Identification and Characterization of a Non-retinoid Ligand for Retinol-binding Protein 4 Which Lowers Serum Retinol-binding Protein 4 Levels in Vivo

Abstract

Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (K i ‫؍‬ 8.3 nM) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4 ؊/؊ mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes. RBP42 is a serum protein that transports retinol (vitamin A) from the liver to extrahepatic tissues (1). The majority of RBP4 is expressed in the liver, with ϳ15-20% expressed in adipose (2). In the serum, RBP4 is present as a complex with transthyretin (TTR), which effectively increases the molecular weight of RBP4 and protects it from glomerular filtration. Thus disruption of the RBP4⅐TTR complex in vivo by administration of the synthetic retinoid fenretinide (N-(4-hydroxyphenyl)retinamide) results in a rapid reduction in serum RBP4 levels (3, 4).Although the major physiological ligand for RBP4 appears to be retinol, RBP4 can bind to other endogenous and synthetic retinoids. For example, using biochemical assays, RBP4 has been shown to bind to retinol, all-trans-and 13-cis-retinoic acid, retinyl acetate, N-(ethyl)retinamide, and fenretinide (4 -6). In addition, x-ray diffraction analysis of a variety of RBP4-retinoid co-crystal structures has demonstrated that these retinoids bind to the same site as retinol, with the cyclohexene ring buried within the internal cavity and the polar head group pointing toward the exterior of the protein (7-9). The loop regions of RBP4 surrounding the entrance of the binding cavity form the binding site for TTR, with the binding of retinol (in particular the presence of the hydroxyl group) increasing the affinity of RBP4 for TTR by a factor of ϳ4 (10). The binding of fenretinide, however, has the opposite effect. Through a combination of steric hindrance (from the bulky phenylamide head group) and changes in the position of the loop regions of RBP4 located at the TTR binding interface, fenretinide completely disrupts the binding of RBP4 to TTR (4, 9).Recent reports have suggested that, in addition to its role in vitamin A transport, RBP4 may also be involved in the development of insulin resistance. For example, Yang et al. reported that mice overexpressing an RBP4 transgene and mice injected with recombinant RBP4 protein become insulin-resistant, and that reduction of RBP4 levels in mice either by gene ablation or treatment with fenretinide improves insulin sensitivity (11). In huma...

A1120, a Nonretinoid RBP4 Antagonist, Inhibits Formation of Cytotoxic Bisretinoids in the Animal Model of Enhanced Retinal Lipofuscinogenesis

“…Nonlinear regression analysis conducted following saturation binding revealed a dissociation constant (K d ) of 62.5 nM. The K d for retinol binding to RBP4 defined in our saturation binding experiments is in line with two previously reported values, 70 33 and 60 11 nM, while being somewhat different from the two other literature values, 190 34 and 29 27 nM. To compare A1120 and fenretinide potency, we conducted competitive binding experiments with A1120 and fenretinide at the fixed 10 nM concentration of 3 H-retinol (Fig.…”

“…A1120, a nonretinoid RBP4 ligand, originally was identified in a screen for compounds that may improve insulin sensitivity. 27 However, administration of A1120 did not improve insulin sensitivity, making it an unlikely drug candidate for diabetes treatment. In our study, we confirmed the ability of A1120 to displace retinol from RBP4, disrupt retinol-induced RBP4-TTR interaction, and reduce serum RBP4 levels.…”

“…Given the excellent correlation between serum RBP4 and serum retinol across wide range of retinol concentrations, 31 and taking into account the absolute correlation between A1120-induced changes in serum RBP4 and serum retinol in mice, 27 we used serum RBP4 a surrogate marker for serum retinol in this study. Blood samples were collected from a tail vein at days 0, 21, and 42 of the A1120 dosing.…”

Fenretinide Ameliorates Insulin Resistance and Fatty Liver in Obese Mice

“…7,11,12,18) Short-term treatment with fenretinide decreases circulating RBP4 levels only, 18) whereas 16 weeks of treatment enhances insulin sensitivity. 7,8) Long-term treatment (34 weeks) also decreases the severity of obesity.…”

Patent Highlights

“…The inventors present 2-(4-(2-(trifluoromethyl)phenyl)piperidine-1-carboxamido)benzoic acid (A1120), a high affinity (Ki = 8.3 nM) nonretinoid ligand for retinol-binding protein 4 which disrupts its interaction with transthyretin. A1120 has been reported before in this mechanistic context [7], but this had not been applied to the treatment dry AMD or Stargardt Disease. In the Abca4(-/-) mouse future science group Patent Highlights Mucke model that manifests accelerated accumulation of lipofuscin in the retinal pigment epithelium [8], the levels of A2E and other bisretinoids were reduced by approximately 50%.…”