SUMMARY Hepatocyte Nuclear Factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β-cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.

The principal finding of this study is that two drugs, alverine and benfluorex, used in vastly different clinical settings and previously unknown to share mechanistic or structural similarity, activated the nuclear receptor transcription factor HNF4α. Both were hits in a high-throughput screen for compounds that reversed the inhibitory effect of the fatty acid palmitate on human insulin promoter activity. Alverine is used in the treatment of irritable bowel syndrome, while benfluorex (Mediator) was used to treat hyperlipidemia and type II diabetes. Benfluorex was withdrawn from the market recently because of serious cardiovascular side effects related to fenfluramine-like activity. Strikingly, alverine and benfluorex have a previously unrecognized structural similarity, consistent with a common mechanism of action. Gene expression and biochemical studies revealed that they both activate HNF4α. This novel mechanism of action should lead to a reinterpretation of previous studies with these drugs and suggests a path towards the development of therapies for diseases such as inflammatory bowel and diabetes that may respond to HNF4α activators.

Cell penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3β-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). This synthetic receptor inserts into cellular plasma membranes, projects NTA headgroups from the cell surface, and rapidly cycles between the plasma membrane and intracellular endosomes. Jurkat lymphocytes treated with the synthetic receptor (10 micromolar) for one hour displayed ~ 8,400,000 NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 micromolar) and Ni(OAc) 2 (100 micromolar) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within four hours at 37 °C. No adverse effects on cellular proliferation or morphology were observed under these conditions. By enabling common oligohistidine affinity tags to function as cell penetrating peptides, this metal-chelating cell surface receptor provides a useful tool for studies of cellular biology.Cell penetrating peptides and proteins (CPPs) have emerged as important new tools for the delivery of impermeable molecules into living mammalian cells. These delivery systems have been constructed from basic segments of HIV Tat, 1 oligoguanidine-containing peptoids, 2 β-peptides, 3 and other oligocationic motifs. 4 The mechanism of cellular penetration by CPPs is primarily thought to involve endocytosis, 5 particularly with larger cargo, 6 but varies depending on the delivery system and the cell type. 7Oligohistidine peptides are often fused to proteins to provide affinity tags ("His tags") that facilitate protein purification. To enable these peptides to function as CPPs, we synthesized an artificial cell surface receptor (1) comprising the plasma membrane anchor N-alkyl-3β-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). N-Alkyl-3β-cholesterylamine derivatives can function as prosthetic molecules active on the surface of living mammalian cells because this steroid can insert into cellular plasma membranes, project linked headgroups from the cell surface, and rapidly cycle between the plasma membrane and intracellular endosomes, similar to many naturally-occurring cell surface receptors. 8,9 The NTA motif binds tightly to nickel, cobalt, copper, and zinc dications, and immobilized metal chelate chromatography (IMAC) with NTA-linked supports is widely used to purify proteins fused to His tags. 10 Other lipids 11,12 and polymers 13 linked to NTA headgroups have also been reported. Receptor 1 was synthesized from 3β-cholesterylamine (2) 9 as shown in Scheme 1. Alkylation of 2 with ethyl 5-bromovalerate, followed by Boc protection to afford 3, provided an improved route to protected N-alkyl derivativ...

Chemical inducers of dimerization (CIDs) are powerful tools for controlling diverse cellular processes. These small molecules typically form strong noncovalent interactions with proteins. We report a related approach involving covalent acylation of a specific lysine residue of a target protein by the small molecule biotin. To control protein-protein interactions with biotin, the biotin protein ligase BirA from E. coli was coexpressed in yeast with a streptavidin-LexA fusion protein and Avitag or BCCP biotin acceptor peptides fused to the B42 activation domain. The addition of biotin (10 nM) resulted in BirA-mediated biotinylation of the biotin acceptor protein, recruitment to LexA DNA sites, and maximal activation of reporter gene expression in this yeast tribrid system. The high potency, low toxicity, and low molecular weight of biotin as a covalent CID are attractive properties for controlling cellular processes.