Optimization of Highly Kinase Selective Bis-anilino Pyrimidine PAK1 Inhibitors

Abstract

Group I p21-activated kinase (PAK) inhibitors are indicated as important in cancer progression, but achieving high kinase selectivity has been challenging. A bis-anilino pyrimidine PAK1 inhibitor was identified and optimized through structurebased drug design to improve PAK1 potency and achieve high kinase selectivity, giving in vitro probe compound AZ13705339 (18). Reduction of lipophilicity to lower clearance afforded AZ13711265 (14) as an in vivo probe compound with oral exposure in mouse. Such probes will allow further investigation of PAK1 biology. KEYWORDS: PAK, LLE, kinase selectivity, probe, bis-anilino pyrimidine T he p21-activated kinases (PAKs) are a family of six serine/ threonine-specific intracellular protein kinases that are positioned at the intersection of multiple signaling pathways of importance in cancer progression. 1,2 The PAK family comprises two subgroups based on sequence homology: group I (PAKs 1−3) and group II (PAKs 4−6). Group I PAKs have high sequence identity in the kinase domain and possess an autoinhibitory domain, which is relieved by binding of the GTP-binding proteins Rac or Cdc42, while group II PAKs have lower kinase domain homology and are not activated by Rho GTPases. 3 Group I PAKs are overexpressed in a wide variety of cancers, and PAK1 is commonly overexpressed in breast tumors with poor prognosis. 4 In ovarian cancer cells characterized by PAK1 amplification, treatment with a PAK1 inhibitor decreased proliferation and migration. 5 In tumors characterized by neurofibromatosis type 2 (NF2) inactivation, group I PAKs have been shown to be hyperactivated. 6 Consequently, there is increasing interest in identifying potent and selective small molecule inhibitors of PAK1 for therapeutic use in tumors characterized by PAK activation. 7 There are relatively few chemotypes described in the literature for PAK1 inhibitors 8 and only pan-PAK inhibitor PF-3578309 9 progressed into clinical trials but is now stopped. Ourselves 10 and others 11,12 have recently disclosed PAK1 inhibitors but achieving high kinase selectivity with a chemotype amenable to achieving oral exposure has been challenging. Without high kinase selectivity it is difficult to discern whether PAK1 is driving efficacy; thus, new alternative chemotypes for selective PAK1 inhibition are required.Following a kinase-focused subset screen (120k compounds) of the AstraZeneca compound collection, bis-anilino pyrimidine 1 (Figure 1) was identified as a modestly potent PAK1 inhibitor (IC 50 = 100 nM) with 11-fold selectivity over PAK4 (Table 1).While selectivity within the group I PAKs was not expected, PAK4 was utilized to monitor selectivity against group II PAKs. Selectivity against some key kinase antitargets, KDR and FGFR1, 13 was modest. Since 1 originated from an EphB4 kinase project, 14 a member of the Src kinase family, high activity for Src kinase was expected. Src family kinase activity was of less concern to us but was monitored as a measure of overall kinase selectivity. A crystal structure of 1 bound...

Sterically stabilized liposomes targeting P21 (RAC1) activated kinase‑1 and secreted phospholipase A₂ suppress prostate cancer growth and metastasis

“…Several PAK inhibitors have been studied for their anti-cancer efficacies in vitro and in vivo ( 10 , 14 , 15 , 44 , 45 ). Several PAK1 inhibitors, such as G-5555 ( 46 ), FL172 ( 47 ), PF-3758309 ( 48 ), and AZ13705339 ( 49 ) have demonstrated PAK1 activity suppression in vitro ; however, these compounds also exhibited off-target effects on several other kinases, such as the Src family of kinases, Akt1, AMP Kinases, Cyclin-dependent kinase-7 and serum glucocorticoid kinase, due to their competition for the ATP-binding site, that is conserved in several kinases ( 44 , 45 ). Among these, PF-3758309 and PF-03758309 are specific inhibitors of PAK4, a group II PAK isoform ( 50 ), and a clinical trial on PF-03758309 for advanced solid tumors (NCT00932126) was terminated, due to the undesirable pharmacokinetic characteristics, such as unfavorable levels of the drug in the plasma and the lack of a dose-response association.…”

Targeting PAK1

“…AstraZeneca has recently disclosed ATP-competitive kinase inhibitors with exceptional potency against PAK1 [51]. Overlay of two chemotypes (bis-anilino pyrimidine and previously described 7-azaindole Pak inhibitor [52]) bound to PAK1 allowed the visualization and design small molecules with improved binding mode.…”

Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents

“…Bis-anilinopyrimidine was reported as potent and selective PAK1 inhibitor and as highly selective group I p21-activated kinase (PAK1) inhibitor [146]. Additionally, N-phenyl-N′-[4-(pyrimidin-4-ylamino)phenyl] urea derivatives (see (27) at Figure 10) exhibit selective inhibition to class III receptor tyrosine kinase subfamily [147].…”