Novel triazines as potent and selective phosphodiesterase 10A inhibitors

The identification of highly potent and orally active triazines for the inhibition of PDE10A is reported. The new analogs exhibit low-nanomolar potency for PDE10A, demonstrate high selectivity against all other members of the PDE family, and show desired drug-like properties. Employing structure-based drug design approaches, we investigated the selectivity of PDE10A inhibitors against other known PDE isoforms, by methodically exploring the various sub-regions of the PDE10A ligand binding pocket. A systematic assessment of the ADME and pharmacokinetic properties of the newly synthesized compounds has led to the design of drug-like candidates with good brain permeability and desirable drug kinetics (t(1/2), bioavailability, clearance). Compound 66 was highly potent for PDE10A (IC(50)=1.4nM), demonstrated high selectivity (>200×) for the other PDEs, and was efficacious in animal models of psychoses; reversal of MK-801 induced hyperactivity (MED=0.1mg/kg) and conditioned avoidance responding (CAR; ID(50)=0.2mg/kg).     

Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model

Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.     

In vitro interaction between ceruloplasmin and human serum transferrin

The thermodynamics of the interactions of serum apotransferrin (T) and holotransferrin (TFe(2)) with ceruloplasmin (Cp), as well as those of human lactoferrin (Lf), were assessed by fluorescence emission spectroscopy. Cp interacts with two Lf molecules. The first interaction depends on pH and μ, whereas the second does not. Dissociation constants were as follows: K(11Lf) = 1.5 ± 0.2 μM, and K(12Lf) = 11 ± 2 μM. Two slightly different interactions of T or TFe(2) with Cp are detected for the first time. They are both independent of pH and μ and occur with 1:1 stoichiometry: K(1T) = 19 ± 7 μM, and K(1TFe2) = 12 ± 4 μM. These results can improve our understanding of the probable process of the transfer of iron from Cp to T in iron and copper transport and homeostasis.     

Biointeraction analysis by high-performance affinity chromatography: Kinetic studies of immobilized antibodies

A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody–antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7–12 × 10⁶ M⁻¹ at pH 7.0 and 25 °C. Split-peak analysis gave association rate constants of 1.4–12 × 10⁵ M⁻¹ s⁻¹ and calculated dissociation rate constants of 0.01–0.4 s⁻¹ under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056–0.17 s⁻¹. A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4 × 10⁻⁴ s⁻¹. This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports.     

Chromatographic analysis of acetohexamide binding to glycated human serum albumin

Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide–HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K_a) for the high affinity interactions was in the range of 1.2–2.0 × 10⁵ M⁻¹ and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K_a for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K_a for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.