Steven G. Blanchard

Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation.

A Unique PPARγ Ligand with Potent Insulin-Sensitizing yet Weak Adipogenic Activity

FMOC-L-Leucine (F-L-Leu) is a chemically distinct PPARgamma ligand. Two molecules of F-L-Leu bind to the ligand binding domain of a single PPARgamma molecule, making its mode of receptor interaction distinct from that of other nuclear receptor ligands. F-L-Leu induces a particular allosteric configuration of PPARgamma, resulting in differential cofactor recruitment and translating in distinct pharmacological properties. F-L-Leu activates PPARgamma with a lower potency, but a similar maximal efficacy, than rosiglitazone. The particular PPARgamma configuration induced by F-L-Leu leads to a modified pattern of target gene activation. F-L-Leu improves insulin sensitivity in normal, diet-induced glucose-intolerant, and in diabetic db/db mice, yet it has a lower adipogenic activity. These biological effects suggest that F-L-Leu is a selective PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.

HIV protease inhibitors block adipogenesis and increase lipolysis in vitro

AIDS therapies employing HIV protease inhibitors (PIs) are associated with changes in fat metabolism. However, the cellular mechanisms affected by PIs are not clear. Thus, the affects of PIs on adipocyte differentiation were examined in vitro using C3H10T1/2 stem cells. In these cells the PIs, nelfinavir, saquinavir, and ritonavir, reduced triglyceride accumulation, lipogenesis, and expression of the adipose markers, aP2 and LPL. Histological analysis revealed nelfinavir, saquinavir and ritonavir treatment decreased oil red O-staining of cytoplasmic fat droplets. Inhibition occurred in the presence of the RXR agonist LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Moreover, these three PIs increased acute lipolysis in adipocytes. In contrast, two HIV PIs, amprenavir and indinavir, had little effect on lipolysis, lipogenesis, or expression of aP2 and LPL. Although, saquinavir, inhibited ligand-binding to PPARgamma with an IC(50) of 12.7+/-3.2 microM, none of the other PIs bound to the nuclear receptors RXRalpha or PPARgamma, (IC(50)s>20 microM), suggesting that inhibition of adipogenesis is not due to antagonism of ligand binding to RXRalpha or PPARgamma. Taken together, the results suggest that some, but not all, PIs block adipogenesis and stimulate fat catabolism in vitro and this may contribute to the effects of PIs on metabolism in the clinic.

Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library

BACKGROUND The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown. RESULTS A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.

Conjugated linoleic acid is an activator and ligand for peroxisome proliferator-activated receptor-gamma (PPARγ)

Abstract The goal of the present study was to elucidate the extent that CLA binds to and activates PPARγ. All of the isomers of CLA tested activated PPARγ in a manner similar to the polyunsaturated fatty acid, linoleic acid (18:2n6). When the binding affinity of CLA to PPARγ was determined using a scintillation proximity assay (SPA), isomers of CLA were ligands for PPARγ with micromolar affinity. To determine the extent that metabolism of CLA via Δ6 desaturase could alter activation of PPARγ, we used a Δ6 desaturase inhibitor, SC-26,196, to block Δ6 desaturase metabolism. Blocking Δ6 desaturase significantly reduced activation of PPARγ by c9t11-CLA. These data suggest that the ability of CLA to induce PPAR-responsive genes may be via both direct binding of CLA to the nuclear hormone receptor, PPARγ, as well as active metabolites of CLA via Δ6 desaturase. Further work is needed to determine the ability of Δ6 desaturase metabolites of CLA to bind and activate PPARγ.

Differential activity of rosiglitazone enantiomers at PPARγ

Anal. of the enantiomers of rosiglitazone in a PPARγ binding assay suggests that the (S)-(-)- isomer is responsible for the antidiabetic activity.

Synthesis and biological activity of a novel series of indole-derived PPARγ agonists

Abstract The synthesis and structure-activity relationships of a novel series of indole 5-carboxylic acids that bind and activate peroxisome proliferator-activated receptor gamma (PPARγ) are reported. These new analogs are selective for PPARγ vs the other PPAR subtypes, and the most potent compounds in this series are comparable to in vitro potencies at PPARγ reported for the thiazolidinedione-based antidiabetic drugs currently in clinical use. The synthesis and structure-activity relationships of a novel series of indole 5-carboxylic acids that bind and activate peroxisome proliferator-activated receptor gamma (PPARγ) are reported. These compounds are selective for PPARγ vs the other PPAR subtypes, and the most potent compounds have in vitro potencies at PPARγ comparable to those reported for the thiazolidinedione-based antidiabetic drugs currently in clinical use.

Early postnatal development of calmodulin-dependent protein kinase II in rat brain

The postnatal levels of the alpha- (Mr 50,000) and beta- (Mr 58,000/60,000) subunits of type II calmodulin-dependent protein kinase were investigated in cytosolic, cytoskeletal and nuclear subfractions of rat brain. Substantial amounts of the beta-subunit were present neonatally, especially in the cytoplasm; both subunits increased markedly during the subsequent three weeks with alpha-preponderance in the cortex and midbrain. The development of the alpha-subunit was attenuated in the cerebellum and brain stem, resulting in a relative excess of the beta-subunit. Primary neuronal cultures from fetal brain initially displayed low enzyme content, followed by an increase in the levels of the beta- but not alpha-subunit. Regulation of the cellular content of type II calmodulin-dependent protein kinase may be relevant to neuronal differentiation.

Iterative size-exclusion chromatography coupled with liquid chromatographic mass spectrometry to enrich and identify tight-binding ligands from complex mixtures

Abstract A method is described for the enrichment of tight-binding ligands from complex mixtures. The mixture is equilibrated with a protein or receptor, and protein: ligand complexes are separated from unbound ligands by size-exclusion. If the resulting mixture is allowed to re-establish its equilibrium and then passed again through the size-exclusion column, significant enrichment of tight-binding ligands is realized, even in cases where the weak-binders are present in large excess. Ligands are identified by liquid chromatography and mass spectrometry following the size-exclusion step. The process can be repeated multiple times to further enrich ligands. Analysis of pools of ligands generated by combinatorial chemistry is an obvious application of the technique.

Discovery of Bioavailable Inhibitors of Secretory Phospholipase A2

Substrate-mimetic inhibitors of sPLA2 with submicromolar in vitro potency were discovered by use of a novel dual substrate screening strategy. In vivo evaluation of selected inhibitors in the rat carrageenan paw edema model of inflammation, however, indicated that in vitro potency was not a good predictor of in vivo activity. Studies of the metabolic stability of early examples of these inhibitors suggested that the metabolic lability of these compounds was a major contributing factor to the observed weak in vivo activity. In an attempt to achieve improved in vivo activity, we prepared and tested compounds designed to overcome the observed metabolic instability. The design of the new compounds involved two types of changes in the inhibitor molecules. First, the C-2 ester moiety was replaced with an amide function so that direct cleavage by stomach acid and blood esterases at this site was minimized. Second, omega-oxidation of the decanamide moiety was eliminated by substitution of hydrogen with fluorine in this position. Compounds containing fluorine in the terminal positions of the alkyl chain retained sPLA2 inhibitory activity and also possessed improved in vitro metabolic stability and pharmacokinetic parameters relative to nonfluorinated inhibitors in this series. As exemplified by GW 4776, improvements in metabolic stability alone, however, were not sufficient to ensure oral activity. Thus, GW 4776 did not show oral activity in the carrageenan edema model and had only modest activity after i.v. dosing in the same model. In fact, the results for GW 9624 and GW 8219 suggested that factors in addition to potency of sPLA2 inhibition and metabolism affect the observed in vivo activity. Despite the fact that these two compounds varied only by a single oxygen-to-sulfur substitution, one was active whereas the other was not. One possible explanation for the observed variability is a compound-dependent difference in the rate of equilibration into tissue. This possibility is relevant as both the carrageenan paw edema model and the phorbol ester edema model involve a localized inflammation. No measurements were made to assess differences in the distribution of the different inhibitors between the blood and the localized site of inflammation. In summary, a series of bioavailable inhibitors of sPLA2 was prepared using an iterative approach that combined medicinal chemistry, in vitro and in vivo evaluation of biological activity, and metabolic and pharmacokinetic studies. Although some compounds in the series showed in vivo activity, the anti-inflammatory effect observed in animal models was modest and a decision was made to abandon sPLA2 as a molecular target for the development of anti-inflammatory agents.