Richard L. Tolman

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.

Immunogenicity of synthetic HIV-1 gp120 V3-loop peptide-conjugate immunogens.

A successful prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine must elicit an immune response that will prevent establishment of the persistent viral infection. The only response shown to be effective in this regard is virus-neutralizing antibody directed against the viral gp120 hypervariable V3-loop region. Conjugate immunogens, containing cyclic peptides representing the V3 determinant covalently bound to a carrier protein, were capable of eliciting virus-neutralizing antibodies. The consistency of the response was related to peptide size. The smaller cyclic peptides, expressing relatively conserved sequences from the V3-loop apex, were poor inducers of neutralizing activity. In contrast, the largest cyclic peptides mediated neutralizing responses that were similar to those observed and previously reported for intact gp120 immunogens. A cyclic synthetic peptide expressing most of the prototypic HIV-1 MN variant V3 determinant warrants further study as a potentially effective vaccine immunogen.

Synthesis of the chiral acyclonucleoside antiherpetic agent (S)-9-(2,3-dihydroxy-1-propoxymethyl)guanine

Abstract A new synthesis of the potential antiviral agent, (S)-9-(2,3-dihydroxy-1-propoxymethyl)guanine is described, starting from the readily available methyl 2,3,4-tri- O -benzyl-α-(=D)-glucopyranoside. The sequence utilizes the absolute configuration defined by carbons 4, 5 and 6 of the (=D)-glucose ring and provides a ready synthesis of the single enantiomer without recourse to many chromatographic separations.

5-Aryl thiazolidine-2,4-diones as selective PPARγ agonists

A series of 5-aryl thiazolidine-2,4-diones containing 4-phenoxyphenyl side chains was designed, synthesized, and evaluated for PPAR agonist activities. One such compound 28 exhibited comparable levels of glucose correction to rosiglitazone in the db/db mouse type 2 diabetes animal model.

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.

MK386 : a potent, selective inhibitor of the human type 1 5α-reductase

Abstract Steroid 5α-reductase is required for the conversion of testosterone to dihydrotestosterone. Localization of type 1 5α-reductase in the sebaceous gland of skin offers the possibility for selective inhibition of this isozyme as a treatment for acne. The goals of these studies are to demonstrate the mechanism of inhibition of MK386 and its selectivity for type 1 5α-reductase. The apparent potency of MK386 differed depending on the source of the enzyme (i.e. recombinant vs. native), yet selectivity for type 1 5α-reductase was unchanged. Our results indicate that the apparent potency of MK386 is modulated by the membrane concentration of the assay. These results suggest that MK386 has a high affinity for the lipid-rich membrane environment of 5α-reductase. MK386 was also found to be a slow binding inhibitor of type 1 5α-reductase. However, the cause of this time-dependent inhibition is unrelated to partitioning of the inhibitor into the membrane because similar studies with type 2 5α-reductase indicate that MK386 is a reversible, competitive inhibitor. A number of counterscreens were developed to demonstrate that MK386 is a poor inhibitor of other steroid metabolizing enzymes.

The synthesis of certain derivatives of 2-fluoro-D-ribose, 2-deoxy-2-fluoro-D-ribose, and 2-deoxy-2-fluoro-D-arabinose

Abstract Addition or trifluoromethyl hypofluorite to 3,4-di- O -acetyl- D -arabinal ( 1 ) gave three crystalline products. The reaction products (one F,OCF 3 adduct and two F,F adducts) were determined, by spectroscopic and chemical methods, to be trifluoromethyl 3,4-di- O -acetyl-2-deoxy-2-fluoro-β- D -arabinopyranoside ( 2 ), 3,4-di- O -acetyl-2-deoxy-2-fluoro-β- D -arabinopyranosyl fluoride ( 3 ), and 3,4-di- O -acetyl-2-deoxy-2-fluoro-α- D -ribopyranosyl fluoride ( 4 ). Acid hydrolysis of 2 and 3 furnished 2-deoxy-2-fluoro- D -arabinose ( 5 ); the reaction sequence is particularly well-suited for large-scale preparation of this fluoro sugar. Similarly, hydrolysis of 4 gave 2-deoxy-2-fluoro- D -ribose ( 6 ). Crystalline 2,3,4-tri- O -acetyl-1,5-anhydro- D - erythro -pent-1-enitol ( 8 ) was prepared from tri- O -acetyl-β- D -arabinopyranosyl bromide; it gave two adducts on treatment with trifluoromethyl hypofluorite in the presence of calcium oxide; the structures assigned to them, namely, trifluoromethyl 2,3,4-tri- O -acetyl-2-fluoro-β- D -ribopyranoside ( 9 ) and 2,3,4-tri- O -acetyl-2-fluoro-β- D -ribopyranosyl fluoride ( 10 ), were confirmed by 90-MHz n.m.r. spectral studies and spin-decoupling experiments.

Syntheses of all four possible diastereomers of the acyclonucleoside 9-(1,3,4-trihydroxy-2-butoxymethyl)guanine from carbohydrate precursors

Abstract The syntheses of all four possible diastereomers of 9-(1,3,4-trihydroxy-2-butoxymethyl)guanine, starting from D- and L-xylose and from D- and L-arabinose derivatives are described.

Inhibition of RNA Virus Replication in Vitro by 3-Deazacytidine and 3-Deazauridine

Summary Investigations conducted on in vitro antiviral activity of 3-deazacytidine and 3-deazauridine indicated that these nucleosides possess significant antiviral activity against rhino 1A, 13 and 56; influenza A and B; parainfluenza 1 (Sendai) and vesicular stomatis viruses. 3-Deazacytidine was found to possess superior antiviral activity against influenza B and Sendai viruses. Preliminary studies performed on the mode of antiviral action showed that the analogs did not have any virucidal property, and exerted their inhibitory action during the viral infective process. The antiviral activity of the two 3-deazapyrimidine nucleosides could be reversed by the corresponding naturally occurring pyrimidine nucleosides.