Discovery of 3-(trifluoromethyl)-1H-pyrazole-5-carboxamide activators of the M2 isoform of pyruvate kinase (PKM2).

Activators of the pyruvate kinase M2 (PKM2) are currently attracting significant interest as potential anticancer therapies. They may achieve a novel antiproliferation response in cancer cells through modulation of the classic 'Warburg effect' characteristic of aberrant metabolism. In this Letter, we describe the optimization of a weakly active screening hit to a structurally novel series of small molecule 3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as potent PKM2 activators.

High molecular weight complex analysis of Epstein-Barr virus Latent Membrane Protein 1 (LMP-1): structural insights into LMP-1's homo-oligomerization and lipid raft association.

LMP-1 is a constitutively active Tumor Necrosis Factor Receptor analog encoded by Epstein-Barr virus. LMP-1 activation correlates with oligomerization and raft localization, but direct evidence of LMP-1 oligomers is limited. We report that LMP-1 forms multiple high molecular weight native LMP-1 complexes when analyzed by BN-PAGE, the largest of which are enriched in detergent resistant membranes. The largest of these high molecular weight complexes are not formed by purified LMP-1 or by loss of function LMP-1 mutants. Consistent with these results we find a dimeric form of LMP-1 that can be stabilized by disulfide crosslinking. We identify cysteine 238 in the C-terminus of LMP-1 as the crosslinked cysteine. Disulfide crosslinking occurs post-lysis but the dimer can be crosslinked in intact cells with membrane permeable crosslinkers. LMP-1/C238A retains wild type LMP-1 NF-κB activity. LMP-1's TRAF binding, raft association and oligomerization are associated with the dimeric form of LMP-1. Our results suggest the possibility that the observed dimeric species results from inter-oligomeric crosslinking of LMP-1 molecules in adjacent core LMP-1 oligomers.

Pharmacologic activation of PKM2 slows lung tumor xenograft growth.

Inactivation of the M2 form of pyruvate kinase (PKM2) in cancer cells is associated with increased tumorigenicity. To test the hypothesis that tumor growth may be inhibited through the PKM2 pathway, we generated a series of small-molecule PKM2 activators. The compounds exhibited low nanomolar activity in both biochemical and cell-based PKM2 activity assays. These compounds did not affect the growth of cancer cell lines under normal conditions in vitro, but strongly inhibited the proliferation of multiple lung cancer cell lines when serine was absent from the cell culture media. In addition, PKM2 activators inhibited the growth of an aggressive lung adenocarcinoma xenograft. These findings show that PKM2 activation by small molecules influences the growth of cancer cells in vitro and in vivo, and suggest that such compounds may augment cancer therapies.

Epigenetic drug discovery: targeting DNA methyltransferases.

Epigenetic modification of DNA leads to changes in gene expression. DNA methyltransferases (DNMTs) comprise a family of nuclear enzymes that catalyze the methylation of CpG dinucleotides, resulting in an epigenetic methylome distinguished between normal cells and those in disease states such as cancer. Disrupting gene expression patterns through promoter methylation has been implicated in many malignancies and supports DNMTs as attractive therapeutic targets. This review focuses on the rationale of targeting DNMTs in cancer, the historical approach to DNMT inhibition, and current marketed hypomethylating therapeutics azacytidine and decitabine. In addition, we address novel DNMT inhibitory agents emerging in development, including CP-4200 and SGI-110, analogs of azacytidine and decitabine, respectively; the oligonucleotides MG98 and miR29a; and a number of reversible inhibitors, some of which appear to be selective against particular DNMT isoforms. Finally, we discuss future opportunities and challenges for next-generation therapeutics.

Chronic murine typhoid fever is a natural model of secondary hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory clinical syndrome associated with neoplastic disorders especially lymphoma, autoimmune conditions, and infectious agents including bacteria, viruses, protozoa and fungi. In both human and veterinary medicine, hemophagocytic histiocytic disorders are clinically important and frequently fatal. HLH in humans can be a primary (familial, autosomal recessive) or secondary (acquired) condition, with both types generally precipitated by an infectious agent. Previously, no mouse model for secondary HLH has been reported. Using Salmonella enterica serotype Typhimurium by oral gavage to mimic naturally-occurring infection in Sv129S6 mice, we characterized the clinical, hematologic and morphologic host responses to disease thereby describing an animal model with the clinico-pathologic features of secondary HLH as set forth by the Histiocyte Society: fever, splenomegaly, cytopenias (anemia, thrombocytopenia), hemophagocytosis in bone marrow and spleen, hyperferritinemia, and hypofibrinogenemia. Disease severity correlates with high splenic and hepatic bacterial load, and we show disease course can be monitored and tracked in live animals. Whereby secondary HLH is known to occur in human patients with typhoid fever and other infectious diseases, our characterization of a viable natural disease model of secondary HLH offers an important means to elucidate pathogenesis of poorly understood mechanisms of secondary HLH and investigation of novel therapies. We characterize previously unreported secondary HLH in a chronic mouse model of typhoid fever, and novel changes in hematology including decreased tissue ferric iron storage that differs from classically described anemia of chronic disease. Our studies demonstrate S. Typhimurium infection of mice is a natural infectious disease model of secondary HLH that may have utility for elucidating disease pathogenesis and developing novel therapies.

Hemophagocytic macrophages harbor Salmonella enterica during persistent infection.

Salmonella enterica subspecies can establish persistent, systemic infections in mammals, including human typhoid fever. Persistent S. enterica disease is characterized by an initial acute infection that develops into an asymptomatic chronic infection. During both the acute and persistent stages, the bacteria generally reside within professional phagocytes, usually macrophages. It is unclear how salmonellae can survive within macrophages, cells that evolved, in part, to destroy pathogens. Evidence is presented that during the establishment of persistent murine infection, macrophages that contain S. enterica serotype Typhimurium are hemophagocytic. Hemophagocytic macrophages are characterized by the ingestion of non-apoptotic cells of the hematopoietic lineage and are a clinical marker of typhoid fever as well as certain other infectious and genetic diseases. Cell culture assays were developed to evaluate bacterial survival in hemophagocytic macrophages. S. Typhimurium preferentially replicated in macrophages that pre-phagocytosed viable cells, but the bacteria were killed in macrophages that pre-phagocytosed beads or dead cells. These data suggest that during persistent infection hemophagocytic macrophages may provide S. Typhimurium with a survival niche.