Androgenic steroids marketed online as nutraceuticals are a growing concern in sport doping. The inability of conventional mass spectrometry (MS)-based techniques to detect structurally novel androgens has led to the development of in vitro androgen bioassays to identify such designer androgens by their bioactivity. The objective of this study was to determine the androgenic bioactivity of novel steroidal compounds isolated from nutraceuticals using both yeast and mammalian cell-based androgen bioassays. We developed two new in vitro androgen bioassays by stably transfecting HEK293 and HuH7 cells with the human androgen receptor (hAR) expression plasmid together with a novel reporter gene vector (enhancer/ARE/SEAP). The yeast β-galactosidase androgen bioassay was used for comparison. Our new bioassay featuring the enhancer/ARE/SEAP construct (-S) displayed simpler assay format and higher specificity with lower sensitivity compared with the commonly used mouse mammary tumour virus (MMTV)-luciferase. The relative potencies (RP), defined as [EC(50)] of testosterone/[EC(50)] of steroid, of nutraceutical extracts in the yeast, HEK293-S, and HuH7-S, were 34, 333, and 80,000 for Hemapolin; 208, 250, and 80 for Furazadrol; 0.38, 10, and 106 for Oxyguno; 2.7, 0.28, and 15 for Trena; and 4.5, 0.1, and 0.4 for Formadrol, respectively. The wide discrepancies in rank RP of these compounds was reconciled into a consistent potency ranking when the cells were treated with meclofenamic acid, a nonselective inhibitor of steroid metabolizing enzymes. These findings indicate that steroids extracted from nutraceuticals can be converted in vitro into more or less potent androgens in mammalian but not in yeast cells. We conclude that the putative androgenic bioactivity of a new compound may depend on the bioassay cellular format and that mammalian cell bioassays may have an added benefit in screening for proandrogens but sacrifice specificity for sensitivity in quantitation.

Protein-protein interactions (PPI) are a hallmark of cellular signaling.

E levated low-density lipoprotein cholesterol (LDLC) levels in the plasma is the most important causative factor of atherosclerosis and associated ischemic cardiovascular diseases. The LDL receptor (LDLR) is the preferential pathway through which LDLs are cleared from the circulation. LDLs bound to the LDLR are internalized into clathrin-coated pits and subsequently undergo lysosomal degradation, whereas the LDLR is recycled back to the plasma membrane. See accompanying article on page 1333Familial hypercholesterolemia (FH) is an autosomal dominant disorder associated with elevated LDL levels and premature coronary heart disease. FH is caused primarily by mutations of the LDLR or of apolipoprotein B100 (apoB100), the protein component of LDL that interacts with the LDLR. In 2003, "gain of function" mutations on a newly identified gene, proprotein convertase subtilisin/kexin type 9 (PCSK9), were associated with FH. In 2005, a causative association was established between "loss of function" mutations in PCSK9 and low LDLC levels in 2% of the African-American population. The coronary heart disease risk in these individuals was reduced by 88%. As a result of these landmark studies (reviewed in Reference 1), PCSK9 became the subject of intensive research to discover the underlying mechanisms.PCSK9 is a serine protease mainly expressed in the liver and the intestine. It acts by reducing the amount of LDLR in hepatocytes. This was demonstrated in vitro and in mouse models and inferred by genetic studies in patients with PCSK9 mutations (reviewed in Reference 2). In brief, PCSK9 enzymatic activity permits its intracellular maturation, followed by secretion ( Figure). Circulating PCSK9 binds the LDLR on the cell surface and is subsequently cointernalized together with the LDLR. This promotes the degradation of the receptor in the lysosome, rather than recycling to the plasma membrane. PCSK9 can also bind the LDLR intracellularly. 3 Thus, by virtue of its role as a major inhibitor of the LDLR, PCSK9 has emerged as a hot new drug target to treat hypercholesterolemia and coronary heart disease.PCSK9 inhibition has been intensively studied in cellbased systems. A peptide, which mimics the interaction domain of the LDLR with PCSK9, can inhibit PCSK9 binding to the LDLR and prevent its degradation. 4 Likewise, an anti-PCSK9 antibody 5 and an anti-PCSK9 antigen binding fragment 6 disrupt the interaction between PCSK9 and the LDLR, thus restoring cellular LDL-uptake. In vivo, PCSK9 has been inhibited using antisense oligonucleotides 7 or small interfering RNA (siRNA). 8 These treatments dramatically increase hepatic LDLR and lower plasma LDLC in rodents and monkeys. Another approach has involved infusions of humanized anti-PCSK9 antibodies. 9 A single injection of these antibodies reduced LDLC by 80% in monkeys. This study also showed that anti-PCSK9 antibodies act synergistically with statins to increase LDLR cell surface expression, indicating that blocking PCSK9 in statin-treated patients will most likely further reduce their...