Discovery of non-covalent dipeptidyl peptidase IV inhibitors which induce a conformational change in the active site

A series of non-covalent inhibitors of the serine protease dipeptidyl peptidase IV (DPP-IV) were found to adopt a U-shaped binding conformation in X-ray co-crystallization studies. Remarkably, Tyr547 undergoes a 70 degrees side-chain rotation to accommodate the inhibitor and allows access to a previously unexposed area of the protein backbone for hydrogen bonding.     

Synthesis and biological evaluation of a new type of 99mtechnetium-labeled Fatty Acid for myocardial metabolism imaging

Technetium-labeled fatty acids intended for myocardial metabolism imaging and the respective rhenium model complexes were synthesized according to the "4 + 1" mixed-ligand approach and investigated in vitro and in vivo. The non-radioactive rhenium model complexes were characterized by NMR, IR, and EA, and the geometrical impact of the chelate unit on the integrity of the fatty acid head structure was determined by single-crystal X-ray analyses. To estimate the diagnostic value of the 99mTc-labeled fatty acids, the compounds were investigated in experiments in vitro and in biodistribution studies using male Wistar rats. The new fatty acid tracers contain the metal core in the oxidation states +3, well-wrapped in a trigonal-bipyramidal coordination moiety, which is attached at the omega-position of a fatty acid chain. This structural feature is considered to be a good imitation of the well-established iodinated phenyl fatty acids. High heart extraction in perfused heart studies (up to 26% injected dose (ID)) and noticeable heart uptake of the 99mTc tracers in vivo being in the order of 2% ID/g at 5 min (postinjection, pi.), accompanied by a good heart to blood ratio of 8, confirms that the new Tc compounds are suitable as fatty acid tracers.     

Magnetic Resonance Imaging of Soft Tissue Infection with Iron Oxide Labeled Granulocytes in a Rat Model

Object

We sought to detect an acute soft tissue infection in rats by magnetic resonance imaging (MRI) using granulocytes, previously labeled with superparamagnetic particles of iron oxide (SPIO).

Materials and Methods

Parasternal infection was induced by subcutaneous inoculation of Staphylococcus aureus suspension in rats. Granulocytes isolated from isogenic donor rats were labeled with SPIO. Infected rats were imaged by MRI before, 6 and 12 hours after intravenous injection of SPIO-labeled or unlabeled granulocytes. MR findings were correlated with histological analysis by Prussian blue staining and with re-isolated SPIO-labeled granulocytes from the infectious area by magnetic cell separation.

Results

Susceptibility effects were present in infected sites on post-contrast T2*-weighted MR images in all animals of the experimental group. Regions of decreased signal intensity (SI) in MRI were detected at 6 hours after granulocyte administration and were more pronounced at 12 hours. SPIO-labeled granulocytes were identified by Prussian blue staining in the infected tissue and could be successfully re-isolated from the infected area by magnetic cell separation.

Conclusion

The application of SPIO-labeled granulocytes in MRI offers new perspectives in diagnostic specificity and sensitifity to detect early infectious processes.     

Mechanisms for abnormal postprandial glucose metabolism in type 2 diabetes

To assess mechanisms for postprandial hyperglycemia, we used a triple-isotope technique ([\3-(3)H]glucose and [(14)C]bicarbonate and oral [6,6-dideutero]glucose iv) and indirect calorimetry to compare components of glucose release and pathways for glucose disposal in 26 subjects with type 2 diabetes and 15 age-, weight-, and sex-matched normal volunteers after a standard meal. The results were as follows: 1) diabetic subjects had greater postprandial glucose release (P<0.001) because of both increased endogenous and meal-glucose release; 2) the greater endogenous glucose release (P<0.001) was due to increased gluconeogenesis (P<0.001) and glycogenolysis (P=0.01); 3) overall tissue glucose uptake, glycolysis, and storage were comparable in both groups (P>0.3); 4) glucose clearance (P<0.001) and oxidation (P=0.004) were reduced, whereas nonoxidative glycolysis was increased (P=0.04); and 5) net splanchnic glucose storage was reduced by approximately 45% (P=0.008) because of increased glycogen cycling (P=0.03). Thus in type 2 diabetes, postprandial hyperglycemia is primarily due to increased glucose release; hyperglycemia overcomes the effects of impaired insulin secretion and sensitivity on glucose transport, but intracellular defects persist so that pathways of glucose metabolism are abnormal and glucose is shunted away from normal sites of storage (e.g., liver and muscle) into other tissues.     

Preparation and biological characterization of isomeric 188Re(V) oxocomplexes with tetradentate S4 ligands derived from meso-dimercaptosuccinic acid for labeling of biomolecules

A new type of tetradentate S4 ligand has been synthesized by bridging two molecules of meso-2,3-dimercaptosuccinic acid for stable binding and easy conjugation of rhenium-188 to tumor targeting structures. The stereoisomeric tetrathiolato S4 ligands form very robust anionic five-coordinated oxorhenium(V) and oxotechnetium(V) complexes. Two routes for the preparation of the (188)Re(V) oxocomplexes with (iBu)2N(O)C-C(SH)C(SH)C(O)NH(CH2)3NH(CH2)3NHC(O)C(SH)C(SH)C(O)N(iBu)2 (ligand 1) and its hydrophilic crown ether derivative (ligand 2) were tested and optimized. Several isomers were separated by HPLC from the preparation solutions and characterized in vitro and in vivo. The identity of the species obtained was determined by comparison with the HPLC profiles of reference (185/187)Re analogues and (99/99m)Tc complexes which were characterized by ESI-MS. All of them were absolutely stable in rat and human plasma solutions. Challenge experiments with cysteine corroborated the high inertness of the isomers toward ligand exchange reactions. Various in vivo samples, taken off at different times from blood, intestine, and urine of rats, confirmed the high in vivo stability of the (188)Re-S4 complexes. Biodistribution studies using male Wistar rats were performed and exhibited a high uptake and fast clearance from the liver of the more lipophilic cis and trans isomers of complex I (log P(o/w) between 1.5 and 1.7), whereas the isomers of the hydrophilic complex II (log P(o/w) about -1.75) were rapidly excreted via the renal and the hepatobiliary pathway. The low level of activity in the stomach confirms good in vivo stability. Thus, these new (188)Re-S4 complexes fulfill the requirements for a stable and high specific activity labeling of biomolecules with rhenium-188.     

Synthesis,characterization, and biological evaluation of technetium(III) complexes with tridentate/bidentate S,E,S/P,S coordination (E = O,N(CH(3)), S): a novel approach to robust technetium chelates suitable for linking the metal to biomolecules

A novel type of mixed-ligand Tc(III) complexes, [Tc(SCH(2)CH(2)-E-CH(2)CH(2)S)(PR(2)S)] (E = S, N(CH(3)); PR(2)S = phosphinothiolate with R = aryl, alkyl) is described. These "3+2"-coordinated complexes can be prepared in a two-step reduction/substitution procedure via the appropriate chloro-containing oxotechnetium(V) complex [TcO(SES)Cl] [E=S, N(CH(3)]. Tc(III) compounds have been fully characterized both in solid and solution states and found to adopt the trigonal-bipyramidal coordination geometry. The equatorial trigonal plane is formed by three thiolate sulfur atoms, whereas the phosphorus of the bidentate P,S ligand and the neutral donor of the tridentate chelator occupy the apical positions. The (99)Tc(III) complexes have been proven to be identical with the (99m)Tc agents prepared at the no-carrier-added level by comparison of the corresponding UV/vis and radiometric HPLC profiles. Challenge experiments with glutathione clearly indicate that this tripeptide has no effect on the stability of the (99m)Tc complexes in solutions. Biodistribution studies have been carried out in rats at 5 and 120 min postinjection. The substituents at the bidentate P,S ligand significantly influence the biodistribution pattern. Remarkable differences are observed especially in brain, blood, lungs, and liver. All the complexes are able to penetrate the blood-brain barrier of rats and showed a relatively fast washout from the brain.     

MyD88-dependent immune activation mediated by human immunodeficiency virus type 1-encoded Toll-like receptor ligands

Immune activation is a major characteristic of human immunodeficiency virus type 1 (HIV-1) infection and a strong prognostic factor for HIV-1 disease progression. The underlying mechanisms leading to immune activation in viremic HIV-1 infection, however, are not fully understood. Here we show that, following the initiation of highly active antiretroviral therapy, the immediate decline of immune activation is closely associated with the reduction of HIV-1 viremia, which suggests a direct contribution of HIV-1 itself to immune activation. To propose a mechanism, we demonstrate that the single-stranded RNA of HIV-1 encodes multiple uridine-rich Toll-like receptor 7/8 (TLR7/8) ligands that induce strong MyD88-dependent plasmacytoid dendritic cell and monocyte activation, as well as accessory cell-dependent T-cell activation. HIV-1-encoded TLR ligands may, therefore, directly contribute to the immune activation observed during viremic HIV-1 infection. These data provide an initial rationale for inhibiting the TLR pathway to directly reduce the chronic immune activation induced by HIV-1 and the associated immune pathogenesis.