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New crystal structures of human CYP2D6 and CYP3A4 have recently been reported, and in this study, we wanted to compare them with previously used homology models with respect to predictions of site of metabolism and ligand-enzyme interactions. The data set consisted of a family of synthetic opioid analgesics with the aim to cover both CYP2D6 and CYP3A4, as most of these compounds are metabolized by both isoforms. The program MetaSite was used for the site of metabolism predictions, and the results were validated by experimental assessment of the major metabolites formed with recombinant CYP450s. This was made on a selection of 14 compounds in the data set. The prediction rates for MetaSite were 79-100% except for the CYP3A4 homology model, which picked the correct site in half of the cases. Despite differences in orientation of some important amino acids in the active sites, the MetaSite-predicted sites were the same for the different structures, with the exception of the CYP3A4 homology model. Further exploration of interactions with ligands was done by docking substrates/inhibitors in the different structures with the docking program GLUE. To address the challenge in interpreting patterns of enzyme-ligand interactions for the large number of different docking poses, a new computational tool to handle the results from the dockings was developed, in which the output highlights the relative importance of amino acids in CYP450-substrate/inhibitor interactions. The method is based on calculations of the interaction energies for each pose with the surrounding amino acids. For the CYP3A4 structures, this method was compared with consensus principal component analysis (CPCA), a commonly used method for structural comparison to evaluate the usefulness of the new method. The results from the two methods were comparable with each other, and the highlighted amino acids resemble those that were identified to have a different orientation in the compared structures. The new method has clear advantages over CPCA in that it is far simpler to interpret and there is no need for protein alignment. The methodology enables structural comparison but also gives insights on important amino acid substrate/inhibitor interactions and can therefore be very useful when suggesting modifications of new chemical entities to improve their metabolic profiles.

In drug design, it is crucial to have reliable information on how a chemical entity behaves in the presence of metabolizing enzymes. This requires substantial experimental efforts. Consequently, being able to predict the likely site/s of metabolism in any compound, synthesized or virtual, would be highly beneficial and time efficient. In this work, six different methodologies for predictions of the site of metabolism (SOM) have been compared and validated using structurally diverse data sets of drug-like molecules with well-established metabolic pattern in CYP3A4, CYP2C9, or both. Three of the methods predict the SOM based on the ligand's chemical structure, two additional methods use structural information of the enzymes, and the sixth method combines structure and ligand similarity and reactivity. The SOM is correctly predicted in 50 to 90% of the cases, depending on method and enzyme, which is an encouraging rate. We also discuss the underlying mechanisms of cytochrome P450 metabolism in the light of the results from this comparison.

To determine the prevalence of renal amyloidosis of the AA-type in a defined population, formalin-fixed specimens from the kidneys of all the cases autopsied in 1983 at The General Hospital of Malmö, Sweden, were investigated using immunohistochemical techniques. Amyloid deposits of protein AA were found in 10 of 1,158 investigated cases and the calculated prevalence was 0.86 per cent. The mean age at death of the individuals with the AA-type of amyloidosis was 79 years. Six of the cases with amyloidosis had rheumatoid arthritis. The avidin-biotin-peroxidase complex technique was found to be superior to the immunofluorescence method and a high sensitivity and specificity was achieved when sequence-specific antibodies against a synthetized nonapeptide corresponding to a hydrophilic segment of the polypeptide chain of protein AA were used in the assay. Nine cases with other types of amyloid deposits in the kidneys were also detected. None of these cases showed any AA immunoreactivity but all of them demonstrated Congophilic deposits which were immunohistochemically stained by antibodies against the amyloid P-component. The prevalence of renal amyloidosis comprising all types of amyloid protein deposits was 1.64 per cent.

gamma-Trace, a microprotein occurring in neuroendocrine cells, was demonstrated by immunohistochemical technique to be present in the calcitonin-producing C-cells of normal simian thyroid gland and of human medullary thyroid carcinoma. A comparatively high concentration of gamma-trace was demonstrated in tissue extract of neoplastic C-cells. The immunoreactive protein found in the extract had a molecular weight and electrophoretic mobility characteristic for gamma-trace.