G-5555 : ATP-competitive inhibitor of PAK1, PAK2, PAK3
Protein target names: PAK1, PAK2, PAK3
Mechanism of action: ATP-competitive inhibitor
In Vitro Validations
In Cell Validations
In Vivo Data
This is a potent inhibitor of Group I PAK enzymes (PAK1, 2 and 3; PAK1 Ki = 4 nM) that has been screened versus 235 kinases at 100 nM, with biochemical inhibition of 5 kinases (IC50 <50 nM) observed. Expanded off-target kinase screening would be helpful to enhance the selectivity profile. Parallel use of the orthogonal PAK1-selective probe NVS-PAK1-1 is suggested to aid interpretation of cellular effects between PAK family members. The selectivity of G-5555 for Group I PAK enzymes over non-kinase targets (IC50 >10 uM for 36/42 diverse receptors, ion channels, enzymes and transporters) supports its use as a cellular probe. On-target activity in cells was demonstrated (inhibition of pMEK S298, IC50 = 69 nM) and supports the suggested concentration range for cellular studies. Compound pharmacokinetics are suitable for in vivo studies and on-target modulation of PAK-dependent pMEK S298 was demonstrated in H292 xenografts following 20 and 30 mg/kg single, oral doses. A caveat to the use of the compound in vivo for high doses or repeat dosing is the reported narrow therapeutic index due to acute cardiotoxicity of pan-PAK inhibitors (attributed to PAK2 inhibition with possible exacerbation by PAK1 inhibition) that limits tolerability and exposure (J. Med. Chem. 2016, 59, 5520).
Starting with an advanced inhibitor, the authors optimized a PAK1-inhibitory compound for potency and selectivity. Based on analysis of their SAR tables, the authors did a good job balancing trade-offs between different types of potential liabilities, including 2 log(P) units, 7-fold better potency in vitro and 3-fold more activity in cells. Reducing hERG inhibition was a stated goal, and the reported compound has about half the activity against hERG compared with the predecessor; notably, some compounds tested were even better relative to hERG, but they were less attractive in other tested parameters. Based on a comparison of the kinase-profiling data reported by the authors, there were a handful of other kinases with IC50 values near that for PAK1, some within a few (2-3)-fold and 7 within 15-fold. On the other hand, the EC50 in cells is reported ~17-fold higher (69 nM) than the IC50 against PAK1. Based on this analysis, the recommended concentration range provided (70-250 nM) might be on the high side. While 70 nM is appropriate, if the ratio of EC50/IC50 is preserved for the "off-target" kinases, then 250 nM in cells might be above the EC50 for 2-3 of these other kinases.
G-5555 is one of a number of quality compounds now available for the study of the function of group 1 PAKs, and its use in cells is recommended, especially if used alongside other chemotypes such as FRAX-1036 (ACS Med Chem Lett 2015, p1241), the Novartis allosteric inhibitor (ACS Med Chem Lett 2015, p776) and the AstraZeneca inhibitor (ACS Med Chem Lett 2016, p1118). Although G-5555 has pharmacokinetic properties that make it suitable for in vivo studies, caution should be exercised for acute doses >30 mg/kg or repeat dosing due to the reported narrow therapeutic index in a later J. Med. Chem. paper.