G-5555

Reagent Authentication Certificate:

Chemical Probes Portal G-5555.pdf

Protein target name:

PAK1, PAK2, PAK3

Mechanism of action:

ATP-competitive inhibitor

Recommended Concentration for use in cells:

70 nM-250 nM

Probe Availability

MedChem Express (1648863-90-4)

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

STE

HUGO ID:

HGNC:8590 HGNC:8591 HGNC:8592

Entrez gene ID:

5058 5062 5063

UNIPROT ID:

Q13153 Q13177 O75914

Target alias:

p21 (RAC1) activated kinase 1, p21 (RAC1) activated kinase 2, p21 (RAC1) activated kinase 3, "mental retardation, X-linked 47", MRX30, MRX47,

Probe Details

Chemical Probes Portal ID:

CPP790

Probe alias:

G-5555; CS-5608; HY-19635

PubChem CID:

91664373

SMILES string:

ClC(C=C(C1=NC(C)=CC=C1)C=C2)=C2C3=CC4=CN=C(NC)N=C4N(C[C@@H]5OC[C@@H](N)CO5)C3=O

InChi Key:

ZBCMHWUFWQFPLV-UHFFFAOYSA-N

Control compound:

(R)-6-(2-Chloro-4-(4-methyl-2-oxopyridin-1(2H)-yl)phenyl)- 2-(methylamino)-8-(morpholin-2-ylmethyl)pyrido[2,3-d]- pyrimidin-7(8H)-one

Orthogonal probe:

FRAX1036NVS-PAK1-1

Physico-chemical properties

NMR:

1H NMR (400 MHz, DMSO-d6) δ 8.68 (d, 1H, rotamer), 8.22 (d, J = 1.7 Hz, 1H), 8.08
(dd, J = 8.1, 1.8 Hz, 1H), 7.92 – 7.77 (m, 4H), 7.51 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 7.4
Hz, 1H), 5.07 – 4.85 (m, 1H), 4.52 – 4.37 (m, 2H), 4.00 – 3.84 (m, 2H), 3.11 (t, J = 10.6
Hz, 2H), 2.91 (d, J = 4.7 Hz, 3H), 2.85 – 2.70 (m, 1H), 2.57 (s, 3H).

Storage Recommendations

Storage (dry powder):

room temperature

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

3.7 nM

Potency assay:

Measuring the phosphorylation of a FRET peptide substrate (Ser/Thr19) labeled with Coumarin and Fluorescein

PDB ID for probe-target interaction (3D structure):

5DEY

Structure-activity relationship:

SAR described in "Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pKa Polar Moiety, ACS Med. Chem. Lett. 2015, 6, 1241" and Chemically diverse Group I PAK inhibitors impart acute cardiovascular toxicity with a narrow therapeutic window, J. Med. Chem. 2016, 59, 5520. SAR assay used is described above.

Off target activity

Potency assay (off target):

IC50s: SIK2: 9 nM KHS1: 10 nM MST4: 20 nM MST3: 43 nM YSK1: 34 nM LCK: 52 nM

Probe Selectivity in Vitro:

5HT1B: 84% inhibition at 10 uM 5HT2B: 92% at 10 uM

In cell validation

On target activity in cells

Potency in cells:

IC50

69 nM

Potency assay (cells):

pMEK (S298) phosphorylation assay using EBC-1 cells

Target engagement assay (cells):

see above

In vivo validation

Organism:

Mouse

Dose:

10, 20, and 30 mg/kg

Route of delivery:

Oral

Plasma half life:

53 min

Systemic clearance:

24.2 mL/min/kg

Fraction of probe bound to plasma protein:

99.7

Cmax:

~10 uM (following 25 mg/kg dose)

Tmax:

30 min

Primary Reference

Reference (doi):

10.1021/acsmedchemlett.5b00398

Reference (PMID):

26713112

Endorsed probe

The Chemical Probes Portal endorses this compound as chemical probe for use in cells.

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

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).

Jan 9 2017 - 1:50pm

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.

Jan 10 2017 - 12:30pm

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.

Jun 13 2017 - 2:41pm

G-5555

Probe Availability

Probe Information

Target Details

p21 (RAC1) activated kinase 1, p21 (RAC1) activated kinase 2, p21 (RAC1) activated kinase 3, "mental retardation, X-linked 47", MRX30, MRX47,

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

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Search form

G-5555

Probe Availability

Probe Information

Target Details

p21 (RAC1) activated kinase 1, p21 (RAC1) activated kinase 2, p21 (RAC1) activated kinase 3, "mental retardation, X-linked 47", MRX30, MRX47,

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?