Combinatorial Chemistry & High Throughput Screening - Volume 13, Issue 4, 2010

Improvement of the one-step production of L-ribose from ribitol using a recombinant Escherichia coli is described. The gene encoding the enzyme mannitol-1-dehydrogenase (MDH) from Apium graveolens has previously been codon-optimized, cloned into the constitutive pZuc10 vector, and expressed in E. coli. This MDH catalyzes the NAD-dependent conversion of mannitol to D-mannose and has the ability to convert several polyols to their L-sugar counterparts, including ribitol to L-ribose. Here, three rounds of directed evolution using libraries generated through errorprone PCR and screened using a dinitrosalicylate reagent. Mutants were selected for improved conversion of L-ribose, and the best mutant was isolated by combining two round 2 mutations. Libraries were also selected for thermal stability and screened at increasingly higher temperatures with each round of mutagenesis. An overall 19.2-fold improvement was observed with a final conversion of 46.6 ± 1.7% and a productivity of 3.88 ± 0.14 gL-1d-1 in 50 mL shaken flasks at 34°C. Further characterization of the mutants suggests that increased enzyme thermal stability and expression are responsible for the increase in L-ribose production. The mutant E. coli production strain isolated represents an improved system for largescale production of L-ribose.

An overview of the usefulness of different nanoparticles to improve the features of high throughput separation and individual and multiplexed detection bioassays is presented. Although the development of microarray and microfluidic systems has expanded the capabilities of these high throughput assays, the combined use of NPs and these devices has provided them with new applications in drug discovery, proteomic and genomic studies, and clinical diagnosis. This article reviews the wide application field of magnetic, gold, silver, semiconductor and other nanoparticles in high throughput bioassays. Also, the versatility of the detection systems described shows that NPs are useful alternatives to fluorescent dyes, which are often used in these assays.

State-of-art water gas shift catalysts (FeCr for high temperature shift and CuZn for low temperature shift) are not active enough to be used in fuel processors for the production of hydrogen from hydrocarbon fuels for fuel cells. The need for drastically lower catalyst volumes has triggered a search for novel WGS catalysts that are an order of magnitude more active than current systems. Novel catalytic materials for the high, medium and low temperature water gas shift reactions have been discovered by application of combinatorial methodologies. Catalyst libraries were synthesized on 4 inch wafers in 16x16 arrays and screened in a high throughput scanning mass spectrometer in the temperature range 200°C to 400°C. More than 200 wafers were screened under various conditions and more than 250,000 experiments were conducted to comprehensively examine catalyst performance for various binary, ternary and higher-order compositions.

A calorimetric assay is described for the high throughput screening of enzymes that produce inorganic phosphate. In the current example, cellobiose phosphorylase (EC 2.4.1.20) is tested for its ability to synthesize rare disaccharides. The generated phosphate is measured in a high throughput calorimeter by coupling the reaction to pyruvate oxidase and catalase. This procedure allows for the simultaneous analysis of 48 reactions in microtiter plate format and has been validated by comparison with a colorimetric phosphate assay. The proposed assay has a coefficient of variation of 3.14% and is useful for screening enzyme libraries for enhanced activity and substrate libraries for enzyme promiscuity.

Recently, we identified a novel class of potent cathepsin L inhibitors, characterized by a thiocarbazate warhead. Given the potential of these compounds to inhibit other cysteine proteases, we designed and synthesized a library of thiocarbazates containing diversity elements at three positions. Biological characterization of this library for activity against a panel of proteases indicated a significant preference for members of the papain family of cysteine proteases over serine, metallo-, and certain classes of cysteine proteases, such as caspases. Several potent inhibitors of cathepsin L and S were identified. The SAR data were employed in docking studies in an effort to understand the structural elements required for cathepsin S inhibition. This study provides the basis for the design of highly potent and selective inhibitors of the papain family of cysteine proteases.

Cell-based assays for the inhibition of viral infections most commonly couple a positive signal (e.g., an increase in fluorescence) to the infection itself and not to its inhibition. When performing drug screens, compounds decreasing the signal are therefore considered as putative inhibitors. However, this approach can cause the selection of many false positives, since, for example, both killing of the host cell and inhibiting viral cell-entry results in the same signal. Using a model system based on murine leukemia virus (MLV) particles pseudotyped with the G-protein of vesicular stomatitis virus (VSV-G), we have developed generic assays coupling a positive readout to the inhibition of viral transduction. Consequently, the system favors drug candidates (and concentrations thereof) that do not harm human cells and significantly decreases the probability for selecting false positives. The assay allows Z-factors of ∼0.9, takes cytotoxic side effects into account and could in theory be adapted for high-throughput screening of inhibitors against further viral species.

Fluorescence resonance energy transfer substrates were designed and tested as substrates for ADAM9. The donor/quencher pair used were 5-carboxyfluorescein (Fam) and 4-(4-dimethyl-aminophenylazo)benzoyl (Dabcyl) since they have been well studied sensitive fluorescent probes. The peptides based on precursor TNF-alpha, Dabcyl-Ser-Pro- Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Fam)-NH2 and Dabcyl-Leu-Ala-Gln-Ala-HomoPhe-Arg-Ser-Lys(Fam)-NH2, and C-terminal TGF-alpha, Dabcyl-Glu-His-Ala-Asp-Leu-Leu-Ala-Val-Val-Ala-Ala-Lys(Fam)-NH2 cleavage sites were effectively processed by ADAM9 with turnover numbers of 100 ± 20 x 10-2 min-1, 20 ± 10 x 10-2 min-1, and 10 ± 3 x 10-2 min-1. In addition, a peptide based on the 33kDa cleavage site of the low affinity receptor for IgE, CD23, Dabcyl-Leu- Arg-Ala-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(Fam)- NH2 was processed as well but with less efficiency. A more selective substrate for ADAM9 was found based on the betacellulin cleavage site. However, the valine containing precursor TNF-alpha based substrate was used to measure IC50 values of metalloproteinase inhibitors against ADAM9 since it was processed most efficiently. The tightest binding inhibitor was the Wyeth Aerst compound, TMI-1, with an IC50 of 2.1 ± 0.3 nM. In addition, GI254023, previously identified as a selective inhibitor of ADAM10, also inhibited ADAM9 with an IC50 of 280 ± 110 nM. These results demonstrate that sensitive substrates for ADAM9 can be developed that are useful in high throughput screening assays for ADAM9.

Computer simulations play a crucial role in contemporary chemical investigations, generating enormous amounts of data. The constraint of sharing data and results is regarded as a major impediment in drug discovery. Among the steepest barriers to overcome in the high throughput screening studies is the limited number of suitable, freely accessible repositories for storing drug and drug target data. By offering a uniform data storage and retrieval mechanism, various data might be compared and exchanged easily. This paper presents the stages of the MoStBioDat software platform development, originally designed for the efficient storage, management and access of SDF and PDB data. The detailed architecture and software implementation of this project are described, indicating also the disadvantages of the solutions chosen. The current implementation of the first prototype is written in Python, an open-source, high-level, object-oriented scripting language. The modular architecture of the package enables future extension with the necessary functionalities. The main objective of the MoStBioDat is to serve as an alternative, extensible open-source database derived partly from SDF and PDB files.