Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor

Abstract

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase.

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

“…For one of these, PAK1 (PDB: 3Q4Z), Wang et al . described the autophosphorylation complex, a head-to-head asymmetric complex of PAK1 in the crystal asymmetric unit (11), whereas the others were not shown or discussed in the relevant publications (25-28). Wang et al .…”

“…The newly identified autophosphorylation events include: (i) the activation loop Tyr of human nonreceptor tyrosine kinase LCK [PDB: 2PL0 (20)], which is similar to the IGF1R structure (10); (ii) a second tyrosine autophosphorylation site (Tyr 1166 ) in the activation loop of human IGF1R [PDB: 3LVP (21)]; (iii) a Tyr in the N-terminal juxtamembrane region of human colony stimulating factor 1 receptor (CSF1R) [PDB: 3LCD (22)] that is homologous to the tyrosine observed in the c-KIT complex (7); (iv) a Tyr in the N-terminal juxtamembrane region of Ephrin receptor A2 (EPHA2) [PDB: 4PDO (23)] that represents a phosphorylation site near but distinct from that in the c-KIT and CSF1R complexes; and (v) a Ser in the C-terminal tail of human CDC2/CDC28-like kinase 2 (CLK2) [PDB: 3NR9 (24)]. We also identified several additional structures of autophosphorylation complexes of PAK1: PDB: 4O0R, 4O0T (25); 4P90 (26); 4ZY4, 4ZY5, 4ZY6 (27); and 4ZLO, 4ZJI, and 4ZJJ (28). Two of these structures, PDB: 4ZY4 and 4ZY5, contain an autophosphorylation complex with a fully ordered activation loop in the substrate kinase; whereas the others, including PDB: 3Q4Z (11), are missing the coordinates of 8-11 residues within the activation loop.…”

Targeting p21 activated kinase 1 (Pak1) to PAKup Pancreatic Cancer

“…One possible impeding factor which is difficult to achieve is the selectivity within the PAK family, because of sequence homology among the family members. Although some Pak1 selective inhibitors like IPA-3 [9,[16][17][18][19], FRAX-597 [11,20], G-5555 [12], and Novartis compound 3 [13] have been disclosed, the successful in vivo dose optimization for target specificity and stability is yet to be tested on larger group of animals. …”

“…As the earlier pharmacologic inhibitors of Pak1 are not very specific, new Pak1 selective inhibitors such as FRAX-597, G-5555, Novartis compound 3 etc. are been synthesized and are under evaluation for clinical efficacy [11][12][13]. In addition, as suggested by Baker et al [14], it would be worthwhile to identify a reliable Pak1 substrate-specific biomarker for Pak1 inhibitor antitumor activity.…”

Contributions of Biomolecular NMR to Allosteric Drug Discovery

“…These allosteric ligands were further optimized into potent and highly selective Pak1 inhibitors, which could be used as tool compounds to investigate the role of Pak1 in tumor maintenance. [20] …”