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...

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Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure−activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent (K i = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.

A high-throughput screen resulted in the discovery of benzoxazepine 1, an EP2 antagonist possessing low microsomal stability and potent CYP3A4 inhibition. Modular optimization of lead compound 1 resulted in the discovery of benzoxazepine 52, a molecule with single-digit nM binding affinity for the EP2 receptor and significantly improved microsomal stability. It was devoid of CYP inhibition and was ∼4000-fold selective against the other EP receptors. Compound 52 was shown to have good PK properties in CD-1 mice and high CNS permeability in C57Bl/6s mice and Sprague-Dawley rats. In an ex vivo assay, it demonstrated the ability to increase the macrophage-mediated clearance of amyloid-beta plaques from brain slices in a dose-dependent manner.