Saturation transfer difference (STD), inter-ligand NOEs (INPHARMA NMR), and docking calculations are reported for investigating specific binding sites of the high-affinity synthetic ...7-nitrobenz-2-oxa-1,3-diazoyl-4-C
fatty acid (NBD-C
FA) with non-labeled human serum albumin (HSA) and in competition with the drugs warfarin and ibuprofen. A limited number of negative interligand NOEs between NBD-C
FA and warfarin were interpreted in terms of a short-range allosteric competitive binding in the wide Sudlow's binding site II (FA7) of NBD-C
FA with Ser-202, Lys-199, and Trp-214 and warfarin with Arg-218 and Arg-222. In contrast, the significant number of interligand NOEs between NBD-C
FA and ibuprofen were interpreted in terms of a competitive binding mode in Sudlow's binding site I (FA3 and FA4) with Ser-342, Arg-348, Arg-485, Arg-410, and Tyr-411. NBD-C
FA has the unique structural properties, compared to short-, medium-, and long-chain saturated and unsaturated natural free fatty acids, of interacting with well-defined structures with amino acids of both the internal and external polar anchor sites in Sudlow's binding site I and with amino acids in both FA3 and FA4 in Sudlow's binding site II. The NBD-C
FA, therefore, interacts with novel structural characteristics in the drug binding sites I and II and can be regarded as a prototype molecule for drug development.
Energy homeostasis, a fundamental property of all organisms, depends on the ability to control the storage and mobilization of fat, mainly triacylglycerols (TAG), in special organs such as mammalian ...adipose tissue or the fat body of flies. Malregulation of energy homeostasis underlies the pathogenesis of obesity in mammals including human. We performed a screen to identify nutritionally regulated genes that control energy storage in the model organism
Drosophila. The
brummer (
bmm) gene encodes the lipid storage droplet-associated TAG lipase Brummer, a homolog of human adipocyte triglyceride lipase (ATGL). Food deprivation or chronic
bmm overexpression depletes organismal fat stores in vivo, whereas loss of
bmm activity causes obesity in flies. Our study identifies a key factor of insect energy homeostasis control. Their evolutionary conservation suggests Brummer/ATGL family members to be implicated in human obesity and establishes a basis for modeling mechanistic and therapeutic aspects of this disease in the fly.
Human glucose transporters (GLUTs) are responsible for cellular uptake of hexoses. Elevated expression of GLUTs, particularly GLUT1 and GLUT3, is required to fuel the hyperproliferation of cancer ...cells, making GLUT inhibitors potential anticancer therapeutics. Meanwhile, GLUT inhibitor-conjugated insulin is being explored to mitigate the hypoglycemia side effect of insulin therapy in type 1 diabetes. Reasoning that exofacial inhibitors of GLUT1/3 may be favored for therapeutic applications, we report here the engineering of a GLUT3 variant, designated GLUT3exo, that can be probed for screening and validating exofacial inhibitors. We identify an exofacial GLUT3 inhibitor SA47 and elucidate its mode of action by a 2.3 Å resolution crystal structure of SA47-bound GLUT3. Our studies serve as a framework for the discovery of GLUTs exofacial inhibitors for therapeutic development.
Introduction:
9-PAHSA belongs to a class of endogenous mammalian bioactive lipids, fatty acid esters of hydroxy fatty acids (FAHFA), that are present in circulation at nanomolar concentrations in ...mice and humans. Published preclinical data suggest beneficial effects of 9-PAHSA treatment on glucose metabolism as well as modulation of immune function. However, receptor molecules with high affinity towards these lipids have not been identified so far.
Methods:
In a broad screen of a panel of G protein-coupled receptors (GPCRs) we discovered that 9-PAHSA displays antagonist activity with an IC
50
in the micromolar range on selected chemokine receptors, namely, CCR6, CCR7, CXCR4, and CXCR5. The potential immunomodulatory activities in a human cellular model of innate immunity were then investigated.
Results and discussion:
In our
in vitro
experiments, a weak anti-inflammatory potential for high concentrations of 9-PAHSA (10–100 µM) could be detected, as treatment reduced the LPS-induced secretion of certain chemokines, such as CXCL10, MIP-1 beta and MCP. Regarding metabolic effects, we re-investigated 9-PAHSA on glucose metabolism and insulin sensitivity
in vitro
and in mice confirming conclusions from our earlier study that FAHFAs lack glucoregulatory activity following an acute treatment. In conclusion, the specific interactions with a subset of chemokine receptors may contribute to weak anti-inflammatory properties of 9-PAHSA, but further studies are needed to confirm its in anti-inflammatory potential
in vivo
.
The financial institution that issues a structured retail product (SRP) becomes the dealer for that security. Issuer-dealer funding constraints can directly impact price quotes for SRPs. The ...2007-2009 financial crisis diminished issuer funding liquidity, and both bid and ask quotes declined, with the decrease in bids being significantly greater than that for the asks. A reduction in the bid (ask) discourages (encourages) investor selling (buying) and helps preserve dealer capital. The SRP's intrinsic value places a bound on how far the ask needs to be reduced to induce investor buying. High-leverage (low-leverage) issuers are the most (least) financially constrained and decrease their bids by a significant 167% (nonsignificant 41%) compared to the pre-crisis average. The decrease in asks is nonsignificant for both groups.
Several countries legally mandate representation of workers on boards of directors. The evidence on the shareholder wealth effects of such a corporate governance design is mixed. I examine abnormal ...announcement returns around major milestones leading to the passing of the German Codetermination Act in 1976. I find that news about the act causes an average decline in the equity value of firms that are certain to have been affected by the new law of up to 1.5% relative to the control firms. Firms close to the regulatory threshold of 2,000 employees remain unaffected implying an expectation of avoiding compliance.
We find evidence of systematic optimism and pessimism among credit analysts, comparing contemporaneous ratings of the same firm across rating agencies. These differences in perspectives carry through ...to debt prices and negatively predict future changes in credit spreads, consistent with mispricing. Moreover, the pricing effects are the largest among firms that are the most opaque, likely exacerbating financing constraints. We find that masters of business administration (MBAs) provide higher quality ratings. However, optimism increases and accuracy decreases with tenure covering the firm. Our analysis demonstrates the role analysts play in shaping investor expectations and its effect on corporate debt markets.
Lipid metabolism is a growing area of biochemical research because understanding these pathways could lead to treatments for metabolic disorders such as obesity and type 2 diabetes. To study lipid ...metabolism, researchers need tools to identify and quantitate glycerides, the main component of animal fat. However, it can be difficult to tell one glyceride apart from another subtly different glyceride using current analytical methods such as mass spectrometry. Thus, we developed a method of discriminating glycerides using an array of cross-reactive proteins in conjunction with pattern recognition algorithms. By incorporating an olefin metathesis pretreatment step, we were able to distinguish glyceride regio- and stereoisomers and to predict these structural features. Finally, we achieved quantitation of glycerides in mixtures.
Glycerides are of interest to the areas of food science and medicine because they are the main component of fat. From a chemical sensing perspective, glycerides are challenging analytes because they are structurally similar to one another and lack diversity in terms of functional groups. Furthermore, because animal and plant fat consists of a number of stereo- and regioisomeric acylglycerols, their components remain challenging analytes for chromatographic and mass spectrometric determination, particularly the quantitation of species in mixtures. In this study, we demonstrated the use of an array of cross-reactive serum albumins and fluorescent indicators with chemometric analysis to differentiate a panel of mono-, di-, and triglycerides. Due to the difficulties in identifying the regio- and stereochemistry of the unsaturated glycerides, a sample pretreatment consisting of olefin cross-metathesis with an allyl fluorescein species was used before array analysis. Using this simple assay, we successfully discriminated 20 glycerides via principal component analysis and linear discriminant analysis (PCA and LDA, respectively), including stereo- and regioisomeric pairs. The resulting chemometric patterns were used as a training space for which the structural characteristics of unknown glycerides were identified. In addition, by using our array to perform a standard addition analysis on a mixture of triglycerides and using a method introduced herein, we demonstrated the ability to quantitate glyceride components in a mixture.
The fluorogenic substrate 6,8-difluoro-4-methylumbiliferyl phosphate (DIFMUP) has been widely used for the detection of serine and threonine phosphatase activities. Here we describe the use of this ...substrate for the characterization of protein tyrosine phosphatases (PTPs) and for the screening for PTP inhibitors. The measured kinetic and inhibitor constants for DIFMUP cleavage were comparable with those of the widely used but less discriminative and practicable substrates,
para-nitrophenylphosphate and phosphotyrosine-containing peptides, respectively. Furthermore, the continuous and highly sensitive assay allows fast and accurate investigations of the type, kinetic behavior, and binding mode of small-molecule inhibitors. We discuss the validation of this assay system for various PTPs and its use in inhibitor screening for PTP1B.
A single high‐affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐C12 fatty ...acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site‐specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA‐binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow‐sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X‐ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug‐protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.
It's not a hydrophobic sponge: The identification and characterization of a single high‐affinity fatty acid binding site in human serum albumin has been achieved using a labeled fatty acid with highly specific binding. An X‐ray crystal structure locates the binding site in subdomain IIA. The identified binding site helps in the understanding of drug protein binding, modulation of pharmacokinetic profiles, and drug depot development.
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