Englerins: A Comprehensive Review

In the decade since the discovery of englerin A (<b>1</b>) and its potent activity in cancer models, this natural product and its analogues have been the subject of numerous chemical, biological, and preclinical studies by many research groups. This review summarizes published findings and proposes further research directions required for entry of an englerin analogue into clinical trials for kidney cancer and other conditions

Englerin A Selectively Induces Necrosis in Human Renal Cancer Cells

<div><p>The number of renal cancers has increased over the last ten years and patient survival in advanced stages remains very poor. Therefore, new therapeutic approaches for renal cancer are essential. Englerin A is a natural product with a very potent and selective cytotoxicity against renal cancer cells. This makes it a promising drug candidate that may improve current treatment standards for patients with renal cancers in all stages. However, little is known about englerin A's mode of action in targeting specifically renal cancer cells. Our study is the first to investigate the biological mechanism of englerin A action in detail. We report that englerin A is specific for renal tumor cells and does not affect normal kidney cells. We find that englerin A treatment induces necrotic cell death in renal cancer cells but not in normal kidney cells. We further show that autophagic and pyroptotic proteins are unaffected by the compound and that necrotic signaling in these cells coincided with production of reactive oxygen species and calcium influx into the cytoplasm. As the first study to analyze the biological effects of englerin A, our work provides an important basis for the evaluation and validation of the compound's use as an anti-tumor drug. It also provides a context in which to identify the specific target or targets of englerin A in renal cancer cells.</p> </div

"Novel idebenone analogs block Shc\u2019s access to insulin receptor to improveinsulin sensitivity"

There has been little innovation in identifying novel insulin sensitizers. Metformin, developed in the 1920s, is still used first for most Type 2 diabetes patients. Mice with genetic reduction of p52Shc protein have improved insulin sensitivity and glucose tolerance. By high-throughput screening, idebenone was isolated as the first small molecule 'Shc Blocker'. Idebenone blocks p52Shc's access to Insulin Receptor to increase insulin sensitivity. In this work the avidity of 34 novel idebenone analogs and 3 metabolites to bind p52Shc, and to block the interaction of p52Shc with the Insulin receptor was tested. Our hypothesis was that if an idebenone analog bound and blocked p52Shc's access to insulin receptor better than idebenone, it should be a more effective insulin sensitizing agent than idebenone itself. Of 34 analogs tested, only 2 both bound p52Shc more tightly and/or blocked the p52Shc-Insulin Receptor interaction more effectively than idebenone. Of those 2 only idebenone analog #11 was a superior insulin sensitizer to idebenone. Also, the long-lasting insulin-sensitizing potency of idebenone in rodents over many hours had been puzzling, as the parent molecule degrades to metabolites within 1\u2009h. We observed that two of the idebenone\u2019s three metabolites are insulin sensitizing almost as potently as idebenone itself, explaining the persistent insulin sensitization of this rapidly metabolized molecule. These results help to identify key SAR\u2009=\u2009structure-activity relationship requirements for more potent small molecule Shc inhibitors as Shc-targeted insulin sensitizers for type 2 diabetes

Englerin A does not induce cleavage of caspase 3, PARP, caspase 1 or the autophagic markers LC-3 and Beclin-1.

<p>Cells were treated with either 1 μM englerin A, carrier DMSO or 5 μM staurosporine for the indicated amount of time. (A) After the incubation, cells were lysed and lysates were analyzed by immunoblotting for PARP cleavage or full-length and cleaved caspase 3 (Casp3-fl, Casp3-cl). Equal protein loading was confirmed by probing for GAPDH. Full-length and cleaved bands are indicated. The experiment was repeated three times. (B) Alternatively, after incubation cells were lysed and caspase 3 activity was tested using a caspase 3 activity assay kit. Values shown are means ± SEM (n = 6), statistically significant differences are marked with asterisks (*** p<0.001). (C) Cells were treated with either 1 μM englerin A, carrier DMSO for 60min or 50 μM chloroquine diphosphate (Chloro) for 18 h. After the incubation, cells were lysed and lysates were analyzed by immunoblotting for Beclin-1, LC3-I/II and caspase 1 cleavage (proenzyme p45 and cleaved active subunit p20). Equal protein loading was confirmed by probing for GAPDH. All membranes were analyzed using IRDye secondary antibodies and a Licor Odyssey system. Membranes shown are from representative experiments.</p

Novel idebenone analogs block Shc's access to insulin receptor to improve insulin sensitivity.

There has been little innovation in identifying novel insulin sensitizers. Metformin, developed in the 1920s, is still used first for most Type 2 diabetes patients. Mice with genetic reduction of p52Shc protein have improved insulin sensitivity and glucose tolerance. By high-throughput screening, idebenone was isolated as the first small molecule 'Shc Blocker'. Idebenone blocks p52Shc's access to Insulin Receptor to increase insulin sensitivity. In this work the avidity of 34 novel idebenone analogs and 3 metabolites to bind p52Shc, and to block the interaction of p52Shc with the Insulin receptor was tested. Our hypothesis was that if an idebenone analog bound and blocked p52Shc's access to insulin receptor better than idebenone, it should be a more effective insulin sensitizing agent than idebenone itself. Of 34 analogs tested, only 2 both bound p52Shc more tightly and/or blocked the p52Shc-Insulin Receptor interaction more effectively than idebenone. Of those 2 only idebenone analog #11 was a superior insulin sensitizer to idebenone. Also, the long-lasting insulin-sensitizing potency of idebenone in rodents over many hours had been puzzling, as the parent molecule degrades to metabolites within 1 h. We observed that two of the idebenone's three metabolites are insulin sensitizing almost as potently as idebenone itself, explaining the persistent insulin sensitization of this rapidly metabolized molecule. These results help to identify key SAR = structure-activity relationship requirements for more potent small molecule Shc inhibitors as Shc-targeted insulin sensitizers for type 2 diabetes

Englerin A induces production of reactive oxygen species and increased concentration of intracellular Ca²⁺.

<p>(A) Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min. The relative change in reactive oxygen (ROS) or reactive nitrogen species (RNS) compared to cells treated with the carrier DMSO was measured using the Total ROS detection kit. Histograms show fluorescence intensities in a representative experiment (left panel). Quantified relative changes in ROS/RNS shown (right panel) are means ± SEM (n = 5), statistically significant differences are marked with asterisks (* p<0.05). (B) Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min, or 10 μM ionomycin for 50 min. Fluo-3 binding to Ca²⁺ ions was measured through an increased fluorescence emission of the dye at 520 nm upon excitation at 485 nm. Histograms show fluorescence intensities in a representative experiment (left panel). Quantified relative changes in intracellular calcium ions shown (right panel) are means ± SEM (n = 3), statistically significant differences are marked with asterisks (* p<0.05, *** p<0.001).</p

Englerin A selectively reduces cell viability in renal cancer cells.

<p>(A) Chemical Structure of englerin A. (B) Glioblastoma (SF-295), normal immortalized kidney cells (HEK-293), renal proximal tubule cells (RPTC) and renal cancer cells (UO-31, A-498) were incubated with the indicated concentration of englerin A for 48 h. Cell viability was analyzed using an XTT Cell Proliferation Assay. Results are shown in % viability compared to a cell sample treated with the carrier DMSO. Values shown represent the mean ± SEM of all experiments (n≥6). IC50 values were calculated with Prism 5 using a non-linear regression fit (log(inhibitor) vs. normalized response – variable slope).</p

Englerin A does not lead to up-regulation of extracellular phosphatidyl serine.

<p>Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min, or 5 μM staurosporine for 3 h. After incubation, cells were trypsinized and stained for extracellular phosphatidyl serine expression using FITC-tagged Annexin V and propidium iodide (PI) as co-stain to test cell membrane integrity. Shown is a result representative of three independent experimental repeats. Quantifications and statistics of all data are depicted as bar graphs and show the distribution of cells testing positive for Annexin V binding (early apoptotic stages) or Annexin V binding and propidium iodide uptake (late apoptotic stages/necrotic death). Values shown are mean ± SEM (n = 3), statistically significant differences are marked with asterisks (*** p<0.001), n.s. = not significant.</p

Englerin A induces cell death morphologically distinct from staurosporine induced apoptosis.

<p>Micrographs show the morphology of cells treated with englerin A or staurosporine, a known inducer of apoptosis. Cells were treated with either 1 μM englerin A or carrier DMSO for 60 min, or 1 μM staurosporine for 5 h. Pictures were taken using a Zeiss Axiovert200 M microscope with a 40× phase objective. For every treatment, 5–10 random fields of vision were acquired. The experiment was repeated three times, micrographs shown are representative of the average cell morphology upon treatment. Scale bars represent 20 μm.</p