Inhibitor Report for: Ebelactone-B

Fusarium graminearum (sexual stage: Gibberella zeae) is the causative agent of Fusarium Head Blight (FHB), which is one of the most destructive plant disease of cereals, accounting for high grain yield losses, especially for wheat and maize. Like other fungal pathogens, several extracellular enzymes secreted by G. zeae are known to be involved in host infection. Among these secreted lipases, G. zeae lipase (GZEL), which is encoded by the FGL1 gene, was demonstrated to be crucial to G. zeae pathogenicity. However, the precise mechanism of GZEL remains unclear due to a lack of detailed structural information. In this study, we report the crystal structure of GZEL at the atomic level. The structure of GZEL displays distinct structural differences compared to reported homologues and indicates a unique "double lock" enzymatic mechanism. To gain insight into substrate/inhibitor recognition, we proposed a model of GZEL in complex with substrate and the lipase inhibitor ebelactone B (based on the reported structures of GZEL homologues), which defines possible substrate binding sites within the catalytic cleft and suggests an "anti sn-l" binding mode. These results pave the way to elucidating the mechanism of GZEL and thus provide clues for the design of anti-FHB inhibitors.

Cathepsin A (CatA) is a serine carboxypeptidase distributed between lysosomes, cell membrane, and extracellular space. Several peptide hormones including bradykinin and angiotensin I have been described as substrates. Therefore, the inhibition of CatA has the potential for beneficial effects in cardiovascular diseases. Pharmacological inhibition of CatA by the natural product ebelactone B increased renal bradykinin levels and prevented the development of salt-induced hypertension. However, so far no small molecule inhibitors of CatA with oral bioavailability have been described to allow further pharmacological profiling. In our work we identified novel beta-amino acid derivatives as inhibitors of CatA after a HTS analysis based on a project adapted fragment approach. The new inhibitors showed beneficial ADME and pharmacokinetic profiles, and their binding modes were established by X-ray crystallography. Further investigations led to the identification of a hitherto unknown pathophysiological role of CatA in cardiac hypertrophy. One of our inhibitors is currently undergoing phase I clinical trials.

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.

Fusarium graminearum (sexual stage: Gibberella zeae) is the causative agent of Fusarium Head Blight (FHB), which is one of the most destructive plant disease of cereals, accounting for high grain yield losses, especially for wheat and maize. Like other fungal pathogens, several extracellular enzymes secreted by G. zeae are known to be involved in host infection. Among these secreted lipases, G. zeae lipase (GZEL), which is encoded by the FGL1 gene, was demonstrated to be crucial to G. zeae pathogenicity. However, the precise mechanism of GZEL remains unclear due to a lack of detailed structural information. In this study, we report the crystal structure of GZEL at the atomic level. The structure of GZEL displays distinct structural differences compared to reported homologues and indicates a unique "double lock" enzymatic mechanism. To gain insight into substrate/inhibitor recognition, we proposed a model of GZEL in complex with substrate and the lipase inhibitor ebelactone B (based on the reported structures of GZEL homologues), which defines possible substrate binding sites within the catalytic cleft and suggests an "anti sn-l" binding mode. These results pave the way to elucidating the mechanism of GZEL and thus provide clues for the design of anti-FHB inhibitors.

The present study was undertaken to investigate whether ebelactone B, an inhibitor of bradykinin and angiotensin I hydrolysis by serine carboxypeptidase Y-like enzymes, could influence platelet aggregation ex vivo in renovascular hypertensive rats (2-kidney, 1-clip Goldblatt model, 2K1C). We found that ebelactone B (5 mg/kg) administrated subcutaneously once a day for 5 days, 5 weeks after the development of hypertension, or a single dose of ebelactone B (0.5 mg/kg) injected intravenously into 2K1C hypertensive rats before the induction of arterial thrombosis, both markedly suppressed collagen-induced platelet aggregation in whole blood. In contrast, inhibition of collagen-induced platelet aggregation was not evident in vitro after pretreatment of the blood with ebelactone B, indicating that ex vivo the antiaggregatory action of this compound can proceed through an indirect mechanism. The injection of ebelactone B did not affect the mean blood pressure of 2K1C hypertensive rats but lowered an elevated extracellular serine carboxypeptidase cathepsin A-like activity. Thus, the data indicate that ebelactone B may be a promising antiaggregatory agent in renovascular hypertension and suggest that 1 of the possible mechanisms through which it exerts this effect may be related to the suppression of cathepsin A-like activity released locally during the development of renovascular hypertension.

Ebelactones A and B, natural products from Streptomyces aburaviensis are potent inhibitors of pancreatic lipase. Lipase is the key enzyme required for the absorption of dietary triglycerides (TG). Ebelactone B inhibited, in a dose-dependent manner, the intestinal absorption of fat after fat-feeding in the rat. The most effective inhibition was observed when the inhibitor was administered at 60 min prior to fat-feeding. When ebelactone B (10 mg/kg) was administered, the serum levels of TG (58%) and cholesterol (36%) were decreased. Since ebelactone B effectively inhibitors absorption of dietary fat, if may provide a promising means for prophylaxis or therapeutics of hyperlipidemia and obesity.

Incubation of bradykinin with human urine resulted in a successive degradation of bradykinin-(1-8), bradykinin-(1-7), bradykinin-(1-6), and bradykinin-(1-5). Although D,L-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (100 microM) and captopril (100 microM) did not have any significant effect on bradykinin degradation in human urine, the neutral endopeptidase inhibitor phosphoramidon (100 microM), a carboxypeptidase Y-like exopeptidase inhibitor ebelactone B (100 microM), and o-phenanthroline (100 microM) significantly inhibited bradykinin degradation by 36%, 38% and 48% respectively. The combination of phosphoramidon and ebelactone B completely (by 95%) inhibited bradykinin degradation in human urine. At pH 5, bradykinin degradation was performed by carboxypeptidase Y-like exopeptidase; at pH 7, this degradation was performed by neutral endopeptidase in addition to carboxypeptidase Y-like exopeptidase. From these results, it can be concluded that carboxypeptidase Y-like exopeptidase and/or neutral endopeptidase certainly have a role in kinin degradation in human urine under neutral and acid pH conditions.