Branched-chain aminotransferases (BCAT) are enzymes that initiate the catabolism of branched-chain amino acids (BCAA), such as leucine, thereby providing macromolecule precursors; however, the function of BCATs in macrophages is unknown. Here we show that BCAT1 is the predominant BCAT isoform in human primary macrophages. We identify ERG240 as a leucine analogue that blocks BCAT1 activity. Selective inhibition of BCAT1 activity results in decreased oxygen consumption and glycolysis. This decrease is associated with reduced IRG1 levels and itaconate synthesis, suggesting involvement of BCAA catabolism through the IRG1/itaconate axis within the tricarboxylic acid cycle in activated macrophages. ERG240 suppresses production of IRG1 and itaconate in mice and contributes to a less proinflammatory transcriptome signature. Oral administration of ERG240 reduces the severity of collagen-induced arthritis in mice and crescentic glomerulonephritis in rats, in part by decreasing macrophage infiltration. These results establish a regulatory role for BCAT1 in macrophage function with therapeutic implications for inflammatory conditions.

Tissue factor pathway inhibitor (TFPI) contains three Kunitz-type proteinase inhibitor domains and is a potent inhibitor of tissue factor-mediated coagulation. Here, we report that TFPI inhibits the proliferation of basic fibroblast growth factor-stimulated endothelial cells. A truncated form of TFPI, containing only the first two Kunitz-type proteinase inhibitor domains, has very little antiproliferative activity, suggesting that the carboxyl-terminal region of TFPI is responsible for this activity. Binding studies revealed that full-length TFPI, but not the truncated TFPI molecule, is recognized by the very low density lipoprotein receptor (VLDL receptor) indicating that this receptor is a novel high affinity endothelial cell receptor for TFPI. The antiproliferative activity of TFPI on endothelial cells is inhibited by the receptor-associated protein, a known antagonist of ligand binding by the VLDL receptor, and by anti-VLDL receptor antibodies. These results confirm that the antiproliferative activity of TFPI is mediated by the VLDL receptor and suggest that this receptor-ligand system may be a useful target for the development of new antiangiogenic and antitumor agents.The extrinsic pathway of blood coagulation is initiated when factor VII (fVII) 1 binds to its cellular receptor, tissue factor (TF). The fVIIa⅐TF complex then functions as a potent enzyme, activating factor X which leads to thrombin generation. In addition to initiating coagulation, recent research suggests that fVIIa⅐TF complexes play an important role in angiogenesis (1-3). Zhang et al. (1) correlated TF expression in tumor cells with the ability of the tumors to secrete vascular endothelial growth factor (VEGF) and, in turn, to induce an angiogenic response when implanted in immunodeficient mice. Although TF is not normally expressed on the surface of vascular endothelial cells, in situ hybridization studies have detected TF mRNA in tumor-associated endothelial cells from patients with invasive breast cancer (2). Finally, in a TF-dependent metastasis model, the binding and proteolytic activity of VIIa was shown to be necessary for the initial steps of tumor metastasis (3).The tissue factor pathway is regulated by a potent inhibitor termed tissue factor pathway inhibitor (TFPI). TFPI forms a tight complex with both fXa and fVIIa leading to their inhibition. The TFPI molecule contains three Kunitz-type domains and a basic carboxyl-terminal region. By employing site-directed mutagenesis, Girard et al. (4) demonstrated that the second Kunitz domain is required for efficient binding and inhibition of fXa, and both Kunitz domains 1 and 2 are required for the inhibition of fVIIa/TF activity. Mutation of the predicted inhibitory residues of the third Kunitz domain had no significant effect on either function of TFPI. Rather, this portion of the molecule binds to the cell surfaces by interacting with cell-surface glycosaminoglycans (5). TFPI can also bind to the low density lipoprotein receptor-related protein (LRP) (6) and, in doing so, mediates ...

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In response to environmental stimuli, macrophages change their nutrient consumption and undergo an early metabolic adaptation that progressively shapes their polarization state. During the transient, early phase of pro-inflammatory macrophage activation, an increase in tricarboxylic acid (TCA) cycle activity has been reported but the relative contribution of branched chain amino acid (BCAA) leucine remain to be determined. Here we show that glucose but not glutamine is a major contributor of the increase in TCA cycle metabolites during early macrophage activation in humans. We then show that, although BCAA uptake is not altered, their transamination by BCAT1 is increased following 8h lipopolysaccharide (LPS) stimulation. Of note, leucine is not metabolized to integrate the TCA cycle in neither basal nor stimulated human macrophages. Surprisingly, the pharmacological inhibition of BCAT1 reduced glucose-derived itaconate, α-ketoglutarate, and 2-hydroxyglutarate levels, without affecting succinate and citrate levels, indicating a partial inhibition of TCA cycle. This indirect effect is associated with NRF2 activation and anti-oxidant responses. These results suggest a moonlighting role of BCAT1 through redox-mediated control of mitochondrial function during early macrophage activation.

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Trypsin is inactivated by the levorotatory enantiomers (most likely PS) of 4-nitrophenyl 4-H-, 4-CH3-,4-OCH3-, and 4-Cl-phenacyl methylphosphonates (PMNs) with second-order rate constants between 231 and 884 M-1 s-1. 4-NO2-PMN hydrolyzes before inhibiting the enzyme. The second-order rate constants for the inactivation of alpha-chymotrypsin by the levorotatory enantiomers of the five PMNs are between 37,000 and 770,000 M-1 s-1, and those for the dextrorotatory enantiomers are between 400 and 640 M-1 s-1; the enantioselectivity is 90-1880. Specific rotation [alpha]22D of the faster-reacting enantiomer of 4-CH3-PMN with trypsin and alpha-chymotrypsin is -30 +/- 6 degrees. 31P NMR of the adducts shows a signal at 41.0 ppm, 10 ppm downfield from the parent compound. Results of molecular mechanics and dynamics calculations show that the principal interactions are between the phosphonyl group and constituents of the oxyanion hole and between the aromatic fragment and residues in the binding regions of the enzymes. Trypsin activity returns from its phenacyl methylphosphonyl adducts on the hour time scale and in reversed order to the rates of inactivation within the series. Recovery of alpha-chymotrypsin activity from the adducts formed with the (-) enantiomers is on a slower time scale still, whereas its recovery from the adducts formed with the (+) enantiomers is on the second to minute time scale. The data support a mechanism of reactivation involving rate-determining intramolecular displacement of Ser by the carbonyl hydrate of the phenacyl moiety. The pH-rate profiles for trypsin reactivation from its adducts indicate involvement of an ionizable group with pKa approximately 8.0. The pH dependence and solvent isotope effects are small in most cases. The compounds demonstrate favorable properties for controllable and temporary modulation of enzyme activity.

Efrapeptins (EF), a family of fungal peptides, inhibit proteasomal enzymatic activities and the in vitro and in vivo growth of HT-29 cells. They are also known inhibitors of F 1 F 0 -ATPase, a mitochondrial enzyme that functions as an Hsp90 co-chaperone. We have previously shown that treatment of cancer cells with EF results in disruption of the Hsp90:F 1 F 0 -ATPase complex and inhibition of Hsp90 chaperone activity. The present study examines the effect of EF on breast cancer growth in vitro and in vivo. As a monotherapy, EF inhibited cell proliferation in vitro with an IC 50 value ranging from 6 nM to 3.4 μM. Inhibition of Hsp90 chaperone function appeared to be the dominant mechanism of action and the factor determining cellular sensitivity to EF. In vitro inhibition of proteasome became prominent in the absence of adequate levels of Hsp90 and F 1 F 0 -ATPase as in the case of the relatively EF-resistant MDA-MB-231 cell line. In vivo, EF inhibited MCF-7 and MDA-MB-231 xenograft growth with a maximal inhibition of 60% after administration of 0.15 and 0.3 mg/kg EF, respectively. 2-Deoxyglucose (2DG), a known inhibitor of glycolysis, acted synergistically with EF in vitro and antagonistically in vivo. In vitro, the synergistic effect was attributed to a prolonged endoplasmic reticulum (ER) stress. In vivo, the antagonistic effect was ascribed to the downregulation of tumoral and/or stromal F 1 F 0 -ATPase by 2DG.