ERK5 kinase activity is dispensable for cellular immune response and proliferation.

Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.

Amides of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid as zinc-dependent inhibitors of Lp-PLA2.

AX10479, the phenyl amide of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid, was identified as a Zn(2+)-dependent, 27nM inhibitor of human plasma Lp-PLA(2). Structure-activity relationship studies focused on the AX10479 2-phenylamide group identified equipotent cycloaliphatic amides, an enantioselective preference for chiral amides, and phenyl substitution patterns (e.g., 2-methyl-3-fluoro) that increased potency.

Synthesis and structure-activity relationship of (1-halo-2-naphthyl) carbamate-based inhibitors of KIAA1363 (NCEH1/AADACL1).

KIAA1363 is a serine hydrolase whose activity has been shown to be positively associated with tumor cell invasiveness. Thus, inhibitors of KIAA1363 represent a novel targeted therapy approach towards cancer. AX11890 ((1-bromo-2-naphthyl) N,N-dimethylcarbamate) was identified as a KIAA1363 inhibitor with an IC(50) value of 1.2 µM and was shown using ESI-MS to carbamylate the catalytic residue Ser(191). SAR studies explored both substitution of the 1-bromo group and derivatization of the 6-position. Activity-based protein profiling demonstrated AX13057 inhibited tumor-localized KIAA1363 in SK-OV-3 xenograft-bearing mice.

Amides of xanthurenic acid as zinc-dependent inhibitors of Lp-PLA(2).

AX10185, the phenyl amide of xanthurenic acid, was found to be a sub-100nM inhibitor of Lp-PLA(2). However, in the presence of EDTA the inhibitory activity of AX10185 was extinguished while the enzymatic activity of Lp-PLA(2) did not change. Subsequent metal screening experiments determined the inhibition to be Zn(2+) dependent. Structure-activity relationship studies indicated the presence of the 4-hydroxy group to be critical and selected substituted phenyl, polycyclic, and cycloaliphatic amides of xanthurenic acid to be well tolerated.

Synthesis and optimization of 2-pyridin-3-yl-benzo[d][1,3]oxazin-4-one based inhibitors of human neutrophil elastase.

The hit-to-lead optimization of the HNE inhibitor 5-methyl-2-(2-phenoxy-pyridin-3-yl)-benzo[d][1,3]oxazin-4-one is described. A structure-activity relationship study that focused on the 5 and 7 benzoxazinone positions yielded the optimized 5-ethyl-7-methoxy-benzo[d][1,3]oxazin-4-one core structure. 2-[2-(4-Methyl-piperazin-1-yl)-pyridin-3-yl] derivatives of this core were shown to yield HNE inhibitors of similar potency with significantly different stabilities in rat plasma.

Extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontin.

BACKGROUND: Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS: Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS: In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSIONS: The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies.

Identification of elongation factor 1alpha as a potential associated binding partner for Akt2.

Akt protein kinase has been shown to play a pivotal role in diverse cell functions, including motility, apoptosis, growth and metabolism. How it differentially regulates these diverse functions is of significant interest. Three isoforms have been well characterized, Akt1, 2, and 3, encoded by separate genes, but showing high homology over the entire coding sequence (> 80%). An area of variability between the three isoforms is the C-terminal tail. To find potentially regulating binding partners of Akt2, the isoform implicated in metabolic control, we used a glutathione-S-transferase (GST) fusion protein expressing the C-terminal 75 residues of Akt2 (GST-Akt2 tail) to screen for proteins that specifically bound the Akt2 tail. Elongation factor 1alpha (EF1alpha) and beta-tubulin were identified as binding partners for the Akt2 tail by peptide mass fingerprinting. These two proteins have themselves been previously identified as interacting partners (Nakazawa et al.FEBS Lett. 453,29-34, 1999). Using CHOT cells that overexpress insulin receptors and HA-tagged Akt2, we showed that EF1alpha co-immunoprecipitated with HA-tagged Akt2. It is thus possible that these proteins colocalise as part of a regulatory signaling complex with the cytoskeleton directing them to sites of cell activity.

Gene expression profiling of tumor xenografts: In vivo analysis of organ-specific metastasis.

Mammary carcinoma frequently metastasizes to specific organs, including the regional lymph nodes, the lung and bone marrow. The mechanisms that guide organ-specific metastasis and the molecular players that are involved remain to be established. To gain insight into this problem, we used an orthotopic model of breast cancer in which the MDA-MB-435 cells are implanted into the mouse mammary fat pad. Sublines that preferentially metastasized to specific sites were isolated by excising metastatic tumors and growing explants of these tumors in culture. Cells lines that preferentially metastasize to the lymph node and thoracic cavity were obtained. The gene expression profiles of primary tumors from these sublines were then compared with cDNA arrays containing 5,800 known genes. In tumors that preferentially metastasize to the lymph node, several genes encoding adhesion and matrix proteins were upregulated. Genes encoding proteins involved in metabolism were downregulated. One of the upregulated genes in lymph-homing tumors was CD73. Immunohistochemistry showed that the CD73 protein is also upregulated in primary tumors of this cell line and that its expression is elevated in the lymph node metastases. CD73 is a transmembrane protein that has previously been implicated in the homing of normal lymphocytes to the nodes. This raises the hypothesis that tumors preferentially metastasize to lymph nodes by using CD73 to mimic part of the lymphocyte homing process.