A novel inhibitor of mitogen-activated protein kinase kinase (MEK) was identified as U0126, which inhibits AP-1 transactivation in a cell-based reporter assay. U0126 specifically inhibits MEK-1 and MEK-2, two members of the MEK family, without affecting other kinases such as protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis showed that U0126 and PD098059 are noncompetitive inhibitors of MEK with respect to both substrates, ATP and ERK. U0126 has approximately 100-fold higher affinity for the ΔN3-S218E/S222D MEK mutant than PD098059. However, the affinity of both compounds for wild-type MEK is significantly lower than for the mutant enzyme, suggesting that subtle conformational differences between the two activated MEK forms influence their binding affinity. U0126 was also shown to inhibit the induction of c-Fos and c-Jun in TPA-stimulated cells, which are components of the AP-1 complex. This inhibition was not due to a direct blockage of AP-1 DNA binding but rather to a suppression of the up-regulation of c-Fos and c-Jun mRNA and protein levels. U0126 inhibits the MAP kinase cascade by directly inhibiting MEK, which is essential for the activation of ERK. This inhibition is selective for MEK over other kinases and is effective in cellular systems. U0126 was found to be a potent and specific inhibitor of MEK-1 and MEK-2 in vitro and in vivo. It inhibits the phosphorylation and activation of ERK, which in turn prevents the downstream phosphorylation of factors such as p62 (Elk-1), thereby preventing the induction of c-Fos and c-Jun, components of the AP-1 complex. The identification of U0126 was based on its ability to block AP-1 activation, which relies on this activation pathway. U0126 is a noncompetitive inhibitor of ΔN3-S218E/S222D MEK-1 with a Ki of 41–109 nM. Steady-state kinetic analysis indicates that PD098059 is also a noncompetitive inhibitor of this enzyme. The noncompetitive nature of U0126 and PD098059 inhibition of MEK may be a pharmacological advantage for compounds of this type. The binding site for these compounds on MEK is clearly distinct from those for the two substrate molecules, ATP and ERK. Hence, such compounds mayA novel inhibitor of mitogen-activated protein kinase kinase (MEK) was identified as U0126, which inhibits AP-1 transactivation in a cell-based reporter assay. U0126 specifically inhibits MEK-1 and MEK-2, two members of the MEK family, without affecting other kinases such as protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis showed that U0126 and PD098059 are noncompetitive inhibitors of MEK with respect to both substrates, ATP and ERK. U0126 has approximately 100-fold higher affinity for the ΔN3-S218E/S222D MEK mutant than PD098059. However, the affinity of both compounds for wild-type MEK is significantly lower than for the mutant enzyme, suggesting that subtle conformational differences between the two activated MEK forms influence their binding affinity. U0126 was also shown to inhibit the induction of c-Fos and c-Jun in TPA-stimulated cells, which are components of the AP-1 complex. This inhibition was not due to a direct blockage of AP-1 DNA binding but rather to a suppression of the up-regulation of c-Fos and c-Jun mRNA and protein levels. U0126 inhibits the MAP kinase cascade by directly inhibiting MEK, which is essential for the activation of ERK. This inhibition is selective for MEK over other kinases and is effective in cellular systems. U0126 was found to be a potent and specific inhibitor of MEK-1 and MEK-2 in vitro and in vivo. It inhibits the phosphorylation and activation of ERK, which in turn prevents the downstream phosphorylation of factors such as p62 (Elk-1), thereby preventing the induction of c-Fos and c-Jun, components of the AP-1 complex. The identification of U0126 was based on its ability to block AP-1 activation, which relies on this activation pathway. U0126 is a noncompetitive inhibitor of ΔN3-S218E/S222D MEK-1 with a Ki of 41–109 nM. Steady-state kinetic analysis indicates that PD098059 is also a noncompetitive inhibitor of this enzyme. The noncompetitive nature of U0126 and PD098059 inhibition of MEK may be a pharmacological advantage for compounds of this type. The binding site for these compounds on MEK is clearly distinct from those for the two substrate molecules, ATP and ERK. Hence, such compounds may