Combinatorial Chemistry & High Throughput Screening - Volume 11, Issue 10, 2008

Pharmaceuticals are omnipresent in waste-water world-wide. Research has shown that many pharmaceuticals are not completely removed during wastewater treatment, and as a result, this has led to their occurrence being reported in waste water treatment plant effluents, rivers and lakes, and more rarely in groundwater and in drinking water. Hence, it is only logical that pharmaceutical residues in the environment and their potential toxic effects have been recognized as one of the emerging research areas in environmental chemistry. A lack of data, especially ecotoxicological and fate data on pharmaceuticals, is evident. The extent to which data are missing should therefore be looked upon in more detail in order to trigger further political steps in performing studies concerning the risk assessment of pharmaceuticals in the environment. In this investigation, we evaluate the data-availability of 16 pharmaceuticals in 17 Internet databases which means we examine a 16 (objects) x 17 (attributes) data-matrix. The consideration of the chosen pharmaceutical in databases is coded 0 = not available, or 1 = available. For the evaluation of the data-matrix, we apply the multi-criteria decision method named METEOR (Method of Evaluation by Order Theory). In contrast to conventional multi-criteria decision aids, like the wellknown PROMETHEE, AHP, SMART, ORESTE as well as different versions of ELECTRE, we support the basic consideration of environmetrics: let first the data speak and let us then include subjective preferences in order to get a unique decision. The basis of METEOR is a data-matrix in which the objects are characterized by a set of attributes (indicators). By means of the attributes, a partial order is derived. In the subsequent steps, attributes are aggregated by a weighting procedure, allowing a high degree of involvement of experts, stakeholders and other participants. All conducted approaches show that the data-situation on the chosen test-set of 16 well-known and highly produced pharmaceuticals is far from being satisfactory. For the two well-known pharmaceuticals roxithomycin (antibiotic) and diatrizoate (contrast media), the data-situation is extremely bad, independent of how the weighting is performed. The dataavailability for diatrizoate is a little better. The best data coverage is detected for the chemicals carbamazepine, diazepam, ethinyl estradiol, 5-fluorouracil, and phenazone. The issue of pharmaceuticals in the environment and the unavailability of data necessitate much closer communication between science and medical healthcare and politicians in the future.

There exists a great amount of information on ranking methods provided in the literature, a new appealing methodology that is generally dealt in a so mathematical fashion. Ranking strategies are applied in a wide range of scientific fields, such as Decision Support, Toxicology, Environmental Research, Analytical Chemistry, Food Chemistry, QSPR-QSAR, etc. When contrasted to other multi-criteria data analyzes, Partial Order Ranking results in a very transparent and suitable way to perform comparisons among a set of objects (such as molecules) according to their attributes (molecular descriptors) values. The scope of this review is to explore such sorting ideas and to provide the non-specialist reader with insights into this formalism applied to QSPR-QSAR studies. Several concepts are explained, revising some of the research developed by different experts on the topic.

This review summarizes the use of partial order ranking (POR) techniques for the assessment of chemicals. Simple partial order ranking may advantageously be applied to give the single chemicals investigated an identity in relation to other substances. Thus, it constitutes an effective tool for the prioritization of chemicals, e.g., based on their PBT (Persistence, Bioaccumulating, Toxicity) characteristics. In more elaborate cases where a larger number of descriptors are taken into account, e.g., comprising physico-chemical characteristics, atmospheric parameters, geospecific factors, and possibly socio-economic factors, hierarchical partial order ranking (HPOR) may be applied. Thus, in a first ordering step, a series of meta-descriptors are generated that later subsequently may be used as descriptors in subsequent ordering. HPOR allows a sensible ranking model even if a relatively high number of descriptors are included. Finally, accumulation partial order ranking (APOR) is illustrated. Accumulating partial APOR is a technique where data from a series of individual tests of various characteristics are aggregated while maintaining the basics of the partial order ranking methodology. APOR offers prioritization based on mutual probabilities derived from the aggregated data. Alternatively, prioritization may be achieved based on averaged ranks derived from the APOR. The application APOR is demonstrated by an assessment of a series of potential PBT substances. In all cases, an absolute ranking can be achieved based on the average ranks of the single substances. Alternatively, ranking probabilities can be derived.

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A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved 509VSG511 tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The Km^O2 value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the 509VSG511 tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.

Glucocerebrosidase (GC) catalyzes the hydrolysis of β-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene (GBA) result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some inhibitors of GC have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. High throughput screening (HTS) of small molecule libraries for such compounds with potential for chaperone therapy requires an accurate, reproducible and sensitive assay method. We have adapted and optimized two fluorogenic GC enzyme assays and miniaturized them into the 1536-well plate format for HTS. The two substrates, 4-methylumbelliferyl β-Dglucopyranoside and resorufin β-D-glucopyranoside, have Km values of 768 μM and 33 μM, respectively, and different emission spectra. Paired screening with the two assays helps to eliminate false inference of activity due to autofluorescence or fluorescence quenching by the screened compounds. Test screens with the LOPAC library indicated that both assays were robust for HTS, and gave comparable results for GC inhibitor activities. These two assays can be used to identify both GC activators and inhibitors with potential therapeutic value.

The demand for organized storage concepts to maintain, collect and distribute compounds has grown not only at pharmaceutical companies, but also at smaller research organizations and academic laboratories where there is the demand to store and retrieve substances systematically. However, budget limitations have prevented these smaller groups from buying costly storage systems offered by specialized commercial vendors. On the other hand, within pharmaceutical companies a need for inexpensive and flexible storage concepts has developed and complements the existing automated archives. For reasons of efficiency, most companies have built centralized facilities holding large collections of internal medicinal chemistry compounds to assist various, globally distributed research programs. This standardization and centralization though is not always ideal for a global organization. Therefore, site specific and localized requirements need to be addressed to ensure quick on site access to compounds without losing the global accessibility to them. In this article, we describe an approach towards a low cost and highly flexible store concept with manual compound stores of variable design addressing local needs, created to complement the existing automated stores. A key component of our implementation is the Compound Store Manager software which is capable of administering the different global stores. The developed backend system and centralized data management facilitates the operation and integration of the stores into an existing store environment.

Procedures for cytomic screening were developed for identifying compounds with immuno-modulating properties from the crude extracts of natural products. Human peripheral blood mononuclear cells (hPB-MNCs) were first cultured with different natural crude extracts for 12 hours in culture media. By analyzing the expression of early activation CD69 marker, the potential immuno-activating properties of ethanol extracts of Calocedrus formosana were observed. By the double staining of antibodies recognizing CD69 and specific cell type markers, the increase of CD69 expressions was observed in CD3 and CD14 cell populations. To examine the immuno-activating properties in CD3 T cells and CD14 monocytes, the extracts were further purified. From NMR and mass spectra, sugiol was identified as a pure functional compound, and its immuno-enhancing activities were confirmed by CD69 expressions in the affected cell populations. Furthermore, to clarify the sugiol-affected subpopulations in CD3 T cells, CD3 T cell activation in association with increase in CD8 cytotoxic T cells subpopulation was observed. To address the effect of sugiol on each isolated cell population, we found that the expression of CD69, CD80, and CD86 increased in CD14 monocytes upon exposure to sugiol, whereas for CD3 T cells, sugiol failed to induce the expressions of CD69 and CD25. However, T cell activation by coculturing monocytes and T cells suggests that the sugiol activation of T cells in hPB-MNCs involved the accessory mechanisms of sugiol-primed monocytes. Therefore, cytomic screening as a multiple-parameter screening strategy reveals the plasticity for immuno-functional studies, leading to the applications to discover new drugs of specific immunomodulating activities.

Several applications of polystyrene-supported 1,1,3,3-tetramethylguanidine (PS-TMG) in synthetic organic chemistry have been explored. This study evidenced the effectiveness and versatility of this new member of the supported guanidine superbases as an attractive candidate to replace the bases usually employed in organic synthesis during the implementation of environmentally friendly preparative processes.