PD98059

PD98059 inhibits either basal MEK1 or a partially activated MEK produced by mutation of serine at residues 218 and 222 to glutamate (MEK-2E) with IC50 of 2 μM. PD98059 does not inhibit the MAPK homologues JNK and P38. PD98059 is highly selective against MEK, as it does not inhibit a number of other kinase activities including Raf kinase, cAMP-dependent kinase, protein kinase C, v-Src, epidermal growth factor (EGF) receptor kinase, insulin receptor kinase, PDGF receptor kinase, and phosphatidylinositol 3-kinase. PD98059 inhibits PDGF-stimulated activation of MAPK and thymidine incorporation into 3T3 cells with IC50 of ~10 μM and ~7 μM, respectively. ^[1] PD98059 potently prevents the activation of MEK1 by Raf or MEK kinase with IC50 of 4 μM, and weakly inhibits the activation of MEK2 by Raf with IC50 of 50 μM. PD98059 does not inhibit the activation of MEK homologues MKK4 and RK kinase that participate in stress and interleukin-1-stimulated kinase cascades in KB and PC12 cells, and the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. ^[2] PD98059 completely blocks the nerve growth factor (NGF)-induced differentiation of PC12 cells without altering cell viability. ^[3] PD98059 inhibits the proliferation of RAW264.7 cells in the culture containing RANKL in a dose-dependent manner, resulting in an apparent decrease of TRAP-positive cells. ^[4]

Treatment of mice 30 minutes before focal cerebral ischemia with PD98059 protects against damage, resulting in a decrease in infarct volume. ^[5] Pretreated with PD98059 (10 mg/kg per i.v. injection) 30 minutes before and then together with hourly cerulein injections for 3 hours significantly ameliorates cerulein-induced acute pancreatitis ipancreatitis on the basis of pancreatic wet weight and histology. ^[6] Administration of PD98059 (10 mg/kg) in mice 1 hour after carrageenan causes a reduction in all the parameters of inflammation measured. ^[7]

• In vitro MEK-inhibitory activity

  Incorporation of ³²P into myelin basic protein (MBP) is assayed in the presence of glutathione S-transferase (GST) fusion proteins containing the 44-kDa MAPK (GST-MAPK) or the 45-kDa MEK (GST-MEK1). Assays are conducted in 50 μL of 50 mM Tris, pH 7.4/10 mM MgCl₂/2 mM EGTA/10 μM [γ-³²P]ATP containing 10 μg of GST-MEK1, 0.5 μg of GST-MAPK, and 40 μg of MBP. After incubation at 30°C for 15 minutes, reactions are stopped by addition of Laemmli SDS sample buffer. Phosphorylated MBP is resolved by SDS/10% PAGE.

• Cell lines

  K-Balb, KNRK, v-raf-3Y1, SRA/3Y1, EGFR/3T3, and K562

• Concentrations

  Dissolved in DMSO, final concentrations ~100 μM

• Incubation Time

  3 dyas, or 7-10 days

• Method

  For monolayer growth, cells are plated into multi-well plates at 10,000-20,000/mL. Forty-eight hours later, various concentrations of PD98059 are added to the cell growth medium and incubation is continued for an additional 3 days. Cells are then removed from the wells by incubation with trypsin and enumerated with a Coulter Counter. For growth in soft agar, cells are seeded into 35-mm dishes at 5,000-10,000 cells per dish with growth medium containing 0.3% agar and desired concentrations of PD98059. After 7-10 days of growth, visible colonies are manually enumerated with the aid of a dissecting microscope.

• Animal Models

  Male Sprague–Dawley rats with acute pancreatitis

• Dosages

  10 mg/kg

• Administration

  Injection i.v.