Combinatorial Chemical Libraries Research Articles

The Design of Leadlike Combinatorial Libraries.

The optimization of low-potency leads into drugs is often accompanied by an increase in molecular weight (M(r)) and lipophilicity, as a consequence of affinity enhancement. Hits with affinity at µM levels discovered by screening leadlike libraries allow scope for this optimization process, as shown schematically by the distributions of M(r) for a leadlike library (1), oral drugs (2), and a typical combinatorial chemistry library (3). y=percentage with a particular molecular weight.

Efficient split synthesis for targeted libraries.

We propose a new approach for fabricating more sophisticated combinatorial chemistry libraries via split synthesis and evaluate its potential through extensive simulation. Our algorithmically intensive method promises to reduce the time and materials costs of synthesizing libraries which are (1) too large to synthesize economically by sequential or parallel synthesis, (2) too long or irregular for conventional split synthesis generation techniques, and (3) not used in sufficient quantity to justify the setup costs of array makers. It also encourages the design of more focused and interesting libraries than are typically constructed using split synthesis. Our algorithms automate the design of efficient synthesis procedures for motif-based libraries which are too complex to design by hand. Our software allows the user to select the most desirable tradeoff between minimizing the number of steps in the synthesis process and containing the combinatorial explosion of the number of compounds synthesized.

Efficient Discovery of Inhibitory Ligands for Diverse Targets from a Small Combinatorial Chemical Library of Chimeric Molecules

Living systems are mainly composed and regulated by compounds in four biochemical classes and their polymers—nucleotides, carbohydrates, lipids, and amino acids. Early combinatorial chemistry libraries consisted of peptides. The present report describes the general bioactivity and biophysical properties of a combinatorial chemical library that used glyco, nucleotidyl, and lipid building blocks. The resulting chimeric combinatorial library of 361 compounds had a confirmed cumulative hit rate of 0.16%, which is 8-fold higher than a commonly claimed industrial benchmark of 0.02%. It produced 7 structurally confirmed hits for a third of 12 proprietary drug discovery projects, and these comprised a variety of molecular targets. Diversity analyses demonstrated that despite the small number of compounds, a wider range of diversity space was covered by this library of biochemical chimeras than by a branched tripeptide library of the same size and similar generic formula.

Toward designing drug-like libraries: a novel computational approach for prediction of drug feasibility of compounds.

Prediction of the degree of drug-like character in small molecules is of great industrial interest. The major barrier, however, is the lack of a definition for drug-like character. We used the concept of the multilevel chemical compatibility (MLCC) between a compound and a drug library as a measure of the drug-like character of a compound. The rationale is that the local chemical environment of each atom or group of atoms in a compound largely contributes to the stability, toxicity, and metabolism in vivo. A systematic comparison of the local environments within a compound and those within the existing drugs provides a basis for determining whether and how much a compound is drug-like. We applied the MLCC calculations to four test sets: top selling drugs, compounds under biological testing prior to the preclinical test, anticancer drugs, and compounds known to have poor drug-like character. The following conclusions were obtained: (1) A convergent number of unique local structure types were found in the analysis of the library of the existing drugs. It suggests that the current drug library contains about 80% of all the viable types; therefore, discovery of a drug with new local structures is only an event of relatively small probability. (2) The method is highly selective in discerning drug-like compounds: most of the top drugs are predicted to be drug-like, about one-quarter of the biological testing compounds are drug-like, and about one-fifth of the anticancer drugs are drug-like. (3) The method also correctly predicted that none of the known problematic compounds are drug-like. (4) The method is fast enough for computational screening of virtual combinatorial chemistry libraries and databases of available compounds.

Selection of gp41-mediated HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements.

The trimeric, alpha-helical coiled-coil core of the HIV-1 gp41 ectodomain is thought to be part of a transient, receptor-triggered intermediate in the refolding of the envelope glycoprotein into a fusion-active conformation. In an effort to discover small organic inhibitors that block gp41 activation, we have generated a biased combinatorial chemical library of non-natural binding elements targeted to the gp41 core. From this library of 61,275 potential ligands, we have identified elements that, when covalently attached to a peptide derived from the gp41 outer-layer alpha-helix, contribute to the formation of a stable complex with the inner core and to inhibition of gp41-mediated cell fusion.

A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases.

Oxygenases catalyze the hydroxylation of a wide variety of organic substrates. An ability to alter oxygenase substrate specificities and improve their activities and stabilities using recombinant DNA techniques would expand their use in processes such as chemical synthesis and bioremediation. Discovery and directed evolution of oxygenases require efficient screens that are sensitive to the activities of interest and can be applied to large numbers of crude enzyme samples. Horseradish peroxidase (HRP) couples the phenolic products of hydroxylation of aromatic substrates to generate colored and/or fluorescent compounds that are easily detected spectroscopically in high-throughput screening. Coexpression of the coupling enzyme with a functional mono- or dioxygenase creates a pathway for the conversion of aromatic substrates into fluorescent compounds in vivo. We used this approach for detecting the products of the toluene-dioxygenase-catalyzed hydroxylation of chlorobenzene and to screen large mutant libraries of Pseudomonas putida cytochrome P450cam by fluorescence digital imaging. Colors generated by the HRP coupling reaction are sensitive to the site of oxygenase-catalyzed hydroxylation, allowing the screen to be used to identify catalysts with new or altered regiospecificities. The coupled oxygenase-peroxidase reaction system is well suited for screening oxygenase libraries to identify mutants with desired features, including higher activity or stability and altered reaction specificity. This approach should also be useful for screening expressed DNA libraries and combinatorial chemical libraries for hydroxylation catalysts and for optimizing oxygenase reaction conditions.

Combinatorial discovery process yields antimicrobial peptoids

N-alkylated glycine trimers are generically referred to as peptoids. The identification of antimicrobial peptoids from a statistically unbiased diverse combinatorial chemistry library led to the design of the optimization peptoid library that we describe in this manuscript. This optimization library was designed using structural information from the most active peptoids in the unbiased library. Screening of the optimization library for antimicrobial activity identified a single pool of peptoids with activity against both Staphylococcus aureus and Escherichia coli. The active peptoids from this pool were active against drug sensitive and drug resistant organisms and represent novel antibacterial compounds.

A planning module for performing grid search, factorial design, and related combinatorial studies on an automated chemistry workstation

The investigation of combinations of factors in a defined search space has broad application in chemistry experimentation. The factors can involve continuous variables such as concentration and temperature, or discrete variables such as chemical reactants or reagents. The number of data points rapidly becomes quite large in such factorial experimentation, which can be adequately handled through the use of an automated chemistry workstation. The automated chemistry workstation utilizes experiment templates for implementation of factorial designs. Experimental templates represent generic plans that are used repetitively with slight variation of designated parameters for the chemical system under investigation. A linkage is established between the dimensions of the search space and the variables in the experimental template. Various patterns of points can be selected, enabling the composition of full factorial or partial factorial designs. The search space points are inscribed in the experimental template, giving rise to one experimental plan per point in the search space. The experimental plans are assessed for resource availability and those for which resources are sufficient are then scheduled for parallel implementation on a microscale automated chemistry workstation. An adaptive feature allows unproductive experiments to be terminated early, de-allocating resources for use in other experiments. Experimental plans can be readily composed and implemented for fundamental studies of chemical reactions, generating response surfaces, performing parallel screening procedures, and creating indexed combinatorial chemical libraries.

Encoding of Combinatorial Chemistry Libraries by Fluorine-19 NMR

Encoding of Combinatorial Chemistry Libraries by Fluorine-19 NMR

Development of a p38 Kinase Binding Assay for High Throughput Screening.

p38 is a member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, which is activated by cellular stressors and has been shown to be a critical enzyme in the synthesis and action of proinflammatory cytokines, tumor necrosis factor-a (TNF-alpha) and interleukin-1beta (IL-1beta). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to the ATP pocket of the enzyme. These compounds inhibit the p38 kinase activity, block TNF-alpha and IL-1beta secretion both in vivo and in vitro and are found to be effective in animal models of arthritis, bone resorption, and endotoxin shock. We postulated that other classes of compounds capable of competing the binding of pyridinyl imidazole with p38 enzyme could have efficacy in the treatment of inflammatory diseases. Therefore, a simple and robust assay was developed to measure the ability of small molecules to inhibit the binding of tritium-labeled pyridinyl imidazole, SB202190, to recombinant p38 kinase. For assay development, the human p38 gene was cloned in baculovirus and then expressed in insect cells. Tritiated SB202190 was synthesized and used as the p38 ligand for a competitive filter binding assay. This assay has been used successfully to screen both synthetic and combinatorial chemical libraries for other classes of p38 kinase inhibitors.

Characterization of novel antimicrobial peptoids.

Peptoids differ from peptides in that peptoids are composed of N-substituted rather than alpha-carbon-substituted glycine units. In this paper we report the in vitro and in vivo antibacterial activities of several antibacterial peptoids discovered by screening combinatorial chemistry libraries for bacterial growth inhibition. In vitro, the peptoid CHIR29498 and some of its analogues were active in the range of 3 to 12 microg/ml against a panel of gram-positive and gram-negative bacteria which included isolates which were resistant to known antibiotics. Peptoid antimicrobial activity against Staphylococcus aureus was rapid, bactericidal, and independent of protein synthesis. beta-Galactosidase and propidium iodide leakage assays indicated that the membrane is the most likely target of activity. Positional isomers of an active peptoid were also active, consistent with a mode of action, such as membrane disruption, that does not require a specific fit between the molecule and its target. In vivo, CHIR29498 protected S. aureus-infected mice in a simple infection model.

Purine Based Combinatorial Chemistry: Solution Phase Simultaneous Addition of Functionalities. Iterative Deconvolution by Orthogonal Protection to a Single Compound with Potent Antibacterial Activity

We have recently described a method to prepare combinatorial chemistry libraries by solution phase simultaneous addition of functionalities (SPSAF).1–2 SPSAF has been used to create libraries based on the purine heterocycle. The nucleophilic sites (secondary nitrogens) in the planer heteroaromatic purine scaffold were built in via linkers. Thus, to continue the use of electophilic functionalities, as in previous libraries, a bifunctional nucleophilic linker was required. Piperazines readily served this purpose. Nucleophilic displacement of the chloro groups on 2,6-dichloropurine with piperazines provides reactive, constrained secondary amines for combinatorialization (Figure 1, 1). An additional piperazine was placed in the 9-position by alkylation of 2,6-dipiperazinylpurine. In this manner, the functionality that differentiates each pool (sublibrary) could be placed last in the synthetic scheme (fix last concept).1

NMR tools for biotechnology

Recent developments in NMR spectroscopy verify that NMR continues to be an exciting area of research. These advances can be placed into three general categories: new hardware; new techniques; and novel applications. The hardware developments include many advances in the area of flow NMR and some new probe designs. The new techniques include several ways to edit the NMR spectra of mixtures without using chromatographic separation. These new NMR tools are now allowing us to analyze complex mixtures, combinatorial-chemistry libraries, bound drugs, unstable compounds, very small samples, and heterogeneous samples.

Computational screening: large-scale drug discovery

Computational screening methods can be used to identify and to design biologically active compounds by searching large databases of compounds and can also be used in the design or mining of combinatorial chemical libraries. Computational screening methods can be used to identify and to design biologically active compounds by searching large databases of compounds and can also be used in the design or mining of combinatorial chemical libraries.

Elemental Analysis of Individual Combinatorial Chemistry Library Members by Energy-Dispersive X-Ray Spectroscopy

Elemental analysis of single combinatorial chemistry library members bound to polystyrene beads was studied by energy-dispersive X-ray spectroscopy (EDS) on a scanning electron microscope (SEM). The effects of bead geometry and electron beam exposure time on the elemental analysis results were evaluated. Elemental analysis data were highly variable on round beads, but reliable concentration data could be obtained from flattened beads held in silicon wafer bead holders. The duration of exposure time on flattened beads had no effect on elemental concentration data for the compounds studied. EDS analysis of single beads provided results similar to those obtained by combustion analysis. In summary, elemental analysis of single combinatorial chemistry library members by EDS was shown to be a reliable and fast method of analysis of single beads.

A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases.

The discovery of various protein/receptor targets from genomic research is expanding rapidly. Along with the automation of organic synthesis and biochemical screening, this is bringing a major change in the whole field of drug discovery research. In the traditional drug discovery process, the industry tests compounds in the thousands. With automated synthesis, the number of compounds to be tested could be in the millions. This two-dimensional expansion will lead to a major demand for resources, unless the chemical libraries are made wisely. The objective of this work is to provide both quantitative and qualitative characterization of known drugs which will help to generate "drug-like" libraries. In this work we analyzed the Comprehensive Medicinal Chemistry (CMC) database and seven different subsets belonging to different classes of drug molecules. These include some central nervous system active drugs and cardiovascular, cancer, inflammation, and infection disease states. A quantitative characterization based on computed physicochemical property profiles such as log P, molar refractivity, molecular weight, and number of atoms as well as a qualitative characterization based on the occurrence of functional groups and important substructures are developed here. For the CMC database, the qualifying range (covering more than 80% of the compounds) of the calculated log P is between -0.4 and 5.6, with an average value of 2.52. For molecular weight, the qualifying range is between 160 and 480, with an average value of 357. For molar refractivity, the qualifying range is between 40 and 130, with an average value of 97. For the total number of atoms, the qualifying range is between 20 and 70, with an average value of 48. Benzene is by far the most abundant substructure in this drug database, slightly more abundant than all the heterocyclic rings combined. Nonaromatic heterocyclic rings are twice as abundant as the aromatic heterocycles. Tertiary aliphatic amines, alcoholic OH and carboxamides are the most abundant functional groups in the drug database. The effective range of physicochemical properties presented here can be used in the design of drug-like combinatorial libraries as well as in developing a more efficient corporate medicinal chemistry library.

ChemInform Abstract: New Methods for Obtaining High‐Resolution NMR Spectra of Solid‐Phase Synthesis Resins, Natural Products, and Solution‐State Combinatorial Chemistry Libraries

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.

Synthesis and evaluation of paclitaxel C7 derivatives: Solution phase synthesis of combinatorial libraries

A novel and efficient two-step, automated solution phase synthesis of a 26-membered combinatorial chemistry library of paclitaxel C7 esters was accomplished using the HP 7686 Solution Phase Synthesizer. Results of combinatorial synthesis, purification, analysis, and biological evaluation are described.

A Case Study of Employing Spectroscopic Tools for Monitoring Reactions in the Developmental Stage of a Combinatorial Chemistry Library

Organic synthesis on solid-phase resins requires new applications of nondestructive analytical tools to monitor the progress of reactions. Herein we describe our novel approach to validate a five-step reaction sequence on resin. We have developed a protocol list that allows us to follow reactions step-by-step using different analytical procedures in the order of increasing experiment time. Results from these various techniques for each reaction step are contrasted and compared. The key to achieve definitive analytical data for the entire reaction sequence is the combined application of more than one analytical technique.

Detection System for Reaction-Rate Analysis in a Low-Volume Proteinase-Inhibition Assay

High-throughput screening of large combinatorial chemical libraries in biochemical assays will benefit from reduced reagent volume and increased speed of measurement. Standard assays typically are performed in 96-well microtiter plates having 200-μL well volumes and up to an hour of incubation time. In this paper, we demonstrate a technique for precise and rapid measurement of the progress of an enzymatic reaction and its inhibition with reduced volume and time (for this work, the assay was mixed at the 200-μL level and detected in 2-μL volumes with minutes of total assay time). Directly measuring the enzyme activity in the small volume format yields a precise value for the median inhibitory concentration (IC50) of an inhibitor compound. The model assay is the endoproteolytic cleavage of a small fluorogenic peptide by human neutrophil collagenase (MMP-8). The fluorogenic peptide was labeled at one end with a UV/blue fluorophore (N-methylanthranilyl) and at the other end with a quencher (dinitrophenol). To generate inhibition data, a hydroxamate peptide analog inhibitor of collagenase, actinonin, was included in the reaction. The experiments were performed using ultraviolet laser illumination (325 nm wavelength) and parallel fluorescence detection by a cooled, charge-coupled-device camera system to increase sensitivity and speed. The assay volume was reduced to 2 μL for data collection, and the total time for mixing, incubation, and measurement was less than 6 min. For comparison to a standard format, the same assay was performed in a 96-well microtiter plate in 200 μL using 30 min of incubation and measurement in a microtiter plate fluorimeter. Median inhibitory concentrations (IC50) for actinonin of 73 ± 16 and 100 ± 14 nM were obtained in the 2- and 200-μL assays, respectively. One concern with assay miniaturization and increases in throughput is a potential loss of precision and accuracy. Laser excitation and parallel detection of fluorescence is a promising approach for increased speed and reduced cost without loss of precision for proteinase inhibition assays.