BioInfoBank Institute

Squalene epoxidase (SE) is a key flavin adenine dinucleotide (FAD)-dependent enzyme of ergosterol and cholesterol biosynthetic pathways and an attractive potential target for drugs used to inhibit the growth of pathogenic fungi or to lower cholesterol level. Although many studies on allylamine drugs activity have been published during the last 30 years, up until now no detailed mechanism of the squalene epoxidase inhibition has been presented. Our study brings such a model at atomic resolution in the case of yeast Saccharomyces cerevisiae . Presented data resulting from modeling studies are in excellent agreement with experimental findings. A fully atomic three-dimensional (3D) model of squalene epoxidase (EC 1.14.99.7) from S. cerevisiae was built with the help of 3D-Jury approach and further screened based on data known from mutation experiments leading to terbinafine resistance. Docking studies followed by molecular dynamics simulations and quantum interaction energy calculations [MP2/6-31G(d)] resulted in the identification of the terbinafine-squalene epoxidase mode of interaction. In the energetically most likely orientation of terbinafine its interaction energy with the protein is ca. 120 kJ/mol. In the favorable position the terbinafine lipophilic moiety is located vertically inside the squalene epoxidase binding pocket with the tert-butyl group oriented toward its center. Such a position results in the SE conformational changes and prevents the natural substrate from being able to bind to the enzyme's active site. That would explain the noncompetitive manner of SE inhibition. We found that the strongest interaction between terbinafine and SE stems from hydrogen bonding between hydrogen-bond donors, hydroxyl group of Tyr90 and amine nitrogen atom of terbinafine. Moreover, strong attractive interactions were recorded for amino acids whose mutations resulted in terbinafine resistance. Our results, elucidating at a molecular level the mode of terbinafine inhibitory activity, can be utilized in designing more potent or selective antifungal drugs or even medicines lowering cholesterol in humans.

Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17β-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17β-HSD types 1, 2, 4, 5 and 7 but also against 17β-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17β-HSDs analyzed were observed. Especially, the rodent 17β-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17β-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.

In reply to the letter from Behrman (1), we want to clarify the following. We have shown that hydrolysis of the N-formylmaleamic acid generated by the ring cleavage of 2,5-dihydroxypyridine by a dioxygenase enzyme (NicX) in Pseudomonas putida KT2440 is carried out by a dedicated deformylase enzyme (NicD) (2) and is not due to an additional activity of the dioxygenase enzyme, as was previously suggested (3). Although it had been shown earlier that N-formylmaleamic acid can undergo nonenzymatic hydrolysis and that this might explain why this compound was not detected by Gauthier and Rittenberg (3), under our assay conditions the N-formylmaleamic acid generated by NicX was readily detected, thereby rendering unlikely the possibility of nonenzymatic hydrolysis as the primary mechanism of decarboxylation. On the basis of these results, we suggested in our article that the previously implied dioxygenase-mediated decarboxylation of N-formylmaleamic acid can be now explained as a result of a deformylase activity that contaminated the purified dioxygenase (2). It is of course well known that sequentially acting enzymes sometimes copurify, and, indeed, the possibility of a minor contamination in the dioxygenase preparation was allowed by Gauthier and Rittenberg to explain their own results, and the lack of N-formylmaleamic acid hydrolysis observed (3) could be due to the fact that, according to Behrman, they might have used N-formylfumaramic instead of N-formylmaleamic acid as substrate (4). We do not therefore consider that we have misstated conclusions or impugned previous work, as indicated by Behrman (1), but simply have eliminated an earlier hypothesis and demonstrated that the hydrolysis of N-formylmaleamic acid in P. putida is not a dioxygenase-mediated reaction or a nonenzymatic process but rather a NicD-catalyzed reaction.

The aerobic catabolism of nicotinic acid (NA) is considered a model system for degradation of N-heterocyclic aromatic compounds, some of which are major environmental pollutants; however, the complete set of genes as well as the structural-functional relationships of most of the enzymes involved in this process are still unknown. We have characterized a gene cluster (nic genes) from Pseudomonas putida KT2440 responsible for the aerobic NA degradation in this bacterium and when expressed in heterologous hosts. The biochemistry of the NA degradation through the formation of 2,5-dihydroxypyridine and maleamic acid has been revisited, and some gene products become the prototype of new types of enzymes with unprecedented molecular architectures. Thus, the initial hydroxylation of NA is catalyzed by a two-component hydroxylase (NicAB) that constitutes the first member of the xanthine dehydrogenase family whose electron transport chain to molecular oxygen includes a cytochrome c domain. The Fe(2+)-dependent dioxygenase (NicX) converts 2,5-dihydroxypyridine into N-formylmaleamic acid, and it becomes the founding member of a new family of extradiol ring-cleavage dioxygenases. Further conversion of N-formylmaleamic acid to formic and maleamic acid is catalyzed by the NicD protein, the only deformylase described so far whose catalytic triad is similar to that of some members of the alpha/beta-hydrolase fold superfamily. This work allows exploration of the existence of orthologous gene clusters in saprophytic bacteria and some pathogens, where they might stimulate studies on their role in virulence, and it provides a framework to develop new biotechnological processes for detoxification/biotransformation of N-heterocyclic aromatic compounds.

We present here the estimation of the upper limit of the number of molecular targets in the human genome that represent an opportunity for further therapeutic treatment. We select around approximately 6300 human proteins that are similar to sequences of known protein targets collected from DrugBank database. Our bioinformatics study estimates the size of 'druggable' human genome to be around 20% of human proteome, i.e. the number of the possible protein targets for small-molecule drug design in medicinal chemistry. We do not take into account any toxicity prediction, the three-dimensional characteristics of the active site in the predicted 'druggable' protein families, or detailed chemical analysis of known inhibitors/drugs. Instead we rely on remote homology detection method Meta-BASIC, which is based on sequence and structural similarity. The prepared dataset of all predicted protein targets from human genome presents the unique opportunity for developing and benchmarking various in silico chemo/bio-informatics methods in the context of the virtual high throughput screening.

We present here the recent update of AutoMotif Server (AMS 2.0) that predicts post-translational modification sites in protein sequences. The support vector machine (SVM) algorithm was trained on data gathered in 2007 from various sets of proteins containing experimentally verified chemical modifications of proteins. Short sequence segments around a modification site were dissected from a parent protein, and represented in the training set as binary or profile vectors. The updated efficiency of the SVM classification for each type of modification and the predictive power of both representations were estimated using leave-one-out tests for model of general phosphorylation and for modifications catalyzed by several specific protein kinases. The accuracy of the method was improved in comparison to the previous version of the service (Plewczynski et al., "AutoMotif server: prediction of single residue post-translational modifications in proteins", Bioinformatics 21: 2525-7, 2005). The precision of the updated version reached over 90% for selected types of phosphorylation and was optimized in trade of lower recall value of the classification model. The AutoMotif Server version 2007 is freely available at http://ams2.bioinfo.pl/ . Additionally, the reference dataset for optimization of prediction of phosphorylation sites, collected from the UniProtKB was also provided and can be accessed at http://ams2.bioinfo.pl/data/ .

No abstract currently available.

In their report “transmission of equine influenza virus to dogs” (21 Oct. 2005, p. [482][1]), P. C. Crawford et al. observed an unprecedented interspecies transfer of a complete equine influenza virus to the dog and the emergence of a new canine-specific influenza virus associated with acute respiratory disease. They noticed that a viral hemagglutinin (HA), a critical determinant of host species specificity of influenza virus, differs mainly in four residues (N83S, W222L, I328T, and N483T) between the equine and canine HA orthologs, out of which only one (W222L) is exposed to the serum and is most likely involved in receptor binding. Our analysis revealed an additional important mutation (N54K) located in the antibody-binding region of HA ([1][2]). This residue is highly conserved in all noncanine (94) HA sequences of the subtype H3N8 (see multiple sequence alignment at ). In contrast, a leucine residue observed in the canine HA at position 222 is also present in three equine orthologs deposited in GenBank. The figure presents a comparison of the three-dimensional models of the equine and the canine HAs created with 1HA0 ([2][3]) and 1KEN ([3][4]) structures as templates. Highlighted areas show that the N54K mutation changes the electrostatic potential on the protein surface significantly. Moreover, it is placed in the middle of an N glycosylation motif (Asn-X-Ser) and likely increases the probability of the posttranslational modification of the preceding asparagine ([4][5]). The glycosylation of HA has been shown to enable the virus to mask its antigenic sites ([5][6]). We suggest that this mutation may help the virus escape the dog's immune defense and may be part of the minimal repertoire of changes required for the host specificity transition in the observed case. ![Figure][7] The ribbon representation ( A ) and the protein surface colored by electrostatic potential ( B , C ) of 3D models of the canine (A, B) and the equine (C) influenza hemagglutinins. Five dog-specific mutations are marked (A) with visible amino acid side chains. Highlighted areas (B, C) show the highest differences in electrostatic potential caused by the N54K mutation. This picture was created with Swiss PDB Viewer. 1. 1.[↵][8] 1. D. Fleury, 2. R. S. Daniels, 3. J. J. Skehel, 4. M. Knossow, 5. T. Bizebard , Proteins (2000) 40, 572. 2. 2.[↵][9] 1. J. Chen 2. et al. , Cell (1998) 95, 409. 3. 3.[↵][10] 1. C. Barbey-Martin 2. et al. , Virology (2002) 294, 70. 4. 4.[↵][11] 1. S. H. Shakin-Eshleman, 2. S. L. Spitalnik, 3. L. Kasturi , J. Biol. Chem. (1996) 271, 6363. 5. 5.[↵][12] 1. J. J. Skehel 2. et al. , Proc. Natl. Acad. Sci. U.S.A. (1984) 81, 1779. 6. 6. M.v.G. is a fellow of Foundation for Polish Science. # Response {#article-title-2} Glycan camouflaging of HA antigenic sites is certainly a successful strategy of the human influenza virus in evasion of antibody responses elicited by previous influenza infections in adult populations. Canine influenza is a newly emerging pathogen and dogs are immunologically naive to the virus. Without the selective pressure applied by preexisting antibodies, the role of the amino acid substitution at position 54 in virus escape from antibody neutralization is probably not as important in either adaptation to or maintenance of the virus in the canine population at this time. We agree with von Grotthuss and Rychlewski that, in addition to the four amino acid substitutions we described, the N54K substitution in the HA may have contributed to the successful transfer of equine H3N8 virus to the dog. However, the effects of these amino acid mutations on HA function are undefined and are likely multifactorial. It will be very interesting to monitor the evolution of these five sites of the HA as the virus becomes endemic in the dog population and herd immunity develops from infection or vaccination. [1]: /lookup/doi/10.1126/science.1117950 [2]: #ref-1 [3]: #ref-2 [4]: #ref-3 [5]: #ref-4 [6]: #ref-5 [7]: pending:yes [8]: #xref-ref-1-1 View reference 1. in text [9]: #xref-ref-2-1 View reference 2. in text [10]: #xref-ref-3-1 View reference 3. in text [11]: #xref-ref-4-1 View reference 4. in text [12]: #xref-ref-5-1 View reference 5. in text