Combinatorial Chemical Libraries Research Articles

Dynamic high throughput screening of chemical libraries using acoustic-wave sensor system

We report a novel sensor-based high throughput screening (HTS) system for identification and quantitation of volatile substances in combinatorial chemical libraries. The measurement method employs a combination of a periodic introduction of a minute amount of a liquid sample into the HTS system, rapid evaporation of volatile components in the sample at room temperature, and dynamic measurement of a generated vapor pulse. These measurements are performed using an array of four 10 MHz acoustic-wave thickness-shear mode sensors coated with different chemically sensitive films. Developed HTS system is applied for screening of multiple samples such as those created in combinatorial chemical libraries of catalyst candidates in an industrially important arene oxidation process. The temporal modulation of the concentration of analyte vapors and measurement of both the temporal profile and the magnitude of the response improves sensor selectivity and makes possible robust identification and quantitation of arene oxidation components such as cresol and benzoquinone in multicomponent combinatorial mixtures with reduced number of sensors in the array. Different solvents such as water, acetonitrile, benzene, and toluene do not alter the response of sensors to analytes. Depending on the gas flow rate, quantitative measurements are performed 10–150 s after the sample introduction and provide significant throughput advantage over gas-chromatographic instruments. Determinations of mixtures of analytes in a variety of solvents are performed using multivariate locally weighted regression. This data analysis method provides the root mean squared error of prediction of less than 2 μg when measurements of cresol and benzoquinone amounts ranging from 0 to 50 μg are performed in 2 μL samples. This method of dynamic sensor-based measurements allows for instrument miniaturization and increases the usefulness of the instrument in space-limited applications. Upon operation of multiple sensors in parallel, effective analysis of dense combinatorial libraries of materials is possible.

Development of a system to evaluate compound identity, purity, and concentration in a single experiment and its application in quality assessment of combinatorial libraries and screening hits.

The development and use of a new assay system for the simultaneous determination of identity, purity, and concentration of sample components from combinatorial libraries produced by parallel synthesis are described. The system makes use of high-performance liquid chromatography with UV/vis photodiode array (PDA), evaporative light scattering (ELSD), chemiluminescent nitrogen (CLND), and time-of-flight mass spectrometer (TOFMS) detectors (HPLC-PDA-ELSD-CLND-TOFMS). Although these detectors have previously been utilized separately for the analysis of combinatorial chemistry libraries, the use of TOFMS along with CLND provides a synergistic combination enabling target and side-product structures to be identified and their concentrations and purities determined in a single experiment from a solution containing microgram levels of material. The CLND was found to give a linear response based on the number of moles of nitrogen present. Therefore, if the number of nitrogens per molecule is known, the concentration of each nitrogen-containing sample component may be determined utilizing an unrelated co-injected standard. A molecular formula for an impurity may often be calculated from the exact mass determined by the TOFMS and knowledge of the chemistry involved. Thus, if the sample components contain nitrogen, the concentration of every identified HPLC peak may be determined even in the absence of primary standards. This combination of detectors enabled the characterization of both target compounds and byproducts in combinatorial libraries, allowing the optimization of library synthetic procedures. This system was also used to survey the quality of libraries, enabling the selection of the best libraries for screening. This method also facilitated the characterization of samples from combinatorial libraries found as hits in high-throughput screening to establish the potency of the leads based on their actual concentration. In addition, concentrations and potencies of impurities were determined after identification of their structures, utilizing exact mass data, determination of charge states, and knowledge of the synthetic chemistry.

On-bead and solution screening approaches for genomically derived targets. Discovery of surrogate ligands and substrates using combinatorial chemistry libraries.

On-bead and solution screening approaches for genomically derived targets. Discovery of surrogate ligands and substrates using combinatorial chemistry libraries.

Natural product-derived building blocks for combinatorial synthesis. Part 1. Fragmentation of natural products from myxobacteria

Novel and unique chiral building blocks of high structural diversity were obtained by selective chemical fragmentation of natural products from myxobacteria. Subsequent modification reactions provided primary alcohol and carboxylic acid derivatives, which are suitable for the construction of combinatorial chemical libraries. The single SPOT synthesis of a hybrid structure on a polypropylene membrane was employed to demonstrate the chemical recombination of such rare building blocks on a micro-scale.

Predictive Array Design™. A method for sampling combinatorial chemistry library space

A method, Predictive Array Design™, is presented for sampling combinatorial chemistry space and selecting a sub-array for synthesis based on the experimental design method of Latin Squares. The method is appropriate for libraries with three sites of variation. Libraries with four sites of variation can be designed using the Graeco-Latin Square. Simulated annealing is used to optimise the physicochemical property profile of the sub-array. The sub-array can be used to make predictions of the activity of compounds in the all combinations array if we assume each monomer has a relatively constant contribution to activity and that the activity of a compound is composed of the sum of the activities of its constitutive monomers.

Identification of novel Ah receptor agonists using a high-throughput green fluorescent protein-based recombinant cell bioassay.

The Ah receptor is a ligand-dependent transcription factor that mediates the biological and toxic effects of polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Recent evidence also suggests a role for the AhR in normal physiology and development. Although a variety of structurally diverse chemicals are reported to bind to and activate the AhR, the full spectrum of structural chemical classes that can interact with the AhR remains to be elucidated. Large-scale analysis of the ligand binding specificity of the AhR requires the use of a high-throughput AhR bioassay system for chemical screening. We have utilized a recombinant mouse hepatoma cell line (H1G1.1c3) containing a stably integrated TCDD- and AhR-responsive enhanced green fluorescent protein (EGFP) reporter gene to screen a 1,5-dialkylamino-2,4-dinitrobenzene combinatorial chemical library consisting of 155 parental amines and up to 12 090 combinatorial products in less than 7 days for novel AhR agonists. These analyses have identified numerous parental amines as relatively potent inducers of EGFP (with EC(50)s between 8 and 1000 microM) and also have revealed several novel products of the combinatorial chemical library synthesis with EC(50)s between 10 and 100 microM. Overall, these results have not only allowed the identification of novel activators of the AhR but also demonstrate the utility of the recombinant H1G1.1c3 cell bioassay for high-throughput chemical screening.

ChemInform Abstract: Identification of a Potent and Selective Oxytocin Antagonist, from Screening a Fully Encoded Differential Release Combinatorial Chemical Library.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Characterization of small combinatorial chemistry libraries by (1)H NMR. Quantitation with a convenient and novel internal standard.

A novel silane standard, 1,4-bis(trimethylsilyl)benzene (BTMSB), is introduced for the generic quantitation of small organic molecules in DMSO-d(6) solution by (1)H NMR. This standard is an easily weighable solid and is stable for at least 1 month in DMSO solution, and its (1)H NMR spectrum contains a strong singlet in a region usually free of signals. With a set of certified standards, concentration determination with about 2% precision and accuracy is verified after solution preparation with fully automated procedures, thus making very effective the characterization of small combinatorial chemistry libraries for identity and purity when combined with other physicochemical or biochemical tests. As an example, for a set of about 400 compounds, results of (1)H NMR characterization are compared to the more customary LC-UV-MS method. NMR and MS data agree for identity on the vast majority of cases (84% positive and 5% negative), whereas the remaining cases (11%) are marked as highly impure only after NMR spectra analysis. Most importantly, determination of concentration rather than that of relative purity appears the right choice for a correct evaluation of biochemical potency.

Analysis of combinatorial chemistry samples by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method has been developed that can evaluate the purity of samples generated in combinatorial chemistry libraries. This method uses an open tube capillary (27 cm x 50 microm) along with a run buffer composed of sodium dodecyl sulfate (SDS), hydroxypropyl-beta-cyclodextrin, and sodium tetraborate coupled with UV detection. Neutral compounds and compounds that were insoluble in aqueous buffers could be analyzed under these conditions in approximately 3 min. The concentration of SDS and the concentration of hydroxypropyl-beta-cyclodextrin effected the separation. The affect on selectivity resulting from the addition of an organic modifier to the run buffer was examined. The low background absorbency of the run buffer made for easy detection of compounds that absorbed at low UV wavelengths. The quick analysis time made this suitable for analysis of combinatorial chemistry samples.

Identification of a potent and selective oxytocin antagonist, from screening a fully encoded differential release combinatorial chemical library

A library of 1296 1,4-benzodiazepines was prepared on 160 μM Tentagel beads. Compounds are attached to the beads using orthogonally cleavable linkers. The library was first screened as pools of 30 beads where 50% of the material is released and screened. GW405212X, a selective oxytocin antagonist, was identified by picking single beads from active pools.

On-line Sample Preparation for High Throughput Reversed-phase LC / MS Analysis of Combinatorial Chemistry Libraries.

An on-line sample preparation method utilizing a time-programmed autosampler is described for high throughput liquid chromatography/mass spectrometry (LC/MS). This approach is particularly helpful for the LC/MS analysis of samples which require solvents incompatible with HPLC in the sample preparation process. The on-line sample preparation approach minimizes a bottleneck in throughput and improves sample recovery under some circumstances.

Electrospray ionization gas-phase electrophoresis under ambient conditions and it's potential or high-speed separations.

A moderately high resolution nanoelectrospray ionization gas-phase electrophoresis instrument was constructed and evaluated for simple high-speed separations of several groups of compounds. The insertion of a plate containing a 1.6 cm diameter exit orifice, 2.5 cm from the location of electrospray, allowed ions to be created and desolvated under ambient conditions with minimal solvent contamination to the drift tube. Ion separation selectivity is discussed and shown to be slightly altered by changing the drift gas flow rate. Issues of using gas-phase electrophoresis as a high-speed separation technique are discussed. Gas-phase electrophoresis-spectra of selected benzodiazepines, triazine herbicides, and simple combinatorial chemistry libraries are demonstrated.

LIPID-BASED FORMULATIONS FOR ORAL ADMINISTRATION: OPPORTUNITIES FOR BIOAVAILABILITY ENHANCEMENT AND LIPOPROTEIN TARGETING OF LIPOPHILIC DRUGS

The identification of increasingly complex intracellular drug targets, escalating requirements in terms of drug potency and the use of combinatorial chemistry libraries and in vitro receptor-based ...

Techniques for increasing the throughput of flow injection mass spectrometry.

Improvements to the design and operation of a Gilson 215 multiprobe liquid-handling system have resulted in a significant increase in the throughput for flow injection molecular weight characterization of combinatorial chemistry libraries. The rapid injection sequence, and subsequent increased sample throughput, is effected by directing the entire mobile-phase flow through each of the injection loops sequentially while isolating or "dead-ending" the remaining nonactive loops. This mode of operation was accomplished by incorporating column-switching valves prior to and following the set of eight parallel injectors. Analysis rates are achieved without sacrificing the integrity of the flow injection peak profile as baseline resolution is maintained for all samples. Using this system, the total analysis time for a 96-well microtiter plate has been reduced to approximately 5 min.

PLUMS: a program for the rapid optimization of focused libraries.

PLUMS is a new method to perform rational monomer selection for combinatorial chemistry libraries. The algorithm has been developed to optimize focused libraries with specific two-dimensional and/or three-dimensional properties. A preliminary step is the identification of those molecules in the initial virtual library which satisfy the imposed property constraints; we define these molecules as the virtual hits. From the virtual hits, PLUMS generates a starting library, which is the true combinatorial library that includes all the virtual hits. Monomers are then removed in an iterative fashion, thus reducing the size of the library. At each iteration, the worst monomer is removed. Each sublibrary is selected using a global scoring function, which balances effectiveness and efficiency. The iterative process continues until one is left with a library that consists entirely of virtual hits. The optimal library, which is the best compromise between effectiveness and efficiency, can then be selected according to the score. During the iterative process, equivalent solutions may well occur and are taken into account by the algorithm, according to a user-defined parameter. The number of monomers for each substitution site and the size of the library are parameters that can be either optimized or used to constrain the selection. The results obtained on two test libraries are presented. PLUMS was compared with genetic algorithms (GA) and monomer frequency analysis (MFA), which are widely used for monomer selection. For the two test libraries, PLUMS and GA gave equivalent results. MFA is the fastest method, but it can give misleading solutions. Possible advantages and disadvantages of the different methods are discussed.

Directly coupled HPLC–NMR and HPLC–NMR–MS in pharmaceutical research and development

The methodology for the direct coupling of HPLC with NMR spectroscopy and the simultaneous double coupling of HPLC with NMR and mass spectrometry (MS) is described. Indications of the necessary technical developments to achieve this are given, and the applications of these new techniques to studies of pharmaceutical relevance are reviewed. These include studies of combinatorial chemistry libraries, synthetic chemical impurities, characterisation of drug mixtures, identification of natural products of possible pharmaceutical interest and identification of xenobiotic metabolites in human, animal and in vitro systems. In addition, HPLC–NMR has been used to investigate xenobiotic metabolite reactivity. Finally, the potential future directions of the techniques are discussed.

Available Pathways Database (APD): An Essential Resource for Combinatorial Biology

A relational database, the Available Pathways Database (APD), has been constructed of microbial natural products, their producing strains, and their biosynthetic pathways. The database allows the ready selection of donor strains for combinatorial biology experiments. It provides the same type of resource for combinatorial biology as the Available Chemicals Directory (ACD) does for combinatorial chemical library generation. Its cataloging ability can also provide insight into novel aspects of biosynthetic routes. In particular, no 10-unit Type I polyketides were found in the compilation of this edition of the APD (Version I).

Euclidean shape-encoded combinatorial chemical libraries.

A method for the encoding of split/mix combinatorial chemical libraries based on Euclidean shapes is described. The shapes are fashioned from a polymeric matrix designed to swell in common organic solvents while retaining their unique forms, and exhibit good mechanical strength. The lightly crosslinked gel-type polymer was processed into an array of Euclidean forms that serve as encoding elements in the synthesis of combinatorial chemical libraries by using the split/pool methodology. To assess the viability of this approach, a library of compounds based on a urea scaffold was prepared. The validity of this methodology was demonstrated through correct deconvolution of the library mixture by shape discrimination. Furthermore, because the shapes used have a large surface area to volume ratio, each monolith can act as an independent chemical reactor. This simplifies the analytical identification process because each compound can be prepared in significant quantities and isolated as single entities. Given the high loading capacity of the monoliths and the conceptually simple encoding strategy, it is envisioned that these Euclidean forms will find significant application in combinatorial and high-throughput synthetic chemistry.

Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

Solution-phase combinatorial synthesis and evaluation of piperazine-2,5-dione derivatives

An efficient one-pot synthesis of a 61-membered combinatorial chemistry library of piperazine-2,5-diones was accomplished. Results of combinatorial synthesis, purification, analysis, and biological evaluation are described.