is dependent on PAK4.14 In contrast with the dramatic neuronal phenotype of PAK4, single gene disruption of either PAK5 or PAK6 did not result in a noticeable phenotype. However,

Figure 2. Percent sequence similarity in the kinase domain of different PAK isoforms. The boxes highlighted in blue indicate the degree of homology of isoforms within each group.

channel and actomyosin function.7 More recent studies have revealed that PAK1-deficient mice were vulnerable to cardiac hypertrophy and readily progress to heart failure under sustained pressure overload and were susceptible to ischemia/

Table 1. Tissue Expression, Mouse Knockout Phenotypes, and Species Conservation of the Six PAK Isoforms isoform

tissue expression

mouse knockout phenotype

PAK1

Wide expression; high in brain, muscle, and spleen

Viable, fertile, and normal life span. Immune defects, glucose homeostasis defects, neuronal defects, compromised cardiac function upon challenge

PAK2

Ubiquitous; high in endothelial cells

Embryonic lethal; multiple developmental abnormalities, most prominently involving defective vascularization

PAK3

Mostly neuronal tissues

Viable, fertile. Impaired synaptic plasticity, defects in learning and memoryb

PAK4

Ubiquitous

Embryonic lethal (heart defect); neuronal defects, improperly formed neural tube

PAK5


Viable, fertile, deficits in locomotion, learning and memory

PAK6


Viable, fertile, deficits in locomotion, learning and memory

species conservation [%]a,17 M 99.26 R 99.08 D 96.87 M 97.14 R 96.56 D 95.8 M 98.53 R 98.71 D 95.29 M 93.22 R 93.22 D 95.59 M 93.88 R 94.57 D 96.94 M 92.66 R 92.95 D 94.42

a

Percent homology to human isoform: M: mouse; R: rat; D: dog. bLoss-of-function mutations in PAK3 are associated with familial cognitive disorders in humans. B



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combined deletion of both PAK5 and PAK6 revealed deficits in learning and memory.15 It is unclear why the phenotypes between PAK4 and PAK5/PAK6 knockout mice differ so significantly, but it may result from temporal differences in expression during neuronal and brain development.16 Taken together, this extensive body of work using genetically engineered mouse models has considerably increased our knowledge of PAK family function in development and homeostasis. To date, however, no knock-in mouse models have been created to delineate the specific contribution of PAK catalytic activity, as compared to potential scaffolding functions, in these biological processes. Information on tissue expression, mouse knockout phenotypes, and species conservation is summarized in Table 1.

In 2011, it was reported that focal genomic amplification of PAK1 at 11q14.1 is prevalent in the luminal subtype of breast adenocarcinoma when examined in 216 human breast carcinoma specimens.19 These findings were supported by a subsequent study20 and expanded upon by the large-scale breast cancer genomic initiatives, The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (The Cancer Genome Atlas Network).21 Focal amplification of PAK1 is now known to occur in approximately 7% of breast cancer and is enriched in the poor prognosis, luminal B subtype. In addition to amplification, PAK1 protein levels were determined by immunohistochemical staining in 274 breast tumor tissue specimens.19 In this study, low PAK1 protein levels were found in normal mammary epithelium; however, high cytoplasmic protein levels were observed in 39% of primary adenocarcinomas. Consistent with the evolutionarily conserved role for PAK1 in regulating cell motility, high PAK1 expression was associated with lymph node invasion and occurred more frequently in nodal metastases compared to primary tumors. It is interesting that the frequency of dysregulated expression of PAK1 was greater (39%) than predicted by genomic amplification alone (7%) indicating that additional regulatory mechanisms may increase PAK1 expression in breast cancer, e.g., microRNA regulation.22 Analysis of breast cancer cell lines with PAK1 genomic copy number gain revealed dependence on PAK1 expression and activity for cell survival19 and transformation.20 These PAK1associated functions might also be a contributing factor to the association of elevated PAK1 expression with reduced clinical benefit that has been reported for patients treated with tamoxifen.23 Consistent with these findings, functional studies using transgenic mouse models demonstrated that overexpression of PAK1 in the murine mammary gland promotes the formation of preneoplastic lesions and breast tumors24 and that PAK1 contributes to human EGFR receptor 2 (HER2)/ Neu-driven tumorigenesis.25 Breast cancer is not the only tumor indication with PAK1 amplification, and there is evidence for PAK1 dysregulation in additional tumor types, including ovarian cancer and melanoma. In the case of melanoma, copy number gain and increased protein expression of PAK1 negatively associate with activating mutations of the BRAF oncogene.26 PAK1 knockdown or inhibition attenuates signaling through MAPK and cytoskeleton-regulating pathways and modulates the proliferation and migration of BRAF wildtype melanoma cells. Taken together, these findings indicate that PAK1 amplification and overexpression may drive tumorigenesis in some disease contexts. There also is evidence for genomic amplification of PAK4. Copy number gain of PAK4 at 19q13.2 has been demonstrated in approximately 22% and 11% of pancreatic and colorectal cancers, respectively;27,28 however, this amplicon is relatively broad and the dominant driver genes still remain to be validated. In PAK4-amplified pancreatic adenocarcinoma cell lines, the role of PAK4 is primarily related to induction of tumor cell motility.28 Unlike other kinases implicated in cancer, there have been few reports of nonsynonymous somatic mutations that correlate with enhanced PAK activity, perhaps owing to the complex activation mechanism of this kinase family. Recently, PAK5 mutation was reported in a targeted genetic dependency screen of non-small-cell lung cancer cell lines29 and PAK5 contributed to tumor cell proliferation and survival. Interestingly, the identified PAK5 (T538N) mutation

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ROLE OF PAKS IN CANCER Much of the interest in understanding the role of PAKs in human disease has been focused on cancer. Multiple PAK family members have been shown to maintain cell transformation in vitro by stimulating signaling pathways leading to proliferation, survival, motility, and angiogenesis (Figure 3).1,18 With respect to molecular driver events, PAKs are amplified, overexpressed, or hyperactivated in several tumor subtypes.1,4

Figure 3. Signaling for PAK family kinases. Receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and integrins activate PAKs via guanosine triphosphate (GTP) bound Rac and Cdc42 small GTPases. Direct and indirect PAK effectors mediating cellular effects on survival, proliferation, and motility are shown. Abbreviations are as follows: BAD, Bcl-2-associated death promoter; CDC42, cell division control protein 42; CRAF, v-raf-1 murine leukemia viral oncogene homolog; ERα, estrogen receptor α; ERK, extracellular signalregulated kinase; GPCR, G-protein-coupled receptor; LIMK, LIM domain kinase; MEK1, MAPK/ERK kinase 1; Merlin, moesin-ezrinradixin-like protein; MLCK, myosin light chain kinase; NF-κB, nuclear factor κB; PAK, p21-activated kinase; Rac, Ras-related C3 botulinum toxin substrate; RTK, receptor tyrosine kinase; STMN1, stathmin 1. Solid arrow indicates direct substrate. Dotted arrow indicates indirect substrate. C



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coordinate protein translation-dependent synaptic plasticity.43 A PAK small molecule inhibitor in this indication could be efficacious but might also be expected to require a very safe profile in order to treat pediatric patients with mild to moderate cognitive impairment. Taken together, it is clear that PAK family members significantly contribute to cognitive function, and opportunities for therapeutic targeting of PAKs in neurological disorders are currently being investigated by a number of laboratories. PAKs also play a role in the life cycle of eukaryotic, prokaryotic, and viral pathogens, and inhibiting PAKs may provide an effective strategy to modulate host−pathogen responses. For example, in the cases of Helicobacter pylori, Gram-negative bacteria found in the stomach, and Pseudomonas aeruginosa, Gram-negative bacteria found in soil, bacterial proteins are translocated into host cells to subvert cellular functions such as actin cytoskeleton rearrangement, and this process is in part mediated by activation of the small GTPases Rac and Cdc42.44,45 PAKs would therefore be possible targets to inhibit bacterial internalization, and published data support this hypothesis.44,46 PAKs have also been implicated in promoting viral replication and pathogenesis. Human immunodeficiency virus (HIV) encodes and secretes negative factor (Nef) protein that contributes to pathogenesis,47 and both PAK1 and PAK2 associate with and can be activated by Nef as part of the virus life cycle.48,49 PAK1 activity is also stimulated by influenza A virus replication, and while PAK1-specific siRNA knockdown decreased viral titers by 10- to 100-fold,50 in vivo data confirming the requirement of PAKs for robust replication of this pathogen are still required. In summary, the involvement of PAKs in promoting pathogen survival and proliferation may offer new avenues for therapeutic approaches to mitigate infectious diseases in the future.

is located in the substrate recruitment domain, a novel category of tumor-specific, kinase activating mutations.30 In addition to direct dysregulation by genomic amplification, overexpression, or mutation, PAKs are also key effectors of well-established oncogenes, such as the Ras small monomeric GTPase. Somatic gain-of-function mutations of Ras occur in approximately 30% of human tumors.31 To date, it has been challenging to directly target Ras, and hence, drug discovery efforts have focused on targeting effector pathways. Given that Rac and Cdc42 lie downstream of Ras,32,33 several groups have evaluated the contribution of PAKs to Ras-driven cellular transformation and in vivo tumorigenesis. For instance, PAK1 deletion in a mouse model of Ras-driven cutaneous squamous cell carcinoma led to markedly decreased tumorigenesis and disease progression, which was accompanied by attenuated signaling through the MAPK and phosphoinositide 3-kinase (PI3K) pathways.34 In lung cancer, KRas signaling to lymphocyte-specific tyrosine kinase (LCK) and PAK1 has been implicated in regulation of antiapoptotic pathways in KRas-dependent tumor cells.35 PAK4 also plays an important role in mediating Ras-driven xenograft tumor formation and signaling.36,37 It has yet to be determined which PAK isoform(s) inhibitors will be most effective in terms of efficacy and safety in Ras-mutant tumors. Taken together, there is emerging evidence for targeting PAKs in several cancer indications, and generating tool compounds with varying PAK family selectivity profiles will shed further light on their roles in preclinical and clinical studies.

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ROLE OF PAKS IN NONONCOLOGY INDICATIONS There are additional therapeutic opportunities for targeting PAKs outside of oncology, including neurological disorders and infectious diseases. Given that cognitive decline in both nonsyndromic X-linked mental retardation (which has been linked to loss of PAK3 function) and Alzheimer disease (suggested to involve PAK1 dysregulation) is associated with decreased synaptic function and dendritic spine loss, it has been hypothesized that altered PAK signaling and regulation of the cytoskeleton may underlie this related cellular pathology.38 For example, in Alzheimer disease the decreased cytoplasmic localization and aberrant activation of PAK1 was observed in brain tissue from patients.39 In these specimens, PAK1 was present in membrane-cytoskeletal fractions where it might presumably interact with upstream activators, such as Rac and Cdc42. This phenotype was recapitulated in cultured hippocampal neurons treated with β-amyloid oligomers, and the rapid loss of dendritic spines could be rescued by ectopic expression of wild-type but not catalytically inactive PAK1. Although these results suggest that PAK1 activation might contribute to early stages of Alzheimer disease, the loss of PAK1 and PAK3 expression and signaling has been observed in advanced patient tissues40 and could therefore limit the therapeutic window and utility of PAK inhibitors in this indication.38 Another neurological disorder that has generated considerable interest with respect to PAK-specific intervention is X chromosome-linked mental retardation. Missense and nonsense mutations of the PAK3 gene have been found in the nonsyndromic form of this disease,41 and inhibition of PAK activity alleviated symptoms in the syndromic form, Fragile X syndrome (FXS), in mice.42 PAK1 has also been linked to FXS via direct interaction and phosphorylation of Fragile X mental retardation protein 1 (FMR1) and its close homologs, which

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ATP BINDING POCKET OF GROUP I AND II PAKS The PAK1 gene encodes a multidomain protein. The amino terminal end of the PAK1 protein harbors several sequence motifs responsible for interacting with partner proteins such as Nck, PIX, and p21s (Cdc42/Rac). Inactive PAK1 exists as autoinhibited homodimers held together by an autoregulatory motif (residues 87−136) named “inhibitory switch (IS) domain”.51 In a PAK1 dimer, the IS domain of each molecule binds to the kinase domain of the partner molecule. This dimer interface is reinforced by an antiparallel β-ribbon formed between the two IS domains. A “kinase inhibitor (KI) segment” (residues 137−149) traverses through the kinase cleft of the dimer partner forcing its activation loop to tuck into the ATP binding site.51 PAK1 can be activated by p21 proteins such as Cdc42 and Rac. These p21 proteins exhibit high affinity to the p21-binding domain (PBD) of PAK1, which partially overlaps with the IS domain. P21 binding disrupts the IS domain structure, resulting in dissociation of the PAK1 dimer. The PAK1 monomers are prone to phosphorylation and become activated in the cellular environment.52,53 Other group I PAKs are reported to be regulated in a similar manner.51 The mechanisms of activation of group II PAKs are less clear. In contrast to group I PAKs, the kinase domain of the group II PAKs is constitutively phosphorylated.54 Hence, transition to the active form likely depends on conformational changes. Their detailed mode of activation is ambiguous, and several models have been proposed.54,55 The ATP binding pocket of PAK1 has been extensively studied and found to be conspicuously open and malleable, and D



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specifically the large size of the cleft between the N-lobe and Clobe, as well as the high mobility of the N-lobe, was pointed out.52 This was supported by the subsequently published PAK1 structures. Figure 4 illustrates a view of global dynamics in the

With a view to taking advantage of the large ATP-binding pocket in PAK1, Meggers and co-workers designed octahedral ruthenium complexes around the “staurosporine-inspired” metallopyridocarbazole scaffold.60 Identified through screening of a focused library of 48 such ruthenium complexes, one of the initial leads was the GSK3/Pim1 “half-sandwich” inhibitor DW12 (2, Figure 5)60 with modest PAK1 activity (IC50 ≈ 1 μM). A crystal structure confirmed the expected hinge interaction (four hydrogen bonds from the maleimide and indole OH groups) as well as van der Waals contacts between the CO ligand and the P-loop similar to that shown for ΛFL172 (compound 3, Figure 5)60 in Figure 7A. The ruthenium center is located within the ribose binding site, while the η5cyclopentadienyl group does not make any important interactions and is instead projected into the solvent-exposed region. The fact that this comparatively large and threedimensional metal−ligand complex fits within the PAK1 ATPbinding pocket is demonstrative of its size and/or flexibility. To take advantage of this additional size, bulkier substituents were added in place of the Cp− motif, such as the bidentate phenyliminopyridine ligand in 3. A cocrystal structure of 3 bound to PAK1 revealed that the pyridocarbazole moiety and CO ligand maintain their interactions, while the larger iminopyridine moiety now better fills the available space (Figure 7B). The result of this was a gain in PAK1 potency (IC50 of 130 nM) and selectivity; when 3 was tested against a panel of 264 kinases (3 μM ligand concentration and 10 μM ATP concentration; racemic mixture tested), only 15 kinases (5.7%) were inhibited by >80%. In subsequent efforts, a simplified analog was designed, compound 4 (Figure 5),56 with improved PAK1 potency (IC50 of 83 nM for the racemic mixture at 1 μM ATP), albeit with significantly reduced kinase selectivity.56 With four fewer aromatic rings and one fewer Hbond to the hinge, this compound is synthetically more accessible and more ligand-efficient. Interestingly, the uncomplexed ligand 15 (Figure 6)56 showed no PAK1 activity (IC50 > 100 μM), confirming the important role of the Ru− ligand complex. Additionally, despite retaining important binding motifs and structural features, there is a significant change in the binding mode between 3 and 4, with the pyridine and CO/SCN ligands oriented differently in the two structures. This, in addition to the reduced steric bulk, may account for the reduced kinase selectivity observed with 4. To date, no further information on cell activity, efficacy, or pharmacokinetics have been reported for this organometallic conjugate series, and their drug properties and toxicity concerns render the utility of this class as in vivo tool compounds questionable. Pyrrolopyrazole Series. Pfizer researchers were perhaps the pioneers in seeking PAK inhibitors, with reports in the patent literature stretching back to 2004.61−64 A particularly prominent structural motif is the pyrrolopyrazole hinge binding scaffold (Figure 8) that was inspired by earlier described Aurora-2 kinase inhibitors containing pyrazole cores integrated into a fused bicyclic system.61 Following a high-throughput screen against PAK4 (1.3 million compound library) and subsequent kinase counterscreening, benzimidazole-indazole, and pyrrolopyrazole scaffolds were identified (Figure 9). Because of kinase selectivity concerns with these scaffolds (for the benzimidazoles, such as 16)65 and poor ligand efficiency (LE = 0.14 for 6),65 respectively, optimization efforts were undertaken leading to superior compounds. An example is compound 17, 65

Figure 4. PAK1 kinase domain structure. Fourteen structures currently available from PDB are superimposed based on the C-terminal lobe, where the structures are well conserved. The N-terminal lobe displays a high degree of conformational heterogeneity. The structural variations include concerted N-lobe shift relative to C-lobe and localized conformational changes more frequently seen in P-loop, helix-C, and activation loop.

PAK1 kinase domain, where the currently available 14 PAK1 entries in Protein Data Bank (PDB) are structurally aligned according to their conserved C-lobe region, with the exception of the activation loop (A-loop). The alignment reveals a high degree of conformational heterogeneity in the N-lobe; particularly large dynamic ranges are associated with the phosphate-binding loop (P-loop) and C-helix. Such characteristics appear functionally relevant, as in the autoinhibitory state eight amino acid residues of the kinase A-loop need to tuck inside its own ATP-cleft. It has been suggested that these binding site characteristics impeded the identification of highaffinity inhibitors.56 Fourteen ligand cocrystal structures have been reported to date, seven with PAK1 and seven with PAK4 (Figure 5). In line with the observations described above, the PAK1 structures reflect a highly accommodating character of the ATP binding pocket, with ligand-dependent backbone displacements predominantly occurring in the β1 and β2 strands of the N-lobe.51 Similar to PAK1, the kinase domain of PAK4 as a representative of the group II PAKs was found to be highly plastic and rich in conformationally distinct states.57

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ATP COMPETITIVE PAK INHIBITORS Oxindole/Maleimide-Based Inhibitors. The indolocarbazole-based natural product staurosporine (14, Figure 6)58 and analogs, such as hydroxy derivative ST2001 (1, Figure 5),80 are prototypical ATP-competitive kinase inhibitors with a high affinity for a broad range of kinases, particularly STE20 family kinases, such as the MST, GCK, and PAK subfamilies. 14 inhibits PAK1 with an IC50 of 0.6 nM, rendering it one of the most potent PAK1 inhibitors reported to date.59 E



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Figure 5. ATP competitive PAK inhibitors with published cocrystal structures. The molecule portions rendered in red indicate the atoms participating in kinase hinge hydrogen bonding contacts.

possessing a PAK4 Ki of 30 nM and enhanced ligand efficiency (LE = 0.32). A key modification was removal of the solvent exposed phenoxyl tail (Figure 9). Compound 17 inhibited phosphorylation of the PAK4 substrate GEF-H1 (EC50 = 32 nM) but suffered from poor oral bioavailability, likely due to poor permeability and/or high efflux as suggested by CACO-2 assay data (AB 0.1; BA/AB 326). By use of continuous infusion (via an implanted subcutaneous pump, 480 mg/kg/day), 17 was tested in an animal tumor model (HCT116), wherein pGEF-H1 was downregulated by ∼61% after 18 h. The primary strategy for

improving the permeability of 17 was to reduce the basicity and the number of hydrogen bond donors. It was postulated that substituting the carbamate for a urea function could achieve both goals with a single atom change. Additionally, it was observed that the arylamide moiety was not making any key interactions and that this might be better replaced with a bulky alkyl group. These changes resulted in compound 18,65 which is less basic and shows an increased log D of 2.52 (vs 1.77 for 17). While permeability for 18 was only moderate, efflux was significantly reduced (CACO-2 AB 1.7; BA/AB 9.9). A more dramatic improvement was seen in cellular potency, with a F



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Figure 8. 3-Aminotetrahydropyrrolo[3,4-c]pyrazole (group II) substructure of Pfizer PAK inhibitors.

based on the pan-PAK activity of related compounds, this compound is unlikely to possess any PAK isoform selectivity. Inhibitor 18 was progressed into animal pharmacokinetic and efficacy studies where the oral bioavailability in rat was found to be low (15%), consistent with its relatively high in vivo rat clearance (52 mL min−1 kg−1). Compound 18 was further evaluated in dog, where oral bioavailability was again moderate (20%), this time with clearance at 12 mL min−1 kg−1 and a halflife of 2.2 h. Metabolite identification studies on this and related compounds revealed the dealkylation product of the dimethylamine moiety as the major metabolite. In an in vivo HCT-116 colon cell xenograft mouse tumor model, 18 showed appreciable efficacy as tumor growth inhibition was observed at 12 mg/kg (b.i.d., TGI = 37%) and more significantly at 20 mg/kg (b.i.d., TGI = 52%). Furthermore, in an M24met melanoma tumor model, 18 showed robust tumor growth inhibition at a top oral dose of 15 mg/kg (b.i.d., TGI = 68%, unbound cave = 50.5 nM). The first PAK inhibitor to proceed into clinical trials also came from Pfizer, PF-3758309 (5, Figure 5),66 and is derived from the same series. This compound progressed into phase 1 clinical trials in patients with advanced/metastatic solid tumors.67 Compound 5 is a pan-PAK inhibitor, equipotent against all group II PAK kinase domains (PAKs 4−6) at around 20 nM, with variable activity against the group I PAKs (PAK1 Ki = 13.7 nM, PAK2 IC50 = 190 nM, PAK3 IC50 = 99 nM).64,66 The key structural difference from 18 is the substituent attached to the exocyclic amine of the aminopyrazole motif (thienopyrimidine vs pivaloyl group). Information on the binding mode of this series, and in particular, 5, has been published,65,67,68 and cocrystal structures bound to both PAK1 and PAK4 are shown in Figure 10. Apart from the generic three-point donor−acceptor−donor hydrogen bond interactions with the kinase hinge, there are a number of important interactions that drive PAK4 potency as well as selectivity. These include a hydrogen bond to water from the urea carbonyl oxygen and an ionic interaction of the dimethylamino group with Asp-458. The geminal dimethyl group of the pyrrolopyrazole is crucial for kinase selectivity, as it clashes with the residues present in the ATP-binding of other kinases site but packs well with Val-335 and Met-395 in PAK4. Of particular note is the methyl group at the 2-position of the thienopyrimidine. This group has a dramatic effect on both potency (5-fold improvement in vitro and 87-fold in pGEFH1) and efflux (a 10-fold improvement relative to the des-Me analogue, with AB/BA ratio of 9).68 As a result of these interactions, 5 is a potent PAK4 inhibitor (PAK4 Ki = 22 nM). As the functional cellular activity of 5 (1.3−3.9 nM) is greater than its biochemical activity (20 nM) against PAK family members, off-target activity presumably contributes to its cell activity especially since the higher ATP concentration in cellbased assays should cause a loss in potency. With a t1/2 of 68 s (koff = 0.010 s−1) against PAK4 as determined by SPR (Kd = 2.7 nM), binding kinetics would also not appear to contribute to the observed shift in cellular activity. A key to the high cell

Figure 6. Staurosporine (14) and uncomplexed ligand 15 of ruthenium complex 4.

Figure 7. Indolocarbazole based compound 3 bound to PAK1.60 (A) Compound 3 is rendered in yellow, and PAK1 protein is displayed as a backbone cartoon shown with surface rendering. The compound makes four hydrogen bonds (red dotted lines) with the hinge. (B) Compound 3 pushes the P-loop up (between the white and orange cartoon trace) to create space for the large headgroup. The ribbon drawing in white shows the P-loop position when compound 5 is bound.86

pGEF-H1 EC50 of 4 nM. Given the reported PAK4 Ki,63 which is 75 nM for this compound, off-target kinase activity might be contributing to its cellular activity. Screening 18 against 119 kinases at 1 μM concentration revealed >70% inhibition of AMPK, CDK7, ERK2, PBK, GSK3β, RSK, PKA, MARK, and MST4. Although not reported specifically for compound 18, G



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Figure 9. Evolution of Pfizer PAK4 inhibitors.

(>70% TGI) in Ras-driven models,70,71 such as colon (HCT116 and Colo205), breast (MDA-MB-231), lung (A549), and melanoma (M24met). Unfortunately, clinical investigation of 5 revealed major liabilities. First, very low oral bioavailability (∼1%) was observed upon dosing in patients (n = 33).67 This was not necessarily expected based on clearance data from animal in vivo studies (CL in dog was 15.4 mL min−1 kg−1, and in rat it was 155 mL min−1 kg−1) and moderate oral bioavailability (dog, F = 39−76%; rat, F ≈ 20%), which translated to an acceptable predicted human pharmacokinetic profile: CL = 3.8 mL min−1 kg−1; T1/2 = 6.6 h; F = 35%; Vd = 2.1 L/kg.68 Concomitant with the poor bioavailability, no tumor responses were observed at any of the eight dose levels from 1 mg q.d. to 60 mg b.i.d., and no PD data were reported. Second, adverse events were reported, including grade 4 neutropenia and numerous gastrointestinal findings.67 As a result, clinical investigation of 5 was placed on hold67 and no conclusions could be drawn about the effect of PAK inhibition on solid tumors in patients. Monocyclic Aminopyrazole Series. In 2006, Pfizer disclosed a series of 5-substituted monocyclic aminopyrazoles.62 The corresponding patent application includes a number of examples with high PAK4 activities. An interesting SAR feature in this disclosure is the frequent use of the 3azabicyclo[3.1.0]hexan-6-amine tail group, which seems to impart high potency across a number of substituted pyrimidine cores. One such example is the highly ligand-efficient PAK4 inhibitor 1962 (Figure 11, PAK4 IC50 = 4 nM, LE = 0.46). This is a relatively common scaffold present in other reported kinase inhibitors, such as the Aurora inhibitor N-[4[[4-(4-methylpiperazin-1-yl)-6-[(5-methyl-1H-pyrazol-3-yl)amino]pyrimidin-2- yl]sulfanyl]phenyl]cyclopropanecarboxamide (2S)-2-hydroxypropanoate (MK-0457) and the JAK2 inhibitor (S)-5-chloro-N2-(1-(5-fluoropyrimidin-2-yl)ethyl)-N4-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (AZD1480).72,73 Genentech reported PAK1 inhibitors using the same aminopyrazole-based scaffold for use in cancer treatment in a 2013 patent application, this time as inhibitors of PAK1.74,75 Biochemical and cellular (pMEK1 S298) activity data for 26 example compounds were reported, including compound 20,74 the most potent PAK1 inhibitor. With a PAK1 Ki of 1.6 nM and pMEK IC50 of 43 nM, this, to the best of our knowledge, represents the most ligand-efficient76 PAK1 inhibitor published to date (LE = 0.42). While no further details regarding kinase selectivity, ADME properties, or efficacy of this series have been reported to date, one can postulate that their pendent, generally bicyclic aromatic substituent from the 2-position of the pyrimidine ring may be

Figure 10. Crystal structures of compound 5 bound to PAK1 (in blue) and PAK4 (in yellow).86,57

potency of this series is likely its off-target activity against kinases involved in the cell-cycle mechanism such as CDK7 (IC50 = 7 nM).69 HCT116 cells treated with 100 nM compound exhibited cell-cycle arrest and apoptosis after 48 h.66 5 also showed activity against other cell types, including p53-containing cell lines (IC50(p53/Pin1) = 5.7 nM; IC50(p53/ Mdm2) = 5.6 nM). Treatment of cells with DNA damageinducing agents led to an increase in phospho-PAK4, and coadministration of 5 attenuated the induction of p53 regulated genes, such as p21waf/cip1. 5 was also tested in a panel of human xenograft tumor models. Oral dosing (7.5−30 mg/kg, b.i.d.) for 9−18 days resulted in significant tumor growth inhibition H



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Figure 11. Monocyclic aminopyrazole PAK inhibitors from Pfizer and Genentech.

The 2-amino substituted pyrido[2,3-d]pyrimidine-7-one core of the Afraxis PAK inhibitors is a common scaffold described in the kinase inhibitor literature, and its binding in the ATP binding pocket of kinases is well understood and structurally characterized (see, for example, PD0332991 (palbociclib), a CDK4/6 inhibitor in phase III trials with a pyrido[2,3d]pyrimidine-7-one core, bound to CDK6 (PDB code 2EUF)).79 Therefore, as expected, the crystal structure of 7 bound to PAK1 (Figure 16) shows the characteristic hydrogen bonds between the N3 nitrogen of the core to the backbone NH of Leu-347 and between the NH moiety of the ligand and the backbone carbonyl of the same Leu residue. Clearly the most interesting aspect of this X-ray structure is the binding of the C6 substituent (“headgroup”) of the inhibitor, 2-chloro-4(thiazoly-5yl)phenyl, which penetrates deeply into the back pocket of the ATP binding site. The aromatic ring of the chlorophenyl moiety fits tightly into the cleft formed by the hydrophobic side chains of the Met-344 gatekeeper residue and the aliphatic carbons of Arg-299 (a K299R mutation was introduced to enable crystallographic studies), and the thiazole moiety protrudes into the deep pocket of the ATP binding site formed by Glu-315, Ile-316, and Val-342. This pocket is not sampled by other structurally characterized PAK inhibitors, such as 180 or 2.52 Superimposition of the structure of 7 bound to PAK1 with PAK4−ligand cocrystal structures suggests significant constriction in this area in PAK4, presumably because of different conformational preferences of the gatekeeper Met-395 and Lys-350 side chains compared to the corresponding residues in PAK1.78 This provides a potential explanation of the high degree of PAK1 vs PAK4 selectivity of 7 and other compounds with similarly extended headgroups. Another feature of the 7-PAK1 cocrystal structure worth pointing out is the orientation of the phenylpiperazine moiety, a frequently found motif in the PAK inhibitor patent applications from Afraxis. According to the structure, this moiety is directed toward the bulk solvent region with no apparent specific binding interactions with the protein. Given the abundance of aromatic and heteroaromatic rings in the structure of 7, it is not surprising that later reports by Afraxis reveal efforts to remove aromaticity, presumably with the goal of improving drug properties. A result of this work is FRAX1036 (25, Figure 13),81,82 which bears an ethylpiperazine in place of a phenylpiperidine side chain. This change does not compromise PAK1 potency, and likewise high selectivity against PAK4 (>100-fold) is retained. Interestingly, not only does this modification address drug properties but it also imparts significant improvement in kinome selectivity. As illustrated by the kinome tree in Figure 14 and the Venn diagram comparison in Figure 15, 25 spares a number of

oriented toward the sugar pocket, perhaps in a similar manner to the 3-azabicyclo[3.1.0]hexan-6-amine group used in the compounds described earlier (albeit for PAK4). In a subsequent filing, Genentech reported on related compounds, this time with aza-bicyclic heterocycles at the pyrimidine 2position.77 Again, no further data beyond PAK1 IC50 and pMEK were provided. The aminopyrazole substituent was almost exclusively cyclopropyl or fluorocyclopropyl, suggesting that this is important for binding affinity. A representative compound, 21,77 a 4-azaindole tail analog with a transfluorocyclopropyl headgroup (PAK1 Ki = 8 nM) is shown in Figure 11. Pyrido[2,3-d]pyrimidine-7-one Series (Group I PAK Inhibitors). Afraxis, a biotechnology company founded in 2007, described in a series of patent applications PAK inhibitors based on a pyrido[2,3-d]pyrimidine-7-one core (Figure12).

Figure 12. 2-Aminopyrido[2,3-d]pyrimidine-7(8H)-one core of Afraxis PAK inhibitors.

Initial disclosures focused on the use of these compounds for the treatment of CNS disorders, particularly Fragile X syndrome, but the scope of use was later expanded to include oncology indications.2 This compound class was identified in a high-throughput screen of a 12 000-membered kinase-focused compound library and then optimized for PAK group I vs group II selectivity.78 As shown in Figure 13, appending a 2,4di-Cl-phenyl ring at the 6-position of the core of FRAX019 (22, Figure 13) increased PAK1 activity greater than 20-fold without imparting a change in PAK4 activity (FRAX414, 23), and replacement of the p-Cl group of the latter compound with a heterocycle, as exemplified by the thiazole analog FRAX597 (7, Figure 13), completely ablated PAK4 activity. In an expanded kinase panel (140 kinases) at a concentration of 1 μM, 7 showed moderate selectivity, with >75% inhibition against 23 of the 52 tyrosine receptor kinases tested but significantly better selectivity against kinases from other families. As expected from the high homology in the ATP binding pocket within the two PAK groups, the group I PAKs (1, 2, and 3) are all inhibited to a similar extent while the group II PAKs (4, 5, and 6) are spared. Both its PAK group I vs group II selectivity and its selectivity against AGC and CAMK kinase family members render the selectivity profile of this compound very different from the profile of 5 (see kinome trees in Figure 14). I



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Figure 13. Evolution of Afraxis PAK inhibitors. Extension of the headgroup at the 6-position of the pyrido[2,3-d]pyrimidine-7(8H)-one core imparts a gain in potency and PAK group I vs group II selectivity (group 1 represented by PAK1 and group II represented by PAK4). 24 and 26 are structural analogs of 23 and 7, respectively, and 25 is a representative of a new, more kinase selective subseries devoid a phenyl ring at the left-hand side.

that interfering with PAK group I activity could produce significant antitumor activity in KRas-driven tumors. Interestingly, 7 reduced PAK1 and PAK2 protein expression levels in treated animals, a finding reproduced in vitro. This is suggestive of a dual inhibitory role of this compound, ATP-competitive inhibition and destabilization, potentially by binding to an “open” form of the PAK kinase domain.34 The occurrence of mutational inactivation of the tumor suppressor NF2 in about 50% of sporadic meningiomas and the fact that group I PAKs are inhibited by the NF2-encoded protein Merlin prompted the investigation of Afraxis PAK inhibitors in meningioma models. Indeed, studies using compounds 7, 25, and 26 as tool compounds revealed antiproliferative and antimotility effects of these compounds in both benign (Ben-Men1) and malignant (KT21-MG1) meningioma cells. In addition, strong reduction in phosphorylation of MEK and S6 as well as decreased cyclin D1 expression in both cell lines after treatment with PAK inhibitors was observed. Using intracranial injection of luciferaseexpressing KT21-MG1 cells as an in vivo model, significant tumor suppression was observed in mice dosed orally with all three PAK inhibitors. Tumors dissected from treated animals exhibited an increase in apoptosis without notable change in proliferation. Collectively, these results suggest that PAK inhibitors might be useful agents in the treatment of NF2deficient meningiomas.82 In another study, compound 25 induced apoptosis of PAK1amplified hormone-receptor positive breast cancer cell lines, consistent with earlier genetic PAK1 knock-down studies.81 Given the evolutionarily conserved role of PAK1 in regulating cytoskeletal dynamics and the common use of microtubule inhibitors in later lines of breast cancer treatment, a combination of 25 with taxanes was also investigated. Administration of docetaxel with either 25 or PAK1 siRNA oligonucleotides dramatically altered signaling to cytoskeletal-

kinases inhibited by 7, particularly in the RTK family. The kinases that are still potently inhibited by 25 are spread across multiple kinase families, albeit with some enrichment in the STE20 kinase family that the PAKs are part of. The ethylpiperazine moiety of 25 is located at the E0 region of the protein, the common entrance area for ligands in kinases. This area has previously been used to increase selectivity in the design of kinase inhibitors because of its diversity in sequence and conformation,83 and in fact a recent report on p38 inhibitors bearing a pyrido[2,3-d]pyrimidine-7-one core described a successful example of kinase selectivity improvement through replacement of an aromatic group at this position by an aliphatic moiety.84 No clinical development on pyrido[2,3-d]pyrimidine-7-one-based PAK inhibitors has been reported to date, but the compounds described have been used in a number of biological studies outlined in the following section. On the basis of high PAK1 potency and favorable pharmacokinetic properties upon subcutaneous (sc) injection, including high total brain levels, FRAX486 (24, Figure 13) was used as a tool compound for Fragile X in vivo studies.85 Unbound brain levels were not reported, but considering a high calculated log P (>5) and presence of a basic amine with a pKa ≈ 9, brain tissue binding is expected to be substantial. Nevertheless, following sc injection of a 20 mg/kg dose, this compound imparted specific reversal of the dendritic spine phenotype in Fragile X mental retardation 1 (Fmr1) mice in addition to a beneficial effect on neurological and behavioral symptoms. The discovery that a small-molecule PAK inhibitor rescues all of the Fmr1 KO mouse behavioral phenotypes is remarkable and mandates further preclinical and clinical investigation. Treatment of KrasG12D mice with 7 induced regression of squamous cell carcinomas and decreased MAPK and PI3K/ AKT pathway activity.34 These findings with 7 support the idea J



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Figure 14. Kinome trees for five PAK inhibitors tested at a compound concentration of 1 μM (5, 7, 25) and 0.1 μM (13, 28). Inhibition of >90% is represented by red, 75−90% by orange, 50−75% by yellow, and 75% at a compound concentration of 1 μM (compounds 7 and 25) or 0.1 μM (compound 28). 7 was tested in an abbreviated kinase panel of 151 kinases, 28 in a panel of 114 kinases, and 25 in a panel of 240 kinases. Detailed information is shown in the Supporting Information.

Figure 17. 7-Azaindole group I PAK selective inhibitors from AstraZeneca.

This compound binds as expected, with the donor−acceptor pair of the azaindole forming hydrogen bonds with the hinge of the kinase, while the 2-Cl-phenyl moiety resides in a selectivity pocket adjacent to the methionine gatekeeper residue, as seen with the deep-pocket binding compounds from Afraxis (Figure 18A). In fact, superimposition of the PAK1 cocrystal structures of azaindole 8 and the pyrido[2,3-d]pyrimidine-7-one 7 reveals a similar positioning of the chlorophenyl moiety pointing toward the deep pocket (Figure 18B). The importance of the 2chloro group was underscored by the observation that placement of the chloro atom to the meta or para positions caused a significant drop in potency (111- and 14-fold less potent than the ortho-Cl analog, respectively). Subsequent SAR investigations around the 2-Cl-Ph ring and the basic pyrrolopiperazine motif led to the identification of 28 as a more permeable and cell-potent PAK1 inhibitor, with an IC50 of 1 nM and a EC50 of 140 nM in a phospho-PAK1 cell assay. Kinase selectivity of 28 is comparable to the earlier generation Afraxis PAK inhibitors, such as 7, and interestingly, the overlap of kinase activities is extensive (Figure 15) which is perhaps not surprising given the similar binding mode of both compounds. In vitro and in vivo pharmacokinetic data have not been disclosed for this compound class to date. Benzimidazole Core Series (Group II PAK Inhibitors). Researchers from Genentech recently succeeded in identifying a highly potent and group II PAK-selective inhibitor series with exquisite kinome selectivity.87 Compound 29 (Figure 19),87 a 2-methylbenzimidazole core analog with moderate PAK4

Figure 16. Crystal structure of compound 7 bound to PAK1.78

combination as a viable strategy to increase antitumor efficacy.81 Taken together, pyrido[2,3-d]pyrimidine-7-one scaffold based PAK inhibitors with extended headgroups are potent and the most kinase selective examples of ATP-competitive group I selective PAK inhibitors described to date, and these compounds have proven useful in a variety of in vitro and in vivo biological validation studies. On the basis of the selectivity progress made and proven preclinical utility, further development of this compound class seems warranted. Biaryl Ketone Azaindole Series (Group I PAK Inhibitors). AstraZeneca recently reported the identification of the 7-azaindole PAK1 inhibitor 28 (Figure 17).86 Following a screen of a kinase-focused subset of the AZ compound collection (∼120 000 compounds), biaryl ketone azaindoles were found to be potent against PAK1 and exhibit high PAK1 vs PAK4 selectivity. 2-Cl ketone 2786 was identified as a group I selective PAK inhibitor (Figure 17, PAK1 IC50 = 20 nM; PAK4 IC50 = 550 nM) with a high LLE of 6.1 (based on measured log D), a very good starting point for a drug discovery program. A close analog of 27, the 2-Cl, 5-hydroxymethylaryl inhibitor 8,86 was cocrystallized with the human PAK1 kinase domain. L



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and the alkyne directing the propargyl substituent past the gatekeeper residue (Met-395) into a lipophilic back pocket. Given the PAK4 DFG motif and α-C-helix are “in” and yet the ligand extends past the gatekeeper, this binding mode fits the recently reported type I 1/2 description88 with the exception that a hydrogen bond is accepted from the Phe rather than Asp residue backbone NH (Figure 20). This binding mode is

Figure 20. Crystal structure of compound 12 bound to PAK4.86

unprecedented in the PAK literature; however, propargyl alcohol-driven type I 1/2 binding has been demonstrated for AKT and NIK (both also possessing methionine gatekeepers).87 Further exploration of preferred hinge binding contacts through modifications of both core and hinge binding heterocycle culminated in the identification of compound 13,87 a highly potent and kinase selective PAK4 inhibitor. In a 222kinase panel at 0.1 μM concentration, besides PAK4, PAK5, and PAK6, it inhibited only EphB1 at >60% (Figure 14). The basis for PAK4 over PAK1 selectivity was elucidated by structural analysis. Compared to compound 7, 12 extends into the back pocket with a different vector. As shown in Figure 20, the propargyl alcohol anchors to the floor of the ATP pocket via a hydrogen bond interaction to the main chain NH of Phe478. A concomitant hydrogen bond with the side chain of Glu366 on helix C also seems critical for stabilizing this conformation. As a result, the cyclohexyl moiety of 12 readily enters a pocket adjacent to the C-terminus of helix-C in PAK4. The equivalent site in PAK1 is significantly smaller and cannot accommodate the cyclohexane moiety. This size difference in the selectivity pocket was attributed to main chain flexibility in the C-terminal end of helix-C, where the sequence homology between PAK1 and PAK4 is lower. Asn-322PAK1 is particularly

Figure 18. Crystal structure of compound 8 bound to PAK1 (A) and superimposition of 7 and 8 bound to PAK1, respectively. (B) The chlorophenyl moiety of 8 presents a vector to expand into the deep pocket.

activity and >10-fold selectivity over PAK1, was identified from database mining and chosen as a starting point for further optimization of potency and selectivity over group I PAKs (Figure 19). SAR studies revealed the propargyl alcohol moiety to be critical for potency; furthermore, potency was highly sensitive to the nature of the substituents at the terminal propargyl alcohol position. Among the substituents investigated, cyclohexyl analog 1287 was preferred and improved PAK1 potency of compound 29 by 7.4-fold. The X-ray structure of compound 12 revealed a novel binding mode among PAK inhibitors known in the literature: two-point hinge binding interaction of the aminotriazine moiety with Leu-98

Figure 19. Identification of potent and selective PAK4 inhibitor 13 starting from compound 29. M



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triple-negative breast cancer (TNBC) cells,21 and therefore, compound 13 was utilized to assess group II PAK-dependent phenotypes in cell lines derived from this tumor subtype.87 Group II PAK catalytic activity was required for efficient migration and invasion of MDA-MB-436 and MCF10APIK3CA(H1047R) cells.89 Of particular note, inhibition of cellular proliferation by compound 13 was correlated with PAK4 mRNA expression level in a small panel of TNBC cell lines, which is consistent with recent reports relying on genetic loss-of-function approaches for PAK4,90 and would not be predicted for less selective PAK family small molecule inhibitors.

important, as it acts as a capping residue and rigidifies the last turn of helix-C. In contrast, the PAK4 counterpart of Asn322PAK1 is a tyrosine which is less effective in capping the helixC and allows Met-370PAK4 more flexibility to move outside the selectivity pocket (see Figures 20 and 21). By combination of

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ALLOSTERIC PAK INHIBITORS Type II Binders. Dibenzodiazepine Series (PAK1 Selective Inhibitors). Novartis scientists recently disclosed novel dibenzodiazepine PAK inhibitors that bind allosterically in proximity to the ATP binding pocket (Figure 22).91 Compound 30,91 a weakly active PAK1 binder with close structural resemblance to the antipsychotic drug clozapine,92 emerged as a hit in both HTS and NMR fragment screening. NMR studies revealed that this compound was displaced by a type I inhibitor (undisclosed) and was competitive with ATP binding, and early SAR exploration suggested that halogen atoms were necessary for potency. By use of X-ray crystallography and structure-based design, a simple switch from a methyl to an ethyl nitrogen substituent increased PAK1 potency by ∼6-fold, with the resulting analog 3191 demonstrating high PAK isoform selectivity (>100-fold against all other PAK family members tested). Parallel synthesis and further optimization led to compound 32,91 a potent PAK inhibitor that retained high PAK isoform selectivity. Given the sequence similarity of PAK1 and PAK2 in the kinase domain (Figure 2), this high degree of selectivity is remarkable. This series of compounds was found to bind directly in the back pocket of PAK1, as demonstrated by X-ray crystallography, and to inhibit kinase activity by interfering with ATP binding in an allosteric manner.91 Back pocket binding is an effective way of obtaining kinase selectivity because of the greater sequence diversity outside the ATP binding site. In addition, opening up the back pocket requires the kinase to adopt an inactive conformation, which tends to be more variable than the active conformation. It is not surprising that in an expanded kinase panel (Ambit) compound 32 showed exquisite selectivity.93 In the absence of

Figure 21. Overlay of the cocrystal structures of compound 7 bound to PAK1 and compound 12 bound to PAK4. PAK isoform selectivity (group I vs group II PAKs) is achieved through entering the respective deep pockets at different vectors and thus alternative angles, filling different pockets around the less conserved C-terminal ends of the Chelices.

the learning from the Afraxis compound 7 and the propargyl alcohol compound 12, a clearer pattern emerged (Figure 21). PAK isoform selectivity (group 1 vs group 2 PAKs) can be achieved through entering the respective deep pockets from different vectors and thus alternative angles, filling different pockets around the less conserved C-terminal end of helix-C. Elucidation of the structural basis for selectivity is a significant step toward developing PAK inhibitors, as isoform selectivity will be important for discerning efficacy and safety profiles associated with inhibiting individual PAK isoforms. Given its pharmacologic profile, excellent permeability, and satisfactory solubility, compound 13 was chosen for further studies to demonstrate the role of the PAK group II kinase subfamily in disease contexts. PAK4 is often highly expressed in

Figure 22. Identification of potent and selective allosteric PAK1 inhibitor 33 from fragment-screening derived compound 30. N



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phenolic groups and the disulfide moiety, providing an obstacle for both in vitro and in vivo experiments. Cells exposed to 34 were found to rapidly change their redox potential as a consequence of the continuous reduction of 34 and may be the underlying reason for the sensitivity of certain cell types to this compound, independent of any specific effects on PAK. This potential source of data confounding can be managed, at least in part, through the use of the isomeric PIR3.5 (35)94 as a control, a compound with similar redox effect to 34 but no activity against PAK. Unfortunately, SAR around 34 proved to be very steep, and no analogues with similar activity and more favorable drug properties have been reported to date.

high resolution structural information, we are unable to pinpoint specific residues that drive PAK1/2 selectivity. Further modification of compound 32 yielded the more soluble and potent analog 33,91 a compound that retained the selectivity features and cellular potency in a PAK1 autophosphorylation assay; phospho-PAK1 IC50 values were at ∼100 nM in multiple cell lines. Consistent with the greater PAK1 biochemical potency (compared to PAK2), inhibition of PAK1 phosphorylation was greater than inhibition of PAK2 phosphorylation in cells, but interestingly, the extent of selectivity appeared to be diminished (

Inhibitors of p21-Activated Kinases (PAKs) - Journal of Medicinal

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