Francisco de Assis Rocha Neves

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.

Selenoprotein-Related Disease in a Young Girl Caused by Nonsense Mutations in the SBP2 Gene

CONTEXT Selenoproteins are essential for life, and their biosynthesis requires the incorporation of the rare amino acid selenocysteine (Sec) in a process mediated by the Sec insertion sequence-binding protein 2 (SBP2). Although SBP2 is considered a rate-limiting factor mediating Sec incorporation, there has been little evidence so far linking SBP2 dysfunction to widespread selenoprotein-related disease. OBJECTIVE The objective of the study was to report the discovery of novel truncation mutations in the SBP2 gene (R120X/R770X) in a female adolescent and the clinical consequences of the combined deficiency of selenoproteins. SUBJECTS AND METHODS A 12-yr-old girl who presented with a syndrome of abnormal thyroid hormone metabolism, delayed bone maturation, congenital myopathy, and impaired mental and motor coordination development and her family were studied. The coding region of the SBP2 gene was analyzed by sequencing, and gel shift assays were performed to address the in vitro binding properties of the mutant SBP2 protein. RESULTS Serum levels of selenium and glutathione peroxidase in the proband were reduced, and selenoprotein P levels were undetectable. DNA sequencing of the SBP2 gene revealed a compound heterozygous mutation (R120X/R770X). The R120X mutation disrupted all functional motifs and the R770X inhibited the binding of SBP2 to Sec insertion sequence elements. Interestingly, selenium supplementation normalized serum selenium and glutathione peroxidase but not selenoprotein P levels and did not restore thyroid hormone metabolism dysfunction. CONCLUSIONS This distinctive phenotype can only be explained by the combined deficiency of functionally important selenoproteins and pinpoints the clinical relevance of selenoproteins and selenium economy in human development.

GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain.

Background: PPARγ agonists improve insulin sensitivity but also evoke weight gain. Results: GQ-16 is a PPARγ partial agonist that blocks receptor phosphorylation by Cdk5 and improves insulin sensitivity in diabetic mice in the absence of weight gain. Conclusion: The unique binding mode of GQ-16 appears to be responsible for the compounds advantageous pharmacological profile. Significance: Similar compounds could have promise as anti-diabetic therapeutics. The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compounds efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compounds weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby “ideal” PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.

Monitoring renal function: measured and estimated glomerular filtration rates - a review

Chronic kidney disease (CKD) is a world-wide public health problem, with adverse outcomes of kidney failure, cardiovascular disease, and premature death. This finding has led to the hypothesis that earlier recognition of kidney disease and successful intervention may improve outcome. The National Kidney Foundation, through its Kidney Disease Outcomes Quality Initiative (K/DOQI), and other National institutions recommend glomerular filtration rate (GFR) for the definition, classification, screening, and monitoring of CKD. Blood creatinine clearance, the most widely used clinical marker of kidney function, is now recognized as an unreliable measure of GFR because serum creatinine is affected by age, weight, muscle mass, race, various medications, and extra-glomerular elimination. Cystatin C concentration is a new and promising marker for kidney dysfunction in both native and transplanted kidneys. Because of its low molecular weight, cystatin C is freely filtered at the glomerulus and is almost completely reabsorbed and catabolized, but not secreted, by tubular cells. Given these characteristics, cystatin C concentration may be superior to creatinine concentration in detecting chronic kidney disease. This review aims to evaluate from recent literature the clinical efficiency and relevance of these GFR markers in terms of screening CKD.

Gaining ligand selectivity in thyroid hormone receptors via entropy.

Nuclear receptors are important targets for pharmaceuticals, but similarities between family members cause difficulties in obtaining highly selective compounds. Synthetic ligands that are selective for thyroid hormone (TH) receptor β (TRβ) vs. TRα reduce cholesterol and fat without effects on heart rate; thus, it is important to understand TRβ-selective binding. Binding of 3 selective ligands (GC-1, KB141, and GC-24) is characterized at the atomic level; preferential binding depends on a nonconserved residue (Asn-331β) in the TRβ ligand-binding cavity (LBC), and GC-24 gains extra selectivity from insertion of a bulky side group into an extension of the LBC that only opens up with this ligand. Here we report that the natural TH 3,5,3′-triodothyroacetic acid (Triac) exhibits a previously unrecognized mechanism of TRβ selectivity. TR x-ray structures reveal better fit of ligand with the TRα LBC. The TRβ LBC, however, expands relative to TRα in the presence of Triac (549 Å3 vs. 461 Å3), and molecular dynamics simulations reveal that water occupies the extra space. Increased solvation compensates for weaker interactions of ligand with TRβ and permits greater flexibility of the Triac carboxylate group in TRβ than in TRα. We propose that this effect results in lower entropic restraint and decreases free energy of interactions between Triac and TRβ, explaining subtype-selective binding. Similar effects could potentially be exploited in nuclear receptor drug design.

Synthesis and anti-inflammatory activity of new arylidene-thiazolidine-2,4-diones as PPARγ ligands

Eight new 5-arylidene-3-benzyl-thiazolidine-2,4-diones with halide groups on their benzyl rings were synthesized and assayed in vivo to investigate their anti-inflammatory activities. These compounds showed considerable biological efficacy when compared to rosiglitazone, a potent and well-known agonist of PPARgamma, which was used as a reference drug. This suggests that the substituted 5-arylidene and 3-benzylidene groups play important roles in the anti-inflammatory properties of this class of compounds. Docking studies with these compounds indicated that they exhibit specific interactions with key residues located in the site of the PPARgamma structure, which corroborates the hypothesis that these molecules are potential ligands of PPARgamma. In addition, competition binding assays showed that four of these compounds bound directly to the ligand-binding domain of PPARgamma, with reduced affinity when compared to rosiglitazone. An important trend was observed between the docking scores and the anti-inflammatory activities of this set of molecules. The analysis of the docking results, which takes into account the hydrophilic and hydrophobic interactions between the ligands and the target, explained why the 3-(2-bromo-benzyl)-5-(4-methanesulfonyl-benzylidene)-thiazolidine-2,4-dione compound had the best activity and the best docking score. Almost all of the stronger hydrophilic interactions occurred between the substituted 5-arylidene group of this compound and the residues of the binding site.

Negative Regulation of Superoxide Dismutase-1 Promoter by Thyroid Hormone

The role of thyroid hormone [l-3,5,3′-triiodothyronine (T3)] and the thyroid hormone receptor (TR) in regulating growth, development, and metabolic homeostasis is well established. It is also emerging that T3 is associated with oxidative stress through the regulation of the activity of superoxide dismutase-1 (SOD-1), a key enzyme in the metabolism of oxygen free radicals. We found that T3 reverses the activation of the SOD-1 promoter caused by the free radical generators paraquat and phorbol 12-myristate 13-acetate through the direct repression of the SOD-1 promoter by liganded TR. Conversely, the SOD-1 promoter is significantly stimulated by unliganded TRs. This regulation requires the DNA-binding domain of the TR, which is recruited to an inhibitory element between -157 and +17 of the SOD-1 promoter. TR mutations, which abolish recruitment of coactivator proteins, block repression of the SOD-1 promoter. Conversely, a mutation that inhibits corepressor binding to the TR prevents activation. Together, our findings suggest a mechanism of negative regulation in which TR binds to the SOD-1 promoter but coactivator and corepressor binding surfaces have an inverted function. This effect may be important in T3 induction of oxidative stress in thyroid hormone excess.

Claudin-16 Deficiency Impairs Tight Junction Function in Ameloblasts, Leading to Abnormal Enamel Formation.

Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients.

Human thyroid receptor forms tetramers in solution, which dissociate into dimers upon ligand binding

Thyroid hormone nuclear receptors (TRs) bind to DNA and activate transcription as heterodimers with the retinoid X receptor (RXR) or as homodimers or monomers. RXR also binds to DNA and activates transcription as homodimers but can, in addition, self-associate into homotetramers in the absence of ligand and DNA templates. It is thought that homotetramer formation serves to sequester excess RXRs into an inactive pool within the cell. Here, we report systematic studies of the multimeric state of a recombinant human TRβ1 truncation (hTRβ1ΔAB) that encompasses the complete DNA binding domain and ligand binding domain in solution. Native gel electrophoresis, chemical crosslinking, gel filtration, and dynamic light scattering experiments reveal that hTRβ1ΔAB forms a mixture of monomers, dimers, and tetramers. Like RXR, increasing protein concentration shifts the equilibrium between TR multimers toward tetramer formation, whereas binding of cognate thyroid hormone leads to dissociation of tetramers and increased formation of dimers. This work represents the first evidence that apo-hTRβ1 forms homotetramers. The findings raise the possibility that tetramer formation provides an additional, and previously unsuspected, level of control of TR activity and that the capacity for homotetramer formation may be more widespread in the nuclear receptor family than previously thought.

Mechanisms of peroxisome proliferator activated receptor γ regulation by non-steroidal anti-inflammatory drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) display anti-inflammatory, antipyretic and analgesic properties by inhibiting cyclooxygenases and blocking prostaglandin production. Previous studies, however, suggested that some NSAIDs also modulate peroxisome proliferator activated receptors (PPARs), raising the possibility that such off target effects contribute to the spectrum of clinically relevant NSAID actions. In this study, we set out to understand how peroxisome proliferator activated receptor-γ (PPARγ/PPARG) interacts with NSAIDs using X-ray crystallography and to relate ligand binding modes to effects on receptor activity. We find that several NSAIDs (sulindac sulfide, diclofenac, indomethacin and ibuprofen) bind PPARγ and modulate PPARγ activity at pharmacologically relevant concentrations. Diclofenac acts as a partial agonist and binds to the PPARγ ligand binding pocket (LBP) in typical partial agonist mode, near the β-sheets and helix 3. By contrast, two copies of indomethacin and sulindac sulfide bind the LBP and, in aggregate, these ligands engage in LBP contacts that resemble agonists. Accordingly, both compounds, and ibuprofen, act as strong partial agonists. Assessment of NSAID activities in PPARγ-dependent 3T3-L1 cells reveals that NSAIDs display adipogenic activities and exclusively regulate PPARγ-dependent target genes in a manner that is consistent with their observed binding modes. Further, PPARγ knockdown eliminates indomethacin activities at selected endogenous genes, confirming receptor-dependence of observed effects. We propose that it is important to consider how individual NSAIDs interact with PPARγ to understand their activities, and that it will be interesting to determine whether high dose NSAID therapies result in PPAR activation.