Human glucagon receptor antagonists with thiazole cores. A novel series with superior pharmacokinetic properties.

The aim of the work presented here was to design and synthesize potent human glucagon receptor antagonists with improved pharmacokinetic (PK) properties for development of pharmaceuticals for the treatment of type 2 diabetes. We describe the preparation of compounds with cyclic cores (5-aminothiazoles), their binding affinities for the human glucagon and GIP receptors, as well as affinities for rat, mouse, pig, dog, and monkey glucagon receptors. Generally, the compounds had slightly less glucagon receptor affinity compared to compounds of the previous series, but this was compensated for by much improved PK profiles in both rats and dogs with high oral bioavailabilities and sustained high plasma exposures. The compounds generally showed species selectivity for glucagon receptor binding with poor affinities for the rat, mouse, rabbit, and pig receptors. However, dog and monkey glucagon receptor affinities seem to reflect the human situation. One compound of this series, 18, was tested intravenously in an anesthetized glucagon-challenged monkey model of hyperglucagonaemia and hyperglycaemia and was shown dose-dependently to decrease glycaemia. Further, high plasma exposures and a long plasma half-life (5.2 h) were obtained.

Computational prediction of solubilizers' effect on partitioning.

A computational model for the prediction of solubilizers' effect on drug partitioning has been developed. Membrane/water partitioning was evaluated by means of immobilized artificial membrane (IAM) chromatography. Four solubilizers were used to alter the partitioning in the IAM column. Two types of molecular descriptors were calculated: 2D descriptors using the MOE software and 3D descriptors using the Volsurf software. Structure-property relationships between each of the two types of descriptors and partitioning were established using partial least squares, projection to latent structures (PLS) statistics. Statistically significant relationships between the molecular descriptors and the IAM data were identified. Based on the 2D descriptors structure-property relationships R(2)Y=0. 99 and Q(2)=0.82-0.83 were obtained for some of the solubilizers. The most important descriptor was related to logP. For the Volsurf 3D descriptors models with R(2)Y=0.53-0.64 and Q(2)=0.40-0.54 were obtained using five descriptors. The present study showed that it is possible to predict partitioning of substances in an artificial phospholipid membrane, with or without the use of solubilizers.

Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum.

BACKGROUND: The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64-100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64-100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits. RESULTS AND CONCLUSIONS: Serum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.

Differential interactions of nateglinide and repaglinide on the human beta-cell sulphonylurea receptor 1.

Repaglinide and nateglinide represent a new class of insulin secretagogues, structurally unrelated to sulphonylureas, that were developed for the treatment of type 2 diabetes. The inhibitory effect of these drugs was investigated on recombinant wild-type and mutant Kir6.2/SUR1 channels expressed in HEK293 cells. Nateglinide and repaglinide dose-dependently inhibited whole-cell Kir6.2/SUR1 currents with half-maximal inhibitory concentration (IC(50)) values of 800 and 21 nmol/l, respectively. Mutation of serine 1237 in SUR1 to tyrosine (S1237Y) abolished tolbutamide and nateglinide block, suggesting that these drugs share a common point of interaction on the SUR1 subunit of the ATP-sensitive K(+) channel. In contrast, repaglinide inhibition was unaffected by the S1237Y mutation (IC(50) = 23 nmol/l). Radioligand binding studies revealed a single high-affinity binding site for [(3)H]repaglinide on membranes prepared from HEK293 cells expressing wild-type (equilibrium dissociation constant [K(D)] = 0.40 nmol/l) or mutant (K(D) = 0.31 nmol/l) Kir6.2/SUR1 channels. Nateglinide and tolbutamide displaced [(3)H]repaglinide binding to wild-type channels with IC(50) values of 0.7 and 26 micro mol/l, respectively, but produced <10% displacement of [(3)H]repaglinide bound to mutant channels. This is consistent with the idea that binding of nateglinide and tolbutamide, but not repaglinide, is abolished by the SUR1[S1237Y] mutation and that the binding site for repaglinide is not identical to that of nateglinde/tolbutamide. These results are discussed in terms of a conformational analysis of the drug molecules.

Assessing the relative importance of the biophysical properties of amino acid substitutions associated with human genetic disease.

The inclusion of a mutation in a pathology-based database such as the Human Gene Mutation Database (HGMD) is a two-stage process: first, the mutation must occur at the DNA level, then it must cause a clinically detectable disease state. The likelihood of the latter step, termed the relative clinical observation likelihood (RCOL), can be regarded as a function of the structural/functional consequences of a mutation at the protein level. Following this paradigm, we modeled in silico all amino acid replacements that could potentially have arisen from an inherited single base pair substitution in five human genes encoding arylsulphatase A (ARSA), antithrombin III (SERPINC1), protein C (PROC), phenylalanine hydroxylase (PAH), and transthyretin (TTR). These proteins were chosen on the basis of 1) the availability of a crystallographic structure, and 2) a sufficiently large number of amino acid replacements being logged in HGMD. A total of 9,795 possible mutant structures were modeled and 20 different biophysical parameters assessed. Together with the HGMD-derived spectra of clinically detected mutations, these data allowed maximum likelihood estimation of RCOL profiles for the 20 parameters studied. Nine parameters (including energy difference between wild-type and mutant structures, accessibility of the mutated residue, and distance from the binding/active site) exhibited statistically significant variability in their RCOL profiles, indicating that mutation-associated changes affected protein function. As yet, however, a biological meaning could only be attributed to the RCOL profiles of solvent accessibility and, for three proteins, local energy change, disturbed geometry, and distance from the active center. The limited ability of the biophysical properties of mutations to explain clinical consequences is probably due to our current lack of understanding as to which amino acid residues are critical for protein folding. However, since the proteins examined here were unrelated, and our findings consistent, it may nevertheless prove possible to extrapolate to other proteins whose dysfunction underlies inherited disease.