Verifying liquid-handler performance for complex or nonaqueous reagents: a new approach.

Multichannel volume dispensing devices, such as automated liquid handlers, are widely used in drug discovery assays and other high-throughput screening processes. The performance of these systems is heavily based on the ability to deliver proper volumes of specific reagents. Discussed herein is the recent research on broadening existing methods for accurately assessing liquid-handler performance when dispensing complex or nonaqueous reagents. Accurate and reliable adjustment of liquid-handler protocols for varied reagent types could have far-reaching adoption in all scientific communities.

Combinatorial chemistry in cancer research

Among the different strategies to treat cancer, chemotherapy approaches are the subject of intense research efforts. There is still a high demand for new anticancer drugs exhibiting improved efficiency and selectivity for their use in combined therapy strategies. The high development of molecular and cellular biology tools has made possible the set up of simple in vitro assays, susceptible to automation, thus bringing about the possibility of rapid screening of hundreds of compounds. Chemistry has reacted to this challenge by developing a new technology: combinatorial chemistry. By this procedure large collections of compounds, known as chemical libraries, can be prepared in a rapid and efficient manner. In recent years, combinatorial chemistry has had a great impact on drug discovery programmes addressed to tackling cancer pharmaceutical targets. In this review, the contribution of this technology to the discovery of anticancer drugs that are currently in clinical trials or already in the market is discussed (AU)

Potential phosphorus recovery in a WWTP with the BCFS process: interactions with the biological process.

The BCFS process was developed to optimize the activity of denitrifying and P-removing bacteria. In this technology in combination with optimal operating conditions for biological nitrogen removal, chemical precipitation of phosphorus is used to ensure compliance with effluent standards regarding phosphorus. This work addresses the potential of the BCFS technology for phosphorus recovery and the interactions with the biological process. The TUD model calibrated for the Hardenberg WWTP was used. Nitrification was the biological process most influenced by the P stripper operation; however, further research is needed into the effect of limiting phosphate concentrations. Phosphate removal in the anaerobic reactor causes a decrease in the sludge poly-P content. The evaluation of the process operation under dynamic conditions showed that the P stripper use for phosphate recovery does not imply complicated control strategies. The use of the BCFS for phosphate recovery implies a change in the design philosophy not only to achieve the effluent requirements but also to maximize the anaerobic phosphate release and thereby recovery.

Quantitative NMR in synthetic and combinatorial chemistry.

The applications of quantitative NMR to synthetic organic chemistry are reviewed with taking into account both the small libraries (100-150 compounds) and the single, well-characterized substance. The precision and accuracy which are obtained with state of the art instrumentation--both around 1%--rival with other classical tools of quantitative analytics, and qNMR does not require a specific method setup or a standard of the same substance. This characteristic makes it the method of choice in an environment where many different molecules are investigated and reliable quantification is required. NMR may effectively replace other standard characterization tools, such as CHNS analysis, or even complex, multi-determination results as commonly required for the assessment of absolute purity or strength of a substance, when no specific standard is available. Finally, because of the high precision and intrinsic accuracy, quantitative NMR appears the ideal reference method for the validation of other, more rapid, generic techniques for quantitative analysis.

Library design for fragment based screening.

According to Hann's model of molecular complexity an increased probability of detection binding to a target protein can be expected when small, low complex molecular fragments are screened with high sensitivity instead of full-sized ligands with lower sensitivity. Analysis of the HTS summary data of Novartis and comparison with NMR screening results obtained on generic fragment libraries indicate this expectation to be true with hitrates of 0.001% - 0.151% observed in the identification of ligands with an IC(50) threshold in the micromolar range in an HTS setup and hitrates above or equal to 3% observed in NMR screening of fragments with an affinity threshold in the millimolar range. It is however necessary to keep in mind that the sets of target studied were not identical for both method and the experience in NMR screening is too limited for a final conclusion. The term hitrate as used here reflects only the success rate in the observation of ligand binding event. It must not be confused with the overall success rate of fragment and high throughput screening in the lead finding process, which can be entirely different, since the steps required to follow-up a ligand binding event to a lead are different for both methods. A survey of fragment-based lead discovery case studies given in the literature shows that in approximately half of the cases the initial hit fragment was discovered by screening a generic library, whereas in the other cases some knowledge about an initial ligands or the protein binding site has been used, whereas systematic virtual screening of fragment databases has been only rarely reported. As comparatively high hitrates were obtained, further consideration to optimize the generic fragment screening library were directed to the chemical tractability of the fragment. As several functional groups preferred by chemists for modification and linking of the fragments are also preferentially involved in interactions between the fragments and the target protein, a set of screening fragments was derived from chemical building blocks by masking its linker group by a chemical transformation which can be later on used in the chemical follow-up of the fragment hit. For example primary amines can be masked as acetamides. If the screening fragment is active the related building block can then be used for synthesis of a follow-up library.

High-throughput characterization and quality control of small-molecule combinatorial libraries.

To fully realize the potential of combinatorial synthesis and high-throughput screening for increasing the efficiency of the drug discovery and development process, issues related to compound purity must be addressed. Impurities, often present after synthesis, can lead to ambiguous screening results and inhibit the development of quality structure-activity relationships. The demand for high-throughput analytical characterization of combinatorial libraries has prompted the development of more rapid methods to keep pace with compound production. Recent progress has focused upon the development of parallel separation methods, multiplexed detector interfaces, and synergistic combinations of different detectors possessing complementary selectivities.

Quality control in combinatorial chemistry: determination of the quantity, purity, and quantitative purity of compounds in combinatorial libraries.

The quality of combinatorial libraries determines the success of biological screening in drug discovery programs. In this paper, we evaluate and compare various methods for measuring identity, purity, and quantity (yield) of combinatorial libraries. Determination of quantitative purity reveals the true library quality and often indicates potential quality problems before full-scale library production. The relative purity can be determined for every member in a large library in a high-throughput mode, but must be cautiously interpreted. In particular, many impurities are not observable by relative purity measurements using detectors such as UV(214), UV(254), and evaporative light-scattering detection. These "invisible" impurities may constitute a significant portion of the sample weight. We found that TFA, plastic extracts, inorganic compounds, and resin washout are among these impurities. With compelling evidence, we reach a conclusion that purification is the only way to remove "invisible" impurities and improve the quantitative purity of any compound even though some compounds may have a high relative purity before purification.

1H NMR spectroscopy with internal and external standards for the quantification of libraries.

A convenient and easy method based on 1H NMR spectroscopy with both external and internal standards is described for the quantification of members of libraries.

Ionic liquid acceleration of solid-phase suzuki-miyaura cross-coupling reactions.

[reaction: see text] Room-temperature ionic liquids promote various transition metal-catalyzed reactions in the solution phase. Here, for the first time, we show that these effects are translatable to solid-phase reactions. The Suzuki-Miyaura cross-coupling of 4-iodophenol immobilized on polystyrene-Wang resin with various arylboronic acids was significantly accelerated by the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)(-)]).

Quality control in manufacturing oligo arrays: a combinatorial design approach.

The advent of the DNA microarray technology has brought with it the exciting possibility of simultaneously observing the expression levels of all genes in an organism. One such microarray technology, called "oligo arrays," manufactures short single strands of DNA (called probes) onto a glass surface using photolithography. An altered or missed step in such a manufacturing protocol can adversely affect all probes using this failed step and is in general impossible to disentangle from experimental variation when using such a defective array. The idea of designing special quality control probes to detect a failed step was first formulated by Hubbell and Pevzner (1999). We consider an alternative formulation of this problem and use a combinatorial design approach to solve it. Our results improve over prior work in guaranteeing coverage of all protocol steps and in being able to tolerate a greater number of unreliable probe intensities.

A standard operating procedure for assessing liquid handler performance in high-throughput screening.

The thrust of early drug discovery in recent years has been toward the configuration of homogeneous miniaturized assays. This has allowed organizations to contain costs in the face of exponential increases in the number of screening assays that need to be run to remain competitive. Miniaturization brings with it an increasing dependence on instrumentation, which over the past several years has seen the development of nanodispensing capability and sophisticated detection strategies. To maintain confidence in the data generated from miniaturized assays, it is critical to ensure that both compounds and reagents have been delivered as expected to the target wells. The authors have developed a standard operating procedure for liquid-handling quality control that has enabled them to evaluate performance on 2 levels. The first level provides for routine daily testing on existing instrumentation, and the second allows for more rigorous testing of new dispensing technologies. The procedure has shown itself to be useful in identifying both method programming and instrumentation performance shortcomings and has provided a means to harmonizing instrumentation usage by assay development and screening groups. The goal is that this type of procedure be used for facilitating the exchange of liquid handler performance data across the industry.

Quality control in manufacturing oligo arrays: a combinatorial design approach.

The advent of the DNA microarray technology has brought with it the exciting possibility of simultaneously observing the expression levels of all genes in an organism. One such microarray technology, called "oligo arrays", manufactures short single strands of DNA (called probes) onto a glass surface using photolithography. An altered or missed step in such a manufacturing protocol can adversely affect all probes using this failed step, and is in general impossible to disentangle from experimental variation when using such a defective array. The idea of designing special quality control probes to detect a failed step was first formulated by Hubbell and Pevzner. We consider an alternative formulation of this problem and use a combinatorial design approach to solve it. Our results improve over prior work in guaranteeing coverage of all protocol steps and in being able to tolerate a greater number of unreliable probe intensities.

A novel approach to high-throughput quality control of parallel synthesis libraries.

Combinatorial chemistry is a powerful tool to enhance drug discovery efforts in the pharmaceutical industry. One type of combinatorial chemistry, parallel synthesis, is now widely used to prepare numerous compounds of structural diversity. A novel high-throughput method for quality control of parallel synthesis libraries has been developed. The method uses flow injection MS, for proof of structure and estimation of purity, and a novel direct injection CLND technique for quantitation of amount. Following the synthesis of a small molecule library, compounds analyzed using this technique were characterized by mass spectrometry, and an accurate concentration of the compound was assessed by CLND. Characterization of one compound is completed in 60 s, allowing for up to 1000 compounds to be analyzed in a single day. The data is summarized using pass/fail criteria using internally developed software.