Gregory D. Berger

NOVEL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) GAMMA AND PPARDELTA LIGANDS PRODUCE DISTINCT BIOLOGICAL EFFECTS

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARα, PPARδ, and PPARγ. PPARγ has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARγ and PPARδ that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARγ and PPARδ directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARγ agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabeticdb/db mice all PPARγ agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selectivein vivo activation of PPARδ did not significantly affect these parameters. In vivo PPARα activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARγ and PPARδ; 2) ligand-dependent activation of PPARδ involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARγ activation (but not PPARδ or PPARα activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARγ agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARα activation is sufficient to affect triglyceride metabolism, PPARδ activation does not appear to modulate glucose or triglyceride levels.

Activation of PPARδ alters lipid metabolism in db/db mice

Peroxisome proliferator‐activated receptors (PPARs) are nuclear receptors, which heterodimerize with the retinoid X receptor and bind to peroxisome proliferator response elements in the promoters of regulated genes. Despite the wealth of information available on the function of PPARα and PPARγ, relatively little is known about the most widely expressed PPAR subtype, PPARδ. Here we show that treatment of insulin resistant db/db mice with the PPARδ agonist L‐165 041, at doses that had no effect on either glucose or triglycerides, raised total plasma cholesterol concentrations. The increased cholesterol was primarily associated with high density lipoprotein (HDL) particles, as shown by fast protein liquid chromatography analysis. These data were corroborated by the chemical analysis of the lipoproteins isolated by ultracentrifugation, demonstrating that treatment with L‐165 041 produced an increase in circulating HDL without major changes in very low or low density lipoproteins. White adipose tissue lipoprotein lipase activity was reduced following treatment with the PPARδ ligand, but was increased by a PPARγ agonist. These data suggest both that PPARδ is involved in the regulation of cholesterol metabolism in db/db mice and that PPARδ ligands could potentially have therapeutic value.

A novel synthetic approach toward the zaragozic acids core structure

Abstract A novel synthetic approach toward the zaragozic acids core structure is described. New dipolarophiles in the 1,3-dipolar cycloaddition to the carbonyl ylides are reported. The substituent effect of the dipolarophiles is also discussed.

IFITM proteins are incorporated onto HIV-1 virion particles and negatively imprint their infectivity

BackgroundInterferon induced transmembrane proteins 1, 2 and 3 (IFITMs) belong to a family of highly related antiviral factors that have been shown to interfere with a large spectrum of viruses including Filoviruses, Coronaviruses, Influenza virus, Dengue virus and HIV-1. In all these cases, the reported mechanism of antiviral inhibition indicates that the pool of IFITM proteins present in target cells blocks incoming viral particles in endosomal vesicles where they are subsequently degraded.ResultsIn this study, we describe an additional mechanism through which IFITMs block HIV-1. In virus-producing cells, IFITMs coalesce with forming virions and are incorporated into viral particles. Expression of IFITMs during virion assembly leads to the production of virion particles of decreased infectivity that are mostly affected during entry in target cells. This mechanism of inhibition is exerted against different retroviruses and does not seem to be dependent on the type of Envelope present on retroviral particles.ConclusionsThe results described here identify a novel mechanism through which IFITMs affect HIV-1 infectivity during the late phases of the viral life cycle. Put in the context of data obtained by other laboratories, these results indicate that IFITMs can target HIV at two distinct moments of its life cycle, in target cells as well as in virus-producing cells. These results raise the possibility that IFITMs could similarly affect distinct steps of the life cycle of a number of other viruses.

The asymmetric syntheses of the C-1 sidechains of zaragozic acid A and zaragozic acid C

Abstract The asymmetric syntheses of the C-1 sidechains of zaragozic acid A and C are described. Aldol reaction defines the chirality at C-4′and C-5′in two independent routes. Multigram preparation as well as a route amenable to derivatization are highlights of these approaches.

Functional Analysis of the Relationship between Vpx and the Restriction Factor SAMHD1

Background: SAMHD1 is a novel antiviral factor counteracted by the viral protein Vpx Results:Several residues in Vpx affect its ability to increase infection and degrade SAMHD1. Conclusion:Vpx functionality correlates with SAMHD1 degradation, but not with Vpx stability and Vpx-Vpx association. Significance:Several mutants provide further insights into the molecular mechanism of Vpx-induced protection. SAMHD1 is a newly identified restriction factor that targets lentiviruses in myeloid cells and is countered by the SIVSM/HIV-2 Vpx protein. By analyzing a large panel of Vpx mutants, we identify several residues throughout the 3-helix bundle predicted for Vpx that impair both its functionality and its ability to degrade SAMHD1. We determine that SAMHD1 is a strictly non-shuttling nuclear protein and that as expected WT Vpx localizes with it in the nucleus. However, we also identify a functional Vpx mutant with predominant cytoplasmic distribution that colocalizes with SAMHD1 in this location, suggesting that Vpx may also retain SAMHD1 in the cell cytoplasm, prior to its entry into the nucleus. Several mutations in Vpx were shown to affect the stability of Vpx, as well as Vpx:Vpx interactions. However, no strict correlation was observed between these parameters and the functionality of Vpx, implying that neither properties is absolutely required for this function and indicating that even unstable Vpx mutants may be very efficient in inducing SAMHD1 degradation. Overall, our analysis identifies several Vpx residues required for SAMHD1 degradation and points to a very efficient and plastic mechanism through which Vpx depletes this restriction factor.

Phenylacetic acid derivatives as hPPAR agonists.

Beginning with the weakly active lead structure 1, a new series of hPPAR agonists was developed. In vivo glucose and triglyceride lowering activity was obtained by homologation and oxamination to 3, then conversion to substituted benzisoxazoles 4 and 5. Further manipulation afforded benzofurans 6 and 7. Compound 7 was of comparable potency as a glucose and triglyceride lowering agent in insulin resistant rodents to BRL 49653.

The synthesis of C3-methyl, C3-decarboxy-zaragozic acid A : a potent squalene synthase inhibitor

Abstract The title compound and its C4-pivaloyloxy methyl (POM) ester have been synthesized by converting zaragozic acid A to the key intermediate of the protected C3-hydroxymethyl compound 6 . Subsequent radical deoxygenation via the Barton-McCombie procedure, followed by deprotection, afforded the product 11 (L-703,370). This compound possesses squalene biosynthesis inhibitory potency of 22% at 24 mpk and its C4-POM ester, 12 (L-735,142), exhibits an ED 50 of 1.6 mpk in our oral mouse assay.

Chemoselective removal and replacement of the C-4′ and C-6 acyl esters of zaragozic Acid A

Abstract An efficient chemical sequence is described for the modification of the C-4′ and C-6 positions of zaragozic acid A. Key reactions include the specific conditions devised for selective removal of either the C-4′ acetate or the C-6 acyl chain. The resultant hydroxy intermediates were then derivatized as esters, ethers, carbamates, and carbonates.

Diesters of zaragozic acid A: Synthesis and biological activity

Abstract Diesters of zaragozic acid A were synthesized and tested in rat liver squalene synthase and oral mouse cholesterol biosynthesis assays. Of these, 3-isopentyl-4-pivaloyloxymethyl and 3-isopentyl-4-acetoxymethyl esters gave ED50s of 9 mg/kg and 6 mg/kg, respectively, in the oral mouse assay.