Pharmacological characterization and antidiabetic activity of a long-acting glucagon-like peptide-1 analogue conjugated to an antithrombin III-binding pentasaccharide.

AIMS: To examine the biological characteristics of a novel glucagon-like peptide-1 (GLP-1) conjugate, in which an antithrombin III (ATIII)-binding pentasaccharide is conjugated to d-Ala(8) GLP-1 using a tetraethylene glycol linker. METHODS: We assessed GLP-1 receptor binding, cAMP generation and insulin secretory activity of the GLP-1 conjugate in vitro. Circulating half-life, glucose homeostatic and subchronic therapeutic effectiveness were then examined in vivo. RESULTS: The half-life of the GLP-1 conjugate in mice was ∼11 h. In vitro insulin secretion from clonal ß cells and islets was increased (p < 0.001) by the conjugate. The conjugate had half maximum effective concentration values of 1.3 × 10(-7) and 9.9 × 10(-8) M for displacement of (125) I-GLP-1 in competitive GLP-1 receptor binding and cAMP generation, respectively. Glucose tolerance in normal mice, immediately and 4 h after conjugate injection, resulted in significant (p < 0.001) improvements in blood glucose. These effects persisted for >48 h after administration. Daily treatment (21 days) of high-fat-fed and ob/ob mice with 25 nmol/kg conjugate resulted in significant improvement in glucose tolerance (p < 0.001) and reductions in glycated haemoglobin (HbA1c; p < 0.01) equivalent to or better than with exenatide or liraglutide. Treatment of C57BL/KsJ db/db mice for 15 days with 100 nmol/kg conjugate significantly (p < 0.001) reduced glucose and raised plasma insulin. Oral glucose tolerance was significantly (p < 0.001) improved and both 24-h glucose profile (p < 0.001) and HbA1c levels (p < 0.001) were reduced. Islet size (p < 0.001) and pancreatic insulin content were increased without change of islet cell proliferation or apoptosis. CONCLUSION: These data show that d-Ala(8) GLP-1(Lys(37) ) pentasaccharide exerts significant antidiabetic actions and has a projected pharmacokinetic/pharmacodynamic profile that merits further evaluation in humans for a possible once-weekly dosing regimen.

Conformational analysis of cyclophostin and designed analogs in comparison with the potent IP3 receptor agonist adenophostin A.

A conformational analysis of 5'-6"-tethered cyclophostin was carried out in comparison with the mother compound, adenophostin A, which has a potent IP3 receptor agonistic activity. The global minimum 3'-endo/anti conformation of cyclophostin elucidated by a molecular dynamics simulation was in accord with NMR spectroscopic data. In contrast, the 2'-endo/syn conformation was dominant with respect to adenophostin A. Despite the constraint introduced by the tether, the spatial arrangement of the three phosphate groups and the adenine moiety, which are essential for the extremely high potency, was changed only moderately in comparison with adenophostin A. The observed high potency of cyclophostin (EC50 = 38 nM) also indicates that it closely resembles the bioactive conformation of adenophostin A (EC50 = 7 nM). These results led us to estimate the probable active conformation of adenophostin A by comparison with the stable conformations of cyclophostin. Finally, two other tethered analogs were designed and are expected to exhibit high potencies comparable to adenophostin A.

Synthesis of potent agonists of the D-myo-inositol 1,4, 5-trisphosphate receptor based on clustered disaccharide polyphosphate analogues of adenophostin A.

Clustered disaccharide analogues of adenophostin A (2), i.e. mono-, di-, and tetravalent derivatives 6-8, respectively, were synthesized and evaluated as novel ligands for the tetrameric D-myo-inositol 1,4, 5-trisphosphate receptor (IP(3)R). The synthesis was accomplished via Sonogashira coupling of propargyl 2-O-acetyl-5-O-benzyl-3-O-(3, 4-di-O-acetyl-2, 6-di-O-benzyl-alpha-D-glucopyranosyl)-beta-D-ribofuranoside (16) with iodobenzene 18, 22, or 25, followed by deacetylation, phosphorylation, and deprotection. The abilities of the target compounds 6-8, as well as ribophostin 4, propylphostin 5, and IP(3) (1), to evoke Ca(2+) release from permeabilized hepatocytes or displacement of [(3)H]IP(3) from its receptor in hepatic membranes were compared. Although the binding affinities of 4-8 were similar, there were modest though significant differences in their potencies in Ca(2+) release assays: tetraphostin 8 > IP(3) approximately diphostin 7 > phenylphostin 6 > ribophostin 4 approximately propylphostin 5.

Spirophostins: conformationally restricted analogues of adenophostin A.

The synthesis, biological evaluation, and molecular modeling of two conformationally restricted analogues of adenophostinA (1), denominated as spirophostin (3R)-10 and (3S)-11, as novel ligands for the D-myo-inositol 1,4,5-trisphosphate receptor (IP3R), is presented. These diastereoisomeric spiroketals are synthesized by spiroketalization of D-glucose derivatives (2S)-15 and (2R)-16, separation of the protected isomers (3R)-19 and (3S)-20, followed by phosphorylation and deprotection. The spirophostins (3R)-10 and (3S)-11 display comparable biological activity, with a 3H-IP3-displacing and Ca2+-releasing potency less than IP3 and adenophostin A.

The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans.

DNA from Kinetoplastida contains the unusual modified base beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fasciculata and Leishmania tarentolae. We purified the 90 kDa C.fasciculata J-binding protein 50 000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and L.tarentolae. Recombinant proteins expressed in Escherichia coli demonstrated J-specific DNA binding. The J-binding proteins show 43-63% identity and are unlike any known protein. The discovery of a J-binding protein suggests that J, like methylated cytosine in higher eukaryotes, functions via a protein intermediate.

The modified DNA base beta-D-glucosylhydroxymethyluracil confers resistance to micrococcal nuclease and is incompletely recovered by 32P-postlabeling.

The hypermodified DNA base beta-D-glucosylhydroxymethyluracil, also called J, is a naturally occurring DNA modification. J was initially detected by 32P-postlabeling in Trypanosoma brucei and was recently also found in several other eukaryotic parasites. To use 32P-postlabeling as a method to quantitate the absolute levels of J in DNA we have tested the postlabeling efficiency of J using various synthesized standard oligonucleotides containing J. It is known that modified nucleotides, especially bulky ones, are often partially recovered by postlabeling and they are poor substrates for some of the enzymes used. We found that on average only 50% of J is recovered, which shows that the amount of J in T. brucei DNA has been twofold underestimated. Experiments with a short oligomer and defined pyrimidine tracts showed that the incomplete recovery of J is caused at least in part by resistance of J-containing DNA to degradation by micrococcal nuclease.

Quantitative pathogenicity of Marek's disease virus and vaccine virus strains in chickens: macroscopical aspects.

Two virulent strains (JM and K) and one vaccine strain (CVI 988) of Marek's disease virus (MDV), together with two vaccine strains of the herpesvirus of turkeys (HVT) (FC 126 and PB-THV 1), all in the cell-associated state, were administered intramuscularly at 3.7 log TCID50 per dose to day-old SPF White Leghorn chickens. A control group of chicks received uninfected cells. The pathological parameters studied were onset and duration of clinical symptoms, mortality, bird weight and macroscopical lesions of peripheral nerves and visceral organs. Data were obtained from females autopsied at the age of 3, 8 and 20 weeks, and from chickens which died. Virological and serological data were procured mainly from males taken at various ages. The results indicate a clear distinction between virulent and vaccine strains. MD vaccines had no significant influence on bird weight and caused no mortality or macroscopical lesions, whereas the virulent MDV strains produced all these effects. Macroscopical lesions caused by the virulent MDV strains were seen predominantly in nerves (in about 50% of birds succumbing to MD) and gonads (in 0% to 80% of such birds depending on sex and on strain of MDV). Differences between the two virulent strains could be demonstrated. Strain JM induced earlier incidence and shorter duration of clinical disease. With strain JM death occurred earlier in females than in males. Strain K caused significantly more macroscopical lesions in gonads, heart and liver. Under the conditions of the experiment, detection of macroscopical lesions after inoculation with a virulent MDV strain was possible 3 weeks after inoculation.