Discovery, synthesis, and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3.

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson's disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice.

ERK1/2 and p38 cooperate to delay progression through G1 by promoting cyclin D1 protein turnover.

The conditional kinase DeltaMEKK3:ER allows activation of JNK, p38 and ERK1/2 without overt cellular stress or damage and has proved useful in understanding how these pathways regulate apoptosis and cell cycle progression. We have previously shown that activation of DeltaMEKK3:ER causes a sustained G(1) cell cycle arrest which requires p21(CIP1), with ERK1/2 and p38 cooperating to promote p21(CIP1) expression. In cells lacking p21(CIP1), DeltaMEKK3:ER causes only a transient delay in cell cycle re-entry.

The conditional kinase DeltaMEKK1:ER* selectively activates the JNK pathway and protects against serum withdrawal-induced cell death.

The conditional protein kinase DeltaMEKK3:ER* allows activation of the mitogen-activated and stress-activated protein kinases (MAPKs and SAPKs) without imposing a primary cellular stress or damage. Such separation of stress from stress-induced signalling is particularly important in the analysis of apoptosis. Activation of DeltaMEKK3:ER* in cycling CCl39 cells caused a rapid stimulation of the ERK1/2, JNK and p38 pathways but resulted in a slow, delayed apoptotic response.

Carbapenem antibiotic biosynthesis in Erwinia carotovora is regulated by physiological and genetic factors modulating the quorum sensing-dependent control pathway.

Erwinia carotovora produces the beta-lactam antibiotic, carbapenem, in response to a quorum sensing signalling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). We have mapped the OHHL-dependent promoter upstream of the first of the biosynthetic genes, carA. We have also analysed the effect on this promoter of the known genetic regulators of carbapenem expression, carR, carI (encoding homologues of LuxR and LuxI respectively) and hor (encoding a SlyA/MarR-like transcriptional regulator).

ERK1/2 and p38 cooperate to induce a p21CIP1-dependent G1 cell cycle arrest.

To study the mechanisms by which mitogen- and stress-activated protein kinases regulate cell cycle re-entry, we have used a panel of conditional kinases that stimulate defined MAPK or SAPK cascades. Activation of DeltaMEKK3:ER* during serum restimulation of quiescent cells causes a strong activation of JNK1 and p38alpha but only a modest potentiation of serum-stimulated ERK1/2 activity. In CCl39 cells this promoted a sustained G1 arrest that correlated with decreased expression of cyclin D1 and Cdc25A, increased expression of p21CIP1 and inhibition of CDK2 activity.

Selective activation of the c-Jun N-terminal kinase (JNK) pathway fails to elicit Bax activation or apoptosis unless the phosphoinositide 3'-kinase (PI3K) pathway is inhibited.

c-Jun N-terminal kinase (JNK) is activated when cells are exposed to noxious stimuli. The role of JNK in apoptosis is subject to considerable debate; for example, JNK activation may promote or inhibit apoptosis depending on the cell type and stimulus involved. These conflicting results have arisen in part because few studies have successfully separated JNK activation from the primary stress-induced damage or from other stress-induced signalling pathways.

Delta MEKK3:ER* activation induces a p38 alpha/beta 2-dependent cell cycle arrest at the G2 checkpoint.

Whilst many studies have examined the role of the MAP Kinases in regulating the G1-->S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant Delta MEKK3:ER* in asynchronous hamster (CCl39) and rat (Rat-1) fibroblasts resulted in the strong activation of endogenous JNK and p38 but only a weak activation of ERK. Activation of Delta MEKK3:ER* inhibited cell proliferation through a combination of an initial G1 and G2 cell cycle arrest, followed by a delayed onset of apoptosis.